hemorrhagic metalloproteinases from snake venoms
TRANSCRIPT
Pergamon Pharmac. Ther. Vol. 62, pp. 325-372, 1994
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved
0163-7258/94 $26.00
Associate Editor: A. L. HARVEY
HEMORRHAGIC METALLOPROTEINASES FROM SNAKE VENOMS
JON BRAGI BJARNASON*~ a n d JAY WILLIAM Foxt *Science Institute, University of Iceland, Dunhagi 3, IS-I07 Reykjavik,
Iceland t Department of Microbiology, University of Virginia Medical School, Charlottesville, VA 22908,
U.S.A.
Abstract--One of the more significant consequences of crotalid envenomation is hemorrhage. Over the past 50 years of investigation, it is clear that the primary factors responsible for hemorrhage are metalloproteinases present in the venom of these snakes. The biochemical basis for their activity is the proteolytic destruction of basement membrane and extracellular matrix surrounding capillaries and small vessels. These proteinase toxins may also interfere with coagulation, thus complementing loss of blood from the vasculature. Structural studies have shown that these proteinases are synthesized as zymogens and are processed at both the amino and carboxy termini to give the mature protein. The variety of hemorrhagic toxins found in snake venoms is due to the presence of structurally related proteins composed of various domains. The type of domains found in each toxin plays an important role in the hemorrhagic potency of the protein. Recently, structural homologs to the venom hemorrhagic metalloproteinases have been identified in several mammalian reproductive systems. The functional significance of the reproductive proteins is not clear, but in light of the presence of similar domains shared with the venom metalloproteinases, their basic biochemical activities may be similar but with very different consequences. This review discusses the history of hemorrhagic toxin research with emphasis on the Crotalus atrox proteinases. The structural similarities observed among the hemorrhagic toxins are outlined, and the structural relationships of the toxins to the mammalian reproductive proteins are described.
Keywords--Hemorrhage, metalloproteinases, reprolysins, snake toxins, zinc, atrolysins.
C O N T E N T S
1. Preface 326 2. Introduction 327
2.1. A historical perspective 327 2.2. Overview of hemorrhagic toxins 329
3. The Hemorrhagic Effect 334 3.1. Biological effect 334 3.2. Assays of hemorrhagic activity 336
4. The Hemorrhagic Toxins from C. atrox Venom 337 4.1. Introduction 337 4.2. Hemorrhagic toxin e or atrolysin E (EC 3.4.24.44) 339 4.3. Hemorrhagic toxin b (atrolysin B, EC 3.4.24.41) and hemorrhagic
toxins c and d (atrolysin C, EC 3.4.24.42) 346 4.4. Hemorrhagic toxin a or atrolysin A (EC 3.4.24.1) 349 4.5. Hemorrhagic toxins f and g or atrolysin F (EC 3.4.24.45) 350
~Corresponding author. Abbreviations--CD, circular dichroism; EAP-I, Epididimal Apical Protein I; ECM, extracellular matrix;
MMP, matrix metalloproteinase; RBC, red blood cells; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
325
326 J.B. BJARNASON and J. W. Fox
5. Structure and Classification of Hemorrhagic Toxins and their Relationship to Other Metalloproteinases 5.1. General structural characteristics of the C. atrox
hemorrhagic toxins 5.2. Pre- and pro-sequences of the C. atrox hemorrhagic toxins 5.3. Proteinase domain of hemorrhagic toxins 5.4. Disintegrin-like domain of hemorrhagic toxins 5.5. High cysteine domain of the hemorrhagic toxins 5.6. Classification scheme for venom metalloproteinases 5.7. Relationship of venom metalloproteinases to mammalian
reproductive proteases 6. Mechanism of Hemorrhage Production 7. Inhibition of Hemorrhage and Treatment
7.1 ~'" Inhibition of hemorrhage 7.2. Treatment of hemorrhage
8. Conclusions Acknowledgements References
351
351 351 352 354 355 357
358 360 363 363 365 365 366 366
l. PREFACE
Through the course of the history of civilization, venomous snakes have held a unique and particular fascination for humans. This is richly expressed in the arts, the sciences and, indeed, in religion. All peoples and cultures have realized and feared the destructive powers to life that reside in the venomous embrace of a snake's bite. Some have also believed in the healing powers of venoms or its components when used by a learned member of the community. Travel books, so popular during the period of the enlightenment, would not omit a chapter on snakes, even if the land of interest had no snakes. In such cases, this chapter would simply be the shortest one in the book saying only "No snake of any kind are to be met with throughout the whole island", as in the book of travels by N. Horrebow (1758). In more recent times of modern scientific investigation, the study of snake venoms has yielded a vast body of important information on biological systems and insights into medical problems. Indeed, some venom components are thought to hold promise as agents in the treatment of diseases and medical complications.
It is in this spirit of fascination and scientific curiosity that we embark on the task of writing this review on hemorrhagic metalloproteinases from snake venoms. It is particularly relevant now, due to the important advances and leaps in understanding that have been made in this field of investigation during the last six years since we wrote an extensive review article on this topic. In the previous review, we attempted to give a wide overall view of the major investigative efforts in the field of hemorrhagic toxins from snake venoms and cataloged all known hemorrhagic toxins and their characteristics (Bjarnason and Fox, 1988-89). In the present undertaking, we wish to look in depth at the hemorrhagic metalloproteinases that have been studied most extensively during the past decade and attempt to seek a unifying principle for them, and tie them into context with other but related biomedical developments. Our point of reference will be the hemorrhagic toxins from the Cro ta lu s a t r o x venom, the so-called atrolysins (EC 3.4.24.1, 41, 42, 44 and 45--see Table 1), since, in our view, they are the most extensively studied of the hemorrhagic toxins, both individually and as a group from a particular venom, thus forming almost a complete picture. Other extensively studied hemorrhagic toxins will also be described, in context with the atrolysins, but we shall not attempt to describe all the presently observed hemorrhagic venom components (except by' cataloging them in Table 2) since in most instances the little information available on their characteristics does not contribute much to a deeper understanding of them or to the unifying principle about them. For such a general overview, we refer to our previous review (Bjarnason and Fox, 1988-89). Here we seek to find the unifying aspects of the hemorrhagic metalloproteinases from snake venom, and we aspire to describe how these principles apply to them as a group of enzyme-toxins on the protein level, the genetic level and the biomedical level.
Hemorrhagic metalloproteinases from snake venoms 327
2. INTRODUCTION
2.1. A HISTORICAL PERSPECTIVE
The manifestations of local tissue damage, such as hemorrhage and myonecrosis, are among the most dramatic effects of envenomation by crotalid and viperid snakes. In cases of less severe envenomation, the hemorrhagic effect is usually localized at the site of the bite. However, hemorrhage can be found spread widely through a substantial area of the involved extremity. In cases where the envenomation is severe, bleeding in organs distant from the site of envenomation, such as heart, lungs, kidneys and brain, may also occur. From the biochemical investigations on these toxins over the past 30-40 years, the nature of the venom hemorrhagic toxins and their mechanism of activity are now becoming clear. Virtually all of the hemorrhagic toxins isolated and characterized thus far have been determined to be metalloproteinases, and those that have been analyzed for metal content have been found to contain a zinc atom (Table 1) (Bjarnason and Fox, 1988-89; Kini and Evans, 1992).
The venoms of snakes are usually composed of a complex mixture of organic and inorganic components. Insoluble tissue debris is also often noted in the venom from milked snakes. The inorganic constituents of the venoms include: Ca, Cu, Fe, K, Mg, Mn, Na, P, Co, and Zn (Friederich and Tu, 1971). Not all of these metals are found in every type of venom, and the amount of each metal varies with the species of snake. The biological role of each of the metals is not clear; however, it is likely that some of them, such as Ca, Mg, and Mn, are quite important for the stabilization of certain venom proteins, while others, in particular, Zn, Cu, Fe, and Co, may be actual participants in the catalytic mechanisms of certain venom enzyme components, such as the metalloproteinases. It is convenient to divide the organic compounds of the venom into protein- aceous and non-proteinaceous material. The majority of the crude venom is composed of proteinaceous components. The other compounds include carbohydrates (in glycoproteins), lipids (primarily phospholipids), biogenic amines (particularly abundant in Viperidae and Crotalidae venoms), nucleotides and amino acids.
Early investigations of the mechanism of snake venom-induced hemorrhage were greatly hampered by the lack of sufficiently purified hemorrhagic venom components. This was due both to the complexity of the venoms and to the relatively primitive state of protein purification techniques in the 1950s and 1960s. For these reasons, the degree of purity of the hemorrhagic components isolated in the 1960s and early 1970s is, in many cases, suspect. The past two decades, however, have seen a proliferation in the numbers of purified hemorrhagic toxins, to the extent that today five dozen toxins are known. However, fewer than 10 of these toxins have been studied to the extent that their sequences are known. However, two X-ray crystallographic structures of hemorrhagic metalloproteinases, atrolysin C (EC 3.4.24.42) and adamalysin (EC 3.4.24.46), have been solved recently (J. W. Fox, unpublished data and Gomis-Riith et al., 1993, respectively).
In the quest for understanding of the mechanisms of hemorrhage, the search for the biochemical activities, enzymatic or otherwise, associated with the hemorrhagic toxins, and supposedly
TABLE 1. Hemorrhagic Toxins that have Trivial Names and Enzyme Commission Numbers Assigned by the International Union o f Biochemistry
Snake venom Toxin Trivial name EC number
Crotalus atrox Hemorrhagic toxin a Atrolysin A 3.4.24.1 Crotalus atrox Hemorrhagic toxin b Atrolysin B 3.4.24.41 Crotalus atrox Hemorrhagic toxin c Atrolysin C 3.4.24.42 Crotalux atrox Hemorrhagic toxin d Atrolysin C 3.4.24.42 Crotalus atrox Hemorrhagic toxin e Atrolysin E 3.4.24.44 Crotalus atrox Hemorrhagic toxin f Atrolysin F 3.4.24.45 Crotalus adamanteus Proteinase I and II A d a m a l y s i n 3.4.24.46 Crotalus horridus horridus Hemorrhagic protease IV Horrilysin 3.4.24.47 Crotalus ruber tuber Hemorrhagic toxin 2 Rube r ly s in 3.4.24.48 Bothrops jararaca Bothropasin Bothropasin 3.4.24.49 Trimeresurusflavoviridis HR-IA (and IB) Trimerelysin 3.4.24.52 Trimeresurus mucrosquamatus Mucrotoxin A Mucrolysin 3.4.24.54
328 J.B. BJARNASON and J. W. Fox
responsible for their biological effect, has been a primary endeavor of the investigators of these toxins through the years. Since Crotalidae and Viperidae venoms are strongly hemorrhagic and contain proteolytic activities, it was proposed as early as 1930 by Houssay that venom proteinases were responsible for inducing hemorrhage. However, there was little experimental evidence to support this supposition, and the absence of proteolytic activity from a hemorrhagic toxin from the venom of Trimeresurusf lavovir idis was reported as early as 1960 by Ohsaka et al.
For the next two decades, or until the report on five hemorrhagic metalloproteinases appeared in 1978 (Bjarnason and Tu, 1978), there were conflicting reports on the proteolytic properties of hemorrhagic toxins (Bjarnason and Fox, 1988-89). In certain cases, some isolated proteinases from snake venom were found to have hemorrhagic activity (Oshima et al., 1968), but in other cases, the hemorrhagic activity could be separated from the proteolytic activity when casein was used as substrate (Takahashi and Ohsaka, 1970). The proteolytic assay usually used in the early period of hemorrhagic toxin research was the method developed by Kunitz using the milk protein casein as the protein substrate (Kunitz, 1947) and detection of trichloroacetic acid soluble peptide products after timed incubation. In this assay, the proteinase is incubated with casein and after a set reaction time, the mixture is subjected to acid precipitation with trichloroacetic acid. The resultant digestion fragments that remained in solution were then quantitated as absorbance at 280 nm, and the value used as a measure of the proteolytic action of the enzyme on the substrate.
There are, however, two important limitations to this technique as it affects the hemorrhagic peptidases from snake venoms. In our experience, the hemorrhagic toxins are rather specific with respect to their sites of action and substrate preferences and, consequently, few peptide bonds of casein may be cleaved. Furthermore, the bonds that are cleaved may not give rise to soluble peptides, particularly if only a few bonds of casein are cleaved. The second limitation with the Kunitz method is that the monitoring of soluble pepetides by their abaorbance at 280 nm, can yield rather low sensitivity, which is dependent on the amino acid composition of the peptides released. For enzymes with relatively weak activity on casein, TCA precipitation followed by 280 nm absorbance monitoring may, in fact, be so insensitive that the enzyme may not appear to be active on this substrate, leading to the false conclusion that the enzyme is not proteolytic.
The attempt to establish with reasonable certainty whether a purified hemorrhagic toxin contains proteolytic activity requires the application of well-established, sensitive proteolytic activity assay methods. However, it should be understood that it is theoretically impossible to prove that an enzyme is not proteolytic, due to the varied and specialized specificities towards different proteins and the multitude of peptide bonds available for cleavage. In the last 15 years, several methods have been used with success for the detection of the proteolytic activities of hemorrhagic toxins. These generally can be divided into two classes: protein substrates and peptide substrates.
The commonly used protein substrates, which give a rather qualitative assay of proteolysis, include azocoll, azoalbumin, azocasein, and hide powder azure. Various hemorrhagic toxins have been shown to be relatively active towards these substrates (Bjarnason and Fox, 1987, 1988-89; Nikai et al., 1986). The assays are simple to perform and sensitive due to the release of a strongly absorbing dye upon proteolysis of the substrate. Unfortunately, due to the nature of the binding of the dye to the substrate, the insolubility of some of these substrates, and the diversity of peptide bonds cleaved, meaningful kinetic analysis of these digestions cannot be performed. Lin et al. (1969) introduced a very sensitive and more quantitative assay for proteolytic enzymes, which used N,N-dimethylhemoglobin or N,N-dimethylcasein as substrates. They estimated an approximate 100-fold increase in the sensitivity of this assay over that of the Kunitz method. The increase in sensitivity is based on the use of trinitrobenzenesulfonic acid to detect the appearance of new terminal amino groups. This assay was used with success in the isolation and kinetic characteriz- ation of the five hemorrhagic toxins from C. a trox venom (Bjarnason and Tu, 1978; Bjarnason and Fox, 1983).
The use of peptides and small chromogenic substrates has proven advantageous with the hemorrhagic toxins in certain instances, particularly where the substrate specificities are known and kinetic data are sought. The oxidized B chain of insulin has been used with many of the hemorrhagic toxins for analysis of specificity of peptide bond hydrolysis. If properly applied, this method can yield kinetic data (Civello et al., 1983b; Bjarnason and Fox, 1983; Fox et al., 1986; Bjarnason et al., 1988). This method has been used with success to show a hemorrhagic toxin, which
Hemorrhagic metalloproteinases from snake venoms 329
had previously been determined to be devoid of proteolytic activity, to be proteolytic (Nikai et al., 1986, 1987). Due to tradition and convenience, the oxidized B chain of insulin has been the initial choice of peptide substrate in these studies. To date, many of the hemorrhagic toxins have been assayed for proteolysis of the B chain of insulin, and useful comparisons of the cleavage sites among the hemorrhagic toxins and, indeed, with non-hemorrhagic proteinases, can be made (Bjarnason and Fox, 1988, 1988-89).
Other small peptides and chromogenic substrates have been tested for use in proteolytic assays of hemorrhagic toxins (Bjarnason and Tu, 1978; Nikai et al., 1986; Civello et al., 1983b). In general, due to the apparent high degree of substrate specificity of the hemorrhagic toxins and the length requirements that they demonstrate for their substrates, most hemorrhagic toxins have not shown significant activity on these small substrates. These requirements have been determined by the authors of this review with C. a trox hemorrhagic toxins c and d (Fox et al., 1986). These toxins were demonstrated to have both size and sequence specificity in their hydrolysis of small peptides.
One small substrate that has been useful in certain applications is the fluorogenic peptide 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzyl amide, upon which five of the hemorrhagic toxins from C. a trox are active (see Section 4 and Fox et al., 1986). All of these hemorrhagic toxins cleave the -Gly-Leu-bond to yield a change in fluorescence in the reaction mixture. This peptide has proven to be extremely useful to the authors of this review for the routine quantitative analysis of the proteolytic activity of the C. a trox hemorrhagic toxins, as well as in the assays of inhibitors to these enzymes. It may, indeed, be a useful general substrate for all hemorrhagic toxins.
As more specificity data is gained on the various hemorrhagic toxins, better substrates, perhaps similar to the one mentioned above, can be developed. However, to obtain detailed kinetic data and substrate specificity information, similar to that for the hemorrhagic toxins c and d from C. atrox venom, a wide variety of specifically designed peptide substrates of varying lengths and sequences is required. Such knowledge would be instrumental in developing the optimal peptide substrate for a particular hemorrhagic toxin (Fox et al., 1986).
In a review written in 1986-87, we discussed the results of biochemical studies on 42 hemorrhagic toxins from 13 species of snakes (Bjarnason and Fox, 1988-89). At that time, some of these toxins had been under investigation for more than 25 years. The development of this field of research was rather slow during the first half of this period due to the complexity of the snake venoms and the lack of powerful purification techniques. Uncertainties relating to the concept of proteolysis and the methods of detecting proteolytic activity also caused considerable confusion in the field. During the past 15 years, however, we have witnessed rapid progress in our understanding of these toxins, partly due to the extensive research on the function and structure of hemorrhagic toxins from C. atrox venom, and partly due to the sequencing of some hemorrhagic and non-hemorrhagic metalloproteinases from other snake venoms. This will be our main concern in the present review article.
2.2. OVERVIEW OF HEMORRHAGIC TOXINS
The important questions to address, now and in the near future, in the study and comparison of the hemorrhagic toxins from the various families and genera of snakes, are the magnitude and nature of the hemorrhagic and proteolytic activities of the toxins on the one hand, and the chemical composition and protein structure of the toxins on the other. In spite of a wealth of diverse information on the numerous toxins, it was not feasible five years ago for very meaningful comparisons of the toxins to be made, primarily due to the lack of the appropriate data on their sequences and their proteolytic functions on specific protein substrates. Also, in some cases, data on similar phenomena from different laboratories are not readily comparable, for various reasons.
Regardless of these inherent difficulties, we felt that an attempt should be made to draw conclusions and form a generalized picture of the hemorrhagic toxins from the information available. We also acknowledged that this should be refined, modified and corrected as more detailed information became available. We suggested that the hemorrhagic toxins could be assigned into three categories or classes based on their sizes, as expressed by the published molecular masses (Bjarnason and Fox, 1988-89). The relevance of this could naturally be challenged, but as more recent results indicate, it does not appear to have been a totally futile exercise (Hite et al., 1994).
330 J.B. BJARNASON and J. W. Fox
Also, the categories thus formed appeared to be related to the hemorrhagic potencies of the toxins, and, therefore, could possibly serve to identify classes of the hemorrhagic toxins. We had previously suggested the existence of two classes of toxins according to their molecular masses: the large and small hemorrhagic toxins (Fox and Bjarnason, 1983). In light of the additional data available, we proposed the assignment of the hemorrhagic toxins into three classes based on their sizes: class I, the small toxins, having molecular masses of 20-30 kDa; class II, the medium-size toxins, with molecular masses of 30450 kDa; and class III, the large and most potent hemorrhagic toxins, having molecular masses of 60-100 kDa (Bjarnason and Fox, 1988-89).
Unfortunately, sequence data on the toxins were not available at that time, if the short segment of sequence from HT-c and HT-d from C. atrox venom is discounted (Bjarnason and Fox, 1987). Ultimately, the best method of classification will be based on sequence comparisons, which are now becoming feasible and, indeed, substantiate our previous suggestion of three classes of hemorrhagic proteinases. Perhaps even four classes based on size or, as now evident, domain structure, exist (see Sections 4 and 5, Table 2 and Hite et al., 1992b, 1994).
During the last six years, several additions have been made to the list of hemorrhagic metalloproteinases we presented in our previous review, so that we now count more than 60 toxins from 23 species of snakes (Table 2). The important recent developments, in our view, have not been the proliferation in the number of hemorrhagic toxins, but, as previously stated, the closer scrutiny of a few of them, yielding insights into the structure and function of these enzymes and opening vistas previously unseen. We think that it is now important not to purify and study new and hitherto unknown toxins, but rather to study in more detail the 65 hemorrhagic toxins we already know, particularly by sequencing them and studying their mode of action.
The hemorrhagic components from the various snake venoms have been assigned a multitude of different names by the researchers who purified them, such as hemorrhagic toxins, hemorrhagic proteases, hemorrhagins, hemorrhagic principles, hemorrhagic factors or simply proteases, along with designating numbers or letters. Since hemorrhage is one of the most pronounced basic effects of crotalid snake envenomation, we have chosen to refer to the venom components responsible for these effects simply as hemorrhagic toxins. It would probably be beneficial if a consensus could be reached on this issue of nomenclature by the researchers in the field. We would thus suggest that they be termed hemorrhagic toxins along with designating letters and the species name of the snake from which the venom came. Recently, the better known and extensively studied toxin metalloproteinases have been assigned common names and EC numbers by the Enzyme Nomen- clature Committee of the International Union of Biochemistry (Table 1). The names, such as atrolysin for hemorrhagic metalloproteinases from C. atrox venom and trimerolysin for toxins from T. flavoviridis, are not as yet in wide use, but, with time, should gain increased acceptance and usage, due to their simplicity, convenience, and efficacy in classification (Table 1).
As previously stated, we are now aware of 65 hemorrhagic toxins from venoms of 24 species of snakes. Most of these, or 62 toxins, have been found to contain proteolytic activities, and in all likelihood, they are metalloproteinases (Table 2). At least another 29 snake venom metallo- proteinases have been purified and characterized to some extent. Of these, 15 have been determined to be non-hemorrhagic and 14 have not been assayed for hemorrhagic activity (Kini and Evans, 1992). In Table 2, the 65 known hemorrhagic toxins are arranged into four classes according to molecular mass, as previously suggested. These classifications in effect could be similar to those used for the trypsin-like serine proteinases. These are subdivided according to specificities, for instance, into the trypsin class, the chymotrypsin class and the elastase class. This may also be the case for the hemorrhagic metalloproteinases. However, these toxins cannot be classified with certainty according to domain structure, either on the protein or the genetic level, without knowing their protein or nucleic acid sequences. That will be one of the major topics of Section 5.
Of the 65 hemorrhagic toxins mentioned above, about half of them, or 31 toxins, belong to group I (the small toxins), 20 fall into group II (the medium-size toxins), 11 are placed in group III (the large hemorrhagic toxins) and the remaining 3 are put into group IV (the very large but not so active hemorrhagic toxins) (Table 2). It should be recognized, and clearly stated, that these assignments are based primarily on the molecular masses of the hemorrhagic toxins, and, as a secondary consideration, on their hemorrhagic activities, where such considerations are relevant. Thus, large and potent hemorrhagic toxins would belong to class III, whereas medium-size and
Hemorrhagic metalloproteinases from snake venoms 331
fairly large toxins (60 kDa) with relatively low hemorrhagic activities would be assigned to class II, but the distinction between these two classes is presently uncertain (see Section 5).
Many of the large toxins are very potent hemorrhagic agents, according to their published minimum hemorrhagic doses. Indeed, all of the most potent hemorrhagic toxins fall into class III. These toxins include HR-1 from Agkistrodon halys blomhoffii venom, proteinase H from C. adamanteus venom, hemorrhagic toxin a from C. atrox venom and HR-1A and HR-2A from T. flavoviridis venom. From their amino acid compositions among other data, proteinase H and hemorrhagic toxin a may be homologous enzymes from different venoms that define a subclass termed IIIa. Also, HR-1 from A. halys blomhoffii and HR-2A and 2B from T. flavoviridis could be members of this toxin subclass (III-A). However, more information, especially on their amino acid sequences, is needed to corroborate this classification. One might also speculate that the very large toxins of group IV with low hemorrhagic potency, such as HR-II from A. halys blomhoffii venom and mucro toxin A from T. mucrosquamatus, are four-domain toxins and related to RVV-X, coagulation factor X activating enzyme, from Vipera russelli venom, suggestive of a fourth group in this classification scheme. Again, more sequence data is needed to confirm these speculations.
Some of the most active small toxins of class I, with similar amino acid compositions and with isoelectric points in the weakly acidic range, appear to define another subclass of homologous enzymes, which we term I-A. The most obvious members of this subclass are hemorrhagic toxin e from C. atrox venom, which would be the reference for this subclass, and the apparent homologs from A. acutus venom, Ac-1, Ac-2, and AaH-I. Other potential members of this subclass are Ac-5 and AaH-II, also from A. acutus venom, HR-1, from T. gramineus venom, and HR-a, from T. mucrosquamatus venom, although more data on these toxins, especially on their sequences and peptidase substrate specificity, is required for these suggested relationships to be confirmed. The basicity of some of the toxins in class I is not necessarily suggestive of a separate subclass for these toxins. Thus, the basic yet potent hemorrhagic toxins, such as HR-2a and HR-2b from T. flavoviridis venom and possibly even the low-potency HR-b from T. mucrosquamatus venom, may also belong to this same subclass A of class I.
Some of the toxins in class I are so weakly hemorrhagic that it is questionable whether they should be considered hemorrhagic toxins at all. We now assign these toxins to a family designated I-B. Indeed, different biological activities have been associated with some of these toxins. Based on their molecular masses, amino acid compositions, low hemorrhagic activities, substrate specificities and amino acid sequences, the toxins HT-c and HT-d from C. atrox venom define this subclass (Bjarnason and Tu, 1978; Bjarnason and Fox, 1986, 1987; Fox et al., 1986). The basic hemorrhagic toxin HT-b, also from C. atrox venom, clearly belongs to this subclass of metallo- proteinases based on the same criteria as above, in particular the substrate specificity on insulin B chain (Table 4), and its recently obtained amino acid sequence as deduced from the cDNA sequence (Fig. 4, Section 5) (Bjarnason et al., 1988; Hite et al., 1994). Proteinase I and proteinase II, from C. adamanteus venom, as well as moojeni protease A from Bothrops moojeni venom, are also likely members of this subclass. The basic hemorrhagic toxin from A. acutus venom with low hemorrhagic potency, termed AaH-III, and the so-called fibrinolytic protease probably also belong to this I-B subclass of metalloproteinases.
At this point, there are eight published amino acid sequences of metalloproteinases from snake venom to be found in the scientific literature, seven of them hemorrhagic toxins (Hite et al., 1994). In addition to this, two more sequences, those of HT-a and HT-b, are known to the authors of this review and one, that of Jararhagin, has been recently published (Paine et al., 1992; Hite et al., 1994). It is our opinion that toxins that belong to the same subclass from the same venom would have close to 100% sequence identity (99.5% for HT-c and d; 78.3% for HT-b and d). Members of a subclass isolated from venom of different species would probably demonstrate sequence identities between 50-100%. Also, toxins belonging to different subclasses could have considerable sequence identity, such as that observed between HT-d and HT-e (54% homology), with sequence identities in the range of 20-70%, depending on the species similarities and toxin relationships.
Of the 65 hemorrhagic toxins mentioned in this review, proteolytic activities have been found associated with all but two of them. Hemorrhagic toxin HR-3 from V. palaestinae had no detectable gelatinase and caseinolytic activity. As previously explained, this does not constitute a proof that these two toxins are not proteolytic. In this respect, it is noteworthy that hemorrhagic toxins HR-2a
Ven
om
Clr
.~ !
