ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2006

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 250

Sculpted through Time

Evolution and Function of Serine Proteases from theMast Cell Chymase Locus

MAIKE GALLWITZ

ISSN 1651-6214ISBN 91-554-6748-2urn:nbn:se:uu:diva-7379

Tempora mutantur, nos et mutamur in illis.

Times are changing and we are changing with them.

LIST OF PAPERS

This thesis is based on the following papers, which will be referred to in the text by their roman numerals:

I. Gallwitz M and Hellman L. Rapid lineage-specific diversification of the mast cell chy-mase locus during mammalian evolution. Immunogenetics. 2006, 58(8):641-54.

II. Gallwitz M, Reimer JM and Hellman L. Expansion of the mast cell chymase locus over the past 200 million years of mammalian evolution. Immunogenetics. 2006, 58(8):655-69.

III. Gallwitz M, Enoksson M and Hellman L Expression profile of novel members of the rat mast cell pro-tease (rMCP)-2 and -8 families, and functional analyses of mouse mast cell protease (mMCP)-8. Submitted manuscript.

IV. Andersson MK, Enoksson M*, Gallwitz M* and Hellman L The extended substrate specificity of the human chymase re-veals a serine protease with well-defined substrate recognition profile. Manuscript. *contributed equally to this work

V. Gallwitz M, Enoksson M and Hellman L The extended substrate recognition profile of the dog chymase reveals similarities and differences to the human chymase. Manuscript.

Reprints are made with permission from the publisher.

Contents

Introduction....................................................................................................... 11GENERAL OVERVIEW............................................................................ 11I) SERINE PROTEASES............................................................................ 12

Biological functions of serine proteases................................................ 12Catalysis by serine proteases.................................................................. 13Control of proteolytic activity................................................................ 15The chymotrypsin family of serine proteases ....................................... 16Investigation of proteolytic activity....................................................... 19

II) THE MAST CELL CHYMASE LOCUS............................................. 20Serine proteases from the mast cell chymase locus.............................. 20Gene duplications as an evolutionary mechanism generating diversity.................................................................................................................. 23

III) GRANULE EXPRESSION AND FUNCTIONS OF HEMATOPOIETIC SERINE PROTEASES............................................. 26

Cytotoxic granules of NK cells and T cells .......................................... 27Azurophil granules of neutrophils ......................................................... 32Mast cell granules ................................................................................... 36

IV) MAMMALIAN EVOLUTION – A SHORT OVERVIEW .............. 44

PRESENT INVESTIGATIONS...................................................................... 48AIMS............................................................................................................ 48RESULTS AND DISCUSSION................................................................. 48

Repeated duplications have remarkably expanded the mast cell chymase locus of the rat (study I).......................................................... 48The mammalian mast cell chymase locus was probably founded by a single ancestral gene over 215 million years ago (study II) ................ 51Novel members of the Mcpt2- and Mcpt8-families are expressed in rat MMC, and modelling suggests that mMCP-8 is proteolytically active (study III)................................................................................................. 54The human chymase displays a well-defined substrate recognition profile (study IV) .................................................................................... 57Similarities and differences in the substrate recognition profiles of dog and human chymase (study V)............................................................... 58

CONCLUDING REMARKS...................................................................... 60

SVENSK SAMMANFATTNING .................................................................. 61

ACKNOWLEDGEMENTS............................................................................. 65

REFERENCES ................................................................................................. 68

Abbreviations

aa amino acid(s) Asp aspartate Arg arginine Ang angiotensin Cma chymase (gene) CTMC connective tissue mast cell(s) Cts G cathepsin G (protein) Ctsg cathepsin G (gene) Cys cysteine DPPI dipeptidyl peptidase I ECM extra-cellular matrix Gzm granzyme (gene) Gzm granzyme (protein) IL interleukin kb kilobases kDa kilodalton LPS lipopolysaccharide Mb megabases MC mast cell(s) Mcpt mast cell protease (gene) Mcpt mast cell protease (protein) MCT tryptase-positive human mast cells MCTC tryptase- and chymase-positive human

mast cells MMC mucosal mast cell mMCP mouse mast cell protease (protein) Myr million years rMCP rat mast cell protease (protein) Ser serine

11

Introduction

GENERAL OVERVIEW

When an egg is fertilized, a life begins. Life is sustained when we digest food, and life can be saved when our body defeats a virus or when blood coagulation stops a bleeding. All of these processes depend on proteases. These important enzymes are therefore of great scientific interest.

Proteases function basically as a molecular pair of scissors, which cleaves target proteins into smaller parts (peptides). In the different families of pro-teases, different mechanisms are used for cleavage. For example, cleavage by metallo proteases depends on a metal ion, whereas cleavage by serine proteases depends on a reactive serine residue. As many as one third of all proteases are serine proteases. In fact, the serine protease mechanism seems so appealing that it has evolved independently at least in four subgroups.

One of these subgroups contains the so-called Chymotrypsin-like serine proteases. Some of the Chymotrypsin-like serine proteases have important functions in immunology, and their genes are arranged in four clusters in the human genome. I have studied one of these clusters, the mast cell chymase locus. The proteases encoded here are involved in the defence against vi-ruses, tumours and parasites. They are mainly expressed and stored in the granules of NK cells, T cells, neutrophils and mast cells. Interestingly, the mast cell chymase loci in different species display quite striking variations. For example, if we compare mouse and human, the two loci contain a num-ber of corresponding genes, homologs. These homologous genes comprise T cell granzymes B and C/H, mast cell -chymase and neutrophil cathepsin G. However, the mouse locus also holds numerous genes that are not found in humans, namely -chymases, an Mcpt8 gene and additional granzymes. Where do these additional genes come from? What are their functions? Does the presence of different genes result in different functions of the immune cells in human and mouse?