A
. ac
utus
F
P
,4.
aeut
us
Ac-
I A
. ac
utus
A
c-2
A.
acut
us
Ac-
5 A
. ac
utus
A
aH-I
A
. ac
utus
A
aH-I
I A
. ac
utus
A
aH-I
II
Bit
is a
riet
ans
HT
-I
Bot
hrop
s ja
rara
ca
HF
- 1
Bot
hrop
s m
ooje
ni
MP
-A
C.
adam
ante
us
Pro
teas
e I
C.
adam
ante
us
Pro
teas
e II
C
. at
rox
HT
-b
C.
atro
x H
T-c
C
. at
rox
HT
-d
C.
atro
x H
T-e
C.
b. b
asil
iscu
s B
-I
C.
b. b
asil
iscu
s B
-2
C.
rube
r ru
ber
HT
-2
C.
tube
r ru
ber
HT
-3
C.
s. s
cutu
latu
s P-
13
Lac
heri
s m
uta
mut
a L
HF
-II
T. f
lavo
viri
dis
HR
-2a
T. f
lavo
viri
dis
HR
-2B
T
. fla
vovi
ridi
s (O
kina
wa)
H
R-2
a T
. fla
vovi
ridi
s (O
kina
wa)
H
R-2
B
T.
gram
ineu
s H
R-I
T
. m
ucro
squa
mat
us
HR
-a
T.
m, u
cros
quam
atus
H
B-b
V
. le
beti
na
Leb
etas
e C
eras
tes
cera
stes
C
eras
tase
F-4
TAB
LE 2
. L
ist
and
Pro
pose
d C
lass
ific
atio
n o
f K
now
n H
emor
rhag
ic
Tox
ins
Isol
ated
fro
m S
nake
V
enom
Min
imum
P
ropo
sed
Mol
ecul
ar
hem
orrh
agic
su
bcla
ss
mas
s do
se
Pro
teol
ytic
as
sign
men
t.
Tox
in
kDa
/z g
acti
vity
S
ubcl
ass
Ref
eren
ces
24
3.81
+
I-B
24
.5
0.22
+
I-A
25
0.
43
+ I-
A
24
0.37
+
I-A
22
0.
4 +
I-A
22
1.
5 +
I-A
22
10
+
I-B
N
.D.
N.D
. 0.
1 +
22.5
N
.D.
+ I-
B
24.6
N
.D.
+ I-
B
23.7
N
.D.
+ I-
B
24
3 +
I-B
24
8
+ I-
B
24
11
+ I-
B
25.7
1
+ I-
A
27
< 10
+
27.5
<
10
+ 24
0.
3 +
25
1.4
+ 27
1-
2 +
I-A
23
.5
+ 23
0.
07
+ I-
A
24
0.07
+
I-A
24
+
19
+ 24
+
15
1.7
27
1.3
23.7
+
I-B
22
.5
200
+ I-
B
Ouy
ang
and
Hua
ng,
1976
, 19
77
Moi
l et
al.
, 19
84;
Nik
ai e
t al
., 19
91
Mor
i et
al.
, 19
84
Mor
i et
a/.
, 19
84
Xu
et a
L,
1981
X
u et
al.
, 19
81
Xu
et a
l.,
1981
M
ebs
and
Pan
holz
er,
1982
A
ssak
ura
et a
l.,
1986
A
ssak
ura
et a
l.,
1985
K
urec
ki a
nd L
asko
wsk
i, 1
978;
Kur
ecki
et
al.,
1978
K
urec
ki a
nd L
asko
wsk
i, 1
978;
Kur
ecki
et
al.,
1978
B
jarn
ason
and
Tu,
19
78;
Bja
rnas
on e
t al
., 19
88;
Hit
e et
al.
, 19
94
Bja
rnas
on a
nd T
u,
1978
; F
ox e
t al
., 19
86;
Sha
nnon
et
al.,
1989
B
jarn
ason
and
Tu,
19
78;
Bja
rnas
on a
nd F
ox,
1987
; S
hann
on e
t al
., 19
89
Bja
rnas
on a
nd T
u,
1978
; B
jarn
ason
and
Fox
198
7; H
ite
et a
l.,
1992
a M
olin
a et
al.
, 19
90
Mol
ina
et a
l.,
1990
M
oil
et a
l.,
1987
; T
akey
a et
al.
, 19
90b
Mor
i et
al.
, 19
87;
Tak
eya
et a
l.,
1990
b M
arti
nez
et a
l.,
1990
S
anch
ez e
t al
., 19
91
Tak
ahas
hi a
nd O
hsak
a 19
70;
Nik
ai e
t al
., 19
87
Tak
ahas
hi a
nd O
hsak
a 19
70;
Nik
ai e
t al
., 19
87
Yon
aha
eta
/.,
1991
Y
onah
a et
al.
, 19
91
Ouy
ang
and
Hua
ng,
1979
N
ikai
et
al.,
1985
b N
ikai
et
al.,
1985
b S
iigu
r an
d S
iigu
r, 1
991
Dao
ud e
t al
., 19
86a,
b
>.
z ga.
D
X
CIJ
m-l
l A
. ac
utus
A
c-3
57
A.
acut
us
Ac-
4 33
A
. bi
line
atus
B
ilit
oxin
48
B
. ja
rara
ca
Bot
hrop
asin
48
B
. ne
uwie
di
NH
F-a
46
B
. ne
uwie
di
NH
F-b
58
C
allo
sela
sma
roto
stom
a H
P-I
38
C
. at
rox
HT
-f
64
C.
atro
x H
T-g
60
C
. h.
hor
ridu
s H
P-I
V
57
C.
r. r
ubbe
r H
T-1
60
V
. a.
am
mod
ytes
H
T-I
60
V
. a.
am
mod
ytes
H
T-2
60
V
. a.
am
mod
ytes
H
T-3
60
V
. be
rus
beru
s H
MP
56
.3
V.
pala
esti
nae
HR
-!
60
V.
pala
esti
nae
HR
-2
60
V.
pala
esti
nae
HR
-3
60
T.
vlav
ovir
idis
H
R-I
46
A
trac
tasp
is e
ngad
desi
s H
T-I
50
0.95
0.
31
0.5
1.4
4 0.2
+ + + + + + + + + + + + + + + + + N
.D.
+ N
.D.
Cla
~s-l
ll
A.
hali
s bl
omho
ff~i
H
R-I
85
0.
03
+ B
. ja
rara
ca
HF
-2
50
0.02
+
B. j
arar
aca
H F
-3
62
0.0
! 5
N.D
. B
. jar
arac
a Ja
raha
gin
52
+ C
. ad
aman
teus
P
rote
inas
e H
85
.7
0.02
+
C.
atro
x H
T-a
68
0.
04
+
V.
aspi
s as
pis
HT
- 1
67
0.11
+
T. f
lavo
viri
dis
HR
- 1A
60
0.
02
+
T. f
lavo
viri
dis
HR
- 1B
T
. gr
amin
eus
HR
-2
Oph
ioph
agus
han
nah
Han
naht
oxin
60
82
66
95
94
Cla
ss-I
V
A.
h. b
lom
hoff
ii
L.
mut
a m
uta
T.
muc
rosq
uam
atus
N.D
., n
ot d
etec
ted.
0.01
0.7
0.1
0.6
2.3
HR
-II
LH
F-I
M
ucro
toxi
n
+ + + + +
Moi
l et
al.
, 19
84
Mor
i et
aL
, 19
84
Imai
et
aL,
1989
M
ande
lbau
m e
t al
., 19
82
Man
delb
aum
et
aL,
1984
M
ande
lbau
m e
t aL
, 19
84
Ban
do e
t al
., 19
91
Nka
i et
aL,
198
4 N
ikai
et
aL,
1985
a C
ivel
lo e
t aL
, 19
83a,
b M
oil
et a
l.,
1987
J.
W.
Fox
, un
publ
ishe
d da
ta
J. W
. F
ox,
unpu
blis
hed
data
J.
W.
Fox
, un
publ
ishe
d da
ta
Sam
el a
nd S
iigu
r, 1
990
Ova
dia,
197
8a
Ova
dia,
197
8a
Ova
dia,
197
8a
Yon
aha
et a
L, 1
991
Ova
dia,
198
7
Om
ori
et a
l.,
1964
; O
shim
a et
aL,
197
2 A
ssak
ura
et a
l.,
1986
A
ssak
ura
et a
L, 1
986
Pai
ne e
t aL
, 19
92
Kur
ecki
and
Kre
ss,
1985
B
jarn
ason
and
Tu,
19
78;
Bja
rnas
on e
t al
., 19
88,
Hit
e et
al.
, 19
94
Kom
ori
and
Sug
ihar
a, 1
988
Ohs
aka
et a
l.,
1960
; O
mor
i-S
atoh
et
al.,
1967
; O
mor
i-S
atoh
and
Sad
ahir
o, 1
979
Om
ori-
Sat
oh a
nd S
adah
iro,
197
9; T
akey
a et
al.
, 19
90a
Hua
ng e
t al
., 19
84
Tan
and
Sai
fudd
in,
1990
Om
ori
et a
L,
1964
; S
atak
e et
al.
, 19
65;
Iwan
aga
et a
l.,
1965
; O
shim
a et
al.
, 19
68;
1971
S
anch
ez e
t al
., 19
87
Sug
ihar
a et
al.
, 19
83
t~
O
m"
O
,<
O
334 J.B. BJARNASON and J. W. Fox
and HR-2b from T. flavoviridis and HR-I from A. halys blomhoffii, which previously had been considered non-proteolytic, have since been shown to contain proteolytic activities (Nikai et al., 1986, 1987). Thus, it can be fairly safely concluded that snake venom hemorrhagic toxins are proteinases.
All the hemorrhagic toxins examined have shown metal dependency when assayed with metal chelators. Of the 65 hemorrhagic toxins, 12 have been analyzed for their metal content, all have been found to contain zinc and many more are inhibited by metal chelators. Ten of the 12 toxins contained approximately 1 mol of zinc per mol of toxin. Both hemorrhagic and proteolytic activities of most of these toxins are dependent on the presence of the zinc. Also, calcium ions appear to stabilize the hemorrhagic toxins in aqueous solutions. Unfortunately, the metal composition of most of the hemorrhagic toxins has not yet been determined. However, it is reasonable to suggest that all the hemorrhagic toxins are metalloproteinases, and that they are most probably all zinc enzymes. Some of the hemorrhagic toxins have been demonstrated to hydrolyze basement membrane preparations (Ohsaka et al., 1973; Civello et al., 1983b; Bjarnason et al., 1988). Recent results have shown that HT-a,b,c,d, and e from C. atrox venom hydrolyze type IV collagen, fibronectin, nidogen, and laminin, all of which are basement membrane components (Bjarnason et al., 1988, 1993; Baramova et al., 1989, 1990a). It is also noteworthy that these hemorrhagic toxins do not cleave type I, type III, or type V collagens in the native form, but do rapidly digest gelatin.
It is apparent, therefore, that venon-induced hemorrhage is primarily caused by metal-depen- dent, proteolytic activities of the hemorrhagic toxins, probably acting on connective tissue and basement membrane components. The disruption of the intact capillary basement membrane structure results in the escape of capillary contents. The results of many investigators suggest this mechanism of direct proteolytic action of the hemorrhagic toxins on tissue substrates. However, some toxins may induce hemorrhage by a different mechanism, such as activation of endogenous hemorrhagic factors which could themselves cause hemorrhage. No data supporting this mechan- ism has yet been presented. This will be discussed further in Sections 4 and 6.
3. THE HEMORRHAGIC EFFECT
3.1. BIOLOGICAL EFFECT
Hemorrhage or bleeding is a common phenomenon in the victims of Crotalidae and Viperidae envenomation (Arnold, 1982). This is, in most cases, the result of many factors in the venom. The main factors responsible are the hemorrhagic toxins, and there are other secondary factors, such as the venom components that produce non-clotting blood, as well as the enzymes that release kinins from kininogen (Denson, 1969; Cheng and Ouyang, 1967; Bjarnason et al., 1983). The smaller blood vessels appear to be particularly susceptible to the effects of the venom, the result being an alteration of the vessel permeability. The consequences of this altered capillary permeability include the escape of plasma and red blood cells (RBC) into the surrounding tissues, giving rise to ecchymosis, blistering, cyanosis, and edema (Cheng and Ouyang, 1967).
One of the first investigations of the biological effects of isolated hemorrhagic toxins was an electron microscopic study in which the action of a partially purified hemorrhagic toxin (hemorrhagin) from the venom of V. palaestinae was compared with that of a phospholipase isolated from the same venom (McKay et al., 1970). The previously characterized hemorrhagic toxin had an estimated molecular mass of 44 kDa. It exhibited gelatinase activity, which was inhibited by DFP and by soybean trypsin inhibitor, but this did not affect the hemorrhagic activity (Grotto et al., 1967). Thus, as the authors stated, although these two activities can be separated by the use of suitable protease inhibitors, the preparation used in the study contained both activities. The hemorrhagic toxin was injected subcutaneously into the backs of rabbits and samples for microscopic examination were taken at various times. Initially, swelling of the cytoplasm of the endothelial cells was noted, followed by damage of the capillaries and endothelium and leakage of plasma into the connective tissue around the capillary. There was some development of pseudopods of endothelial cytoplasm while the disruption of basement membrane was occurring. The capillaries eventually became occluded with platelets, and at 8 min after injection, the cytoplasmic organelles of many of the endothelial cells were absent, and only the cell membranes
Hemorrhagic metalloproteinases from snake venoms 335
outlined the general anatomy of the cell and tissue architecture. The extravasation of RBC was extensive and many had completely lost their hemoglobin. Throughout the course of observation, no disruption of the intercellular junctions was observed (McKay et al., 1970). Intravenous injection of the hemorrhagic toxin gave rise to visible hemorrhage in the lungs and gastrointestinal tract, as well as damage in the heart, liver, lungs and intestinal tract, as seen under the light microscope. Subcutaneous injection of venom phospholipase did not appear to cause hemorrhage, although interstitial edema was observed, as well as perturbation of the red blood cell membranes.
From the results of these experiments, McKay et al. (1970) concluded that the hemorrhagic toxin preparation acted to disrupt the basement membrane and to cause endothelial cell lysis, allowing RBC and plasma to escape from the capillaries by traversing the cytoplasm of the endothelial cell rather than by moving between cells. The toxin preparation also appeared to cause platelet aggregation at the site of damage (McKay et al., 1970). However, since the hemorrhagic toxin used in these experiments was apparently not pure, interpretation of the data from these experiments must be made with caution (Grotto et al., 1967; Ovadia, 1978a). The results, indeed, may show what effects the venom has, while failing to demonstrate the consequences of a pure hemorrhagic toxin.
The effects of several hemorrhagic toxins isolated from the venom of T.flavoviridis on glomerular basement membranes have been examined (Ohsaka et al., 1973). The three hemorrhagic toxins studied were called hemorrhagic principles HR-1, HR-2a, and HR-2b (Ohsaka et al., 1960; Omori-Satoh and Ohsaka, 1970). At the time of these experiments, HR-1 was not pure and has been further fractionated into two components, HR-la and HR-Ib (Omori-Satoh and Sadahiro, 1979). When HR-1, HR-2a, and HR-2b hemorrhagic toxins were incubated with glomerular basement membrane preparations, both soluble peptides/proteins and carbohydrates were released. The authors also studied the size of the protein- and carbohydrate-containing fragments released, and they observed that HR-2a and HR-2b released similar-sized fragments, which were different in size from the fragments released by HR-1. From this, they concluded that the HR-2a and HR-2b hemorrhagic toxins have a different effect on basement membrane than HR-1 does. Finally, they considered the hemorrhagic effect of the toxins, in general, to be caused by enzymatic disruption of the basement membrane of capillary walls (Ohsaka et al., 1973).
A study, using cinematographic and electron microscopic methods, of the effect of the hemorrhagic toxin HR-1 (from T. flavoviridis) on capillaries from rat mesentery tissue was also carried out (Tsuchiya et al., 1974). The studies revealed that the toxin caused a partial disappearance of basement membrane, followed by RBC oozing out through inter-endothelial gaps. The endothelial cells and pericytes did appear to undergo some functional and organic changes, but the cells did not appear to undergo lysis. The destruction of the basement membrane by the toxin as observed microscopically is in agreement with the aforementioned results (McKay et al., 1970; Ohsaka et al., 1973). However, in the case of the V. palaestinae hemorrhagic toxin, the RBC were noted passing from the capillaries into the surrounding tissue through an endothelial cell space. With the HR-1 toxin, RBC were observed escaping through widened gap junctions. Although it is quite possible that the biological effects of these hemorrhagic toxins may be different, which could result in the observed differences in the pathways of RBC escape from the capillaries, it must be borne in mind that neither the HR-I preparation nor the hemorrhagin from V. palaestinae used in these experiments was homogeneous.
The effects of crude C. atrox venom when injected into mouse muscle tissue have been investigated (Ownby et al., 1974). From electron microscopic observations, the authors noted that the crude venom initially caused the endoplasmic reticulum and the perinuclear space of the endothelial cells to dilate, along with a swelling of the cytoplasm. The endothelial cells eventually began to bleb into the capillary lumen, followed by rupture of the endothelial cell membrane and extravasation of RBC into the surrounding tissue. Disruption of the basement membrane of the capillary was also noted. This sequence of events preceding hemorrhage is similar to that demonstrated by the V. palaestinae hemorrhagic toxin preparation (McKay et al., 1970). The pathological manifestations of the C. atrox crude venom are obviously difficult to attribute to any one particular venom component in light of the wide variety of toxic components present in this venom. However, this study clearly demonstrated, at the microscopic level, the hemorrhagic capability of the crude venom.
JPT 62/3~F
336 J.B. BJARNASON and J. W. Fox
The biological effects on mouse muscle tissue of three purified hemorrhagic toxins isolated from the venom of C. a trox have also been studied (Ownby et al., 1978). The purified toxins, HT-a, HT-b, and HT-e, were each injected into the muscle tissue and then the mice were biopsied at various times for microscopic observation of hemorrhage. Hemorrhagic toxins HT-a and HT-e both produced hemorrhage within 5 min of injection, whereas HT-b-induced hemorrhage appeared after much longer incubation times (5 hr). Tissue necrosis was also noted in the HT-b injected samples. As was seen for all three toxins, the endothelial cells became thin and formed vesicles prior to rupturing. The gap junctions of the endothelium did not appear to be disturbed. The basement membrane around the capillaries, as well as the surrounding connective tissue, was also disrupted, The RBC escaped from the capillary through the lysed endothelial cells and basement membrane into the surrounding tissue. These results are identical to those found by the same authors for the crude venom.
Observations of the biologic effect of hemorrhagic toxins using microscopic techniques gives a somewhat confusing image of the phenomenon. There could be various reasons for this. The different methods of injecting the toxins could have an effect on the experimental results. Varying stages of purity of the toxins used in the investigations might, and probably should, cause some variations in microscopic observations. The different types of tissues used might respond differently to the toxins injected. And, indeed, the inherent difficulties in interpreting the electron microscopic observations, to some extent, could be responsible for some of the differences in conclusions drawn by the different investigators, a phenomenon not unknown in the field of microscopic investi- gations. In addition to these experimental complications, the different venoms and hemorrhgic toxins may well cause different biological effects. Indeed, recent biochemical studies indicate that the various hemorrhagic toxins from C. a trox venom have varying biochemical functions, in particular, proteolytic substrate specificities (see Section 4). Common to all the aforementioned observations is the disruption of the basement membrane. This appears to be the key to understanding the general mechanism of snake venom-induced hemorrhage (see Section 4 and 6).
3.2. ASSAYS OF HEMORRHAGIC ACTIVITY
It was evident to the early investigators of snake venom-induced hemorrhage that a qualitative assay of hemorrhagic activity was essential to their studies, but that a quantitative one was much preferred. The method that most of the quantitative work has been based on allowed for statistical quantitation of the hemorrhagic activity. In this procedure, the backs of rabbits were shaved and injections were made intradermally at evenly spaced intervals across the back (Kondo et al., 1960). One advantage of this method is that as many as 60-70 injections can be made on one rabbit. After 24 hr, when the hemorrhagic response is maximal, the rabbit is killed and skinned. The skin is then placed between two glass plates and the hemorrhagic spots visualized from the visceral side. The authors recommended measuring the transverse diameter of the hemorrhages and using the mean value for quantitation. From these measurements, a minimum hemorrhagic dose can be estimated (Ohsaka et al., 1966), which is the amount of protein in the injection solution that caused a hemorrhagic spot with a defined area.
Many modifications of this technique have been applied since the original report. These include varying (1) the incubation time following the injection, (2) the experimental animal, (3) the route of toxin administration, and (4) the method of estimating the size of the hemorrhagic spot (Bjarnason and Tu, 1978; Civello et al., 1983a; Ownby et al., 1984). Although all of these modifications seem to be useful in their own right, they may complicate matters when minimum hemorrhagic doses for different venoms and toxins are to be compared, since the modifications listed above may affect the minimum hemorrhagic dose.
Rather than visually estimating the hemorrhagic response, another approach is to use mice, which, prior to the intradermal injection of the toxin, had been intravenously infused with Evans Blue dye. After short incubation periods, the animals were killed and the dye from the blue "hemorrhagic spots" was extracted and spectrophotometrically quantitated. Although the method superficially appears adequate, the actual procedure is rather awkward and gives a measure of capillary permeability to serum and possibly erythrocytes rather than a direct measurement of the hemorrhagic response (appearance of erythrocytes).
Hemorrhagic metalloproteinases from snake venoms 337
The method of Ownby et al. (1984) circumvented the above problem of a lack of differentiation between capillary permeability and extravasation of erythrocytes. In their assay, they used an intramuscular injection of venom in the thighs of mice. The mice were killed 3 hr after injection and a portion of the muscle removed and assayed for hemoglobin content. The principle was that the hemoglobin present was a direct result of the escape of erythrocytes into the tissue at the site of capillary damage. However, this hemoglobin assay technique appears somewhat troublesome to perform as compared with the skin response method (Bjarnason and Tu, 1978). Furthermore, it would seem that the amount of hemoglobin detected in the tissue would be very dependent on the precise site from which the tissue sample was taken in relationship to the injection site. The authors did not address this possible complication.
In lieu of a dye, Just et al. (1970) administered to rabbits 5JCr-labeled erythrocytes and t25I-labeled albumin, intravenously, followed by intradermal injections of the hemorrhagic compounds. After 24 hr, the animals were killed and the skin at the injection site assayed for radioactivity. This method is advantageous when one wishes to differentiate the effects of various agents as to their ability to cause varying degrees of capillary permeability.
Another method for the assay of hemorrhage is via the topical application of venom or toxins to the surface of dog lungs (Bonta et al., 1970). In this procedure, the chest cavity of a dog is opened to allow direct application on the exposed lungs of filter disks soaked in the venom or toxin solution. After an arbitrary length of time (usually 3 min), the disks are removed and the time of the appearance of hemorrhage is recorded. This method allows for determination of the onset of hemorrhage, as well as the ability to observe differences in the appearance of the hemorrhagic spot. There are several difficulties associated with this technique, however, including the somewhat high concentration of toxin solution necessary, the surgical requirements for the assay and the variations that are observed with different dogs. These problems make the assay somewhat problematic to perform and not particularly useful for quantitation and standardization.
Of all the methods discussed above, the method of Kondo et al. (1960) is, in some form of modification that suits the individual investigator's particular needs and resources, most commonly used. This is likely due to the high sensitivity of the assay, as well as the high correlation coefficient of the dose-response and the relative ease of performance of the assay.
4. THE HEMORRHAGIC TOXINS FROM C. A T R O X VENOM
4.1. INTRODUCTION
Seven hemorrhagic toxins have been purified from the venom of the western diamondback rattlesnake, C. a t r o x (Table 3). In 1978, Bjarnason and Tu reported the isolation and character- ization of five hemorrhagic toxins and the role of zinc in the action of hemorrhagic toxin e. The toxins were purified by anion and cation exchange chromatographies, as well as gel filtration, and termed hemorrhagic toxins a, b, c, d, and e. Their molecular weights were determined to be 68,000, 24,000, 24,000, 24,000 and 25,700 Da, respectively (see Table 3). From the amino acid compo- sitions, it was evident that HT-c and HT-d were very similar proteins. This was also apparent from the isolation procedure and electrophoresis of HT-c and HT-d, as well as from their proteolytic cleavage progression curves. The high content of cysteine in HT-a was also noteworthy.
All five hemorrhagic toxins were found to lose their hemorrhagic activities after treatment with the metal chelators EDTA and 1,10-phenanthroline. When analyzed for metals, all five were found to contain approximately 1 mol of zinc per mol of enzyme. Hemorrhagic toxin e was also measured for other metals and found to have some calcium associated with it (Bjarnason and Tu, 1978). The toxins were analyzed for proteolytic activity using dimethylcasein and dimethylhemoglobin as substrates, and then reacted with trinitrobenzenesulfonic acid to detect the newly formed amino groups. With this sensitive assay, all five toxins showed cleavage of both substrates. The proteolytic progression curves were clearly different for the different hemorrhagic toxins, suggesting different proteolytic specificities of these toxins. Furthermore, the toxin with the greatest hemorrhagic activity, hemorrhagic toxin a, showed the lowest activity toward dimethylcasein as a substrate. Thus, there did not seem to be a direct relationship between the hemorrhagic activities of the different toxins and their proteolytic activities on dimethylcasein. These observations suggested a
oo
TAB
LE 3
. Su
mm
ary
of
the
Pro
pert
ies
of S
even
Hem
orrh
agic
To
xins
from
the
Ven
om o
f C
. at
rox
Prop
erti
es
HT
-a
HT
-b
HT
-c
HT
-d
HT
-e
HT
-f
HT
-g
Mol
ecul
ar m
ass
(kD
a)
68
24
24
24
25.7
64
60
fr
om e
lect
roph
ores
is
Mol
ecul
ar m
ass
(kD
a)
23.2
23
.2
N.D
. N
.D.
from
a.a
. se
quen
ce
lsoe
lect
ric
poin
t W
eakl
y ac
idic
8.
2 6.
0 6.
1 5.
6 7.
7 6.
8
MH
D (
#g)
0.04
3
8 11
1
0.5
1.4
Met
al c
onte
nt
1 Z
n 1
Zn
l Z
n 1
Zn
1 Z
n l
Zn
N.D
.