I have analysed these questions by mapping and comparing the chymase loci from different mammals. The results from these evolutionary studies help us understand in which order and by what mechanisms the genes in the mast

12

cell chymase locus have evolved over the past 200 million years. By investi-gating which genes that are common to all species and which are not, we also obtain indications for basic vs. species-specific functions. A further step in our analyses is then to clone the concerned proteases and to study their activity. This is done mostly with chromogenic substrates and with random nonapeptides that are displayed on phage surfaces. In order to properly in-terpret the results from these studies, it is important to know what determines the function of a serine protease, and what functions have been described previously for the serine proteases from the mast cell chymase locus. There-fore, the following introduction will outline I) the structure and function of serine proteases, II) the mast cell chymase locus and gene duplications as an evolutionary mechanism, and III) functional data from serine proteases ex-pressed in the granules of NK cells, T cells, neutrophils and mast cells. To provide an evolutionary time frame, a short overview of the evolution of mammals is given in section IV.

I) SERINE PROTEASES

Biological functions of serine proteases

The common feature for all proteases is the cleavage of peptide bonds (Fig. 1). Peptide bonds are very stable, but proteases manage to cleave them by stabilizing transitional states that occur during hydrolysis. A number of cata-lytic mechanisms for this reaction have evolved. However, approximately one third of all proteases make use of a common catalytic mechanism, which involves a highly reactive serine (Ser) residue (24). These evolutionary suc-cessful proteases are called serine proteases.

Serine proteases are represented in all investigated species, from bacteria to mammals. In mammals, the ability of serine proteases to cleave peptide bonds is employed in many contexts. For example, serine proteases are ac-tive in fertilization, in the embryonic development, when we digest food, in our cellular and humoral immune system, and in blood clotting. The proto-typical digestive serine protease is pancreatic trypsin, which cleaves proteins from our food into smaller peptides. This is an example of a serine protease with broad substrate specificity. Other serine proteases have evolved to cleave only one or a few specific targets, e.g. granzyme B. This immune

13

protease hinders the spreading of viruses by inducing cell death (apoptosis) in virus-infected cells. Serine proteases are also part of a number of cas-cades, where cleavage activates a target that can then activate the next target and so on. This is the case, for instance, in the blood coagulation system and in the complement system.

Fig. 1 Hydrolysis of a peptide bond is pictured schematically. A pair of scissors symbolizes a serine protease.

Catalysis by serine proteases

The name-giving residue of serine proteases, the catalytic Ser, is not the only amino acid that is critical for the catalytic mechanism of this protease family. That is, the catalytic Ser is in most members of this protease family assisted by two additional residues, an aspartate (Asp) and a histidine (His). To-gether, the catalytic His, Asp and Ser are called the catalytic triad. The main role of this triad in the hydrolysis of a peptide bond is to stabilize a proton transfer by redistributing charge, i.e. to provide a charge-relay system. In addition, the catalytic His enhances the nucleophilicity of the Ser by acting as a general base.

Within the group of serine proteases, the catalytic triad occurs in four differ-ent structural contexts, where the catalytic His, Asp and Ser are arranged in a specific, conserved order (further groups with atypical catalytic triads are not discussed here). This indicates that the His-Asp-Ser charge-relay system has evolved independently at least four times (reviewed in 37). Thus, serine pro-teases can be divided in four groups (MEROPS, reference (168) with distinct basic structures, namely the Chymotrypsin group (catalytic triad His-Asp-

R1-C N-R2 H2O

O

H

+ R1-CO

OH3N-R2+

peptide acid amine

14

Ser), the Subtilisin group (Asp-His-Ser), the Carboxypeptidase Y group (Ser-Asp-His) and the Clp protease group (Ser-His-Asp).

The first serine protease to be characterized from the Chymotrypsin group was bovine Chymotrypsin (112). Bovine Chymotrypsin and other serine proteases that have arisen from the same evolutionary origin are grouped in the Chymotrypsin clan. All proteases analysed in the studies of this thesis belong to this clan. To facilitate sequence comparisons, the residues in all clan members are numbered according to the corresponding residues in bo-vine Chymotrypsin (Chymotrypsin numbering according to Greer (62)). In this numbering system, the amino acids (aa) of the catalytic triad are referred to as His57, Asp102 and Ser195.

The catalytic hydrolysis of a peptide bond by a serine protease proceeds basically in two steps (reviewed in 109). First, a covalent bond is produced between C1 of the substrate and Ser195 in the enzyme (acylation). This creates an intermediate substrate-enzyme complex where the bonds of the substrate C1 have tetrahedral geometry (tetrahedral transition state). In the transition state, the oxygen in the peptide bond to be cleaved (scissile bond) is nega-tively charged (oxyanion, O-). This oxyanion is stabilized by two NH-groups from the enzyme main chain, glycin (Gly) 193 and Ser195, which form the so-called oxyanion hole. Now, the nitrogen of the scissile bond is protonated, and the peptide thereby cleaved. The amine part of the cleaved peptide leaves the complex, whereas the acid part is still bound (acyl-enzyme com-plex).