Hem
orrh
agic
+
+ +
+ +
+ +
Pro
teol
ytic
+
+ +
+ +
+ +
Oth
er a
ctiv
itie
s C
leav
es:
Cle
aves
: C
leav
es:
Cle
aves
: C
leav
es:
Cle
aves
: C
leav
es:
Bas
emen
t B
asem
ent
Bas
emen
t B
asem
ent
Bas
emen
t F
ibri
noge
n F
ibri
noge
n m
embr
ane
mem
bran
e m
embr
ane
mem
bran
e m
embr
ane
prep
arat
ion
prep
arat
ion
prep
arat
ion
prep
arat
ion
prep
arat
ion
Col
lage
n IV
L
amin
in
Col
lage
n IV
C
olla
gen
IV
Col
lage
n IV
L
amin
in
Nid
ogen
L
amin
in
Lam
inin
L
amin
in
Nid
ogen
F
ibri
noge
n N
idog
en
Nid
ogen
N
idog
en
Fib
rone
ctin
F
ibro
nect
in
Fib
rone
ctin
F
ibro
nect
in
Fib
rino
gen
Fib
rino
gen
Fib
rino
gen
Fib
rino
gen
Gel
atin
I,
III
Gel
atin
I,
III
Gel
atin
I,
III
Gel
atin
I,
III
and
IV
and
IV
and
IV
and
IV
Inhi
bito
rs
ED
TA
; 1,
10-
ED
TA
; 1,
10-
ED
TA
; 1,
10-
ED
TA
; 1,
10-
ED
TA
; 1,
10-
ED
TA
; 1,
10-
ED
TA
; 1,
10-
phen
anth
roli
ne;
phen
anth
roli
ne;
phen
anth
roli
ne;
phen
anth
roli
ne;
phen
anth
roli
ne;
phen
anth
roli
ne
phen
anth
roli
ne
HT
I fr
om
Opr
in;
HT
I fr
om
amin
o ac
id
amin
o ac
id
C.
adam
ante
us
C.
adam
ante
us
hydr
oxam
ate
hydr
oxam
ate
~t 2-
Mac
rogl
obul
in
~t 2-
Mac
rogl
obul
in
~t2-
Mac
rogl
obul
in
Fur
ther
com
men
ts
Cle
aves
ox
insu
lin
Cle
aves
ox
insu
lin
Cle
aves
ox
insu
lin
Cle
aves
ox
insu
lin
Cle
aves
ox
insu
lin
Cle
aves
ox
insu
lin
B c
hain
at
B c
hain
at
B c
hain
at
B c
hain
at
B c
hain
at
B c
hain
at
Asn
3--
Gln
4 H
iss-
Leu
6
His
s--L
eu6
His
s-L
eu 6
A
sn3
--G
ln 4
Val
2-A
sn 9
His
s-L
eu6
His
lo-L
eu,
His
~o-L
eull
His
lo-L
eull
Se
r9-H
is~o
G
ln4-
Asn
s H
islo
-Leu
,~
Ala
14
-Leu
~5
A
la14
-LeU
ls
Ala
,4-L
euls
A
la~4
-Leu
l5
Leu
6-C
ys7
Ala
14-L
euls
T
yrl6
-LeU
l7
Ty
r16
-Leu
17
T
yr16
-Leu
17
His
lo-L
eull
T
yr16
-Leu
17
Gly
23
-Ph
e24
G
ly2
3-P
he2
4
Gly
E3-
Phe
24
Ala
l4-L
euls
T
yr16
-Leu
17
~o
go
O
k¢
N.D
., n
ot d
eter
min
ed.
Hemorrhagic metalloproteinases from snake venoms 339
highly selective specificity of the toxins, especially hemorrhagic toxin a, which was the most potent one (Bjarnason and Tu, 1978).
The five hemorrhagic toxins from C. atrox venom were the first hemorrhagic toxins assayed for zinc and were found to contain approximately l mol of zinc per mol of toxin. Since then, at least seven other hemorrhagic toxins from three different genera of snakes have been assayed for zinc and found to contain this metal ion (Bjarnason and Fox, 1988-89). This is especially noteworthy since zinc is a common prosthetic group for proteolytic enzymes, such as many neutral proteases and collagenases. Hemorrhagic toxin e did not show collagenase activity using native bovine achilles tendon collagen as substrate, nor did it cleave furylacrylolglycyl-L-leucinamide, a substrate for neutral proteases (Bjarnason and Tu, 1978).
Nikai et al. (1984) isolated hemorrhagic toxin f from C. atrox venom in a five-step isolation scheme, three of whose steps are identical to the previously published first steps of HT-a isolation from C. atrox venom. HT-f has a molecular weight of 64 kDa and contains 1 mol of zinc per mol of protein. More recently, Nikai et al. (1985a) reported on the isolation and characterization of hemorrhagic toxin g from C. atrox venom. A five-step purification, of which the first three steps were again identical to the first three steps in the HT-a isolation, yielded 5.9 mg of purified HT-g from 2 g of a crude venom. Amino acid analysis gave a composition of 516 amino acids, based on a molecular weight of 60 kDa in a single polypeptide chain. The most outstanding feature of the amino acid composition of this toxin is, once more, the relatively high cysteine content, 9.3%, similar to that of HT-a and HT-f. For these and other reasons, it seems plausible that HT-f and HT-g are in some way related to HT.a. For instance, they could all be similar but separate gene products. In our cDNA library, made from RNA isolated from the venom glands of western diamond rattlesnakes, we have come across two identical clones that are similar to the clones of HT-a.
In the following subsections, we shall describe the characteristics of the hemorrhagic toxins from C. atrox venom, beginning with HT-e, the most extensively studied toxin, followed by the HT-c/d isozyme, also extensively studied, and finally the other toxins, some of which (HT-f and g) have not been studied in great detail. The description, in general, will be chronological, and in some instances, results for all toxins (except HT-f and g) will be presented in the section on HT-e.
4.2. HEMORRHAGIC TOXIN e OR ATROLYSIN E (EC 3.4.24.44)
Hemorrhagic toxin e was purified in a three-step purification scheme, yielding 320 mg from 20 g of venom. It had a molecular weight of 25,700 Da, an isoelectric point of 5.6, and a minimum hemorrhagic dose of 1/~g. It demonstrated proteolytic activity towards dimethylcasein and dimethylhemoglobin as substrates, and it was found to contain 1.03 mol of zinc per mol of protein. Hemorrhagic toxin e thus appears to be a zinc metalloproteinase (Bjarnason and Tu, 1978). Hemorrhagic toxin e has been the subject of much research from the onset of these investigative efforts due to its abundance in the venom, its relative ease of purification, and its fairly potent hemorrhagic activity. A central, and most significant, issue in the study of hemorrhagic toxin e, and, indeed, all the hemorrhagic toxins, has been the relationship between the hemorrhagic and proteolytic activities, and the effect of the zinc content of the toxin on these activities.
When zinc was removed from hemorrhagic toxin e by dialysis against 1,10-phenanthroline for 24 hr, less than 10% of the original zinc content was found associated with the apotoxin according to atomic absorption measurements. Less than 5% of the original proteolytic activity remained with the apotoxin and approximately 5% of the original hemorrhagic activity was still associated with it. When the apotoxin was incubated with zinc ions using the dialysis method, in an effort to regenerate the holoenzyme, 20% of the original proteolytic activity was regenerated and approximately 25% of the hemorrhagic activity was found associated with the regenerated toxin. Thus, a direct relationship between the hemorrhagic and proteolytic activities of hemorrhagic toxin e was demonstrated, the first time such a relationship had been established for a hemorrhagic toxin (Bjarnason and Tu, 1978).
Spectroscopic methods were used to study the structure of native hemorrhagic toxin e as well as structural changes caused by zinc removal and readdition. With circular dichroism (CD) spectroscopy, the native toxin HT-e was estimated to consist of 23% ,t-helix and 6% fl-structure.
340 J.B. BJARNASON and J. W. Fox
When over 90% of the zinc was removed, the or-helix content appeared to drop from 23% to 7%. When the apotoxin was incubated with zinc, an increase in fl-helix content was observed by evaluation of the peptide region of the CD spectrum. Parallel changes could be seen in the aromatic regions of the CD and UV spectra, which are indicative of changes in the tryptophan environments. These interpretations were also supported by appearances of peaks attributable to tryptophan in the Raman spectrum of the apotoxin which were not visible in the Raman spectrum of the holotoxin. Thus, there appear to be significant conformational changes in hemorrhagic toxin e with the removal of zinc from the toxin (Bjarnason and Tu, 1978).
In 1983, Bjarnason and Fox described the exchange of cobalt for zinc in HT-e and the properties of the cobalt-containing toxin, as well as the proteolytic specificity of hemorrhagic toxin e and cobalt hemorrhagic toxin e. Cobaltous ion was introduced into hemorrhagic toxin e by the method of direct exchange with dialysis. An 80 mM HT-e solution in 5 mM Tris-C1 buffer, pH 8.5, containing 0.1 M NaCI and 2 mM CaCl 2 was dialyzed against a 50-fold volume of 0.2 M cobaltous ion in 0.1 M sodium acetate, pH 5.5 or 36 hr at 2-4°C. The solution was constantly purged with N2. Excess cobalt was removed by extensive dialysis. The cobalt hemorrhagic toxin e thus formed contained 1.1 mol of cobalt per mol of toxin and no measurable zinc. Previous attempts to incorporate cobalt into the apotoxin were unsuccessful. The cobalt-containing toxin had both hemorrhagic and proteolytic activities to a similar, or slightly lesser, degree as the native toxin. The minimum hemorrhagic dose of Co(II)HT-e was found to be 1.2 #g as compared with 1.0 #g for the native toxin. The cobalt-containing toxin was estimated to have a turnover number for dimethylcasein of 10 min -1, whereas the turnover number of the native toxin for the same substrate was 13 min- (Bjarnason and Fox, 1983).
The UV spectra of hemorrhagic toxin e and cobalt hemorrhagic toxin e appeared to be identical, with shoulders at 291 and 284 nm and a peak of 278 nm. Likewise, the CD spectra of the cobalt-containing toxin in the aromatic and peptide regions appeared totally identical to the previously published spectra of the native toxin. Thus, there appears to be no change in structure of hemorrhagic toxin e accompanying the metal exchange. This was in stark contrast to the considerable structural exchanges observed with the same spectroscopic methods upon production of the apotoxin by zinc removal. Thus, zinc removal causes large structural perturbations, whereas exchanging zinc with cobalt is accomplished without observable structural changes.
The absorption and CD spectra of the complex of cobaitous ion and hemorrhagic toxin e in the visible region were also measured. The absorption spectrum has a peak at 505 nm, with a molar absorptivity of 170 i -1 c m -1 . There also appears to be a shoulder at 550 nm and another peak at about 600 nm. The CD spectrum shows minima at 480 nm and 520 nm. These spectra resemble those of certain other cobalt enzyme complexes thought to possess distorted tetrahedral structures with oxygen and nitrogen ligands. However, we have not observed any published spectra with striking similarities to those of cobalt HT-e (Bjarnason and Fox, 1983).
The proteolytic specificity of HT-e and cobalt HT-e was investigated by using the oxidized A and B chains of bovine insulin. The most rapid cleavage of the two oxidized insulin chains occurred at the Alat4-Leu~5 bond on the insulin B chain. This site of cleavage appears to be the primary cleavage site in the insulin B chain for all the hemorrhagic toxins from C. atrox venom, and, indeed, for all hemorrhagic toxins tested so far. A secondary site of cleavage on the B chain was observed at Serg-Hisl0, and a third and very slow cleavage occurred at Asn3-Gln4 (Table 4). The primary site of cleavage on the insulin A chain is the Tyrta-Gln~5 bond, with a secondary site of cleavage at the Alas-Ser 9 bond. The difference in the rates of cleavage at the different sites was about 10-fold.
The rates of the various proteolytic activities of hemorrhagic toxin e and cobalt hemorrhagic toxin e were estimated. The turnover of cleavage for the primary site in the A chain of oxidized insulin, the Tyr~a-Gln~5 bond, was 3.6 min -1 for HT-e and 2.6 min -t for cobaltous HT-e, but for the secondary site, the Alas-Ser9 bond, the turnover of cleavage was 0.46 min- t for both forms of the toxin. Thus, cobaltous ion can be exchanged for zinc in the native toxin without detectable changes in the protein conformation and with only minor functional changes (Bjarnason and Fox, 1983).
It is clear that hemorrhagic toxin e is a zinc-containing metalloproteinase, and that there is a direct relationship between the toxin's hemorrhagic and proteolytic activities. Furthermore, these activities are dependent on the zinc content of the enzyme. The proteolytic cleavage specificities
TABL
E 4.
Sit
es o
f C
leav
age
of
Hem
orrh
agic
Tox
ins
on t
he O
xidi
zed
B C
hain
of
Insu
lin
1 5
! 0
15
20
25
30
Tox
in
Ven
om
F-V
-N-Q
-H-L
-C-G
-S-H
-L-V
-E-A
-L-Y
-L-V
-C-G
- E
-R-G
-F-F
-Y-T
-P-K
-A
Hem
orrh
agic
tox
in a
I C.
atr
ox
~ T
T T
T H
emor
rhag
ic t
oxin
b I
C.
atro
x T
~ T
T H
emor
rhag
ic t
oxin
c a
nd d
2
C.
atro
x T
~ ~
T T
Hem
orrh
agic
tox
in e
3 C
, at
rox
~ T
Hem
orrh
agic
tox
in P
C
. at
rox
T T
T T
~ T
Hem
orrh
agic
pro
tein
ase
IV 5
C
h, h
orri
dus
T Pr
otei
nase
1I 6
1'
1"
T T
1"
1"
HR
-I (
Prot
eina
se b
) 7
~ T
I" T
~ M
ooje
ni p
rote
ase
a s
1' ]'
~ $
T $
1' 1'
$ H
emor
rhag
ic f
acto
r-H
F 9
T T
~"
T H
emor
rhag
ic f
acto
r a 1
° 1'
T T
Hem
orrh
agic
fac
tor
b t°
T
Muc
rotr
oxin
A11
1"
~"
T
T T
Hem
orrh
agic
pro
teas
e-H
RIA
~2
~
A.
h. b
lom
hoff
ii
B.
moo
jeni
B
. jar
arac
a T.
muc
rosq
uam
atus
T.
muc
rosq
uam
atus
7/
'. m
ucro
squa
mat
us
T. fl
avov
irid
is
¢%
¢:r
t~ g r~
JBja
rnas
on e
t al
. (1
988)
. 2F
ox e
t al
. (1
986)
. ~B
jarn
ason
and
Fox
(19
83).
~Nik
ai e
t aL
(19
84).
5Civ
ello
et
aL,
(198
3b).
6Kur
ecki
et
al.,
(197
8).
~Nik
ai e
t al
. (1
986)
. SR
eich
l an
d M
ande
lbau
m (
1993
). 9M
ande
lbau
m e
t aL
(19
76).
I°N
ikai
et
aL (
1985
). II
Kis
hida
et
al.
(198
5b).
~ZY
amak
awa
and
Om
ori-
Sato
h (1
988)
.
t~
.¢
O
342 J.B. BJARNASON and J. W. Fox
of HT-e on the oxidized insulin B chain described above were found to be similar to those of 16 other hemorrhagic toxins, as well as thermolysin and elastase in its primary cleavage of the Alal4-LeUl5 bond (Bjarnason and Fox, 1983).
These results do not include what might be the in vivo substrates of the hemorrhagic toxins, causing hemorrhage upon envenomation. However, other experimental findings (see Section 3) had suggested that components of basement membrane were susceptible to proteolytic degradation by hemorrhagic toxins (Ohsaka et al., 1973; Bjarnason and Fox, 1983). Theoretically, this seems quite reasonable, since it appears almost inevitable that the basement membrane surrounding the endothelial cells of the circulatory system would be degraded in order for erythrocytes to escape into the surrounding tissues when hemorrhage occurs. Therefore, we embarked upon a series of experimental investigations, primarily with HT-e, in an attempt to establish firmly the nature of these activities, thereby illuminating the nature of the mechanism of snake venom-induced hemorrhage.
In the first and somewhat preliminary investigation, all five hemorrhagic toxins (a-e) were shown to be capable of releasing soluble peptides from a basement membrane preparation of Matrigel (Bjarnason et al., 1988). Hemorrhagic toxin a was the most active on the basement membrane, followed closely by HT-e. Hemorrhagic toxins b, c, and d showed activity, but to a much lesser extent. What is particularly interesting about this difference is that the magnitude of hemorrhagic activity, as measured by their minimum hemorrhagic doses, is reflected by the order of activity of toxins on the basement membrane preparation as substrate, but not on common protein substrates. This is further exemplified by results showing HT-b to be 2-10 times more active than HT-a in cleaving the bonds of the oxidized insulin B chain, yet it is much less active towards the basement membrane preparation than HT-a. Therefore, the more potent hemorrhagic toxins may have evolved substrate specificities and catalytic efficiencies for peptide bonds of proteins present in basement membrane. Furthermore, the proteinases HT-a and HT-e (the two hemorrhagic toxins which are most active in producing soluble peptides from the basement membrane preparation and the most potent in their hemorrhagic activities), were also able to cleave band-a (nidogen) and laminin most rapidly, when the reaction products were examined with sodium dodecyl sul- fate-polyacrylamide gel electrophoresis (SDS-PAGE). Although all of the toxins, given sufficient time, were able to digest laminin and nidogen in the Matrigel basement membrane preparation, the products of those digestions were different, except in the case of the HT-c and d isoenzymes, indicating similar but not identical specificities for the toxins. HT-b was not used in these experiments.
In order to determine whether the hemorrhagic toxins were potentially capable of interfering with the blood coagulation system, they were assayed for their proteolytic effect on fibrinogen and fibrin. With respect to fibrinogen, all of the hemorrhagic toxins were able to digest fibrinogen into unclotable products (Bjarnason et al., 1988). Within a 30-min digestion of fibrinogen by HT-a, the Act chain is lost, with the Bfl chain and ~ chain also being readily cleaved, although not quite as rapidly. A similar situation is observed for the action of HT-e on fibrinogen. The A~ chain is cleaved very rapidly followed by the Bfl and ~ chains. The hemorrhagic toxins b, c, and d appear to cleave the fibrinogen chains in a similar fashion as HT-a and e, although more slowly. None of the hemorrhagic toxins demonstrated any proteolytic activity towards fibrin at incubation times of up to 24 hr.
These results prompted further investigations in the direction of basement membrane degra- dations. The proteolytic activities of four of the hemorrhagic toxins (HT-a, c, d, and e) were investigated using isolated extracellular matrix (ECM) proteins (Baramova et al., 1989). The results show that all of the toxins are capable of cleaving fibronectin, laminin, type IV collagen (collagen IV), nidogen, and gelatins I, III and V. However, none of the proteinases degraded the interstitial collagen types I and III, or collagen V. Hemorrhagic toxins c and d produced identical digestion patterns with all the aforementioned substrates, further demonstrating their isoenzyme relation- ship. With fibronectin, HT-a produces a set of products similar to those of HT-c and HT-d, but apparently a different ratio of them, while HT-e generates a unique digestion pattern. HT-a and HT-e produce differing patterns with the laminin/nidogen preparation used, although they shared some similarities with each other, as well as with the patterns generated by HT-c and d. The same can be said for the cleavage of nidogen 150 by these proteinaes. Furthermore, it can be concluded
Hemorrhagic metailoproteinases from snake venoms 343
that HT-e digests nidogen faster and more thoroughly than HT-c/d, while HT-a seems to be intermediate in this respect. The digestion patterns of collagen IV by HT-a and HT-e are similar to the patterns generated by HT-c/d, apparently differing by two bands. The cleavage patterns of the three types of gelatins generated by the three different kinds of toxin proteinases, HT-a, HT-c/d, and HT-e, were all different.
The observation that the interstitial collagen types I and III and also type V collagen were not degraded by the toxins suggests that these enzymes are not analogous to the interstitial collagenases. However, the toxin proteinases were all quite effective in degrading collagen IV, gelatins of the aforementioned collagens, and the glycoproteins laminin, nidogen and fibronectin, albeit with varying specificities. The mammalian proteinase with the greatest degree of substrate similarity with these hemorrhagic proteinases was found to be stromelysin (Baramova et al., 1989). Stromelysin is a metalloproteinase isolated from human fibroblasts and has been demonstrated to degrade fibronectin, laminin, collagen IV, and cartilage proteoglycans (Hibbs et al., 1985; Murphy et al., 1985; Okada et al., 1986). From these data, and the knowledge of the roles these ECM proteins play in maintaining basement membrane structural/functional integrity, one can imagine that the degradation of these ECM proteins could readily lead to loss of capillary integrity, resulting in hemorrhage at these sites.
Further investigations on the mode of cleavage of isolated ECM proteins by hemorrhagic toxin e were conducted (Baramova et al., 1990a, 1991; Bjarnason et al., 1993). The major digestion products generated when collagen IV was incubated with HT-e had molecular masses of 141, 132, 87, 71, 33, and 18 kDa (Baramova et al., 1990a). Sequence analysis of the digestion products revealed that the ~t I(IV) chains are cleaved in a pepsin susceptible triplet interruption of the triple helix at position Ala2~9~31n220, whereas the ct2(IV) chain is cleaved in the same triplet interruption region at the Thr228-Leu229 peptide bond (Bjarnason et al., 1993). These cleavages generate fragments similar in size to the 1/4 and 3/4 chain length products of collagen IV degradation by type IV collagenases. We now consider the cleavage assignments in the previous investigation (Baramova et aL, 1990a) to be incorrect, and the most recent results to be the accurate sequence analysis of the digestion products (Bjarnason et al., 1993). Digestion experiments performed over various time periods indicate that the ct2(IV) chain is cleaved more rapidly than the ~ I(IV) chains.
Generally, type IV collagenases cleave collagen IV at 1/4 the distance from the amino-terminal end, as determined by SDS-PAGE and electron microscopy (Fessler et al., 1984; Tryggvason et al., 1987). It has been suggested that these enzymes are important for the disruption of basement membranes during tumor invasion and in inflammatory processes (Tryggvason et al., 1987). Similarly, the degradation of collagen IV, the major component of basement membranes, by HT-e, could account for its hemorrhagic activity. Collagen IV molecules form the scaffolding structures of basement membranes through their lateral, amino- and carboxyl-terminal interactions. The other major components, laminin, nidogen/entactin, and heparin sulfate proteoglycans, interact with specific sites on collagen IV, located in the NC2 domain and in the triple-helical domain about 250 nm from the NC2 domain (Laurie et al., 1986; Dziadek et al., 1985).
Hemorrhagic toxin e digests laminin and nidogen, both in isolated forms and when present in a purified soluble complex. The only common site of cleavage by HT-e of isolated nidogen and nidogen complexed with laminin is the peptide bond between amino acid residues 335 and 336 in the amino terminal domain of nidogen. Additionally, nidogen in complex with laminin is also cleaved at sites 322, 351, and 840, as determined by sequence analysis, and site 953, as proposed from the molecular mass of a digestion product. Isolated nidogen, on the other hand, was cleaved at amino acid residues 75, 336, 402, and 920, as determined by sequence analysis, and approxi- mately at residues 296, 478, 625, and 702, as proposed from the molecular mass values of the generated polypeptide chains. Products from the proteolytic cleavage of the A and B2 chains of laminin were observed, with sites of cleavage determined to be at position 2666 in the laminin A chain and position 1238 in the laminin B2 chain. The laminin digestion products were identical regardless of whether or not nidogen was present in a complex with the iaminin chains. No cleavage with HT-e was observed in the laminin B 1 chain. Since the laminin/nidogen complex is a ubiquitous component integrated into the basement membrane structures, it probably plays an important role in maintaining their integrity. The evidence that the hemorrhagic proteinase HT-e is capable of
344 J.B. BJARNASON and J. W. Fox
digesting laminin and nidogen in their isolated and complexed forms suggests that such cleavages could contribute to the disruption of capillary basement membrane and hemorrhage.
All proteinases in circulation are subject to influence and regulation by the plasma proteinase inhibitors. Intrinsic enzymes, such as clotting factors, and extrinsic enzymes, such as the hemorrhagic proteinases, may demonstrate little or no activity if the rate of reaction with inhibitors is rapid (Travis and Salvesen, 1983). In the plasma, ~2-macroglobulin is the major metallo- proteinase inhibitor (Starkey and Barrett, 1977; Werb et al., 1974). Therefore, an investigation was conducted on the interactions between human ~ 2-macroglobulin and four of the hemorrhagic toxins, namely HT-a, the isozymes HT-c/d and HT-e (Baramova et al., 1990b). The proteolytic activities of hemorrhagic toxins HT-e and HT-c/d towards the large molecular weight protein substrates, gelatin type I and collagen IV, were completely inhibited by ~t 2-macroglobulin. On the other hand, the activity of HT-a on the same protein substrates was not significantly inhibited. Each mole of ~ 2-macroglobulin bound maximally 2 mol of HT-e and 1.1 mol of HT-c and HT-d. These small proteinases interacted rapidly with ~2-macroglobulin at 22°C. Rate constants based on intrinsic fluorescence measurements were 0.62 x 105 M-~ sec-~ for interaction of ~ 2-macroglobulin with HT-c and d, and 2.3 × 105 M -~ sec -~ for the interaction of ~t2-macroglobulin with HT-e. HT-a interacted very slowly with ~2-macroglobulin at 22°C. Increasing the temperature to 37°C and prolonging the time of interaction with ct2-macroglobulin resulted in the formation of 90 kDa fragments and high molecular mass complexes (large than 180 kDa), in which HT-a is covalently bound to the carboxy-terminal fragment of 0~2-macroglobulin.
The identification of sites of specific proteolysis of ct 2-macroglobulin shows that the cleavage sites for the four hemorrhagic metalloproteinases are within the bait region of the inhibitor. HT-c and d cleave ~2-macroglobulin only at one site, the Arg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin. HT-a cleaves the inhibitor primarily at the same site, but a secondary cleavage site at the His694-Ala695 bond was also identified. HT-e cleaves ~2-macroglobulin at two sites, which are different from those observed with HT-a and HT-c/d. With HT-e, the primary cleavage site is the Va1689-Met690 peptide bond and the secondary site at Gly693-Hi%94. It is noteworthy that HT-a, the most potent of these hemorrhagic toxins, is relatively resistant to inhibition by ct 2-macroglobulin, while the other toxins studied were effectively inhibited. Therefore, it was proposed that the hemorrhagic activities of the toxin proteinases in vivo are partly reflected by their inhibitory interaction with 0~2-macroglobulin (Baramova et al., 1990b).
Most recently, the sequences of two overlapping cDNA clones for hemorrhagic toxin e have been determined (Hite et al., 1992b). The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids (Fig. 1). The mature HT-e protein, as isolated from the crude venom, has a molecular mass of approximately 25 kDa, and, thus, represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal peptide of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase section of 202 amino acids, and a carboxy-terminal domain of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase (MMP) zymogens.
The sequence of the proteinase section of 202 amino acids is similar to known sequences of other snake venom metalloproteinases, with over 57% identity to the low molecular mass HR-2a and H-2 proteinase from the venom of the Habu snake, T. flavoviridis, and a hemorrhagic proteinase LHF-II from the venom of the bushmaster, Lachesis muta muta (Hite et al., 1992a). If only the proteinase section (mature protein) of HT-e is compared with the large hemorrhagic proteinase HT-1 B, a 56.1% identity is obtained, whereas there is a 55.7% identity if both the proteinase section and the carboxy-terminal doamin of HT-e is compared with similar sections of HR-1B. Interest- ingly, of the other snake venom metalloproteinases whose protein sequences had been determined, HT-e was found least similar (54.8%) to HT-d from the same species.
The carboxy-terminal domain of 89 amino acids, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HT-a and HR-IB. It also resembles the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence of this domain bears striking resemblance to a previously reported sequence of the cDNA for the disintegrin trigramin (Neeper and Jacobson, 1990). The translated open reading frames of these
Hemorrhagic metalloproteinases from snake venoms 345
1 21 41 61 81 CTCAGATTG~TTGAAAGCGGG~GAGAT~C~TGTCTT~AGCCAAATC~GCCTCCAAAATGAT~C~GTTCT~TGGTGACT~TAT~TTAGCAGCT
M I Q V L L V T I C L ~ A
101 121 141 161 181 TT~CTTATCAAGGGAGCTCTATAATcCT~TCTGGGAACGTCAATGATTATGAAGT~TTTA~CACGAAAAGTCACTGCATTGCCCAAAGG~AG F P Y Q G S S I I L E S G N V N D Y E V I Y P R K V T A L P K G A
201 221 241 261 281 TT~CCAAAGTATGAAGA~CCATGC~TATG~TTGAA~TG~TGG~AGCC~TGGTccTT~TCT~AAAAAAATAAAGGAcTTTTTTCAAAAGA V Q P K Y E D T M Q Y E L K V N G E P V V L H L E K N K G L F S K D
301 321 341 361 381 TTAC~TGAGACTCATTATTCCTTTGATG~AGAAAAATTAC~CAAACCCTTCAGTTGA~ATC~TGCTATTATCATG~CGCATCGAGAATGATGCT
Y S E T H Y S F D G R K I T T N P S V E D H C Y Y H G R I E N D A
401 421 441 461 481 GACTC~CTGC~G~TCAGTGCA~C~CGGTTTGAAA~ATTTC~TTC~GG~ATGTACCTTATTG~CCCTTGAAGCTT~CGACAGTG D S T A S I S A C N G L K G H F K L Q G E M Y L I E P L K L S D $
501 521 541 561 581 ~GCCCATGCAGTCTTCAAATTGAAAAATGTAGAAAAAG~GAT~GGCCCCCAAAATGTGTG~GT~CCCAG~TTGGGAATCATATG~CCCATC~ E A H A V F K L K N V E K E D E A P K M C G V T Q N W E S Y E P I K
601 621 641 661 681 AAAG~CTCTGATTTAAATCTT~CTG~CATCAAAGATATGTTGAGCTTTTCATTGTTGTGG~cAT~TGTACACAAAATAT~TGGCGATTCA
K A S D L N L N P E H Q R Y V E L F Z V V D H G M Y T K Y N G D S
701 721 741 761 781 GATAAGATAAGACAACGGGT~ATCAAAT~TCAACATTATG~AGAG~ACA~TATATGTA~TCGATATATTACT~CTGGTATAGAAATTTGGT D K I R Q R V H Q M V N I M K E S Y T Y M T I D ~ L L ~ G I E I W
801 821 841 861 881 CCAACGGAGATTTGATT~CGTGC~CCA~ATC~CT~TACTTTG~C~ATT~GAG~TGG~AG~ACAGATTTGCTG~GCAAAAGT~TGA $ N G D L I N V Q P ~ S P N T L N $ F G E W R E T D L L K R K S H D
901 921 941 961 981 T~TGCTC~TTACTCACG~CATTGCCTTCGATGAACAAATTATAGGAC~GCTTATAT~GTG~ATA~CGACCCAAAGCGTTCTAC~GAGTTGTC
N A Q L L T S I ~ F D E Q I I G R A Y I G G I C D P K R S T G V V
1001 1021 1041 1061 1081 CAGGATCAT~CGAAATAAATCTTAGGGTTGCAGTTAC~TGAC~ATGAGCTGGGTCAT~TCT~GCATTCATCATGA~C~ATTCCTGTTCTTGCG Q D H $ E I N L R V A V T M T H E L G H N L G I H H D T D S C S C
1101 1121 1141 1161 1181 GTGGTTACT~TGCATTATGTCTCCTGTGAT~GCGATG~cCTTCCAAATATTTCAGCGATTGTAGTTA~TCC~TGTTG~TTTATTATG~TCA G G Y S C I M S P V I S D E P $ K Y F 5 D C S Y I Q C W E F I M N Q
1201 1221 1241 1261 1281 G~GCCAC~TGCATTCTC~GAAACCCTTGAG~CAGATACTGTTTC~CTCCA~TTCTGGAAATG~TTTGGAGGCGGG~TAG~TGTGACTGT
K P Q C I L K K P L R T D T V S T P V S G N E L L E A G I E C D C
1301 1321 1341 1361 1381 GGCTCTCTTGAAAATCCGT~TGTTATGCTAC~TGTAAAAT~CC~GGT~CAGTGTGC~G~CTGTGTTGTGACC~TGC~ATTTATGA G S L E N P C C Y A T T C K M R P G S Q C A E G L C C D Q C R F M
1401 1421 1441 1461 1481 AAAAAGG~AGTATGCCG~TATC~GTTGATA~TGAT~TACC~CACTGGCC~TCT~TGACTGTC~CAGAAATGG~TCTATGGCTAAAC K K G T V C R V S M V D R N D D T C T G Q S A D C P R N G L Y G
1501 1521 1541 1561 1581 ~C~TGGAGATGGAAAGGTCTGC~C~CAGG~TGTGTTGA~TGACTACAGCCTACT~TC~CCTCTGGCTTCTCTCAGATTTGATCTTGGAGAT
1601 1621 1641 1661 1681 CCTTCTTTc~EAAGGTTCAACTTCCCTTT~TCCAAAGAG~CCACCTGCCTGCATCCTACTAGTAAATCACTCTTACCTTTCATACGG~TCTAAATTC
1701 1721 1741 1761 1781 TGc~TATT~TTCTCCATATTT~TCTGTTTACCTTTT~TGT~TCAAACCTTTTCCCC~CAcAAAGCTCTATGGGCATATACAACACC~G~CTT
1801 1821 1841 1861 1881 ATTTGCTGTC~GAAAAAC~TGGCCATTTTACCGTTTGCC~TTGCAAA~ACATTT~TGC~CAAGTTCTGCCTTTT~GCT~TATATTCGAAATC
1901 1921 1941 1961 ~TGCTTCCTCTTCCAAAATTTCATGCTGCCTTTCC~GATGTA~T~TTCCA~TAAACT~CTATTCTCA
FIG. 1. c D N A sequence of HT-e with the translated open reading ~ame. Both the signal ~quence and the protein sequence, as isolated from the venom, are denoted in bold~ce.
two clones are 70.3% identical overall. The continuation of the open reading frame beyond the end of the observed carboxy terminus of the mature HT-e protein suggests a carboxy-terminal domain, which is processed away from the proteinase. Furthermore, this helps to bridge our understanding of the interrelatedness of the large and small venom metalloproteinases to one another and to the disintegrin family of peptides. It also predicts the existence of this disintegrin-like peptide in C. a t r o x venom.
A cluster of amino acids from residues 92-101 of the proteinase domain of HT-e was noted to be conserved in the hemorrhagic metalloproteinases, but not conserved in the only non-hemor- rhagic snake venom metalloproteinase whose protein sequence had been determined (Hite et al., 1992a). This could be evidence that this region of the proteinase is crucial to its ability to hydrolyze substrates in a manner that results in hemorrhage. The zinc-binding region of HT-e more closely
346 J.B. BJARNASON and J. W. Fox
resembles that of the MMPs than of the thermolysin family of zinc metalloproteinases, which seems reasonable in light of the fact that the substrates of HT-e are more similar to those of the MMPs.
4.3. HEMORRHAGIC TOXIN b (ATROLYSIN B, EC 3.4.24.41) AND HEMORRHAGIC TOXINS C AND d (ATROLYSIN C, EC 3.4.24.42)
Hemorrhagic toxin b (atrolysin B, EC 3.4.24.41) was purified in a four-step purification scheme of gel filtration and anion- and cation-exchange chromatographies, yielding 740 mg from 20 g of venom, thus being the most abundant hemorrhagic toxin in C. atrox venom. It has a molecular mass of 24kDa, a basic isoelectric point, and a minimum hemorrhagic dose of 3~tg. It demonstrated proteolytic activity towards dimethylcasein, and it was found to contain 0.82 mol of zinc per mol of protein (Bjarnason and Tu, 1978). Hemorrhagic toxins c and d (atrolysin c, EC 3.4.24.42) were purified in a concomitant four-step purification scheme of anion exchange and gel filtration chromatographies, yielding 34 mg of HT-c and 62 mg of HT-d from 20 g of C. atrox venom. Both toxins had a molecular mass of 24 kDa, acidic isoelectric points and minimum hemorrhagic doses of 8 #g and 11 pg, respectively, for HT-c and HT-d, making them the least potent hemorrhagic toxins found in C. atrox venom. Both toxins demonstrated proteolytic activity towards dimethylcasein, and both were found to contain 0.86 mol of zinc per mol of protein. The amino acid compositions of HT-c and HT-d are almost identical, indicative, along with other characteristics and their isolation scheme, of a close relationship between them (Bjarnason and Tu, 1978).
Further characterization of hemorrhagic toxins c and d has substantiated this conjecture of a relationship between them (Bjarnason and Fox, 1987). The two toxins were characterized and compared with one another. Their isoelectric points are slightly acidic, HT-c being the more basic of the two, with an isoelectric point of 6.2, whereas HT-d has an isoelectric point of 6.1. The toxins were both demonstrated to be hemorrhagic and proteolytic, using hide powder azure as substrate. Prior treatment of the toxins with ethylenediaminetetraacetic acid and 1,10-phenanthroline eliminated both the hemorrhagic and proteolytic activities. The pH optimum ofproteolysis by HT-c and HT-d on hide powder azure as substrate was between pH 8 and pH 9. The CD spectra for HT-c and HT-d appear almost identical with respect to minima positions and ellipticities, indicative of very similar solution structures for the two enzymes. They also appeared to share identical antigenic structures. These results were corroborated by tryptic mapping of the two toxins. Only one major difference was observed from the maps. In the case of HT-c, it was determined that an aspartate was substituted by an alanine when compared with HT-d. From these characterization studies, the authors concluded that HT-c and HT-d are isoenzymes with only very minor differences in their structures. It is of interest to note that the amino acid compositions of HT-c and HT-d are very similar to those of protease I and protease II from C. adamanteus, suggesting that these might be homologous enzymes from the two species of snakes (Bjarnason and Fox, 1987).
The proteolytic specificities of HT-c and HT-d were investigated by using the oxidized B chain of bovine insulin and synthetic peptide substrates (Fox et aL, 1986). The enzymes cleaved the Alat4-Leu~5 bond of the insulin B chain most rapidly and the Tyr~6-Leul7 bond slightly more slowly. The HiSl0-Leu~t and GlyE3-Phe24 bonds were also cleaved, but at a considerably slower rate. In order to assess the substrate length preferences of the enzymes, peptide analogs of the B chain about the Ala~4-Leu~5 bond were synthesized, ranging in length from four to seven residues. The heptapeptide NHE-Leu-Val-Glu-Aia-Leu-Tyr-Leu-COOH was the best peptide substrate tested in this series, with the other peptides having decreasing k~,t/Km values with decreasing length. The tetrapeptide NH2-Ala-Leu-Tyr-Leu-COOH was not cleaved by the enzymes. On the contrary, this peptide was shown to be a competitive inhibitor of the toxins.
A series of N-acetylated pentapeptides and one hexapeptide, synthesized to probe the active site environment of the enzymes, were significantly better substrates than their unacetylated counter- parts (by approximately two orders of magnitude in kcat/K m values). The N-acetylated peptide Ac-Val-Ala-Leu-Leu-Ala-COOH gave the largest k~at/Km value of all the N-acetylated peptides assayed with both HT-c and HT-d in this series. The peptides were sensitive to substitutions at the peptide sites from position P3 to P'3. A generalized view of the enzymes' substrate binding sites ($3 through S'3) can be envisioned from the cleavage data of the N-acetylated synthetic peptides.
Hemorrhagic metalloproteinases from snake venoms 347
The enzymes seem to prefer hydrophobic residues in their S'~ and S'2 sites. However, an interesting exception is seen in peptide 13, which has an isoleucyl residue at its P'~ site. Apparently, the enzymes cannot accept an isoleucyl residue at S'~ sites, since peptide 13 does not act as a substrate or a competitive inhibitor of the enzymes. The S~ sites in the enzymes are rather selective in that they do not cleave peptides containing polar or charged residues (glutamine, lysine) or bulky aliphatic side chains (valine) at that position. This is in contrast to thermolysin. The $2 sites of the enzymes seem to prefer a bulky aliphatic side chain, like valine, over a smaller side chain (alanine), and the enzymes will not cleave a peptide with a positively charged lysyl residue at that position. Placing a residue in position P3 of a peptide, where nothing was previously, thus having an extended substrate in the substrate binding site, greatly enhances catalysis (20-fold) compared with similar, but truncated, peptides. The best substrate examined, so far, for these toxins is the fluorogenic peptide analog, 2-aminobenzoyI-A-G-L-A-4-nitrobenzoylamide (Fox et aL, 1986).
The sites and rates of peptide bond hydrolysis of the oxidized B chain of bovine insulin by HT-b have been investigated (Bjarnason et al., 1988). Hemorrhagic toxin b was found to cleave the Hiss-Leu6, Hisl0-LeuN, Alal4-Leuls, Tyr16-Leu17 and Gly23-Phe24 bonds. The primary sites of cleavage on the insulin B chain are the Tyr-Leu and Ala-Leu bonds, with turnover numbers of 61.3 min-1 and 48.2 min -1, respectively. The other bonds were cleaved at an order of a magnitude slower than these, thus being secondary sites of cleavage. They showed turnover numbers of 7.9rain -~ for the His~0-LeuH bond, 1.9min -t for the Hiss-Leu6 bond, and 1.6min -l for the Gly23-Phe24 bond. This cleavage specificity of HT-b on the insulin B chain is identical to that of the other hemorrhagic isoenzymes HT-c and HT-d.
As stated in Section 4.2, hemorrhagic toxins b, c, and d have been shown to digest, albeit slowly, a Matrigel preparation of basement membrane, as well as all three chains of fibrinogen. These cleavages of HT-b, c, and d all appear very similar, both in their rates of hydrolysis and in the patterns of digestion products generated. They did not demonstrate any proteolytic activity against fibrin at incubation times up to 24 hr (Bjarnason et aL, 1988). Further investigations into the activities of these toxins on isolated ECM proteins demonstrated that HT-c and d were able to digest collagen IV, nidogen, fibronectin, laminin, and type I, III, and V gelatins, but not collagens of these same types (Baramova et al., 1989). Hemorrhagic toxins c and d produced identical digestion patterns with all aforementioned substrates, further demonstrating their isoenzyme relationship, as described in more detail in Section 4.2.
The amino acid sequence of hemorrhagic toxin d, the first hemorrhagic toxin to be sequenced as a whole, was determined by traditional Edman protein sequencing (Shannon et al., 1989). It contains 203 amino acids in the major form (see Fig. 4, Section 4), and has a molecular mass of 23,234 g mol -~ . Hemorrhagic toxin d was found in the investigation in two forms, one of which has a glutamine in the penultimate position. It was suggested that the enzyme is probably synthesized with glutamine at positions 1 and 2 in the protein sequence, but one of these residues is removed by processing. At position 14, isoleucine was found sometimes instead of valine. The protein used for sequencing was isolated from venom taken from a population of snakes, so the observed microheterogeneity may reflect differences in the genes of different snakes, rather than the presence of two genes within one snake.
During the course of sequencing of the protein, no cycles from the sequenator were blank, suggesting an absence of glycosylated residues (Shannon et al., 1989). Also, the consensus site N-X-S/T was not observed in the sequence, leading to the conclusion that HT-d is not a glycoprotein. This is in agreement with data from previous experiments (Bjarnason and Fox, 1987). Despite the interesting similarities in enzymatic activity that HT-d shares with some enzymes, in particular stromelysin and metalloproteinases from murine tumor cells and phagocytes, the primary structure of HT-d has no significant overall similarity to known sequences of collagenases, other metalloproteinases, or any other available protein sequence, as evaluated by the FASTA (Pearson and Lipman, 1988) program. Therefore, it was proposed that HT-d was a member of a new subfamily of metalloproteinases (Shannon et aL, 1989; Hite et al., 1992a). However, like several other metalloproteinases (McKerrow, 1987; Jongeneel et al., 1989), it has an amino acid sequence at residues 143-148 corresponding to part of the zinc binding site and one of the catalytic glutamates of thermolysin.
348 J.B. BJARNASON and J. W. Fox
Inhibition of the enzymatic activity of HT-d on Abz-A-G-L-A-Nba as substrate by EDTA and cysteine, but not by iodoacetate and phenylmethylsulfonyl fluoride, is typical for metallo- proteinases. The lack of inhibition of HT-d by phosphoramidon distinguishes this enzyme from thermolysin, which is efficiently inhibited by this compound. The collagenase inhibitor SC44463, which inhibits collagenases, including those active towards collagen IV (Reich et al., 1988), is effective in inhibiting HT-d (Shannon et al., 1989). The enkephalinase inhibitor thiorphan (Roques et al., 1980) is capable of inhibiting HT-d, but it is less potent than in its inhibition of enkephalinase. One possible explanation of this is that thiorphan is predicted to place a phenyl moiety at the $1 site of the enzyme. However, it was previously demonstrated that HT-d prefers a leucyl residue at this site (Fox et al., 1986). A likely explanation for this difference in degree of inhibition is that the $1 site of HT-d cannot accommodate the phenyl group of thiorphan as readily as enkephalinase can. Also, as previously described in Section 4.2, the plasma proteinase inhibitor ct 2-macroglobulin is an effective inhibitor of HT-c and HT-d, apparently forming a 1 : 1 complex relatively rapidly with the toxin proteinases. The identification of sites of specific proteolysis of ct2-macroglobulin shows that the cleavage sites for HT-c and d are without the bait region of the inhibitor. The two hemorrhagic toxins cleave ~2-macroglobulin only at one site, the mrg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin (Baramova et al., 1990b).
Most recently, the cDNA sequences of these three hemorrhagic toxins, HT-b, c, and d, have been determined, along with that of HT-a (Hite et al., 1994). With the previously published cDNA sequence of HT-e, this presents the largest collection of metalloproteinase sequences from a single species of snake. Figure 4 shows the translated open reading frames of the five cDNA sequences. The amino acid compositions of HT-b, c, and d based on the cDNA data agree well with the previously published amino acid analysis data (Bjarnason and Tu, 1978; Bjarnason and Fox, 1987). The calculated molecular masses of the toxins are in fairly good agreement with those observed from electrophoretic mobility, with HT-b having a molecular mass of 23.4 kDa and HT-c and d, 23.3 kDa, all containing 203 amino acids.
These sequence data clearly confirm that hemorrhagic toxins c and d are almost identical, differing by only one amino acid at position 181, as previously observed (Bjarnason and Fox, 1987). Hemorrhagic toxin b is the only one of these proteinases that is not an acidic protein. Its pI is calculated to be 8.2. This large difference in net charge of HT-b compared with HT-c and d is due not to a localized region of positively charged residues, but rather to a widespread propensity for more positively charged amino acid residues along the entire length of the protein. Despite these differences, 158 of 203 amino acids (78.3%) are conserved between HT-b and HT-d, probably placing them in a separate class (I-B) of similar or almost identical proteinases (see Section 2.2), bearing also in mind their identical cleavage specificity on the insulin B chain as substrate. This might be somewhat similar to the classification of trypsin-like enzymes or chymotrypsin-like enzymes, which have similar three-dimensional structures, differ in substrate specificities, but exist in both anionic and cationic forms in many species. There is approximately a 40-50% sequence identity between trypsin, chymotrypsin, and elastase from the same species, but a 65% identity between the anionic and cationic forms of bovine trypsinogen, whereas it is 78.3% for the cationic HT-b and the anionic HT-d (LeHuerou et al., 1990). When the amino acid sequence of HT-e is compared with that of HT-b and the HT-c/d isoenzymes, these four toxins share only a 46.5% identity, and if HT-a is included in the comparison, only 40.1% identity is preserved, somewhat similar to that of the three different serin digestive proteinases. This, along with other comparative data, suggests that these five toxins, indeed, may define three groups of hemorrhagic metallo- proteinases within the subclass of snake venom metalloproteinases.
The highly conserved nature of the zymogen domain, 94.7% for the four small toxins, might be indicative of the possibility that it performs an identical function for all of the hemorrhagic proteinases, namely, that of maintaining latency, whereas the less conserved proteinase domains have varied activities and substrate specificities. The cysteine residue approximately 20 amino acids from the beginning of the proteinase domain lies within a region conserved in all snake venom metalloproteinase precursors studied thus far. The sequence around this cysteine is PKMCGVT. Previously (Hite et al., 1992a), we proposed that this region might function in a similar fashion to the "cysteine switch" (Van Wart and Birkedal-Hansen, 1990) or "Velcro mechanism" (Valee and Auld, 1990) in the MMPs. While not an exact match, the similarity to the MMP consensus
Hemorrhagic metalioproteinases from snake venoms 349
sequence and the high degree of conservation among snake venom metalloproteinases lend credence to this hypothesis. Wider implications of sequence comparisons are discussed in Section 5.
4.4. HEMORRHAGIC TOXIN a OR ATROLYSIN A (EC 3.4.24.1)
Hemorrhagic toxin a was purified in a five-step purification scheme of gel filtration and anion exchange chromatographies yielding only 64 nag from 20 g of venom, thus being the least abundant hemorrhagic toxin in C. a trox venom, if the isozymes HT-c/d are counted as one. It has a molecular mass of 68 kDa as judged from SDS-PAGE, an acidic isoelectric point, and a minimum hemorrhagic dose of 0.04/~g, therefore being, by far, the most potent of the hemorrhagic toxins from C. a trox venom. It demonstrated proteolytic activity towards dimethylcasein, and it was found to contain 0.99 mol of zinc per mol of protein. The amino acid composition, as analyzed on a traditional amino acid analyzer, was found to contain an unusually high cysteine content approximately 10.4%.
The sites and rates of peptide bond hydrolysis of the oxidized B chain of bovine insulin by HT-a were investigated (Bjarnason et al., 1988). Hemorrhagic toxin a was found to cleave the Asn3-Gln4, Hiss-Leu6, His~0-Leu11, Ala14-LeUls, and Tyrj6-Leu17 peptide bonds of the B chain. The primary sites of cleavage on the insulin B chain are the Ala-Leu and Tyr-Leu bonds, with turnover numbers of 12.6 min -~ and 6.2 rain -~, respectively. The cleavage of the Hist0-LeUlz bond had a turnover number of 4.0 min-~, while the Hiss-Leu6 bond was cleaved much more slowly, having a turnover number of only 0.54 min -1. This cleavage specificity of HT-a on the insulin B chain is not totally identical to that of any other hemorrhagic toxin analyzed in this way (Table 4). As previously described in Section 4.2, hemorrhagaic toxin a can release soluble peptides from a basement membrane preparation of Matrigel (Bjarnason et al., 1988). Of the five toxins tested, hemorrhagic toxin a was the most active on this basement membrane preparation, although it previously had been found to be the least active on commonly used protease substrates, such as casein (Bjarnason and Tu, 1978), and less active than HT-b on the primary cleavage sites of B-chain insulin (Bjarnason et al., 1988). Furthermore, in a closer scrutiny of this phenomenon, HT-a was shown capable of rapidly and effectively cleaving fibronectin, laminin, collagen IV, nidogen, and gelatins I, III, and V (Baramova et al., 1989). This might be the primary reason for the high potency of HT-a as a hemorrhage-causing proteinase. Another and synergistic reason for its potency might be the fact that it is not effectively inhibited by the plasma proteinase inhibitor ct2-macroglobulin. While the proteolytic activities of hemorrhagic toxins HT-e and HT-c/d towards the large molecular weight protein substrates, gelatin type I and collagen IV, were completely inhibited by ct2-macroglobulin, the activity of HT-a on the same protein substrates was not significantly inhibited (Baramova et al., 1990b).
Most recently, the cDNA sequence of hemorrhagic toxin a has been determined, as well as those for the other hemorrhagic toxins (Hire et al., 1992a, 1994). The HT-a clone is not full length, as it lacks 5' information encoding the amino terminus of the precursor protein; by contrast, the other four sequences contain the complete open reading frames. Figure 4 shows the translated open reading frames of the five cDNA sequences determined to date. The calculated molecular masses of the toxins are in fairly good agreement with those observed from electrophoretic mobility, with the single exception of HT-a, which migrates much more slowly than predicted from the molecular mass deduced from this sequence, most likely due to N-linked glycosylations. Consensus glycosylation sites exist at positions 263, 517, 529, and 533 (Fig. 4). Protein sequence data for the cyanogen bromide peptide, which includes the last three possible sites, suggests that Asnsl 7 and Ash533, indeed, are glycosylated, as they yield blank cycles, while Asns2 9 is not modified, as it yields a normal asparagine signal (Hite et al., 1994).
When the amino acid sequence of the proteinase domain of HT-a is compared with that of HT-b, the HT-c/d isoenzymes and HT-e, these five toxins share only a 40.1% identity. Based on sequence comparisons of the proteinase domains of the snake venom metalloproteinases, along with other comparative data, it can be hypothesized that these five toxins, in general, may define three classes of metalloproteinases within the subfamily of snake venom metalloproteinases, a class of HT-a-like enzymes (Ill-A), a class of HT-c/d-like enzymes (I-B), and a class of HT-e-like enzymes (I-A). This fine classification is based on sequence comparisons of the proteinase domains and functional
350 J.B. BJARNASON and J. W. Fox
aspects, but not domain composition, as is the P-I, 1I, III, and IV classification of the following section (although they may turn out to be one and the same when more information has been gathered). All the proteinase domains contain approximtely 200-205 amino acids, and they share a consensus zinc-binding motif of HEXGHNLGXXHD, with the underline representing strictly conserved residues across all members of the subfamily of venom metalloproteinases whose sequences are known (Hite et al., 1992a, 1994).
Hemorrhagic toxins a and e, while bearing disintegrin-like domains, lack the highly conserved RGD sequence of the disintegrins, having instead RSE and MVD, respectively. Several other protein containing disintegrin-like domains also lack the RGD triad (Hite et al., 1994). The RGD segment in the disintegrins is known to interact with integrins, where it plays an important role in binding to platelet GP lib IIIa. Although the various tripeptide sequences that differ from RGD are not similar in actual sequence, they all contain hydrophilic residues, many of them charged. It remains to be seen whether this tripeptide is important to the function of the snake venom disintegrin-like domains. Finally, HT-a contains a carboxy-terminal cysteine-rich domain of 121 amino acids. It strongly resembles homologous domains in the hemorrhagic proteinases HR-1B and jarahagin, as well as RVV-X from V. russelli venom. The function of this domain is unknown, but it is also similar to the cysteine pattern of the sequence of a novel family of mammalian reproductive proteins. The wider implications of further sequence comparisons are discussed in Section 5.
4.5. HEMORRHAGIC TOXINS f AND g OR ATROLYSIN F (EC 3.4.24.45)
Nikai et al. (1984) isolated hemorrhagic toxin f from C. a t r o x venom in a five-step isolation scheme. Three of the steps are identical to the previously published first steps of the isolation of HT-a from C. a t r o x venom (Bjarnason and Tu, 1978). HT-f has a molecular weight of 64 kDa, contains l mol of zinc per mol of protein, and has a minimum hemorrhagic dose of 0.5 vg- Zinc is essential for the toxin's hemorrhagic activity and for its proteolytic activity. When HT-f was incubated with the B chain of oxidized insulin, it cleaved at the ValE-ASn3, Gln4-Hiss, Leu6-Cys7, His~0-Leu,L, AlalcLeu~5, and Tyrn6-Leun7 bonds. HT-f was also found to hydrolyze the ), chain of fibrinogen without affecting either the A~ or Bfl chains. This was the first time that a hemorrhagic toxin was shown to have fibrinogenase activity, although the reverse had been demonstrated 7 years earlier; i.e. a fibrinogenase and fibrinolytic principle was shown to possess hemorrhagic activity (Ouyang and Huang, 1977). HT-f was also shown to differ immunologicaUy from HT-a and HT-c from C. a t r o x venom. HT-f was found to have a lethal toxicity (LDs0) of 9.8 mg g-~ compared with 3.3 mg g-' for the crude venom. When zinc was removed, HT-f lost its lethal toxicity. From the amino acid composition, it is noteworthy that HT-f, like HT-a and other high molecular weight hemorrhagic toxins, contains an unusually high amount of cysteine of approximately 10% of the total number of amino acid residues.