In the second step, the enzyme is freed from the acyl group (deacylation) in a reaction that is essentially a reverse acylation. In particular, a water molecule hydrolyzes the covalent bond between the acyl group and the enzyme, again generating a tetrahedral intermediate. A proton from the water molecule is then transferred to the enzyme, whereby the enzyme is restored and the acyl group released.

15

TAKE HOME:

• Proteases cleave peptide bonds, thereby generating smaller peptides.

• Many proteases are serine proteases. The proteases in this thesis be-long to the Chymotrypsin clan of serine proteases.

• In order to function properly, serine proteases generally need a cata-lytic triad (Ser195, His57, Asp102) and an oxyanion hole (backbone of Gly193 and Ser195).

Control of proteolytic activity

Considerable damage can be done if proteases cleave the wrong substrates, or if substrates are cleaved at the wrong time or in the wrong place in the body. The activity of proteases is therefore controlled in a number of ways. First, many proteases are stored as inactive enzymes that need to be proc-essed in order to become active. For example, the proteases studied in this thesis are produced in an inactive (pro-) form, which becomes active when a dipeptide is removed from the amino-terminus (N-terminus). Moreover, some proteases act only within a certain pH range or salt concentration, are expressed only in certain tissues or at certain times, or are released from the cell only in certain circumstances. In addition, many proteases can be inacti-vated by specific inhibitors.

Furthermore, proteases generally accept only certain substrates for cleavage, i.e. they have restricted substrate specificity. The degree of specificity for a protease depends on its task. For example, digestive enzymes such as trypsin might be rather unspecific, cleaving after positively charged residues. In contrast, the formation of active trypsin from trypsinogen depends in most mammals on the very specific recognition of a five-residue cleavage site by another serine protease, enterokinase (18). The appropriate specificity for a protease to function well in its context is conferred by the architecture of its substrate-binding site.

In the substrate-binding site, specific residues of the protease interact with the amino acids of the substrate. The interacting residues are numbered as

16

follows: Substrate residues that are amino-terminal of the cleaved bond are numbered P1, P2, P3 etc., starting with the closest neighbour (Fig. 2). The residues on the carboxyterminal side are termed P1´, P2´, P3´etc. In the en-zyme, the corresponding binding-sites are termed S1, S1´ etc. (180). One substrate residue is typically in contact with several residues of the enzyme that are brought together in the three-dimensional structure. The binding sites are often arranged to form a groove or cleft, the substrate-binding cleft. Both the steric accessibility of the cleft and the biochemical properties of the substrate-binding sites restrict the range of accepted substrates. For a prop-erly fitting substrate, the enzyme-substrate interactions position the scissile bond correctly near the catalytic triad. In the case of serine proteases, only peptide bonds within a polypeptide chain can be hydrolyzed, but not those positioned at the ends. This type of proteases is called endopeptidases.

Fig. 2. Scheme depicting the nomenclature for residues in a serine protease substrate (lower part) and the corresponding sites in the protease (upper part). Cleavage oc-curs between residues P1 and P1´.

The chymotrypsin family of serine proteases

My work is focused on serine proteases that belong to the chymotrypsin family (MEROPS peptidase family S1). This family is with more than 240 MEROPS entries the largest of all protease families (168). The family has been named after Chymotrypsin A from cattle (Bos taurus). Most members are first synthesized as pre-pro-enzymes, meaning that they possess an N-terminal pre-peptide followed by a pro-peptide. The pre-peptide is a signal peptide guiding the protease to the secretory pathway. In the endoplasmatic reticulum (ER), the pre-peptide is removed, and the pro-peptide is exposed. The pro-peptide is an activation peptide, the length of which varies between proteases. As mentioned above, the serine proteases addressed in this thesis possess a pro-peptide of two amino acids (dipeptide). The pro-peptide has to be cleaved off in order to enable structural rearrangements that form the S1

+H3N COO-......

S1 S1´S2S3 S2´ S3´

P1 P1´P2P3 P2´ P3´

17

site and the oxyanion hole, which renders the protease active (reviewed in 78).

Chymotrypsin A from cattle consists of 245 amino acids that form two six-stranded -barrels (13). All chymotrypsin-like proteases have a structure similar to this. The active site is situated in the cleft between the barrels, with the catalytic Ser195 on one side, and the catalytic His57 and Asp102 on the other. These residues are part of an extensive hydrogen-bonding network. In the active proteases, the S1-site is shaped as a pocket adjacent to Ser195. It is formed mainly by two loops consisting of residues 182 to 195 and 214 to 228 (159). Three residues lining the S1-pocket, aa 189, 216 and 226, are especially important for the recognition of P1 residues (primary specificity). These residues are therefore called the specificity-conferring triplet. It has been shown that the activity of many extant serine proteases is abrogated when the residues of this triplet are mutated (88, 194, 221). However, a re-constructed ancestral chymase remains active when subjected to the corre-sponding mutations (233). Also other residues can thus support proteolytic activity. For example, structural elements in more distant parts of the en-zyme can contribute to activity and substrate specificity, probably because they function as a scaffold that affects the positions of the aa in the S1-pocket (158). One additional enzyme site that is known to interact with the substrate is the so-called polypeptide-binding site. This site is formed by the backbone of enzyme residues 214 to 216, which builds hydrogen bonds to the backbone of substrate aa P1 to P3 (reviewed in 78). Because these inter-actions involve no side chains of the substrate, they are not substrate-specific. They are however important, as they expose the scissile bond to the catalytic serine, which promotes cleavage (159).