In 1985, Nikai and co-workers reported on the isolation and characterization of hemorrhagic toxin g from C. a t rox venom (Nikai et al., 1985a). A five-step purification, of which the first three steps again were identical to the final three steps in the HT-a isolation, yielded 5.9 mg of purified HT-g from 2 g of crude venom, the purified toxin having a minimum hemorrhagaic dose of 1.4 #g. Amino acid analysis gave a composition of 516 amino acids based on a molecular weight of 60 kDa in a single polypeptide chain. The most outstanding feature of the amino acid composition of this toxin is once more the relatively high cysteine content of 9.3%.
Hemorrhagic toxin g was found to possess proteolytic and lethal activities, which, in addition to the hemorrhagic activity, were inhibited by EDTA and l, 10-phenanthroline, suggesting that the activities are metal ion-dependent. However, no metal analysis was performed on the toxin. Hemorrhagic toxin g also showed fibrinogenase activity, cleaving the A~ and Bfl-chains of fibrinogen, in contrast to HT-f, which only digested the y-chain. Komori et al. (1985) showed that HT-b also contains fibrinogenase activity, hydrolyzing the A0t-chain of fibrinogen first, followed by cleavage of the Bfl-chains. The degradation products of fibrinogen were found to be different from those of thrombin, indicating that the cleavage sites in the A,t- and Bfl-chains are different from those of thrombin. Also, HT-b did not produce a fibrin clot or hydrolyze N-Benzoyl- Phe-Val-Arg-p-nitroanilide, a substrate hydrolyzed by thrombin and reptilase.
Hemorrhagic metalloproteinases from snake venoms 351
5. STRUCTURE AND CLASSIFICATION OF HEMORRHAGIC TOXINS AND THEIR RELATIONSHIP TO OTHER METALLOPROTEINASES
Although the molecular masses of the hemorrhagic toxins vary widely (22-90 kDA), they all appear to be metalloproteinases and thus share a somewhat conserved proteinase domain. The variation and disparity of the molecular masses of the toxins is due to the presence of additional domain structures and varying states of glycosylation on those domains (Hite et al., 1992a, 1994). In this section, we will consider the structural relationships among the C. a t rox toxins, focusing on the proteinase domain, as well as the other domains present in the hemorrhagic toxins of C. atrox venom and other representative members of this toxin family. We will then consider the structures of other venom non-hemorrhagic metalloproteinases. Finally, we will discuss the recently described non-venom proteins that demonstrate very interesting similarities to the venom metallo- proteinases.
5.1. GENERAL STRUCTURAL CHARACTERISTICS OF THE C. A TROX HEMORRHAGIC TOXINS
The primary structure of the C. a t rox hemorrhagic toxin HT-d was reported in 1989 and represented the first complete protein sequence determined for a hemorrhagic toxin (Shannon et al., 1989). One of the most interesting observations arising from the HT-d sequence was that, except for the zinc-binding consensus sequence, no other sequences found in the various data banks at the time shared any significant homology with HT-d. Therefore, it was concluded that the HT-d sequence was the first representative in a novel subfamily of metalloproteinases (Shannon et al., 1989).
Recently, Hite et al. (1992a) reported the first cDNA sequence for a venom hemorrhagic toxin, HT-e from C. a t rox (Fig. 1). These data were significant in several respects: this cDNA sequence was the first reported for a hemorrhagic metalioproteinase toxin; the structure indicated that the metalloproteinases are synthesized with a signal sequence and a pro-enzyme sequence; and that the precursor proteinase can be proteolytically processed to produce a mature protein lacking domains present in the precursor (pro- and disintegrin-like domains). The cDNA sequences for five of the C. a t rox hemorrhagic toxins (HT-a, b, c, d, and e) are now completed, and from these data, in conjunction with protein sequence data, a new classification scheme for the venom metallo- proteinases has been proposed (Hite et al., 1994).
5.2. PRE- AND PRO-SEQUENCES OF THE C. ATROX HEMORRHAGIC TOXINS
Until the report by Hite et al. (1992a), no evidence had been presented that demonstrated that the toxins are translated from their mRNA in venom-producing cells with a signal sequence (pre-sequence) or that the toxins are synthesized in a latent zymogen form containing a pro-sequence, since in the mature form of the toxins, as isolated from the crude venom, neither of these structures had ever been observed. From the translated eDNA sequences of the C. a trox toxins, it can be concluded that the venom metalloproteinases are synthesized with highly conserved signal and pro-sequences (Hite et al., 1994). In Fig. 2, several of the C. a t rox toxin signal sequences are presented. These signal sequences are highly conserved and comprise 18 amino acid residues, with the putative cleavage between the pre- and pro-domains at Gly-18 and Ser-19, following the prediction of Von Heijne (1990).
I0 Ht-e MIQVLLVTICLAAFPYQG Ht-b MIEVLLVTICLAVFPYQG Ht-c . MIEVVLVTICLAVFPYQG Ht-d MIEVLLVTICLAVFPYQG Trig MIQVLLITICLAVFPYQG
FIG. 2. Comparison of the signal sequences translated from the open reading frames of venom metalloproteinases. The HT-b, c, d, and e sequences are from C. atrox toxins and the Trig
(trigramin) sequence is from T. gramineus.
JPT 62/3---G
352 J.B. BJARNASON and J. W. Fox
Ht-e Ht-b Ht-c Ht-d Trig Jara Rhod
i0 20 30 40 50 60 SSIILESGNVNDYEVIYPRKVTALPKGAVQPKYEDTMQYELKVNGEPVVLHLEKNKGLFS SSIILESGNVNDYEVVYPRKVTALPKGAVQPKYEDAMQYELKVNGEPVVLHLEKNKELFS SSIILESGNVNDYEVVYPRKVTALPKGAVQPKYEDAMQYELKVNGEPVVLHLEKNKELFS SSIILESGNVNDYEVVYPRKVTALPKGAVQPKYEDAMQYELKVNGEPVVLHLEKNKELFS SSIILESGNLNDYEVVYPEKVTALPKGAVQQKYEDAMQYEFKVNGEPVVLHLEKNKGLFS
...ATRPKGAVQPKYEDAMQYEFKVNGEPVVLHLEKNKGLFS ...FA
Ht-e Ht-b Ht-c Ht-d Trig Jara Rhod
70 80 90 i00 ii0 120 KDYSETHYSFDGRKITTNPSVEDHCYYHGRIENDADSTASISACNGLKGHFKLQGEMYLI KDYSETHYSPDGRKITTNPSVEDHCYYRGRIENDADSTASISACNGLKGHFKLQGEMYLI KD¥SETHYSPDGRKITTNPSVEDHCYYRGRIENDADSTASISACNGLKGHFKLQGELYLI KDYSETHYSPDGRKITTNPSVEDHCYYRGRIENDADSTASISACNGLKGHFKLQGEMYLI EDYSEIHYSPDGREITAYPSVEDHCYYHGRIENDADSTASISACDGLKGHFKLQGEMYLI KDYSEIHYSPDGREITTYPPVEDHCYYHGRIENDADSTASISACNGLKGYFKLQRETYFI KDYSETHYSPDGTRITTYPSVEDHCYYQGRIHNDADSTASISACNGLKGHFKLQGETYFI
Ht-e Ht-b Ht-c Ht-d Trig Jara Rhod
130 140 150 160 170 EPLKLS DS EAHAVFKLKNVEKE DEAPKMCGVTQ -NWES YE PI KKAS DLNL EPLELSDSEAHAVFKYENVEKEDEAPKMCGVTQ-NWESYEPIKKAS DLNLNPD EPLELSDSEAHAVFKLENVEKEDEAPEMCGVTQ-NWESYEPIKKAS DLNLNPD EPLELSDSEAHAVFKLENVEKEDEAPEMCGVTQ-NWESYEPIKKAS DLNLNPD E PLE LS DS EAHAVFKYENVEKE DE PPEMCGVTQ -NWE S YE S TKKASQ NVTPE EPLKLPDS EAHAVFKYENVEKE DEAPEMCGVTQ -NWKS YE P I KKASQLAFTAE EPMKLPDSEAHAVFKYENIEKEDESPKMCGVTETNWES DEPI KKVSQLNL
FIG. 3. Comparison of pro-(zymogen)sequences from several venom metalloproteinases. HT-e, b, c, and d are from C. atrox toxins. Trig (trigramin) is from T. gramineus, Jara (Jararahagin) is from B. jararaca jararaca and Rhod (Rhodostomin) is from A. rhodostoma. Notes: (1) Rhodostomin and Jararaghin clones are incomplete at the N-terminal end; . . . represents an incomplete end. (2) Positions of the C-terminal ends of some clones were
estimated by comparison with C. atrox processing.
The pro-sequences of the zymogen domain of the C. a t rox toxins are seen in Fig. 3. These sequences are also very highly conserved, and comprise approximately 190 amino acid residues. One very interesting region in the pro-sequence is observed approximately 23 residues amino-ter- minal to the start of the mature protein sequence. It was hypothesized (Hite et al., 1994) that this conserved region containing a cysteinyl residue may serve a function analogous to that of the nine-amino-acid consensus sequence proposed to be involved in the mechanism of pro-enzyme latency in the MMPs (Valee and Auld, 1990; Van Wart and Birkedal-Hansen, 1990). The toxin sequence of this region, P - K - M ~ C - G - V - T - Q , is similar to the MMP consensus sequence P - R ~ C - G - V / N - P - V / L - A / G . The venom sequence has a methionyl residue inserted between the lysyl and cysteinyl residues, but retains the C - G - V sequence of the MMPs. In the MMPs, it is postulated that the cysteinyl residue binds to the active site zinc, preventing substrate and water from entering the active site region, and thus, inhibiting catalysis until displacement of the cysteinyl ligand by either an organomercurial, or in vivo, a proteolytic event. We suggest that a similar mechanism of enzyme latency is present in the venom metalloproteinase zymogens (Hite et al., 1992a, 1994). Synthetic peptides composed of this stretch of amino acids from the putative latency site have been shown to be weakly inhibitory to the MMPs and a venom metalloproteinase toxin (Melchiori et al., 1992 and Fox and Shannon, 1993, respectively), supporting the hypothesis of the mechanism of latency. However, until zymogen forms of the venom metalloproteinases are isolated or constructed, the exact mechanism of enzymatic latency remains speculative.
5.3. PROTEINASE DOMAIN OF HEMORRHAGIC TOXINS
The translated cDNA sequences of the five C. a t rox hemorrhagic toxins are shown in Fig. 4. The sequences are homologous with one another, and all contain a zinc-binding consensus sequence H - E - X - X - H (Jongeneel et al., 1989). Recently, the metalloproteinase family has been subdivided based on specific zinc-binding consensus sequences found in the metalloproteinases (Jiang and Bond, 1992). According to the classification scheme of those authors, there are five subfamilies of metalloproteinases: thermolysin, astacin, serratia, snake venom, and matrixin (MMP). The
Hemorrhagic metalloproteinases from snake venoms 353
distinguishing sequence signature of the snake venom family in the classification scheme is H - E - L - - G - H - N - L - G - M - E - H - D , with the underline representing strictly conserved residues across all the venom metalloproteinases. The toxin consensus sequence is most similar to that of the astacin family sequence, H - E - I - G - H - A - I - G - F - X - H - E .
Previously, we have suggested, based on our sequence studies and those sequences found in the data bases, that the three histidines and water in the venom zinc binding consensus region are the four zinc ligands (Hite et al., 1992a, 1994). Although, as yet, we do not have complete crystal structural data for a hemorrhagic toxin, we hypothesize that there will likely only be four ligands, based on our early CD studies with cobalt-substituted HT-e, which indicated a distorted tetrahedral complex with oxygen and nitrogen ligands (Bjarnason and Fox, 1983). The earlier data correspond to the three histidines and one water molecule as the likely ligands for the zinc. Jiang and Bond (1992) suggested that in the case of the astacin subfamily, there would be five ligands to the zinc ion: the three histidines, the glutamate, and a tyrosine (located 46 amino acids carboxy to first histidine in the consensus sequence). These authors also suggested that a conserved tyrosine at a similar position in the venom metalloproteinases (tyrosine 379, Fig. 2) may serve a similar function as in the astacin family. The crystal structure for astacin, a metalloproteinase from crayfish, has recently been determined (Bode et al., 1992) and confirms the suggestions of Jiang and Bond (1992) for the five zinc ligands in astacin. However, we believe that the zinc ligand structure will be different in the case of the venom metalloproteinases, in that there will likely not be a fifth ligand and the zinc will be tetrahedrally coordinated by the three histidines and the one water molecule of the consensus site. We base this speculation on our previous spectroscopic studies and the fact that in the C. a t r o x toxins at position 379, the toxins HT-a, b, c and d all have a tyrosine, but in HT-e, there is present a cysteine in place of the tyrosine. Although cysteine could conceivably bind to zinc, the fact that the site is not completely conserved across the venom metalloproteinases forces us to doubt its function as a ligand.
The substrate specificity of the venom metalloproteinases is due, in part, to active-site substrate-binding sites and, perhaps in the case of the multiple domain toxins, modulated by those
10 20 30 40 5 0 60 70 80 90 100 H t - e ~VLLVT~C~AFPYQG~I~LESGNVNDYEV~YPRKVTALPKGAV~PKYEDTH~YELKVNGEPVVLHLEKNKGLFSKDY~ETHYSFDGRK~TTNPSVE H t - b ~I~VLLVTICLAVFPYQG~S~LESG~NDYEVVYPRKVTALPKGAV~P~Y~D~H~YELKVNGEPvVLHLEKNKELFSKDY~ETHYSPDGRK~TTNPSVE H t - c ~IE~LVT~CL&VFPYQG~ILESGNVNDYEVVYPRKVT~LPKGAvQPKYEDAHQYELKVNGEP~LHLEKNKELFSKDYSETHY~PDGRKITTNPSVE Ht-d M~EVLLVTICLAVFPYQGSS~LESGNVNDYEVvYPRKVTALPKGAV~PKYEDAMQYELKVNGEP~LHL~KNKELFSKDYSETHYSPDGRK~TTNPSVE
110 120 130 140 150 160 170 180 190 200 Ht-a ...ERLTKRYVE Ht-e DHCYYHGRIENDADSTASISACNG~KGHFKLQGEMYLIEPLKLSDSEAHAVFKLKNVEKEDEAPI~qCGVTQNWESYEPIKKA~DLNLNPEH~ .... RYVE Ht-b DHCYYRGRI~NDRDSTASISACNGLKGHFKI~EMYLIEPLELSD~ERRAVFKYENVEKEDEAPKMCGVTQNWESYEPIKKASDLNLNPDQ~NLP~RYIE Ht-¢ DHcYYRGRIENDADSTA~ISACNGLKGHFKLQGEL¥LIEPLELSDSEAHAVFKLENVEKEDEAP~CGVTQNWE~¥EPIKKASDLNLNPD~QNLPQRYIE Ht-d DH~YYRGRIENDRD~T~SISACNGLKGHFKL~GEMYLIEPLELSD~E~HAVFKLENVEKEDEAPKMCGVTQNWESYEPIKKA~DLNLNPD~NLP~RYI~
210 220 230 240 250 260 270 280 290 300 Ht-a LVIVA~HRMFTKYNGNLKKIRKWIY~IVNTTN~IYIPLNIRVALVRLEIWSNGDLIDVT~AANVTLK~FGNWRVTNLLRRKSHDNAQLLTAIDLDEETLG Ht-e LFIVv~GMYTKYNGDSDKIR~RVH~MVNI~KESYTYMYIDILLAGIEIWsNGDLINVQPA~PNTLNSFGEWRETDLLKRK~HDNA~LLTSIAFDEQIIG Ht-b LvvVRDHRVFMKYNSDLNIIRKRV~ELVNTINGFYRSLNID~LTDLEIWSDQDFITVQSSAKNTLNSFGEW~EADLLRRKSHDHAQLLTAINFEGKIIG Ht-c LVVVADHRVFMKYNSDLNTIRTRVHEIVNFINGF•RSLNIHV•LTDLEIWSNEDQINIQSAS•DTLNAFAEWRETDLLNRKSHDNAQLLTAIELDEETLG Ht-d ~VVvADHRVFM~fNSDLNTIRTRV~EIvNFINGFYRSLNIHv~LTDLEIWSNED~INI~SASSDTLNAFAEWRETDLLNRKSHDNA~LLTAIELDEETLG
310 320 330 340 350 360 370 380 390 400 Ht-a LAPLGTMCDPKLSIGIVQDHSPINLLVAVT~A~ELG~NLGMVHDENRCHCSTPACVMCAVLR~RPSYEFSDCsLN~YRTFIINYNP~C~LNEPL~TDIIS Ht-e RAYIGGICDPKRSTGVVQDHSEINLRV&VTMTHELGHNLGIHHDTD~CSCGGYSCIMSPVISDEPSKYFSDCSYI~CWEFI~KP~CILKK~LRTDTVS Ht-b RAYTS~MCNPRK~VGIVKDHSPINLLVGVT~AHELGHNLG~HDGDKCLRGASLCIMRPGLTPGRS~EFSDDSMGYYQSFLN~YKPQCILNKPLRIDPVS Ht-c LAPLGTMCDPKLSIGIVQDHSPINLL~ELGHNLG/4EHDGKDCLRG~SLcIMRPGLTKGR~LKQYKPQCILNKPLRIDPVS Ht-d LAPLGTMCDPKLSIGIV~DHSPINLLMGVTM~HELGHNLGHE~DGKDCLRG~SLcIMRPGLTKGRS~EFSDDSMH~YERFLK~YKP~CI~NKPLRIDPVs
410 420 430 440 450 460 470 480 490 500 Ht -a PPVCGNELLEvGEECDCGSPRTCRDPCCDAATCKLH~WVECESGECCQQCKFT~AGNVCRPARSECDIAE~CTGQSADCPTDDFHRNGKPCLHNFGYCYN Ht-e TPvSGNELL~AGIECDCGSL---ENPCCYATTC~g~PG~CAEGLCCDQCRFMKKGTVCRV~MVDR~-DDTCTG~SADCP . . . . . RNGL Ht-b TPVSGNELLEAGEE Ht-C TPVSGNELLEAGEE Ht-d TPVSGNELLEAGEE
510 520 530 540 550 560 570 580 590 600 Ht-a GNCPIMYH•CY&LWGSNVTVAPDACFDIN•SGNN•FYCRKENGVNIPCAQEDVKCGRLFCNVNDFLCRHK••DDGMVD•GTKCADGKv•KNR•CVDVTTA
610 Ht-a ~KSTSGFSQI
FIG. 4. Protein sequences of C. atrox hemorrhagic toxins. Signal peptide: 1-18; zymogen: 19-188 (HT-e), 19-190 (HT-b/c/d); mature protein: ?-610 (HT-a), 189-393 (HT-e); 191-393 (HT-b/c/d); HT-a/e disintegrin-like domain: 415-488; HT-a carboxyl cysteine-rich domain:
489-610.
354 J.B. BJARNASON and J. W. Fox
Ht-e LRTDTVSTPVS GNELLEAG ~ E Ht-a LQTDI I SPPV~GNELLEVGEE lIRIB SKTDIVS PPV~NELLEAGEE Jara LGTDIISPPV~NELLEVGEE RVVX LRKDIVSP~NEIWEEGEE
EAPI FI~NFDDFQF~NKKLDEGEE eH~oll ss~v~s~raw~.D Cyrl FVVQPQGGSTNHLLEVPEQ
B i t i s t a t 3 VS PPV~GNKI LEQGED Tri~ram~n EAGED Rhodosto~ln GKE Er~stlooph~n Ech~stat~n
. e . g~sL .... s a ~ Y a ~ R ~ s o ~ g ~ - -aaFaK.G~rJaVS~D-~D~OSADmP ..... ~C InlasParl-am~D~rllKIasw~|ssG~--UKrrs^GsvllaP~s~l~IAns~s~DDFa~u I~liGS PS~I-QYQiI~SlIKLnSm,~SSQ~t~- -pn~rA~TE~SqDZ PSS~rGQSA~Darn~a ~GTPEN~-QNE~DAAT~KLKSGS~GHG~EQ- -~KFSI~GTE~ASMSE~P~SSE~PADVFHKNG
It~PeOsll-a'm~lRD~.nTilv~e~rq~¢GSlisS--I~n~ra~sr I i~PASDSi~rP~aSP~-SPK~ F~V ~IEGSQEi~I-ODT~DAAT~RLKSTSR~QGP~NQ--I~EFKTKGEVmRESTDE~DLPEY~NGSSGAI~-QEDL¥
E~ESG~--~KFLKEGTI IERARGD-DMDD~GKTCDIPRNPHKGPAT
FIG. 5. Disintegrin-like domains. Sequences from snake venom metalloproteinases and mammalian reproductive proteins are compared with true disintegrins. Cysteine residues are shaded to note conserved sites. The RGD tripeptide position is noted by asterisks. HT-e (Hite et al., 1992b), HR-1B (Takeya et al., 1990a), Jara =jararhagin (Paine et al., 1992), RVV-X (Takeya et al., 1992), EAP-I (Perry et al., 1992), PH-30b (Blobel et al., 1992), Cyri = cyritestin (Senftleben et al., 1992), Bitistat3 = bitistatin 3 (Shebuski et al., 1989), Trigramin (Huang et al., 1989), Rhodostomin (Dennis et al., 1989), Eristocophin (Scarborough et al., 1991),
Echistatin (Ganet al., 1988).
additional domains (Hite et al. , 1992a, 1994). As more information on venom metalloproteinases has become available, it is of interest to compare the sequences of the hemorrhagic and non-hemorrhagic proteinases to search for specific amino acid residues that may confer the capacity for generating hemorrhage (Takeya et al . , 1990b; Hite et al. , 1992a,b). However, these types of comparisons have proven less than successful, in that very few residues in the proteinase domains of all of the venom metalloproteinases are conserved exclusively in either the hemorrhagic or non-hemorrhagic metalloproteinases (Hite e t al. , 1994). Thus, it is unclear whether this type of analysis will ever prove conclusive without the addition of many more sequences to the data banks.
From substrate specificity studies of venom metalloproteinases (reviewed in Bjarnason and Fox, 1988-89), there appears to be a general, overall preference for a hydrophobic residue in the S't substrate binding site of these proteinases. This suggests that there should be a moderately conserved hydrophobic sequence or sequences present in the proteinase structures in all of the venom enzymes that could provide such a hydrophobic interaction site for the substrate. When one surveys the sequences of the C. a t r o x toxins, on region may actually fit that description: the X - X ~ - I / V - M - X - X sequence carboxy to the active site zinc (residues 355-361, Fig. 4). This structure will act as a hydrophobic platform or pocket for a substrate. The cysteinyl residue in this stretch of sequence (residue 357) is disulfide-linked to the cysteinyl residue at position 350, which would bring the sequence into close structural proximity to the actie site (recall that one of the zinc histidine ligands is presumed to be at position 345). Confirmation of these speculations will require crystal structure data on the hemorrhagic and non-hemorrhagaic metalloproteinases. These will probably show that, in addition to the general hydrophobic active site/substrate interaction pocket, other specific sequence modifications contribute to the toxic potency of these proteinases by modulating the substrate selectivity and reactivity.
5.4. DISINTEGRIN-LIKE DOMAIN OF HEMORRHAGIC TOXINS
Disintegrins are anticoagulant polypeptides that have been isolated from a variety of crotalid and viperid venoms (Gould et al. , 1990). These peptides have the characteristic biological activity of being inhibitors of platelet aggregation by competing with fibrinogen for binding to the platelet's glycoprotein l ib-Il ia complex. Recently, disintegrin-like domains have been observed at both the protein and cDNA level for several venom hemorrhagic metalloproteinases, as well as non-hem- orrhagic venom metalloproteinases (Takeya et al. , 1990a, 1992; Hite et al. , 1992a, 1994; Paine e t al. , 1992) (Fig. 5). Although these domains found in the venom metalloproteinases share high sequence identity with the disintegrin peptides they have been called disintegrin-like since they do not contain the typical R--G-D sequence typically found in most disintegrin peptides. Thus, the venom disintegrin-like domains found associated with the hemorrhagic toxins appear to form a group of 'non-RGD' disintegrins (Paine et al. , 1992).
Hemorrhagic metalloproteinases from snake venoms 355
There is evidence that hemorrhagic toxins containing the non-RGD disintegrin-like domain can inhibit platelet aggregation. However, these data must carry the caveat that the inhibitory function may also be affected by the proteolytic capacity of the toxin. By what mechanism and with which integrins the non-RGD disintegrins interact is not yet clear. From a functional viewpoint, we suspect that these domains play an important role in the biological activity of the toxins, and, indeed, the toxins that contain the disintegrin-like domain generally display a greater hemorrhagic potency. The manner by which the disintegrin-like domains manifest this enhanced activity in the toxins is not known, but obviously an integrin/disintegrin interaction of some sort may be involved.
5.5. HIGH CYSTEINE DOMAIN OF THE HEMORRHAGIC TOXINS
The higher molecular mass toxins (60-90 kDa), including HT-a from C. atrox venom, have present a domain, carboxy to the disintegrin-like domain, that is remarkable for its relatively high cysteine content. It is assumed that the cysteinyl residues in this domain are actually found in the disulfide bonded form, but the number and arrangement of disulfide structures in this domain have yet to be determined. The hemorrhagic toxins HR-1B from T. flavoviridis and Jararhagin from B. jararaca both have homologous domains with regard to the relative positions of the cysteine residues (Takeya et al., 1990a; Paine et al., 1992). The non-hemorrhagic metalloproteinase RVV-X from V. russelli also possess a homologous high-cysteine domain (Takeya et al., 1992). A schematic diagram of these domains is seen in Fig. 6. From this alignment, a very concise pattern of cysteine positions shared among the hemorrhagic toxins in the first half of the domain is found: Cys-6X-Cys-4X-Cys-14X-Cys-12X-Cys-9X~2ys-4X-Cys-. In the second half of the domain, the pattern is somewhat more variable, with insertions needed for alignment: Cys-6/10X-Cys-15/18/19X--Cys-5X-Cys-4/5X-Cys-. Thus, it seems that the high-cysteine do- main is comprised of two subdomains, in which the first half has a more highly conserved cysteine pattern than has the second half. Since the cysteine pattern of the first subdomain is completely conserved among the venom proteins, it would appear that this region of the high-cysteine domain is more critical in maintaining some functional characteristic of the domain and, consequently, of the protein.
Although the functional significance of the high cysteine domain, at present, is unknown, the domain is only observed when there is a disintegrin-like domain preceding it. This may suggest that the high cysteine subdomain adjacent to the disintegrin-like domain may be of structural significance, perhaps promoting the correct spatial orientation of the disintegrin-like domain present in these proteinases or, as in certain other proteins with domains containing an unusually high cysteine content, these subdomains may be involved in protein-protein interactions. Further studies are required to confirm these speculations.