The proteases in the chymotrypsin family can be subdivided in three main types with different S1 sites. The three types are called chymotrypsin-like, trypsin-like and elastase-like proteases, after the typical representatives chymotrypsin, trypsin and elastase (Fig. 3). These types prefer interactions with different P1 residues and have therefore distinct substrate specificities. The residues of the specificity-conferring triplet (189, 216 and 226) are mainly responsible for these preferences. In chymotrypsin-like and trypsin-like serine proteases, residues 216 and 226 are small and uncharged (159). In chymotrypsin-like proteases, also residue 189 is rather small and uncharged. The S1-site of chymotrypsin like proteases is thereby rather wide and un-charged. This accounts for the fact that chymotrypsin-like proteases cleave adjacent to large, hydrophobic P1 amino acids (Tyr, Trp, Phe, and Met). Trypsin-like proteases also have a rather wide S1-site, but hold a negatively charged aa at the bottom. This type of S1-site favours cleavage after basic (positively charged) P1 residues, such as Arg and Lys. In contrast, the S1-pocket of elastase-like proteases is rather shallow and narrow. This is mainly

18

because residues 216 and 226 are big, aliphatic aa in these enzymes. The S1-pocket of elastase-like proteases thus preferentially admits small P1 aa, e.g. Ala or Val.

In addition, there also other specificities are found within the chymotrypsin family. For example, some of the serine proteases in the granules of immune cells are aspartases (cleaving after Asp) or Met-ases (cleaving after Met). These will be discussed later.

Fig. 3. The S1-pockets of chymotrypsin, trypsin and elastase are depicted schemati-cally. White circles represent carbon atoms other than C , grey circles oxygen at-oms. _ indicates a negative charge. Figure inspired by Mattias Andersson.

TAKE HOME:

• In serine proteases of the chymotrypsin family, mainly residues 189, 216 and 226 determine which amino acid a substrate should have in position P1 in order to be recognized and cleaved.

• Proteases of the chymorypsin family have mainly one of three specificities: Chymotrypsin-like proteases cleave after large, hydro-phobic amino acids (Tyr, Trp, Phe, Met). Trypsin-like proteases cleave after positively charged amino acids (Arg, Lys). Elastase-like proteases cleave after small amino acids (Ala, Val).

Chymotrypsin Trypsin Elastase

large, hydrophobic aa positively charged aa small aa(Phe, Tyr, Trp, Met) (Arg, Lys) (Ala, Val)

216Gly

189Ser

226Gly

216Gly

189Asp

226Gly

216Val

189Ser

226Thr

19

Investigation of proteolytic activity

Different approaches can be used to determine the proteolytic activity of an enzyme. If the primary specificity (preferred P1 residue) of the investigated protease is already known or can be predicted with some certainty, a quick and easy way to confirm that it is actually active is with labelled substrates. In the studies of this thesis, we make use of chromogenic substrates that are derivatives of short peptides (three to four aa) bound to p-nitrophenol. If the bond between p-nitrophenol and the first aa in the peptide (P1) is cleaved, the reaction product absorbs light at 405 nm. This can be monitored with a spectrophotometer. The enzymatic activity can thus be measured very di-rectly. To compare activity against different substrates, one aa can be varied at a time. However, this approach is not feasible to determine the optimal substrate of a protease, to analyse enzyme-substrate interactions on the C-terminal side of the scissile bond, or to investigate proteases of unknown specificity. These questions can better be investigated e.g. using combinato-rial peptide libraries. Combinatorial libraries contain a great number of pep-tide sequences where one position is fixed, while the others are varied. With this approach, it is possible to map the interactions of a protease and its sub-strate N-terminally and C-terminally of the scissile bond, although in sepa-rate reactions. For example, interactions between the human chymase and peptide substrates have been mapped with this method (9). However, there is also a method available where both N- and C-terminal residues are varied simultaneously, substrate phage display. This methodology makes it possible to determine a consensus sequence for the preferred residues throughout the substrate-binding region of a protease (extended cleavage specificity). Moreover, no leaving groups foreign to natural peptides (such as p-nitroanilide) influence the selection of susceptible peptides in this approach. In study IV and V of this thesis, we employ substrate phage display to de-termine the extended cleavage specificity of human chymase and dog chy-mase (unpublished results).

If a clear consensus for an optimal peptide can be predicted from molecular studies, it may be verified by studies of synthesized peptides with this se-quence. Finally, the goal of many protease studies might be reached when a novel natural substrate can be predicted and confirmed experimentally.

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II) THE MAST CELL CHYMASE LOCUS

Serine proteases from the mast cell chymase locus

Chymotrypsin-like proteases cannot only be subdivided in families accord-ing to structural criteria, but also with regard to their evolutionary (phyloge-netic) relationship. The genes of closely related phylogenetic families are often clustered in loci.