One role for the second subdomain in RVV-X is known. The unmatched 'extra' cysteine present in this subdomain serves to covalently link the heavy chain of the protein (containing the metalloproteinase, disintegrin and high-cysteine domains) to a light chain, which is homologous to C-type lectins (Takeya et al., 1992). This particular protein is discussed in more detail in the next section.
s u b d o m a i n a s u b d o m a i n b I
Ht-a Cys-X0- CyeI-X4-Cym-Xs-Cym-XI4-Cys-XI2-CYm -Xs-CYS-Y~-CYS -X4-~s-X e-CYs . . . . . . . . X15-CYs-Xs-C~s-X4-CYs -xle HR1B C~S -X6-C~s-X4-~s-X6-C~8- XI4-C~S-XI2-CYB -Xg-C~s-X6-CFli-X4-~S-X 4-C~s . . . . . . . . X18-C~s-Xs-C~B- X4-C~s -X9 J a r a r C~S -X6-C~s-X4-Cys -X6-c~s-X14-C~s -X12-C~rs -xo-eys-~-cYR -x4-~S-XlO-~s . . . . . . . . Xlll-~s-xs- CYs -x4-c~s-A7 RW-X Cys-Xo-Cys-X4-Cys-Xs-CyS-X:4-Cys-X12-Cys-Xo-Cys-Xe-CyS-X4-CyS-XIo-CyS-X s-Cys-Xls-CYs-Xs-Cys-X4-Cys-X12
s u b d o m a i n a subdolains b ' / b m
eAP-z cy~-xe-cy~-x4-c~-xe-cy~-x14-cy~-~1s-cy~-~-cy~-x6-cy~-~4-cy~-x~-cy~-x~-cy~-xs-cy~ PH-30: CyB -XB-C~I-X4-CYB-XS-CyeI-XI4-CyB-X13-C~ru -Xl I-C~eI-X6-CYa-X4-CYs-~-~s-~-~s - ~ - ~ s t:~-3 op Cys -X6-Cys-X4-Cys-Xe-Cys-XI4-Cys-Xla-CYs -Xm-Cys-Xe-Cys-X4-Cys-X~-Cys-X~-Cys-Xs-CYs ~rit cys-X6-cys-X4-C~s-Xe-cys-X14-cys-xla-cys-x a-cys-xs-cys-x4-cys-X24-cys-x~-cys-X6-~s
FIG. 6. Cysteine consensus of class P-Ill venom metalloproteinases and mammalian reproduc- tive proteins. X, represents a space on n amino acids. Subdomain a is very highly conserved. Subdomain b diverges when compared between the venom and the mammalian structures.
356 J. B. BJARNASON and J. W. Fox
TRANSLATION
P-I*,T-b.c.~.,~ I ,,o~ainas, J N-I class (HT-b,c,d) J Proteolysis - - [ - ~
P-II (HP-I ?' I Proteinase I I Olsin'l N-II class (HT-e, HP-I) I Proteolysia ?
P-Ill (HT-a) I Proteinase II Disin.J Hi-cys J (RSL)
N-Ill class IHT-a) I Proteolysis ? I -as
P-IV |RVV-X) i Proteinase II oi,in.I Hi-cvsl ~ N-IV class (Mucro-A, RVV-X)
NUCLEOTIDE CLASSES M A T U R E PROTEIN CLASSES
FIG. 7. Schematic diagram of the snake venom metalloproteinase class structure. This schematic shows the three known nucleotide classes and a hypothesized fourth class. The right-hand side of the diagram indicates the possible protein products after post-translational processing, including how most (if not all) disintegrin peptides are likely to be produced.
Disin., disintegrin, disintegrin-like.
5.6. CLASSIFICATION SCHEME FOR VENOM METALLOPROTEINASES
From protein and cDNA sequence data of venom metalloproteinases generated from our laboratory, as well as from others, we can now more clearly define a classification scheme for the venom metalloproteinases. This task, until recently, was a questionable one, requiring constant modification as new data became available. We now understand the potential of the metallo- proteinases to be proteolytically processed and glycosylated, factors that previously confused classification schemes. Many of these schemes (see Table 2) were based upon molecular mass of the venom metalloproteinases, with the range of molecular masses described for the hemorrhagic toxins from less than 20 kDa to over 90 kDa (Bjarnason and Fox, 1989). However, we believe that the field is at such a point that we can now propose a scheme that, notwithstanding minor modifications, will be of value for venom scientists, as well as investigators of metalloproteinase structure and function.
Since data are now available on the cDNA structures of the venom metalloproteinases as well as the protein structures, we have developed a two-part, interrelated classification scheme based on these two sets of data (Fig. 7). The first part of the scheme is based on the cDNA sequences of the venom metalloproteinases. The first class, termed N-I (nucleotide class I), is the class of metalloproteinases, which, at the level of the cDNA (mRNA), codes for a signal sequence, pro- (zymogen) sequence and metalloproteinase sequence. Members representing this class from the C. atrox hemorrhagic toxins include HT-b, c, and d. The second nucleotide class, N-II, has signal, pro-, proteinase and disintegrin-like cDNA sequences. Th e representative member of this group from C. atrox venom is HT-e. N-III is the third nucleotide class, and these sequences represent information for signal, pro-, proteinase, disintegrin-like, and high-cysteine domains. The cDNA sequence for HT-a is a member of this class.
We propose a putative N-IV class, which has sequence information for signal, pro-, proteinase, disintegrin-like, high-cysteine and lectin domains. No nucleic acid sequence information is available for this class; however, based on the protein sequence of RVV-X, a high molecular mass metalloproteinase from V. russelli and partial protein sequence for mucrotoxin a from T. mucrosquamatus, we hypothesize that the DNA structure for these proteinases would be represen- tative of this class. This will be considered further below.
When one surveys the protein sequence data of the venom metalloproteinases, one can propose a second, parallel and interrelated classification based on the structure of the mature protein as it occurs in the crude venom. The P-I class (protein class I) has only a proteinase domain in its mature form, i.e. following proteolytic processing of the signal and zymogen structures. Again using the C. atrox hemorrhagic toxins as examples, HT-b, c, d, and e would be members of this class
Hemorrhagic metalloproteinases from snake venoms 357
(class I-B in Section 2). It must be noted that HT-e is a member of the N-II class of nucleotide structures, but the disintegrin domain coded for by the N-II structure is absent in the mature protein with the result of HT-e being a P-I class proteinase (class I-A in Section 2).
There is no venom metalloproteinase, which has been characterized to an extent, that definitively allows it to be classified as a P-II proteinase. However, there are venom proteinases about which there is sufficient data available that makes them strong candidates for membership in this class. For example, HP-I from Calloselasma rhodostoma is a hemorrhagic metalloproteinase with a molecular mass of 38kDa (Bando et al., 1991). From its amino acid composition, it has 311 residues of which 17 are cysteines. These data fit well to the presence of a proteinase domain containing approximately 200 residues of which four are cysteines, a spacer of 21 residues and a disintegrin-like domain of approximately 75-80 residues of which 14 are cysteines. This would comprise a structure similar to that of the HT-e precursor protein in which the nascent disintegrin-like domain was not removed by proteolytic processing (Fig. 7). The complete protein sequence analysis of HP-I is needed to confirm this structure. Certainly, as more data become available on other venom metalloproteinases of this size (class II in Section 2, Table 2), one would anticipate that many of them would be in the N-II/P-II class.
There are three members of the P-III class that have had either their protein and/or cDNA sequences determined: HT-a from C. atrox; HR-1B from T.flavoviridis; and Jararhagin from B. jararaca (Takeya et al., 1990a; Paine et al., 1992; Hite et al., 1994). In their mature form, they all comprise a proteinase domain, a disintegrin-like domain, and a high-cysteine domain. All of these proteinases are potent hemorrhagic toxins, more so than the lower molecular weight toxins, strongly suggesting the importance of the disintegrin-like and the high-cysteine domains for the hemorrhagic activity of these toxins.
The final protein class, P-IV, has two members identified to date, RVV-X from V. russelli (Takeya et al., 1992) and mucrotoxin A from T. mucrosquamatus (Kishida et al., 1985). RVV-X is a 79-kDa protein containing two disulfide-bonded protein chains (59 kDa and 21 kDa), which functions as a coagulation factor X-activating enzyme. The heavy chain is homologous to the structure of the high molecular weight hemorrhagic toxins, such as HT-a and HR-1B, although RVV-X is not hemorrhagic. The light chain is disulfide-bonded to the heavy chain through an extra cysteine present in the second subdomain of the high cysteine domain (Fig. 7). Although it is not known, we would speculate that the protein is translated as a six-domain precursor protein (pre-, pro-, proteinase, disintegrin-like, high-cysteine, and C-lectin) from one long transcript, which is then proteolytically cleaved to produce the two-chain structure. Although this proteinase is not a hemorrhagic toxin, there may be other venom metalloproteinases of comparable size and structure that are hemorrhagic. Mucrotoxin A is a hemorrhagic metalloproteinase from T. mucrosquamatus (Sugihara et al., 1983) with a molecular mass of approximately 94 kDa. This toxin has 39 cysteinyl residues in its amino acid composition, which is similar to the number found in RVV-X (44 cysteines). There is no evidence for mucrotoxin A being a two-chain protein, as is the case for RVV-X. Preliminary protein sequence data on this protein indicate that it is a single-chain protein that has a C-type lectin domain homologous to that in RVV-X. The fact that mucrotoxin A is a single long-chain toxin containing the c-type lectin domain suggests that RVV-X is also translated as a single long-chain protein, which then is processed to release the lectin domain so that it is tethered by a disulfide bond to the high-cysteine domain of the heavy chain. The reason some of the P-IV class of venom metalloproteinases are processed at the carboxy terminal and how this affects the biological activity of the proteinase is uncertain. Furthermore, more data are needed to be confident that only a lectin-like domain is found following the high-cysteine domains in these proteinases, and not some other, as yet, undescribed domain. These concepts will be discussed further in Section 5.7, when we review the structure of the homologous mammalian reproductive proteins.
There certainly will be more examples of the P-IV class of venom metalloproteinases appearing with time. One likely candidate is LHF-I from the venom of the bushmaster, L. muta muta. The purification and partial characterization of this protein was reported several years ago, and it was noted that this proteolytic hemorrhagic toxin has an approximate molecular mass of 100 kDa (Sanchez et al., 1987), which could place it in the P-IV class. Unfortunately, the investigators did not report whether the proteolytic activity could be attributed to a metalloproteinase mechanism,
358 J.B. BJARNASON and J. W. Fox
so it is uncertain as to whether this toxin is, in fact, a metalloproteinase and, thus, fits into the venom metalloproteinase classification scheme.
In the above discussion, we have presented a new classification scheme for the venom metalloproteinases based on either their nucleotide sequences or the mature, processed forms of the proteinases. We feel that the two interrelated classifications are necessary, since there is clear evidence that the protein forms can be proteolytically processed, producing a product different in structure (and perhaps function) from the originally translated precursor. This scheme should allow for a clearer understanding of the ontogeny of the metalloproteinases as they are found in the crude venom, and, thus, ameliorate some of the confusion surrounding the structural relationships of the various venom metalloproteinases.
5.7. RELATIONSHIP OF VENOM METALLOPROTEINASES TO MAMMALIAN REPRODUCTIVE PROTEASES
Until recently, the snake venom metalloproteinases were thought to be representatives of a subfamily of metalloproteinases with no non-venom homologs. However, there are now reports of several mammalian proteins that have clear sequence identities to the venom metalloproteinases. PH-30 is a protein from guinea pig sperm composed of two similar chains, a and b (Blobel et al., 1992). PH-30 is bound to the membrane of guinea pig sperm and is thought to be involved in the sperm-egg fusion process since on chain of the protein contains a viral-like fusion sequence. When the protein sequences of the chains were first reported, the protein was observed to contain only a disintegrin-like domain (lacking the signature R G D sequence found in disintegrin peptides) followed by a high cysteine domain, ending with a transmembrane sequence and cytoplasmic domain. From more recent cDNA sequence studies on this protein, there appears to be a metalloproteinase or metalloproteinase-like sequence amino terminal to the disintegrin-like domain of the chains, thus presenting a structural organization not unlike the N-III/N-IV class of venom metalloproteinases (Fig. 8).
Another example of a similar mammalian reproductive protein is the Epididymal Apical Protein I (EAP-I) from rat (Perry et al., 1992). This is an androgen-induced epididymus-specific protein that is homologous to PH-30. Although it does not appear to have a fusion sequence, it does have a transmembrane domain and overall domain organization similar to PH-30. A mouse cDNA
| | | H |
I Pre- i |
I Pro-
I
i il l.mm
~ m
I Spacer i Proteinue I I DIsI~ tegrin
, ~ ~ !
a b i
• i : - l i l l ,
' I Hl-,cys Trnnsmembrlne
i Lectln i c~o~,smic I
* I I . i b ' ! ,
Class P4 (HT-b,c,d)
Class P-r (FIT-e)
Class PAl (HPJ ?)
Class P411 (HT-a)
Class P-iV (RW-X)
J rAP I
| ! ! 1 1 , i,,.E .. I I ,~,.,.~o ,~ | - - . i i o. P . ~ 0
I Fusion
FIG. 8. Structural relationship among the snake venom metalloproteinases and mammalian reproductive proteins. Shared domain structures are indicated.
Hemorrhagic metalloproteinases from snake venoms 359
sequence coding for a protein named cyritestin shows the same structural organization as PH-30 and EAP-I (Senftleben et al., 1992).
The sequence identity shared between the proteinase domains of these mammalian proteases and the venom proteinase domains is approximately 20-25%, which is somewhat low but does represent continuous homology over a large stretch of sequence and, thus, is likely to be significant. The homology in the disintegrin-like domains is more striking. Interestingly, these disintegrin-like domains in the mammalian proteases also lack the signature RGD sequence found in the disintegrin peptides and, therefore, are more similar to the venom metalloproteinase disintegrin-like domains (Fig. 5). Finally, the cysteine repeat pattern in the first high cysteine sub-domain of the venom metalloproteinases is nearly identical to the same region at the beginning of the high cysteine domain of the mammalian proteases (Fig. 6). The second sub-domain of the venom high-cysteine domain is different from those of the mammalian proteases, suggesting a different function. The fact that all of these proteins share a nearly identical cysteine repeat pattern (Cys-5/6X-Cys-4X-Cys-14X~Cys -12/13X-Cys-9/10/11X-Cys-6X- Cys--4X-Cys-) immediately following the disintegrin-like domain strengthens the argument that this sub-domain is important to the structure/function of the disintegrin-like domain. The second and third cysteine-rich sub-domains following the first cysteine sub-domain of the mammalian proteins are similar for PH-30 and cyritestin, but different from EAP-I, suggesting that these sequences following the first conserved cysteine pattern adjacent to the disintegrin-like domain may influence the different functions of these mammalian reproductive proteins.
Based on the structural similarities of the venom metalloproteinases and the mammalian reproductive proteins, it is tempting to speculate on the evolutionary relationship of their functions. Unfortunately, proteolytic activity for the mammalian proteins has yet to be demonstrated, so whether these metalloproteinase-like sequences can actually function as proteases is unknown. Perhaps they simply represent a vestige of previous proteolytic structure from an earlier time. However, from the viewpoint of biological economy, it is unlikely that the cell would support this non-functional sequence information.
It is not unreasonable to expect the proteinase function and, thus, the biological activity of these related venom and mammalian enzymes discussed above to be modulated by the domains following the proteinase domain. For example, in the case of the hemorrhagic toxins, the disintegrin-like domain may function to alter platelet aggregation via interaction with the GP IIblIIa receptor, and also target the enzyme to regions where platelets may adhere (i.e. disruptions in the capillary basement membrane). In the PH-30 chains, the disintegrin-like domains may also serve to target the protein and, therefore, the sperm to receptors present on the egg. As further investigations progress, more insight into the interactions of these disintegrin-like domains will be gained and, consequently, clarify the biological function of the domain. With regard to the high-cysteine domains found in this group of proteins, it is suggestive that the strongly conserved cysteine repeat found in the first subdomain is important in the functioning of the preceding disintegrin-like domain. In all of these proteins, the sub-domains that follow this first sub-domain probably serve individualized functions specific for the protein. In the case of PH-30b, the second high cysteine sub-domain contains within it the viral-like fusion sequence, and one can speculate that this sub-domain is important in presenting that fusion sequence in a recognizable form to a cell membrane. With the venom protein RVV-X, the second sub-domain serves as the connecting site for the light chain (C-lectin). The functions of these sub-domains in EAP-I and cyritestin are unknown.
In summary, the venom metalloproteinases, regardless of size and function, are structurally related via the classification scheme based on domain structures described above. Furthermore, these venom proteinases, particularly of the P-III and P-IV class, are structurally related to the mammalian reproductive proteins EAP-I, PH-30, and cyritestin. It is expected that additional proteins will be found from snakes and mammals, as well as other organisms, which will strengthen the foundation for the proposal of a class of unique proteins forming a subfamily of the metalloproteinases and related by the structural homologies we have outlined above. We propose that this subfamily be called the reprolysins, a term derived from the 'reptilian and reproductive' origin of these proteins.
360 J.B. BJARNASON and J. W. Fox
6. MECHANISM OF HEMORRHAGE PRODUCTION
Localized hemorrhage and, in some circumstances, disseminated hemorrhage, is the distinguish- ing characteristic of most crotalid and viperid envenomations (Arnold, 1982). Venoms are complex mixtures containing many biologically active proteins. In the case of hemorrhage production, it is likely that several venom components are involved. The primary factors responsible for hemorrhage are, by definition, the hemorrhagic toxins, those proteins that can individually produce the hemorrhagic effect. However, a variety of non-hemorrhagic components, both enzymatic and non-enzymatic, can give rise to poorly-clotting or non-clotting blood. These compounds are considered to act synergistically with the hemorrhagic toxins to enhance the hemorrhagic effect (Denson, 1969; Cheng and Ouyang, 1967; Pandya and Budzynski, 1984; Bjarnason et al., 1983; Ferreira, 1965; Feldberg and Kellaway, 1937). This section will consider only the action of the hemorrhagic toxins.
As previously mentioned, in severe cases of envenoming, disseminated hemorrhage is often observed. In these instances, the venom is carried by the circulatory system to distant sites, such as the kidneys, lungs, and brain, where there are highly vascularized, localized concentrations of basement membranes. Basement membranes, a subset of extracellular matrices, are highly organized sheets of interacting glycoproteins separating epithelial and endothelial cells, and surrounding muscle, fat, and nerve cells (Timpl, 1989). These structures are thought to be involved in regulating cell phenotype, tissue invasion, and molecular filtration (Timpl and Dziadek, 1986; Martin and Timpl, 1987; Liotta and Rao, 1985). Once it was confirmed that hemorrhage is due to the proteolytic function of the toxins (Bjarnason and Tu, 1978), early studies on the biological function of hemorrhagic toxins were then designed to investigate the ability of hemorrhagic toxins to hydrolyze basement membranes. The rationale for this being that disruption of the basement membrane surrounding capillaries and small vessels would allow for the escape of blood into adjacent stroma, thereby producing hemorrhage.
Ohsaka et al. demonstrated that several hemorrhagic proteinases (H-R1, HR-2a, and HR-2b) from T. f lavorviridus were capable of proteolytically releasing soluble peptides from isolated basement membranes (Ohsaka et al., 1973). Furthermore, as described in Section 3, microscopic data were being generated during this same period that indicated that the hemorrhagic toxins, indeed, did disrupt basement membranes surrounding capillaries, with the result of hemorrhage into the stroma. The early studies on basement membrane degradation by hemorrhagic toxins were extended by the work of Bjarnason et al. (1988). In this investigation, they examined the proteolytic disruption of basement membranes isolated from EHS tumor by the hemorrhagic toxins HT-a, e, b, and d from C. a trox venom. Over the time course of the experiment, it was determined that HT-a and e were significantly more proteolytically active on the basement membrane preparation than HT-b and d. The interesting observation from this experiment was that the proteolytic activity of the toxins on this substrate reflected their hemorrhagic activity, in other words, the toxins with the greater hemorrhagic activity also had the greater proteolytic activity on the basement membrane preparation. When the turnover numbers for the hydrolysis of oxidized insulin B chain by HT-a and b were compared, it was seen that the low hemorrhagic activity toxin HT-b produced a greater turnover number on this substrate than HT-a. Thus, it was suggested that hemorrhagic potency was, at least in part, related to the ability of the hemorrhagic toxins to recognize the critical substrate for hemorrhage and its consequent rate of cleavage of specific peptide bonds in the substrate in order for there to be proteolytically productive disruption of the basement membrane, resulting in hemorrhage.
The ability of the C. a trox hemorrhagic toxins to disrupt basement membrane components was also examined by following the course of degradation by SDS-PAGE of the products (Bjarnason et al., 1988). The results from these experiments substantiated those discussed above in that the individual components of basement membranes, collagen IV, laminin, and nidogen were more rapidly degraded by the hemorrhagic toxins a and e than the low hemorrhagic activity toxins b or c. The ability of these hemorrhagic toxins to degrade fibrinogen and fibrin was also investigated. Hemorrhagic toxins a, b, c, d, and e all digested fibrinogen; none of the toxins could digest fibrin. These results demonstrate that a secondary property of these hemorrhagic toxins is their capacity
Hemorrhagic metalloproteinases from snake venoms 361
to defibrinogenate blood, which could increase hemorrhage, following the action of the toxins on the capillary basement membrane, by decreasing the clotting ability of the blood.
The degradation of basement membrane by the C. a trox hemorrhagic toxins was investigated further by examining the proteolysis of individual basement membrane components by the toxins (Baramova et al., 1989). None of the toxins were found to digest collagens, I, III, or V; however, all toxins cleaved fibronectin, laminin, collagen IV, nidogen, and gelatins. Comparison of the rates and patterns of digestion by the hemorrhagic toxins of the basement membrane components allowed for some suggestions to be made regarding the relationship of basement membrane component degradation and the hemorrhagic activity of a toxin. As expected, HT-c and d degraded all of the substrates identically, since these isoenzymes differ by only one amino acid. HT-a and HT-e showed some similarities in their digestion patterns of the various substrates, but the patterns were not identical and were quite dissimilar to the patterns of the low activity toxins HT-c and d. On consideration of all these data, it was noted that the mammalian proteinase with the greatest degree of substrate similarity with the venom hemorrhagic proteinases was stromelysin (MMP-3), a metalloproteinase isolated from human fibroblasts (Okada et al., 1986; Murphy et al., 1985). This proteinase can degrade fibronectin, laminin, collagen IV, and cartilage proteoglycans, and produces a digestion pattern of basement membrane similar to the hemorrhagic toxins (Okada et al., 1986). Stromelysin is very effective at degrading basement membranes, apparently in a manner not unlike the hemorrhagic toxins. Although the hemorrhagic toxins and stromelysin are all metallo- proteinases, they do not share any structural similarities in their proteinase domain structures other than the zinc-binding consensus sequence. Thus, it can be said that, despite differing structures, these proteinases are capable of disrupting basement membranes in a similar manner by degrading the basement membrane proteins in such a way as to produce a similar effect.
One of the current views of basement membrane supramolecular organization is that laminin and collagen IV form homotypic polymeric networks, with nidogen (entactin) serving as a bridging molecule joining the two network systems (Fox et al., 1991; Aumailley et al., 1993; Yurcheno et al., 1992). To elucidate the mechanism of the structural degradation of basement membrane, as related to the hemorrhagic potency, representatives of the C. a trox toxins were examined for their sites of cleavage of several basement membrane components (Baramova et al., 1990a, 1991; Bjarnason et al., 1993).
Isolated EHS tumor collagen IV was observed to be specifically cleaved by HT-e, with the first site of cleavage occurring at position Ala219-Gln220 in the ct I(IV) chains and at Thr228-Leu229 in the 0t2(IV) chains (Bjarnason et al., 1993). Both of these sites are located in the third triple-helical interruption from the N-terminal just carboxy to the 7S domain, which is responsible for homotypic association of the monomers into tetramers. Thus, cleavage of the molecule at this point could seriously compromise the polymeric network structure of collagen IV, since the oligomerization of collagen IV at the 7S region is one of the most important sites of homotypic association for collagen IV, in addition to the lateral association of a region of the triple helical to the NC1 domain and the association of NC1 domains to form dimers. The second specific cleavage, which occurs following the primary cleavage, is located in the carboxy third of the molecule adjacent to the NC1 domain. This may be significant in that the collagen IV lateral associations, as well, and the nidogen/collagen IV associations are thought to occur in this region (Aumailley et al., 1989). Other than these two major sites of cleavage of collagen IV by HT-e, no other sites were observed, indicating that HT-e is rather specific in its degradation of collagen IV.
Specific cleavages of two non-collagenous basement membrane proteins by HT-e have also been investigated (Baramova et al., 1991). Laminin, nidogen, and nidogen in its natural 1 : 1 complex with laminin, were incubated with HT-e, and the digestion products separated by SDS-PAGE, electroblotted onto PVDF membrane and sequenced. Only cleavages in the A chain of laminin at position 2666 and in the B2 chain at position 1238 were observed, whether or not nidogen was present. Hence, laminin appears to be relatively insensitive to degradation by HT-e. From the digestion of nidogen alone, cleavage positions at residues 75, 336, 402, and 920 were identified from sequence analysis and at position 296, 478, 625, and 702, as proposed from the molecular mass of the digestion products (Fig. 9). The nidogen in nidogen/laminin complexes was cleaved at positions 322, 336, 351, and 840, as seen from the sequence, and at residue 953, as proposed from the molecular mass of a digestion product. The cleavage of the nidogen/laminin complex was
362 J. B. BJARNASON and J. W. Fox
HT-e
Collagen IV Laminin
Proteoglycan
FIG. 9. Schematic of HT-e cleavage sites on nidogen in the nidogen/laminin complex. Three cleavage sites are in the link region near the G2 globular domain and one site is in the rod region near the G3 globular domain. The regions that bind collagen IV, proteoglycan, and
laminin are indicated by the horizontal bars.
somewhat more restricted than uncomplexed nidogen alone, which is as expected, since nidogen could be protected from proteolysis by the interaction of its G3 domain with the B2 chain of laminin (Fox et al., 1991). Furthermore, since the isolated nidogen used in the experiment had been exposed to denaturants in order to separate it from laminin, it could be expected to be more susceptible to proteolytic degradation (Fox et al., 1991). Most of the cleavages of nidogen in both circumstances occurred in the flexible link region connecting the G1 and G2 globes of nidogen (Fox et al., 1991). Although the function of the G1 domain is unclear, there is some preliminary data that suggests that this region may be involved in oligomerization of nidogen (Fox et al., 1991).
One of the more interesting cleavage sites of the nidogen/laminin complex by HT-e is at position 840, which is in the rod-like domain of nidogen. This domain separates the G2 from the G3 domains. The G2 domain has been demonstrated to be involved in the binding of collagen IV and perlecan, and the G3 domain in the binding of laminin B2 chain (Fox et al., 1991; Aumailley et al., 1993; Reinhardt et al., 1993). Thus, cleavage in the rod-like domain could disrupt the bridging function of nidogen between laminin and collagen IV and between laminin and perlecan. The disruption of the bridging function of nidogen by the hemorrhagic toxins may well result in a breakdown of the supramolecular organization of the laminin and collagen IV networks and, therefore, be critical in production of hemorrhage.
Thus, the hemorrhagic metalloproteinases can specifically cleave at least three protein com- ponents of basement membranes and degrade basement membranes in such a manner as to allow the escape of blood into surrounding stroma. Yet, non-hemorrhagic toxins in vitro also digest basement membrane components. However, they do so at a rate and substrate specificity that do not give rise to hemorrhage. Currently, the explanation for these differences of basement membrane digestion based on the structural differences of the toxins is not complete, although new sequence information on the hemorrhagic toxins gives some suggestive insights into this phenomenon (see Section 5). Also, it is still uncertain which one, if any, of the basement membrane components is most important for degradation, resulting in hemorrhage. These questions are actively being investigated.
In addition to directly producing hemorrhage via the disruption of basement membrane structure, other characteristics of some of the hemorrhagic toxins may also affect their hemorrhagic potency. Most of the hemorrhagic toxins of molecular masses in the 60-80 kDa range have significantly higher hemorrhagic activities than the lower molecular mass toxins (<60 kDa). Recently, the protein and cDNA sequences for two high molecular mass toxins HT-a and HR-1 B, from C. a t rox and T. f lavovir idus , respectively, have been reported (Takeya et al., 1990a; Hite et al., 1994). In both these proteins, a disintegrin-like domain was observed. As mentioned in Section 5.7, disintegrins are small venom-derived proteins that inhibit platelet aggregation by binding to the GP IIblIIa receptor (Gould et al., 1990). HR-IB also inhibits platelet aggregation. However, it is difficult to dissociate the proteolytic effects from the disintegrin-like effects of the toxin. Further- more, the toxins that contain this domain (see Section 5.4) do not have the typical Arg-Gly-Asp (RGD) tripeptide sequence found in most disintegrin peptides and other integrin ligands (Hite et al., 1992b; 1994). Therefore, the mechanism by which these particular hemorrhagic toxins inhibit
Hemorrhagic metalloproteinases from snake venoms 363
platelet aggregation is not clear. Nevertheless, these toxins may inhibit platelet aggregation due to the presence of the disintegrin-like domain and, thereby, potentiate hemorrhagic activity.
Potential inhibition of the toxins must also be considered when discussing the mechanism of hemorrhage production and the observed hemorrhagic potency of the toxins. Since virtually all assays for hemorrhagic activity involve an in vivo system (see Section 3), the presence of endogenous proteinase inhibitors must be taken into account. Earlier investigations with hemorrhagic toxins from C. adamanteus demonstrated that the venom metalloproteinases were capable of cleaving certain inhibitors of serin proteinases and, thereby, could serve to potentiate the activity of the serine esterases present in most crotalid venoms (Kurecki et al., 1978; Kress and Catanese, 1981; Bjarnason et al., 1983; Kress, 1986; Kress et al., 1983). More important, with regard to the venom hemorrhagic toxins, is the presence of 0t 2-macroglobulin in serum. This high molecular mass, broad specificity proteinase inhibitor was shown to bind 1-2 mol of HT-e, HT-c, or HT-d per mol of inhibitor with a relatively rapid rate (Baramova et al., 1990b). Interestingly, HT-a, the 68 kDa toxin, interacted very slowly with ~2-macroglobulin. Only at longer incubation times at elevated temperatures was HT-a observed to interact with ~t2-macroglobulin by cleaving the bait region and becoming covalently attached to the carboxy terminal fragment of 0t2-macroglobulin (Baramova et al., 1990b). Thus, it was suggested that HT-a may be able to avoid clearance by ~2-macroglobulin and, therefore, have a greater half-life in the system, increasing its likelihood of generating hemorrhage. This could contribute, at least in part, to the relatively high hemorrhagic activity characteristic of the high molecular weight toxins.
In summary, the primary mechanism by which the hemorrhagic toxins cause hemorrhage is via proteolytic disruption of basement membrane structures, allowing the escape of capillary contents into the stroma. The observed differences in hemorrhagic activity among the toxins are likely due to several factors, including substrate specificity and proteolytic activity, rate of inhibition and/or clearance by endogenous inhibitors, and the ability to alter coagulation by the inhibition of platelet aggregation. As more structural and biochemical data becomes available on the toxins and other non-hemorrhagic venom metalloproteinases, it is expected that the remaining questions associated with venom-induced hemorrhage will be answered.
7. INHIBITION OF HEMORRHAGE AND TREATMENT
Treatment of crotalid snake envenoming is potentially a very complex undertaking, due to the many toxic components present in the venom. Although this section will primarily consider hemorrhagic toxins, we must begin with a general discussion of the clinical presentation of crotalid envenoming. Information regarding the type of crotalid snake that bit the victim is very important. Most crotalid venoms can be divided into two groups: those that contain neurotoxins and those that have only low levels of them or none. Generally, the venoms that have significant amounts of neurotoxins, in addition to the typical toxins causing the various coagulatory pathologies, are the more dangerous. An example of a North American snake that contains a potent neurotoxin is C. scutulatus, the mojave rattlesnake. The primary neurotoxin in its venom is mojave toxin which causes a non-depolarizing blockade at the neuromuscular junction, giving rise to a flaccid paralytic state (Bieber et al., 1975). Another example of a snake containing a similar neurotoxin is C. durissus terrificus, which contains the neurotoxin crotoxin in its venom (Slotta, 1938; Slotta and Fraenkel- Conrat, 1938). Most crotalid snakes with neurotoxic venoms are likely to contain toxins similar to crotoxin and mojave toxin. Victims of envenoming by these snakes must be treated for both respiratory paralysis and coagulation dysfunction.
7.1. INHIBITION OF HEMORRHAGE
As mentioned above, many factors in the venom may contribute to the overall presentation of clinical hemorrhage. However, in this section, we will limit the discussion to the inhibition of the actual hemorrhagic toxins, rather than other factors, such as fibrinogenases, etc., which also play a role in the generation of hemorrhage in an envenomation.
364 J.B. BJARNASON and J. W. Fox
The principle that must be remembered when considering the inhibition of hemorrhagic toxins is that nearly all of these toxins are metalloproteinases and the hemorrhagic activity of these toxins is dependent on their proteolytic activity (Bjarnason and Tu, 1978; Fox and Bjarnason, 1983). Therefore, most considerations of hemorrhagic toxin inhibition involve inhibition of the proteolytic activity. Many synthetic compounds have been developed, which inhibit metalloproteinase activity by interacting with the active site and liganding to the zinc ion so that substrate binding is blocked and peptide bond hydrolysis is prevented (Holmquist and Vallee, 1979). Most of these compounds are peptide-based structures, which specifically interact with the enzyme via side chains of peptide amino acid residues. Some of the zinc ligands incorporated into these structures include: phosphonamidate, carboxyl, sulhydryl, chloromethyl, pyro-glutamate, and hydroxamate groups (Mookhtiar et al., 1987; Orlowski et al., 1988; Schwartz et al., 1991; Fox et al., 1986; Robeva et al., 1991; Nishino and Powers, 1978).
Several of these liganding groups have been used in synthetic inhibitors designed for use against the hemorrhagic toxins. Using substrate specificities determined for the hemorrhagic toxins of C. atrox , peptides were designed and synthesized that contained hydroxamate, chloromethyl, and pyro-glutamate groups, and they were tested for inhibitory potential against the hemorrhagic toxins (Fox et al., 1986; Robeva et al., 1991). Most of these compounds, depending on their peptide sequence and liganding moiety, were able to inhibit in the low micromolar range. Recently, new compounds have been reported, which appear to be even more active against the hemorrhagic toxins (Barelli et al., 1992).
Interestingly, pyro-glutamate peptides are found in the crude venom of most crotalid snakes. These peptides were originally reported to be inhibitors of kininases responsible for the hydrolysis of bradykinin and also angiotensin-converting enzyme, both factors that could contribute to venom-induced hypotensive shock (Ferreira, 1965; Cheung and Chrisman, 1973). However, a more reasonable alternative function for these peptides in the venom may be to inhibit the hemorrhagic toxins in situ prior to their injection into the victim. The concentration of these inhibitors in the crude venom has been estimated to be in the millimolar to micromolar range, and in vivo would be at concentrations that could effectively inhibit the toxins while in the venom gland (Robeva et al., 1991). Once injected, the localized concentration of the inhibitors would fall to levels such that they would no longer sufficiently inhibit the toxins to prevent their activity. Preliminary X-ray crystallographic studies have suggested that these pyro-glutamate peptide inhibitors bind to the active site of the hemorrhagic toxins so that the nitrogen atom in the pyro-ring structure of the glutamate might bind to the active site zinc.*
It has long been known that certain reptiles and mammals are resistant to certain snake venoms, particularly crotalid and viperid venoms (Straight et al., 1976; Perez et al., 1978; Ovadia, 1978b). This would suggest that the endogenous inhibitors of these organisms are, in fact, protease inhibitors with relatively broad specificity, given the complexity of protease activity in crotalid venoms. Some of these endogenous serum inhibitors have been identified. For example, the serum from the hedgehog, Erinacaeus europaeus, has been demonstrated to have a wide variety of high and moderate molecular weight proteins that inhibit proteases (De Wit and Westrom, 1987a,b). Some of these, such as the macroglobulins, could conceivably inhibit hemorrhagic metallo- proteinases. Baramova et al. (1990b) reported on the ability of human ~t2-macroglobulin to inhibit the hemorrhagic metalloproteinases from C. atrox. The human • 2-macroglobulin could inhibit the low molecular weight toxins from hydrolyzing protein substrates, but did not significantly inhibit the high molecular weight toxin HT-a.
Protein inhibitors of venom hemorrhagic activity have been purified from the sera of several resistant mammals (Ovadia, 1978b; Menchaca and Perez, 1981; Pichyangkul and Perez, 1981; Garcia and Perez, 1984; Weissenberg et al., 1991; Tanizaki et al., 1991; Catanese and Kress, 1992). One of the best characterized of these inhibitors is oprin, isolated from the serum of the opossum, Didelphis virginiana (Catanese and Kress, 1992; Kress and Catanese, 1985). This protein of molecular mass 52 kDa (211 amino acid residues) was demonstrated to inhibit a low molecular mass (~ 24 kDa) hemorrhagic toxin from C. atrox, but did not inhibit the high molecular weight
*MEYER, E., BJARNASON, J. B. and Fox, J. W. (1994) Manuscript submitted for publication.
Hemorrhagic metalloproteinases from snake venoms 365
toxin HT-a from the same venom. However, other fractions from the opossum serum did effectively inhibit HT-a, but hve yet to be characterized.
The sequence of the eDNA for oprin has been determined and showed homology to human 0tlB-glycoprotein, a human protein with unknown function (Catanese and Kress, 1992). It is likely that as more of the medium molecular mass (50-90 kDa), serum-derived hemorrhagic metalloproteinase inhibitors are sequenced, they will fall into a similar structural category as oprin.
7.2. TREATMENT OF HEMORRHAGE
Often snake bites result in a negligible injection of venom and, therefore, no significant danger, other than infection, is present. However, crotalid envenoming by those species with limited neurotoxic activity primarily leads to severe venom-induced coagulopathy. Within several hours after envenoming, disseminated intravascular coagulation can appear along with severe fibrinogenolysis and defibrination (Van Mierop and Kitchens, 1980; Budzynski et al., 1984; Hasiba et al., 1975). In the past, many unusual treatments have been reported, most of which have been proven to have little effectiveness and in fact can be dangerous in themselves. These include cryotherapy, steroids, surgical therapy, and electrical shock (Stahnke et aL, 1957; Wood et al., 1955; Glass, 1976; Huang et al., 1974).
Most of the current treatment protocols for severely envenomated patients from crotalid bites are directed toward re-establishing coagulatory homeostasis. This can be approached by replace- ment of clotting factors, fibrinogen, and fluids, with or without the co-infusion of antivenin (Crotalidae polyvalent antivenin, Wyeth) (Bates et al., 1988; Burch et al., 1988). The extent to which the treatment is prosecuted depends on the apparent severity of the envenoming and, if known, the species of the snake. Often the rattlesnakes produce more severe symptoms than, for example, copperheads, but this is not always the case, for it also depends on many other factors, such as the size of snake, the amount of venom injected, and the condition of the patient, etc. With regard to the use of antivenin in conjunction with coagulatory homeostasis therapy, there is the risk of serum sickness or acute anaphylactic reaction, and this must be considered as a decision factor in its use. Furthermore, some physicians suggest that antivenin does not significantly alter the outcome of the patient if the coagulopathologies are appropriately treated (Burch et al., 1988). Therefore, there still seems to be some controversy in the treatment protocols for crotalid envenoming. However, it is clear that early intervention to reverse the loss of coagulatory hemostasis is critical to the successful outcome of the treatment.
8. CONCLUSIONS
The purpose of this review was to give a historical background of the hemorrhagic metallo- proteinases isolated from snake venoms, with emphasis on the toxins isolated from the western Diamondback rattlesnake, C. atrox . Clearly, a significant amount of information has been produced on this subject over the past 50-60 years, much of which was either controversial or confusing. However, as this review has illustrated, the recent advances in the structural understand- ing of the venom metalloproteinases have served to clarify the structural relationships among these proteinases and identify previously unknown homologies with other members of the metallo- proteinase family. The homology of the venom metalloproteinases with various mammalian reproductive proteins is certainly one of the most exciting discoveries of the field in the past few years. The structural similarities shared by these metalloproteinases may, as one might reasonably expect, extend to certain functional properties of the proteinases, such as substrate specificities and localization. No doubt these discoveries will advance both fields and increase the efforts directed toward understanding the biochemical basis for hemorrhagic production by the toxins. As many investigators in the field of venom research have stated, much of the information gained on the proteinases in venom is of broad importance in that these proteinases will likely have analogous and homologous counterparts in other biological systems. This prediction has been demonstrated to be true, especially in the case of the venom metalloproteinases.
366 J.B. BJARNASON and J. W. Fox
The advances made over the years have contributed to a greater understanding of the factors involved and mechanisms of metalloproteinase-induced hemorrhage resulting from crotalid snake envenoming. Much of the recent knowledge gained in these areas is poised to be exploited for developing more efficacious treatment regimes to lessen the often debilitating consequences of crotalid envenoming. The development of inhibitors of the venom metalloproteinases may eventually find some usefulness in the area of the reproductive metalloproteinases, for example, as a method of contraception. Other scientific endeavors that may profit from the research on the venom metalloproteinases could possibly include ECM modulation, basement membrane disrup- tion, tumor cell invasion, wound repair, and protein l igand-matrix interaction. With so many other areas of scientific interest now finding relevance in the field of venom research, we can expect to see an increase in both the quality and quantity of data on snake venom proteinases, particularly the metalloproteinases, appearing in the literature.
In summary, we hope this review has increased the reader's understanding of the history and value of venom hemorrhagic toxin research, in particular, the metalloproteinases, and has exposed many new topics for future research, which will be both of broad importance to protein science, in general, and venom research, in particular.
Acknowledgements--We wish to thank our fathers, Bjarni Bragi J6nsson and William J. Fox, Jr, for their encouragement in the pursuit of this work and Gerlinde Xander for her expert secretarial assistance. This work was supported by a grant from Sb.ttmfilasj6dur of the University of Iceland (J.B.B.), and grant GM31289 from the National Institutes of Health (J.W.F.).
R E F E R E N C E S
ARNOLD, R. E. (1982) Treatment of rattlesnake bites. In: Rattlesnake Venoms: Their Actions and Treatment, pp. 315-333, Tu, A. T. (ed.) Maracel Dekker, New York.
ASSAKURA, M. T., REICHL, A. P., ASPERTI, M. C. A. and MANDELBAUM, F. R. (1985) Isolation of the major proteolytic enzyme from the venom of the snake Bothrops moojeni (caissaca). Toxicon 23: 691-706.
ASSAKURA, M. T., REICHL, A. P. and MANDELBAUM, F. R. (1986) Comparison of immunological, biochemical and biophysical properties of three hemorrhagic factors isolated from the venom of Bothrops jararaca (Jararaca). Toxicon 24: 943-946.
AUMAILLEY, M., WIEDEMANN, H., MANN, K. and TIMPL, R. (1989) Binding of nidogen and the laminin-nidogen complex to basement membrane collagen IV. Fur. J. Biochem. 184: 241-248.
AUMAILLEY, M., BATTAGLIA, C., MAYER, U., REINHARDT, D., NISCHT, R., T1MPL, R. and Fox, J. W. (1983) Nidogen mediates the formation of ternary complexes of basement membrane components. Kidney Int. 43: %12.
BANDO, E., NIKAI, T. and SUGIHARA, H. (1991) Hemorrhagic protease from the venom of Calloselasma rhodostoma. Int. J. Biochem. 23:1193-1199.
BARAMOVA, E. N., SHANNON, J. D., BJARNASON, J. B. and Fox, J. W. (1989) Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases. Arch. Biochem. Biophys. 275: 63-71.
BARAMOVA, E. N., SHANNON, J. D., BJARNASON, J. B. and FOX, J. W. (1990a) Identification of the cleavage sites by a hemorrhagic metalloproteinase in Type IV collagen. Matrix 10: 91-97.
BARAMOVA, E. N., SHANNON, J. D., BJARNASON, J. B., GONIAS, S. L. and Fox, J. W. (1990b) Interaction of hemorrhagic metaiioproteinases with human ct2-macroglobulin. Biochemistry 29." 1069-1074.
BARAMOVA, E. N., SHANNON, J. D., FOX, J. W. and BJARNASON, J. B. (199 l) Proteolytic digestion of non-collage- nous basement membrane proteins by the hemorrhagic metalloproteinase HT-e from Crotalus atrox venom. Biomed. Biochim. Acta S0: 763-768.
BARELLI, H., DIVE, V., YIOTAKIS, A., VINCENT, J. P. and CHECLER, F. (1992) Potent inhibition of endopeptidase 24.16 and endopeptidase 24.15 by the phosphonamide peptide N-(phenylethylphosphonyl)-Gly-L-Pro-L- aminohexanoic acid. Biochem. J. 287: 621-625.
BATES, M. C., WEDIN, G. P., WARREN, S. G. and SEIDLER, D. E. (1988) Approach to the patient with severe rattlesnake envenomation. IV. V. Med. J. 84: 398-403.
BIEaER, A. L., To, T. and Tu, A. T. (1975) Studies of an acidic cardiotoxin isolated from the venom of Mojave rattlesnake (Crotalus scutulatus). Biochim. biophys. Acta 400: 178-188.
BJARNASON, J. B. and Fox, J. W. (1983) Proteolytic specificity and cobalt exchange of hemorrhagic toxin e, a zinc protease isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Biochemistry 22: 3770-3778.
BJARNASON, J. B. and Fox, J. W. (1986) Role of zinc in the structure of five hemorrhagic zinc proteases from C. atrox venom. In: Progress in Inorganic Biochemistry and Biophysics. Zinc Enzymes, Vol. l, pp. 249-270, BERTINI, I., LUCHINAT, C., MARAT, W. and ZEPPEZAUER, M. (eds) Birkh/iuscr, Boston.
BJARNASON, J. B. and Fox, J. W. (1987) Characterization of two hemorrhagic zinc proteinascs, toxin c and toxin d, from eastern diamondback rattlesnake (Crotalus atrox) venom. Biochim. biophys. Acta. 911: 356-363.
Hemorrhagic metalloproteinases from snake venoms 367
BJARNASON, J. B. and Fox, J. W. (1988) Characterization and substrate site mapping of two hemorrhagic zinc protease from Crotalus atrox venom. In: Hematology, Vol. 7, Hemostatis and Animal Venoms, pp. 457--477, PIRKLE, H. and MARKLAND, F. S., JR (eds) Marcel Dekker, Inc., New York.
BJARNASON, J. B. and Fox, J. W. (1988-89) Hemorrhagic toxins from snake venoms. J. Toxic. Toxin Rev. 7: 121-209.
BJARNASON, J. B. and Tu, A. T. (1978) Hemorrhagic toxins from Western Diamondback rattlesnake (Crotalus atrox) venom: isolation and characterization of five toxins and the role of zinc in hemorrhagic toxin e. Biochemistry 17: 3395-3404.
BJARNASON, J. B., BARRISH, A., DIRENZO, G. S., CAMPBELL, R. and Fox, J. W. (1983) Kallikrein-like enzymes from Crotalus atrox venom. J. biol. Chem. 258: 12566--12573.
BJARNASON, J. B., HAMILTON, D. and Fox, J. W. (1988) Studies on the mechanism of hemorrhage production by five proteolytic hemorrhagic toxins from Crotalus atrox venom. BioL Chem. Hoppe-Seyler 369: 121-129.
BJARNASON, J. B., HITE, L. A., SHANNON, J. D., TAPANINAHO, S. J. and Fox, J. W. (1993) Atrolysin E (Ht-e, E.C. 3.4.24.44) and the non-hemorrhagaic ~-protease--Comparative properties and sequence of a cDNA clone encoding atrolysin E. Evidence for signal, zymogen and disintegrin-like structures. Toxicon 31: 513.
BLOBEL, C. P., WOLFSBERG, T. G., TURCK, C. W., MYLES, D. G., PRIMAKOFF, P. and WHITE, J. M. (1992) A potential fusion peptide and an integrin ligand domain in a protein active in sperm--egg fusion. Nature 356: 248-252.
BODE, W., GOMIS-ROTn, F. X., HUBER, R., ZWlLLING, R. and STOCKER, W. (1992) Structure of astacin and implication for activation of astacins and zinc-ligation of collagenases. Nature 358: 164-166.
BONTA, I. L., VARGmTIG, B. B., BnARGAVA, N. and DE Vos, C. J. (1970) Method for study of snake venom induced hemorrhage. Toxicon 8:3-11.
BUDZYNSKI, A. Z., PANDYA, B. V. and RUBIN, R. N. (1984) Fibrinolytic afibrinogenemia after envenomation by Western Diamondback rattlesnake (Crotalus atrox). Blood 63: 1-14.
BURCH, J. M., AGARWAL, R., MATTOX, K. L., FELICIANO, D. V. and JORDAN, G. L. (1988) The treatment of crotalid envenomation without antivenin, d. Trauma 28: 35-43.
CATANESE, J. J. and KRESS, L. F. (1992) Isolation of opossum serum of a metalloproteinase inhibitor homologous to human g l-glycoprotein. Biochemistry 31: 410--418.
CHENG, H. C. and OUYANG, C. (1967) Isolation of coagulant and anticoagulant principles from the venom of Agkistrodon acutus. Toxicon 4: 235-243.
CHEUNG, H. S. and CHRISMAN, D. W. (1973) Inhibition of homogeneous angiotensin-converting enzyme of rabbit lung by synthetic venom peptides of Bothrops jararaca. Biochim. biophys. Acta 293: 451-459.
CIVELLO, D. J., DUONG, H. L. and GEREN, C. R. (1983a) Isolation and characterization of a hemorrhagic proteinase from timber rattlesnake venom. Biochemistry 22: 749-755.
CIVELLO, D. J., MORAN, J. B. and GEREN, C. R. (1983b) Substrate specificity of a hemorrhagic proteinase from timber rattlesnake venom. Biochemistry 22: 755-762.
DAOUD, E., TU, A. T. and EL-ASMAR, M. F. (1986a) Isolation and characterization of an anticoagulant proteinase, cerastase F-4, from Cerastes cerastes (Egyptian sand viper) venom. Thromb. Res. 42: 55~2.
DAOUD, E., TU, A. T. and EL-ASMAR, M. F. (1986b) Mechanism of the anticoagulant, cerastase F-4, from Cerastes cerastes (Egyptian sand viper) venom. Thromb. Res. 41: 791-799.
DENNIS, M. S., HENZEL, W. J., PITTI, R. M., LIPARI, M. T., NAPIER, M. A., DEISHER, T. A., BUNTNIG, S. and LAZARUS, R. A. (1989) Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: Evidence for a family of platelet aggregation inhibitors. Proc. natn. Acad. Sei. U.S.A. 87: 2471-2475.
DENSON, K. W. E. (1969) Coagulant and anticoagulant action of snake venoms. Toxicon 7: 5-12. DE WIT, C. A. and WESTROM, B. R. (1987a) Further studies of plasma protease inhibitors in the hedgehog,
Erinaceus europaeus; Collagenase, papain and plasmin inhibitors. Comp. Biochem. Physiol. 86A: 1-5. DE WIT, C. A. and WESTROM, B. R. (1987b) Venom resistance in the hedgehog, Erinaceus europaeus:
purification and identification of macroglobulin inhibitors as plasma antihemorrhagic factors. Toxicon 25: 315-322.
DZIADEK, M., PAULSSON, M. and TIMPL, R. (1985) Identification and interaction repertoire of large forms of the basement membrane protein nidogen. EMBO d. 4: 2513-2518.
FELDBERG, W. and KELLAWAY, C. H. (l 937) Liberation of histamine from the perfused lung by snake venoms. d. Physiol. 90: 257-263.
FERREIRA, S. H. (1965) A bradykinin-potentiating factor (BPF) present in the venom of Bothropsjararaca. Br. d. Pharmae. 24: 163-171.
FESSLER, L. I., DUNCAN, K. G., FESSLER, J. H., SALO, T. and TRYGGVASON, K. (1984) Characterization of the procollagen IV cleavage products produced by a specific tumor collagenase. J. biol. Chem. 259: 9783-9789.
Fox, J. W. and BJARNASON, J. B. (1983) New proteases from Crotalus atrox venom. J. Tox. Toxin Rev. 2: 161-204.
Fox, J. W., CAMPBELL, R., BEGGERLY, L. and BJARNASON, J. B. (1986) Substrate specificities and inhibition of two hemorrhagic zinc proteases Ht-c and Ht-d from C. atrox venom. Fur. J. Biochem. 156: 65-72.
Fox, J. W., MAYER, U., NISCHT, R., AUMAILLEY, M., REINHARDT, D., WIEDMANN, H., MANN, K., TIMPL, R., KREIG, T., ENGEL, J. and CHU, M.-L. (1991) Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen IV. EMBO 3". 10." 3137-3146.
FRIEDERICH, C. and Tu, A. T. (1971) Role of metals in snake venoms for hemorrhage, esterase and proteolytic activities. Biochem. Pharmac. 20:. 1549-1556.
.tiT 62/3--H
368 J.B. BJARNASON and J. W. Fox
GAN, Z.-R., GOULD, R. J., JACOBS, J. W., FRIEDMAN, P. A. and POLOKOFF, M. A. (1988) A potent platelet aggregation inhibitor for the venom of the viper Echis carinatus. J. biol. Chem. 263: 19827-19832.
GARCIA, V. E. and PEREZ, J. C. (1984) The purification and characterization of an antihemorrhagic factor in woodrat (Neotoma micropus). Toxicon 22: 129-138.
GLASS, T. G., JR (1976) Early debridement in pit viper bites. J. Am. Ned. Ass. 235: 2513-2516. GOMIS-ROTH, F.-X., KRESS, L. F. and BODE, W. (1993) First structure of a snake venom metalloproteinase: a
prototype for matrix metalloproteinases/collagenases. EMBO d. 12: 4151-4157. GOULD, R. J., POLOKOFF, M. A., FRIEDMAN, P. A., HUANG, T.-F., HOLT, J. C., COOK, J. J. and NIEWIAROWSKI,
S. (1990) Disintegrins: a family of integrin inhibitory proteins from viper venoms. Proc. Soc. exp. biol. Ned. 195: 168-171.
GROTTO, L., MOROZ, C., DEVRIES, A. and GOLDBLUM, N. (1967) Isolation of Vipera palaestinae hemorrhagin and distinction between its hemorrhagic and proteolytic activities. Biochim. biophys. Acta 133: 356-364.
HASIBA, U., ROSENBACH, L. M. and ROCKWELL, D. (1975) DIC-like syndrome after envenomation by the snake, Crotalus horridus horridus. New Engl. J. Ned. 292: 505-507.
HIBBS, M. S., HASTY, K. A., SEYER, J. M., KANG, A. H. and MA1NARDI, C. L. (1985) Biochemical and immunological characterisation of the secreted forms of human neutrophil gelatinase, d. biol. Chem. 260: 2493-2500.
HIT~, L. A., Fox, J. W. and BJARNASON, J. B. (1992a) A new family of proteinases is defined by several Snake venom metalloproteinases. Biol. Chem. Hoppe-Seyler 373: 381-385.
HITE, L. A., SHANNON, J. D., BJARNASON, J. B. and FOX, J. W. (1992b) Sequence of a cDNA clone encoding zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen and disintegrin-like structures. Biochemistry 31: 6203-6211.
HITE, L. A., JIA, L.-G., BJARNASON, J. B. and Fox, J. W. (1994) cDNA sequence for four snake venom metalloproteinases: structure, classification and their relationship to mammalian reproductive proteins. Arch. Biochem. Biophys. 38: 182-191.
HOLMQUIST, B. and VALLEE, B. L. (1979) Metal-coordinating substrate analogs as inhibitors of metalloenzymes. Proc. hath. Acad. Sci. U.S.A. 76: 6216-6220.