I have studied one such locus of chymotrypsin-like serine proteases, the mast cell (MC) chymase locus or granzyme B cluster. (In the following, the term mast cell chymase locus will be used.) The mast cell chymase locus is situ-ated on chromosome 14 both in human and mouse (29, 69) (Fig. 4a). In the human locus, we find the genes for -chymase (Cma1), granzyme B (Gzmb) and granzyme H (Gzmh), and cathepsin G (Ctsg). Alpha-chymase is ex-pressed in the granules of mast cells, granzymes B and H are found in NK cell and T cell granules, and neutrophil granules contain cathepsin G. This expression in the granules of immune cells has earned the chymase locus-encoded proteases the name graspases (granule-associated proteases).

Common structural features suggest a common evolutionary origin of the graspases. First, virtually all chymotrypsin-like serine proteases hold a disul-phide bridge between two cysteines (Cys) in positions 191 and 220. This bridge is however absent in all graspases (238). The neck of the substrate-binding cleft is therefore wider in graspases, and a new S3 pocket is shaped. This promotes more specific interactions with substrate P3 residues (82, 162, 171, 223). The P3 position is thus of greater importance for many graspases than for other chymotrypsin-like serine proteases. For example, the graspases rat mast cell protease (rMCP)-1 and rMCP-2 cleave chromogenic substrates with optimal P3 side chains 100 times more efficiently than sub-strates with suboptimal P3 residues. This is to compare with chymotrypsin, where little or no difference in hydrolysis is observed with different P3 resi-dues (171).

Next, in graspases, the side chain of aa 226 stretches out from the bottom of the substrate-binding cleft and interacts directly with the substrate P1 side chain. Residue 226 plays therefore an especially important role for the pri-mary substrate specificity of graspases. In human granzyme B, position 226 is occupied by a positively charged arginine (Arg), which confers preference for negatively charged Asp residues in the P1 position (223). The single

21

exchange of this Arg for Gly (Arg226Gly) results in an enzyme that instead prefers aromatic aa in P1 (22). Correspondingly, an Arg226Glu mutant shows preference for basic P1 residues (23). In human cathepsin G (but not in mouse Ctsg), the side chain of Glu226 points slightly away from the posi-tion of the P1 residue, which allows both basic and aromatic side chains to enter the S1 pocket (82, 164).

Research aimed to better understand and characterize the in vivo function of graspases is mainly conducted in animal models, e.g. in the mouse. How-ever, the mouse chymase locus is quite different from its human counterpart, having three times its size and more than three times as many genes (Fig. 4b). These genes and their products need to be characterized thoroughly, if we want to establish how the graspases in human and mouse correspond functionally.

Fig. 4. Chromosomal localization (a) and gene content (b) of the mast cell chymase locus in human and mouse.

Three of the human genes have clear homologs in mouse, i.e. Cma1 (in mouse also called Mcpt5), Ctsg and Gzmb. The three genes are in both loci localized at the borders and close to the middle, respectively. Because their sequence and relative position is conserved, these genes may be phyloge-netically ancient and may encode functions that are basic to the mammalian immune system. The region between Cma1 and Ctsg is completely devoid of genes in the human locus. The mouse locus, in contrast, holds five protease genes in the corresponding region, Mcpt1, -2, -4, -8 and -9. When these genes are compared to other graspase genes in phylogenetic analyses, it be-comes evident that they belong to two gene families that are not found in human.

a bhuman mouse

q11.2

C1/2

chr 14 chr 14

human

mouse

Gzmh

GzmbCtsgCma1

GzmbCtsgCma1/Mcpt5

GzmcGzmf

GzmnGzmgGzme

GzmdMcpt8Mcpt4

Mcpt2Mcpt9

Mcpt1

human

mouse

Cma Ctsg Gzmb

Cma1/Mcpt5 GzmbCtsg

22

Four of the genes, Mcpt1, -2, -4, and –9, are closely related members of a subfamily termed the -chymases (Fig. 5). Beta-chymases have also been described in other rodents, such as rat and hamster, but never in a non-rodent species (reviewed in 26). The closest relative to this group in human is the

-chymase (Cma1). In contrast to -chymases, at least one -chymase has been identified in all studied mammals. Both -chymase and -chymases are expressed in mast cells, and their functional relations are very interesting. The two groups of mast cell chymases are discussed further in section III.

Fig. 5. Dendrogram depicting the phylogenetic relationship between - and -chymases from human (h), mouse (m) and rat (r). The dendrogram is based on se-quences of mature proteins. Tryptase sequences were chosen to provide an outgroup.

The fifth gene, Mcpt8 (122), is in the mouse locus the neighbour of Ctsg. This gene belongs also to a gene family found only in rodents, the Mcpt8-family. Mcpt8 is the single member of the Mcpt8-family in mouse, and this gene is expressed in basophils (124, 165). Three members have previously been identified in the rat, Mcpt8, Mcpt9 and Mcpt10 (125), and these are expressed in mast cells. Interestingly, no proteolytic activity has to date been shown for any member of the Mcpt8-family. Moreover, the mouse locus holds six granzyme genes in the region between Ctsg and Gzmb, i.e. Gzmc,

d, e, f, g and n. Only one gene is present in the corresponding region in hu-man, Gzmh. Sequence comparisons suggest that both mouse Gzmc and hu-man Gzmh are derived from Gzmb (reviewed in 64). The presence of numerous, phylogenetically related genes in close vicinity to each other is an indication that these genes have been derived by duplica-tions from a common ancestral gene. This important mechanism for the ex-pansion of gene families is discussed in more detail here below.

0.05

outgroup

rMCP-2r vascular chymase

mMCP-1rMCP-1

mMCP-41000

rMCP-4rMCP-3

1000

mMCP-9

658

mMCP-2

764

rMCP-5mMCP-5

h -chymase

10001000

732

999

rMCP-7rMCP-6

h -tryptase

1000

607

23

Gene duplications as an evolutionary mechanism generating diversity

Gene duplications have high prevalence in the genomes of bacteria, archaea and eukaryotes (reviewed in 242). In the mouse and human genome, each gene is duplicated (a copy is “born”) in average every 100 million years (126). This is in the same range as the frequency of nucleotide substitutions (0.1 to 0.5 per site in 100 Myr) in the nuclear genome of vertebrates (re-viewed in 242). Gene duplications have thereby great potential to generate molecular templates for the evolution of novel functions. However, most gene duplicates appear to become silenced or deleted (they “die”) rather than being preserved. Genes can be duplicated by various mechanisms. The most studied of these are unequal crossing-over and retroposition (mRNA is retro-transcribed and then inserted). While unequal crossing-over generates novel genes including introns, introns and regulatory sequences are lost in the case of retroposition. All genes in the mammalian mast cell chymase loci contain introns, and have therefore most likely not undergone retroposition. Novel gene copies also appear when whole chromosomes or genomes are dupli-cated. This is however infrequent in animals, compared to the other duplica-tion mechanisms.

The duplication of genes may not occur entirely randomly, but may be driven by a functional requirement. This is at least suggested by a recent report showing that if a gene is present in only one copy in a certain mam-malian species, but has been duplicated in other species, the single gene has a higher rate of alternative splicing than the duplicates (108). A required variation in transcripts could thus be met either by the presence of multiple gene copies, or by varying the transcripts from a single gene. Generally, it appears that genes involved in physiologic functions that vary greatly be-tween different species, such as immunity, reproduction and sensory sys-tems, have increased birth and death rates. The duplication of these genes appears to contribute to the process of speciation (126). For example, bitter taste receptors (189), and different immune receptor families on NK cells have evolved rapidly via multiple gene duplications (72, 73).

Once a gene is duplicated, the copies face a number of possible fates (re-viewed in 126). First, if the duplication is beneficial per se, the genes may simply be maintained with their original function. Second, because the func-tional constraint on a gene is relaxed as soon as two redundant copies are present, random mutations may become fixed in one of the daughter genes. These mutations can imply beneficial or deleterious functional changes. For

24

example, one of the copies may acquire a new function (neofunctionaliza-tion). The novel function may be only weakly active at first, but if it is ad-vantageous, positive selection can accelerate the fixation of further muta-tions that enhance this activity. Two proteases from the chymase loci of mouse and rat, the -chymases mouse mast cell protease-5 (mMCP-5) and rat mast cell protease-5 (rMCP-5), have apparently undergone a neofunc-tionalization process. In contrast to other -chymases, these two proteases have secondarily acquired elastase function (98, 113). This change in speci-ficity has probably been possible due to the presence of functionally redun-dant -chymases in these species, which reduced the selective pressure on the -chymase.

Next, the function of the ancestral gene can become modified in the copies e.g. by a changed expression pattern, a process called subfunctionalization. This is likely to occur in cases where the ancestral gene had dual functions that can be adopted and refined separately in the two daughter genes, via complementary loss-of-subfunction mutations (reviewed in 45). For exam-ple, the various substrate specificities of the proteases from the MC chymase locus have probably evolved by subfunctionalization from an ancestor with broader specificity (233). Different models for the process of functional di-vergence exist (e.g. (3, 46, 127, 234), but it seems to be clear that both posi-tive selection and periods of relaxed evolution are necessary.

Finally, a gene copy may also become non-functional, i.e. a pseudogene. Mutations that pseudogenize a gene include, for example, frame-shift muta-tions, the loss of the start codon, or loss of activity in the encoded protein. The pressure to delete non-functional genes seems to be notably weaker in eukaryotes than in prokaryotes, and the frequency of pseudogenes in mam-malian genomes is surprisingly high. For example, one pseudogene is found almost for every two functional genes within human chromosomes 21 and 22 (74)! This high incidence is not only explained by the frequent occurrence of deleterious mutations and their subsequent preservation, but also by the du-plication of pseudogenes as such.

To study the processes of subfunctionalization, neofunctionalization and pseudogenization in the mast cell chymase locus, genetic and functional data need to be brought together. The cellular distribution and function of the serine proteases encoded in this locus will therefore be reviewed in the fol-lowing. In particular, the protease content of NK cell and T cell granules (expressing granzymes), neutrophil granules (cathepsin G) and mast cell granules (chymases) will be discussed.

25

TAKE HOME:

• The serine proteases from the mast cell chymase locus are called graspases.

• Unlike other chymotrypsin-like proteases, graspases hold no disul-phide bridge between residues 191 and 220. As a consequence, the substrate P3 residue is especially important for graspases.

• For the primary specificity (preferred substrate residue in P1) of graspases, residue 226 is of major importance.

• The mast cell chymase locus can look rather different in different mammals. In rodents, the locus contains many genes that have no clear homologs in human.

• Many genes in the mast cell chymase locus of e.g. the mouse may have evolved by gene duplications.

• Gene copies can be deleted, can acquire novel functions (neofunc-tionalization), specialize in a subfunction (subfunctionalization), or can loose function (pseudogenization).