HORREBOW, N. (1758) The Natural History o f Iceland. A. Linde, London. HOUSSAY, B. A. (1930) Classification des actions des venius de seppents sur l'organisme animal. C. R. Soc.
Biol. Paris 105: 308-317. HUANG, T. F., CHANG, J. H. and OUYANG, C. (1984) Characterization of hemorrhagic principles from
Trimeresurus gramineus snake venom. Toxicon 22: 45-52. HUANG, T. F., HOLT, J. C., KIRBY, E. P. and NIEWIAROWSKI, S. (1989) Trigramin: primary structure and its
inhibition of yon Willebrand factor binding to glycoprotein IIb/IIIa complex of human platelets. Biochemistry 28: 661-666.
HUANG, T. T., LYNCH, J. B. and LARSON, D. L. (1974) The use of excisional therapy in the management of snakebite. Ann. Surg. 179: 568-605.
IMAI, K., NIKAI, T., SUGIHARA, H. and OWNBY, C. L. (1989) Hemorrhagic toxin from the venom of Agkistrodon bilineatus (common cantil). Int. J. Biochem. 21: 667-673.
IWANAGA, S., OMORI, T., OSHIMA, G. and SUZUKI, T. (1965) Studies on snake venom XVI. Demonstration of a proteinase with hemorrhagic activity in the venom of Agkistrodon halys blomhoffii. J. Biochem. 57: 392-400.
JIANG, W. and BOND, J. S. (1992) Families of metalloendopeptidases and their relationships. FEBS Lett. 312: 110-114.
JONGENEEL, C. V., BOUV1ER, J. and BAIROCH, A. (1989) A unique signature identifies a family of zinc-dependent metailoproteinases. FEBS Lett. 242: 211-214.
JUST, D., URBANITZ, n. and HABERMANN, E. (1970) Pharmakologische Charakterisierung der vaskulaeren Schrankenfunktion gegenueber Erythrocyten und Albumin. Arch. Pharmak. 267: 399-413.
KINI, R. M. and EVANS, H. J. (1992) Structural domains in venom proteins: evidence that metalloproteinases and nonenzymatic platelet aggregation inhibitors (disintegrins) from snake venom are derived from proteolysis from a common precursor. Toxicon 30: 265-293.
KISHIDA, M., NIKAI, T., MORI, N., KOHMURA, S. and SUGIHARA, H. (1985) Characterization of mucrotoxin A from venom of Trimeresurus mucrosguamatus (the Chinese Habu snake). Toxicon 23: 637-645.
KOMORI, Y. and SUGIHARA, H. (1988) Biological study of muscle degenerating hemorrhagic factor from the venom of Vipera aspis aspis (Aspic viper). Int. J. Biochem. 20: 1417-1423.
KOMORI, Y., HAG1HARA, S. and Tu, A. T. (1985) Specificity of hemorrhagic proteinase from Crotalus atrox (western diamondback rattlesnake) venom. Bioehim. biophys. Acta 829: 127-130.
KONDO, H., KONDO, S., IKEZAWA, H., MURATA, R. and OHSAKA, A. (1960) Studies on the quantitative method for the determination of hemorrhagic activity of Habu snake venom. Jap. J. reed. Sci. Biol. 13:43-51.
KRESS, L. F. (1986) Inactivation of human plasma serine proteinase inhibitors (serpins) by limited proteolysis of the reaction site loop with snake venom and bacterial metalloproteinases. J. Cell. Biochem. 24: 51-58.
KRESS, L. F. and CATANESE, J. J. (1981) Identification of cleavage sites resulting from enzymatic inactivation of human antithrombin III by Crotalus adamanteus proteinase II in the presence and absence of heparin. Biochemistry 20: 7432-7438.
KRESS, L. F. and CATANESE, J. J. (1985) Isolation of an inhibitor of snake venom metalloproteinases from opossum serum. Fedn. Proc. 44: 1431.
Hemorrhagic metalloproteinases from snake venoms 369
KRESS, L. F., CATANESE, J. J. and HIRIYAMA, T. (1983) Analysis of the effects of snake venom proteinases on the activity of human plasma Cl-esterase inhibitor, al-antichymotrypsin and a2-antiplasmin. Biochim. biophys. Acta 745: 113-119.
KUNITZ, M. (1947) Crystalline soybean trypsin inhibitor. IIa. General properties. J. gen. Physiol. 30:291-310. KURECKI, T. and KRESS, L. F. (1985) Purification and partial characterization of the hemorrhagic factor from
the venom of Crotalus adamanteus (eastern diamondback rattlesnake). Toxicon 23: 657-668. KURECKI, T. and LASKOWSKI, M., SR (1978) Purification of collagenases from Crotalus adamanteus venom. In:
Toxicon Supplement No. 1, Toxins, Animals, Plant and Microbial, p. 31 l, ROSENBERG, P. and HAaERMEHL, G. (eds) Pergamon Press, Elmsford.
KURECKI, T., LASKOWSKI, M. and KRESS, L. F. (1978) Purification and some properties of two proteinases from Crotalus adamanteus venom that inactive human ~l-proteinase inhibitor. J. biol. Chem. 253: 8340-8345.
LAURIE, G. W., BING, J. T., KLEINMANN, H. K., HASSELL, J. R., AUMAILLEY, M., MARTIN, G. R. and FELDMAN, R. J. D. (1986) Localization of binding sites for laminin, heparan sulfate, proteoglycan and fibronectin on basement membrane (type IV) collagen. J. molec. Biol. 189: 205-216.
LEHUERON, I., WICKER, C., GUILLOTEAU, P., TOULLEC, R. and PUIGSERVER, A. (1990) Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family. Eur. J. Biochem. 193: 767-773.
LIN, Y., MEANS, G. E. and FEENEY, R. E. (1969) The action of proteolytic enzymes on N,N-dimethyl proteins. J. biol. Chem. 244: 789-795.
LIOTTA, L. A. and RAO, C. N. (1985) Role of extracellular matrix in cancer. Ann. N Y Acad. Sci. 460: 333-344. MANDELBAUM, F. R., REICHL, A. P. and ASSAKURA, M. T. (1976) Some physical and biochemical characteristics
of HT2, one of the hemorrhagic factors in the venom of Bothropsjararaca. In: Animal Plant and Microbial Toxins, Vol. 1, pp. I 11-121, OHSAKA, A., HAYASHI, K. and SAWAI, Y. (eds) Plenum Press, London.
MANDELBAUM, F. R., REICHL, A. P. and ASSAKURA, M. T. (1982) Isolation and characterization of a proteolytic enzyme from the venom of the snake Bothrops jararaca, (jaraca). Toxicon 30: 955-972.
MANDELBAUM, F. R., ASSAKURA, M. T. and REICHL, A. P. (1984) Characterization of two hemorrhagic factors isolated from the venom of Bothrops neuwiedi (jararaca pintada). Toxicon 22: 193-206.
MARTIN, G. R. and TIMPL, R. (1987) Laminin and other basement membrane components. Ann. Rev. Cell Biol. 3: 57-85.
MARTINEZ, M., RAEL, E. D. and MADDUX, N. L. (1990) Isolation of a hemorrhagic toxin from Mojave rattlesnake (Crotalus scutulatus scutulatus ) venom. Toxicon 28: 685-694.
MCKAY, D. G., MOROZ, D., DEVRIES, A., CSAVOSSY, I. and CRUSE, V. (1970) The action of hemorrhagin and phospholipase derived from Vipera palaestinae venom on the microcirculation. Lab. Invest. 22: 387-399.
MCKERROW, J. H. (1987) Human fibroblast collagenase contains an amino acid sequence homologous to the zinc-binding site of serratia protease. J. biol. Chem. 262: 5943.
MEBs, D. and PANHOLZER, F. (1982) Isolation of a hemorrhagaic principle from Bitis arietens (puff adder) snake venom. Toxicon 20:509-512.
MELCHIORI, A., ALmNI, A., RAY, J. M. and STETLER-STEVENSON, W. G. (1992) Inhibition of tumor cell invasion by a highly conserved peptide sequence from the matrix metalioproteinase enzyme prosegment. Cancer Res. 52: 2353-2356.
MENCHACA, J. M. and PEREZ, J. C. (1981) Purification and characterization of an antihemorrhagic factor for opossum (Didelphis virginiana) serum. Toxicon 19: 623-632.
MOLINA, O., SERIEL, R. K., MARTINEZ, M., SIERRA, M. L., VARELA-RAMIREZ, A. and RAEL, E. D. (1950) Isolation of two hemorrhagic toxins from Crotalus basiliscus basiliscus (Mexican west coast rattlesnake) venom and their effect on blood clotting and complement. Int. J. Biochem. 22: 253-261.
MOOKHTIAR, K. A., MARLOWE, C. K., BARTLETT, P. A. and VAN WART, H. E. (1987) Phosphonamidate inhibitors of human neutrophil collagenase. Biochemistry 26: 1962-1965.
MORI, N., NIKAI, T. and SUGIHARA, H. (1984) Purification of a proteinase (AcS) and characterization of hemorrhagic toxins from the venom of the hundred-pace snake (Agkistrodon aeutus). Toxicon 22: 451-461.
MORI, N., NIKAI, T., SUGIHARA, H. and Tu, A. T. (1987) Biochemical characterization of hemorrhagic toxins with fibrinogenase activity isolated from Crotalus ruber ruber venom. Arch. Biochem. Biophys. 253: 108-121.
MURPHY, G., McALPINE, C. G., POLL, C. T. and REYNOLDS, J. J. (1985) Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen. Biochim. biophys. Acta 831: 49-52.
NEEPER, M. P. and JACOBSON, i . A. (1990) Sequence of a cDNA encoding the platelet aggregation inhibitor trigramin. Nucl. Acids Res. 18: 4255-4261.
NIKAI, T., MORI, N., KISHIDA, i . , SUGIHARA, H. and Tu, A. T. (1984) Isolation and biochemical characteriz- ation of hemorrhagic toxin f from the venom of Crotalus atrox (western diamondback rattlesnake). Arch. Biochem. Biophys. 231: 309-319.
NIKAI, T., MORI, N., KISHIDA, i . , TSUBOI, M. and SUGIHARA, H. (1985a) Isolation and characterization of hemorrhagic toxin g from the venom of Crotalus atrox (western diamondback rattlesnake). Am. J. Trop. Med. Hyg. 34: 1167-1172.
NIKAI, T., MORI, N., KISHIDA, M., YUKO, K., TAKENAKA, C., MURAMKAMI, T., SHIGEZANE, S. and SUGIHARA, H. (1985b) Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake ( Trimeresurus mucrosquamatus). Biochim. biophys. Acta 838: 122-131.
370 J.B. BJARNASON and J. W. Fox
NIKAI, T., OGURI, E., KISHIDA, M., SUGIHARA, H., MORI, N. and Tu, A. T. (1986) Reevaluation of hemorrhagic toxin, HR-I, from Agkistrodon halys blomhoffii, venom: proof of proteolytic enzyme. Int. J. Biochem. 18: 103-108.
NIKAI, T., NnKAWA, M., KOMORI, Y., SEKOGUCm, S. and SUGIHARA, H. (1987) Proof of proteolytic activity of hemorrhagic toxins, HR-2a and HR-2b, from Trimeresurus flavoviridis venom. Int. J. Biochem. 19: 221-226.
NIKAI, T., KATA, C., KOMOR1, Y., SUGIHARA, H. and HOMr, iA, M. (1991) Characterization of Ac~-proteinase from the venom of Agkistrodon acutus (hundred-pace snake) from Taiwan. Int. J. Biochem. 23:311-315.
NISHINO, N. and POWERS, J. C. (1978) Peptide hydroxamic acids as inhibitors of thermolysin. Biochemistry 17: 2846-2850.
OHSAKA, A., IKEZAWA, H., KONDO, H. and KONDO, S. (1960) Two hemorrhagic principles derived from Habu snake venom and their differences in zone electrophoretic mobility. Jpn J. reed. Sci. Biol. 13: 73-76.
OHSAKA, A., OMORI-SATOH, T., KONDO, H., KONDO, S. and MURATA, R. (1966) Biochemical and pathological aspects of hemorrhagic principles in snake venoms with special reference to Habu (Trimeresurusflavoviridis) venom. Mere. Inst. Butantan, Simp. Int. 33: 193-207.
OHSAKA, A., JUST, M. and HAnERMANN, E. (1973) Action of snake venom hemorrhagic principles on isolated glomerular basement membrane. Biochim. biophys. Acta 323: 415--428.
OHYANG, C. and HUANG, T. F. (1979) Alpha- and beta-fibrinogenases from Trimeresurus gramineus snake venom. Biochim. biophys. Acta 571: 270-283.
OKADA, Y., NAGASE, H. and HARRIS, E. D. (1986) A metalloproteinase from human synovial fibroblasts that digests connective tissue matrix components. Purification and characterization. J. biol. Chem. 261: 14245-14255.
OMORI, T., IWANAGA, S. and SUZUKI, T. (1964) The relationship between the hemorrhagic and lethal activities of Japanese Mamushi (Agkistrodon halys blomhoffi O. Toxicon 2: 1-4.
OMORI-SATOH, T. and OHSAKA, A. (1970) Purification and some properties of the hemorrhagaic principle I in the venom of Trimeresurus flavoviridis. Biochim. biophys. Acta 207: 432-444.
OMORI-SATOH, T. and SADAH1RO, S. (1979) Resolution of the major hemorrhagic component of Trimeresurus flavoviridis venom into two parts. Bioehim. biophys. Acta 580: 392-404.
OMOR1-SATOH, T., OHSAKA, A., KONDO, S. and KONDO, H. (1967) A simple and rapid method for separating two hemorrhagic principles in the venom of Trimeresurus flavoviridis. Toxicon 5: 17-24.
OMORI-SATOH, T., SADAHIRO, S., OHSAKA, A. and MURATA, R. (1972) Purification and characterization of an antihemorrhagic factor in the serum of Trimeresurus flavoviridis, a crotalid. Biochim. biophys. Acta 285: 414-426.
ORLOWSKI, M., MICHAUD, C. and MOLINEAUX, C. J. (1988) Substrate-related potent inhibitors of brain metaUo-endopeptidase. Biochemistry 27: 597-602.
OSHIMA, G., IWANAGA, S. and SUZUKI, T. (1968) Studies on snake venom XVIII. An improved method for purification of the proteinase b from the venom of Agkistrodon halys blomhoffii. J. Biochem. 64: 215-225.
OSHIMA, G., IWANGA, S. and SUZUKI, T. (1971) Some properties of proteinase b in the venom of Agkistrodon halys blomhoffii. Biochim. biophys. Acta 250: 416-427.
OSH1MA, G., OMORI-SATOH, T., IWANAGA, S. and SUZUKI, T. 0972) Studies on snake venom hemorrhagic factor I (HR-I) in the venom of Agkistrodon halys blomhoffii, its purification and biological properties. J. Biochem. 72: 1483-1494.
OUYANG, C. and HUANG, T. (1976) Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom. Biochim. biophys. Acta 439: 146-153.
OUYANG, C. and HUANG, T. (1977) The properties of the fibrinolytic principle of Agkistrodon acutus snake venom. Toxicon 15: 161-167.
OVADIA, M. (1978a) Isolation and characterization of three hemorrhagic factors from the venom of Vipera palaestinae. Toxicon 16: 479--487.
OVADIA, M. (1978b) Purification and characterization of an antihemorrhagic factor from the serum of snake Vipera palestinae. Toxicon 16: 661-672.
OVADIA, M. (1987) Isolation and characterization of a hemorrhagic factor from the venom of the snake Atractaspis engaddensis (Atractasps didae). Toxicon 25: 621-630.
OWNBY, C. L., KAINER, R. A. and Tu, A. T. (1974) Pathogenesis of hemorrhagic induced by rattlesnake venom. Am. J. PathoL 76: 401-408.
OWNBY, C. L., BJARNASON, J. B. and Tu, A. T. (1978) Hemorrhagic toxin from rattlesnake (Crotalus atrox) venom: pathogenesis of hemorrhage induced by three purified toxins. Am. J. Pathol. 93: 201-218.
OWNBV, C. L., COLBENG, T. R. and ODELL, G. V. (1984) A new method for quantitation of hemorrhage induced by rattlesnake venoms: ability of polyvalent antivenom to neutralize hemorrhagic activity. Toxicon 22: 227-233.
PAINE, M. J. I., DESOND, H. P., THEAKSTON, R. D. G. and CRAMPTON, J. M. (1992) Purification, molecular characterization of a high molecular weight hemorrhagic metalloproteinase, jararhagin from Bothrops jararaca venom. J. biol. Chem. 247: 22869--22876.
PANDYA, B. V. and BUDZYNSKI, A. A. (1984) Anticoagulant proteases from western diamondback rattlesnake ( Crotalus atrox ) venom. Biochemistry 23: 460-470.
PEARSON, W. R. and LIPMAN, D. J. (1988) Improved tools for biological sequence comparison. Proc. natn. Acad. Sci. U.S.A. 85: 2444-2448.
Hemorrhagic metalloproteinases from snake venoms 371
PEREZ, J. C., HAWS, W. C., GARCIA, V. E. and JENNINGS, B. M., III (1978) Resistance of warm-blooded animals to snake venoms. Toxicon 16: 375-383.
PERRY, A. C. F., JONES, R., BARKER, P. J. and HALL, L. (1992) A mammalian epididymal protein with remarkable sequence similarity to snake venom hemorrhagaic peptides. Biochem. J. 286: 671-675.
PICHYANGKUL, S. and PEREZ, J. C. (1981) Purification and characterization of a naturally occurring antihem- orrhagic factor in the serum of the hispid cotton rat (Sigmodon hispidus). Toxicon 19: 205-213.
REICH, R., THOMPSON, E. W., IWAMOTO, Y., MARTIN, G. R., DEASON, J. R., FULLER, G. C. and MISKIN, R. (1988) Effects of inhibitors of plasminogen activator, serine proteinases and collagenase IV on the invasion of basement membranes by metastatic cells. Cancer Res. 48: 3307-3312.
REICHL, A. P. and MANDELBAUM, F. R. (1993) Proteolytic specificity of moojeni protease A isolated from the venom of Bothrops moojeni. Toxicon 31: 187-194.
REINHARDT, D., MANN, K., NISCHT, R., FOX, J. W., CHU, M.-L., KRE1G, T. and TIMPL, R. (1993) Mapping of nidogen binding sites for collagen IV, heparan-sulfate proteoglycan and zinc. J. biol. Chem. 268: 10881-10887.
ROBEVA, A., POLITI, V., SHANNON, J. D., BJARNASON, J. B. and Fox, J. W. (1991) Synthetic and endogenous inhibitors of snake venom metalloproteinases. Biomed. biochem. Acta 50: 769-773.
RoQuES, B. P., FOURII~-ZALUSKI, M. C., SOROCA, E., LECOMTE, J. M., MALFROY, B., LORENS, C. and SCHWARTZ, J.-C. (1980) The enkephalin inhibitor thiophan shows antinociceptive activity in mice. Nature 288: 286-288.
SAMEL, K. and SnGUR, J. (1990) Isolation and characterization of hemorrhagaic metalloproteinase from Vipera berus berus (common viper) venom. Comp. Biochem. Physiol. 97C: 209-214.
SANCHEZ, E. F., MAGALHAES, A. and DINIZ, C. R. (1987) Purification of a hemorrhagic factor (LHF-I) from the venom of the bushmaster snake, Lachesis muta mum. Toxicon 25: 611-619.
SANCHEZ, E. F., DINIZ, C. R. and RICHARDSON, M. (1991) The complete amino acid sequence of the hemorrhagic factor LHFII, a metalloproteinase isolated from the venom of the bushmaster snake (Lachesis muta muta). FEBS Left. 282: 178-182.
SATAKE, M., MURATA, Y. and SUZUKI, T. (1965) Studies on snake venom XIII. Chromatographic separation and properties of three proteinases from Agkistrodon halys blomhoffii. J. Biochem. 53: 438-447.
SCARBOROUGH, R. M., RosE, J. W., Hsu, M. A., PHILLIPS, D. R., FRIED, V. A., CAMPBELL, A. M., NANNIZZI, L. and CHARO, I. F. (1991) Barbourin: a GPIIb-IIIa specific integrin antagonist from the venom of Sisstrurus m. barbouri. J. biol. Chem. 226: 9359-9362.
SCHWARTZ, M. A., VENKATARAMAN, S., GHAFFARI, M. A., LIBBY, A., MOOKRTIAR, K. A., MALLYA, S. K., BIRKEDAL-HANSEN, H. and VAN WART, H. E. (1991) Inhibition of human collagenases by sulfur-based substrate analogs. Biochem. biophys. Res. Commun. 176: 173-179.
SENFTLEBEN, A., WALLAT, S., LEMAIRE, L. and HE1NLEIN, U. A. (1992) Pre and post-meiotic germ cell specific expression of TAZ83, a gene encoding a putative, cysteine-rich transmembrane protein. GenBank Accession Number X64227. Unpublished.
SHANNON, J. D., BARAMOVA, E. N., BJARNASON, J. B. and Fox, J. W. (1989) Amino acid sequence of a Crotalus atrox venom metaUoproteinase which cleaves type IV collagen and gelatin. J. biol. Chem. 264:11575-11583.
SHEBUSKI, R. J., RAMJIT, D. R., BENCEN, G. H. and POLOKOFF, M. A. (1989) Characterization and platelet inhibitory activity of bitistatin, a potent arginine-glycine-aspartic acid containing peptide from the venom of the viper Bitis arietens. J. biol. Chem. 264: 21550-21556.
SIIGUR, E. and SIIGUR, J. (1991) Purification and characterization of lebetase, a fibrinolytic enzyme from Vipera lebetina (snake) venom. Biochim. biophys. Acta 1074: 223-229.
SLOTTA, K. H. (1938) P crotaxina, primeira substancia pura dos venenos ofidicos. Ann. Acad. Brazil Sci. Rio. 10: 195-204.
SLOTTA, K. H. and FRAENKEL-CONRAT, H. L. (1938) Two active proteins from rattlesnake venoms. Nature 142: 213-214.
STAHNKE, H. L., ALLEN, F. M. and HORAN, R. V. (1957) The treatment of snakebite. Am. J. Trop. Med. 6: 323-335.
STARKLY, P. M. and BARRETT, A. J. (1977) a 2-Macroglobulin is a physiological regulator of proteinase activity. In: Proteinases in Mammalian Cells and Tissues, pp. 663-692, BARRETT, A. J. (ed.) North-Holland, New York.
STRAIGHT, R. g. , GLENN, G. L. and SNYDER, C. C. (1976) Antivenom activity of rattlesnake blood plasma. Nature 261: 259-260.
SUGIHARA, H., MORIURA, M. and NIKAI, T. (1983) Purification and properties of a lethal, hemorrhagic protein "Mucrotoxin A" from the venom of the Chinese Habu snake (Trimeresurus mucrosquamatus). Toxicon 21: 247-255.
TAKAHASHI, T. and OHSAKA, A. (1970) Purification and some properties of two hemorrhagic principles (HR2a and HR2b) in the venom of Trimeresurusflavoviridis: complete separation of the principles from proteolytic activity. Biochim. biophys. Acta 207: 65-75.
TAKEYA, H., ODA, K., MIYATA, T., OMORI-SATOH, T. and IWANAGA, S. (1990a) The complete amino acid sequence of the high molecular mass hemorrhagic protein HRI B isolated from the venom of Trimeresurus flavoviridis. J. bioL Chem. 265: 16068-16073.
TAKEYA, H., ONIKURA, A., NIK^I, T., SUGIHARA, H. and IWANAGA, S. (1990b) Primary structure of a hemorrhagic metalloproteinase, HT-2, isolated from the venom of Crotalus ruber ruber. J. Biochem. (Tokyo) 108:711-719.
372 J.B. BJARNASON and J. W. Fox
TAKEYA, H., NISHIDA, S., MIYATA, T., KAWADA, S.-I., SAISAKA, Y., MORITA, T. and IWANAGA, S. (1992) Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). J. biol. Chem. 267: 14109-14417.
TAN, N. H. and SAIFUDDIN, M. N. (1990) Isolation and characterization of a hemorrhagin from the venom of Ophiophagus hannah (King cobra). Toxicon 28: 385-392.
TANIZAKI, M. M., KAWASAm, H., SUZUKI, K. and MANDELBAUM, F. R. (1991) Purification of a proteinase inhibitor for the plasma of Bothrops jararaca (jararaca). Toxicon 29:673-681.
TIMPL, R. (1989) Structure and biological activity of basement membrane proteins. Eur. J. Biochem. 180: 487-502.
TIMPL, R. and DZIADEK, M. (1986) Structure, development and molecular pathology of basement membranes. Int. Rev. exp. PathoL 29: 1-112.
TRAVlS, J. and SALVESEN, G. S. (1983) Human proteinase inhibitors. Ann. Rev. Biochem. 52: 655-709. TRYGGVASON, K., HOYHTYA, M. and SALO, T. (1987) Proteolytic degradation of extracellular matrix in tumor
invasion. Biochim. biophys. Acta 907: 191-217. TSUCHIYA, M., OHSH10, C., OHASHI, M., OHSAKA, A., SUZUKI, K. and FUJISHIRO, Y. (1974) Cinematographic and
electron microscopic analysis of the hemorrhage induced by the main hemorrhagaic principle, HR 1, isolated from the venom of Trimeresurusflavovirids. In: Platelets, Thrombosis, Inhibitors, pp. 439-451, D1DESHEIM, P., SHIMAMOTO, T. and YAMAZAKI, H. (eds) Schattauer, Stuttgart.
VALLEE, B. L. and AULD, n. S. (1990) Zinc coordination, function and structure of zinc enzymes and other proteins. Biochemistry 29: 5647-5659.
VAN MIEROP, L. H. S. and KITCHENS, C. S. (1980) Defibrination syndrome following bites by the Eastern Diamondback rattlesnake. 3". Florida Med. Ass. 67: 21-27.
VAN WART, H. E. and BIRKEOAL-HANSEN, H. (1990) The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc. natn. Acad. Sci. U.S.A. 87: 5578-5582.
VON HEIJNE, G. (1990) The signal peptide, d. Memb. Biol. 115: 195-201. WEISSENBERG, S., OVADIA, M., FLEMINGER, G. and KOCHVA, E. (1991) Antihemorrhagic factors from the blood
serum of the Western Diamondback rattlesnake (Crotalus atrox). Toxicon 29: 807-818. WERB, Z., BURLEIGH, i . C., BARRETT, A. J. and STARKEY, P. i . (1974) The interaction of ct2-macroglobulin
with proteinases. Biochem. J. 139: 359-368. WOOD, J. T., HOBACH, W. W. and GREEN, T. W. (1955) Treatment of snake venom poisoning with a CTH and
cortisone. Virginia Med. J. 82: 130-135. Xu, X., WANG, C., LID, J. and Lu, Z. (1981) Purification and characterization of hemorrhagaic components
from Agkistrodon acutus (Hundred pace snake) venom. Toxicon 19: 633-644. YAMAKAWA, Y. and OMOm-SATOH, T. (1988) The sites of cleavage in oxidized insulin B-chain by a hemorrhagic
protease derived from the venom of Habu (Trimeresurusflavoviridis). Toxicon 26: 227-231. YONAHA, K., IHA, M., TOMIHARA, Y., NOZAKI, i . and YAMAKAWA, M. (1991) Characterization of three
hemorrhagic factors from the venom of Okinawa habu (Trimeresurus flavoviridis). Toxicon 29:703-711. YURCHENO, P. D., CHENG, Y.-S. and COLOGNATO, H. (1992) Laminin forms an independent network in
basement membranes. Jr. Cell Biol. 117:1119-1133.