26

III) GRANULE EXPRESSION AND FUNCTIONS OF HEMATOPOIETIC SERINE PROTEASES

Serine proteases from the mast cell chymase locus are stored in the granules of NK cells and T cells, neutrophils, and mast cells. These granules also contain serine proteases encoded from other loci, including the Gzm A/K locus, the Gzm M/elastase locus and the tryptase locus. Proteases encoded from one locus often have similar specificities. For example, many proteases from the mast cell chymase locus function as chymases, whereas most prote-ases from the Gzm M/elastase locus are elastases. Although this correlation is not absolute and many exceptions occur, it indicates that each of the loci could have evolved from one or a few ancestral genes via gene duplications. Also proteases belonging to families other than serine proteases are stored in the immune cell granules. In addition, the granules contain a number of po-tent effector molecules, such as membrane-perturbing molecules in T cells and neutrophils, or biogenic amines in mast cells.

When stored in the granules, the activation dipeptide of graspases has al-ready been removed, but the enzymes are still not proteolytically active. This is because the granule pH is acidic, whereas graspases belong to the group of neutral proteases, which require a near-neutral pH for efficient catalysis. Graspases therefore become active first upon their release into the extra-cellular space. This arrangement ensures that these enzymes do not digest other granule components or each other.

Some of the granule proteases are stored in complex with a proteoglycan matrix (Fig. 6). The proteoglycan consists of a core protein that is attached to a great number of unbranched polysaccharide chains. In cytotoxic T cells (128, 182) and mast cells (103), a prominent core protein is serglycin. This protein contains a central repetitive serine-glycine sequence. The serine resi-dues provide hydroxyl groups, via which the polysaccharide side chains are joined to the core. The chains themselves are glycosaminoglycans that are assembled of repeated disaccharide units. These disaccharide units differ for different types of proteoglycan. They are often carboxylated or sulphated, and thereby highly negatively charged. Thus, they can interact strongly with positively charged granule proteases (239). The most negatively charged known biomolecules are the glycosaminoglycan chains of heparin. Heparin is common in mast cells.

27

serin

glycin

other amino acid

dissaccharide unit, e.g. for heparin:

COO- CH2 OSO3-

OSO3- NHSO3-

OH OH

Fig. 6. Schematic structure of serglycin protoglycans.

Cytotoxic granules of NK cells and T cells

Cytotoxic T cells and NK cells are immune cells that are specialized in kill-ing target cells by inducing apoptosis. This may be necessary when a cell is virus-infected or has mutated into a tumour cell. T cells and NK cells recog-nize their targets in different ways, but are believed to employ similar mechanisms for killing. Two general pathways for the induction of apoptosis are known, one caspase-dependent and one caspase-independent. Both lead finally to the depolarization of mitochondria, to the collapse of the nucleus, and to DNA fragmentation. These pathways are initiated by the action of molecules that are stored in the granules of cytotoxic T cells and NK cells. The apoptosis-inducing molecules comprise granule-specific proteases (granzymes), membrane-perturbing proteins (perforin and granulysin), lysosomal enzymes (cathepsins) and cell surface molecules (FAS ligand). The granzymes will be discussed in the following.

The term “granzyme” refers to the granule-specific expression of an enzymeand does not reflect phylogenetic or functional relationships. Granzymes are therefore a phylogenetically heterogenic group. Known granzymes fall into three phylogenetic families that are encoded from separate loci on different chromosomes, that is, the mast cell chymase locus, the Gzm A/K locus and the Gzm M/elastase locus. I will discuss the three groups in separate sections below. In total, eleven different granzymes have been described in human and mouse (Gzm A, B, C, D, E, F, G, H, K, M and N) (reviewed in 192).

28

Only four of these, Gzm A, B, K and M, are expressed in both species. Gzm H is expressed in human, but not in mice, whereas mice hold six granzymes not found in human, i.e. Gzm C, D, E, F, G and N (reviewed in 190). The different granzymes have very specific substrate preferences, which is con-sistent with a processing rather than degrading function.

Granzymes contribute about 90% of the content of cytotoxic granules. Al-ready during the process of granule packaging, they are activated by dipepti-dyl peptidase I (DPPI), also termed cathepsin C (111, 160, 191). DPPI re-moves the N-terminal activation dipeptide from the granzymes, which re-sults in a structural reorganization that favours enzymatic activity (reviewed in 78). The granzymes finally become fully active when they are released from the acidic granule environment into the neutral cytoplasm. Their pH optimum is about eight (80). In the granules, the granzymes are tightly pack-aged. For example, mouse granzyme B (Gzm B) is one protease that builds complexes with the negatively charged serglycin proteoglycan (65). The proteoclycan-Gzm B complexes reach 40 to 200 nm in diameter, about the size of a virus particle. One complex comprises 30 to 50 granzyme mole-cules that are bound to a 250-kDa proteoglycan molecule (reviewed in 118). When the complexes are released into the extra-cellular fluid, the neutral pH weakens the binding of Gzm B to the serglycin matrix (167), and some Gzm B probably diffuses off.

In order to reach their target molecules in the cytosol of target cells, most granzymes depend on perforin (96, 121). It has long been thought that the role of perforin was to form pores in the cell membrane through which gran-zymes could enter. However, GzmB can enter cells, but not induce apopto-sis, in the absence of perforin (48). The perforin hypothesis has therefore been revised. It now states that perforin is required to release granzymes from endocytic vesicles in a target cell into the cytosol, and for the passage of granzymes into the nucleus.

Granzymes encoded from the mast cell chymase locus

The granzymes encoded from the mast cell chymase locus include human Gzm B and Gzm H, and mouse Gzm B, C, D, E, F, G and N (Table 1). Gzm B is the most abundant granzyme in T cells and has been most thoroughly studied. It is also expressed in NK cells. Gzm B cleaves after Asp residues (163) in different target molecules, which induces caspase-dependent or caspase-independent apoptotic pathways. Molecular targets include classical pro-apoptotic molecules, such as Caspase-3 and Bid (36, 206). Moreover,

29

Gzm B cleaves ICAD, the inhibitor of a caspase-activated DNase (CAD) (212), and PARP (poly(ADP-ribose)polymerase), a sensor of DNA damage (47). Interestingly, it was recently reported that stimulation with IL-3 in-duces mature human basophils to express Gzm B (215). This challenges the general view that expression of Gzm B is restricted to the lymphoid lineage. Moreover, these results indicate that the granule composition of blood granu-locutes is not as fixed as previously thought.

Human Gzm H is closely related to mouse Gzm B and is primarily expressed in NK cells (187). Interestingly, Gzm H displays a different specificity-conferring triplet than Gzm B (179) and has chymotryptic activity (38). Yet, recent findings suggest that also Gzm H induces cell-death, which is medi-ated by caspase-independent pathways (44). Also the mouse granzymes C and F have cytotoxic effect (95, 172). The molecular targets of Gzm C and F are however to date unknown, and also the functions of the mouse Gzm D, E, G and N are very poorly characterized.

Granzymes encoded from the Gzm A/K locus

The most abundant granzyme in human and mouse NK cells is Gzm A (re-viewed in 64). Gzm A is encoded together with Gzm K in the granzyme A/K locus on human chromosome 5 (7) (in mouse on chromosome 13) (136). Both Gzm A and K are tryptases, which cleave after positively charged aa. Mouse Gzm A has been shown to cleave components of an ER-associated protein complex called the SET complex. This induces an alternative caspase-independent apoptotic pathway. This alternative pathway is slower than the caspase-independent pathway activated by Gzm B and generates single-strand damaged DNA (reviewed in 118). Gzm A also directly cleaves targets in the nucleus, i.e. histone protein H1 and nuclear lamins (240, 241). Moreover, Gzm A can cleave a number of ECM proteins and may thereby facilitate the migration of T cells and NK cells through tissues (reviewed (214). Very little is known to date about the functions of Gzm K.

Granzymes encoded from the Gzm M/elastase locus

In addition to Gzm A and Gzm B, NK cells express Gzm M, which is en-coded from the Gzm M/elastase locus on human chromosome 19 (mouse chromosome 10) (161). This locus also holds the neutrophil granule prote-ases azurocidin, neutrophil elastase and proteinase-3. Gzm M, azurocidin

30

and proteinase-3 genes share a common gene structure, where intron 1 di-vides the leader sequence. It is therefore likely that the genes have a close evolutionary relationship, despite their expression in different cell types. Gzm M is a Met-ase, cleaving after Met, Leu and Ile (99, 174). The rather large Ser216 seems to be important for this particular specificity (193). In the presence of perforin, Gzm M mediates caspase-independent cell death [Kelly

TAKE HOME:

• The granules of NK cells and T cells contain large amounts of gran-zymes.

• Granzymes belong to three gene families that are encoded from dif-ferent loci. Gzm B fom the mast cell chymase locus is the most prominent granzyme in cytotoxic T cells.

• Granzymes trigger virus-infected cells or tumour cells to commit suicide (apoptosis). Signals leading to apoptosis are in the target cell mediated via caspase-dependent and caspase-independent pathways.

• Together, the different apoptotic pathways induced by granzymes seem to provide a fail-proof killing system, which is hard to over-come for viruses.

31

Table1. Selected contents of cytotoxic granules.

++: strongly expressed; +: expressed; -: not expressed; ?: expression uncertain, CTL: cytotoxic T lymphocytes.

in NK cells in CTLs Granzymes human mouse human mouse

Function

Gzm B ++ ++ ++ ++ Asp-ase Induces apoptosis

Gzm H + - + - Chymase Induces apoptosis?

Gzm C - ? - + Role against vi-ruses/tumors

Gzm F - ? - + Role against vi-ruses/tumors

Gzm D - ? - + ?Gzm E - ? - + ? Gzm G - ? - + ?

mast cell chymase locus

Gzm N - - - ? ?

Gzm A ++ ++ ++ ++ Tryptase Induces apoptosis ECM re-modeling?

Gzm A/K locus

Gzm K + ? + + Tryptase Role against viruses?

Gzm M/elastase locus

Gzm M + + - - Met-ase Cytotoxic

Other proteases Cathepsin C (DPPI)

+ + + + Gzm activa-tion

Proteoglycan matrix

Serglycin proteo-glycan

+ + + + Gzm storage

(Data from: [Grossman, 2003 #224;Revell, 2005 #287;Lieberman, 2003 #288;Jenne, 1988 #307;Waterhouse, 2004 #311;Grujic, 2005 #327;Schmidt, 1985 #343;MacDermott, 1985 #344;Bade, 2005 #308;Shresta, 1997 #309;Bratke, 2005 #310;Edwards, 1999 #339; Fellows, 2006 #541])

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Azurophil granules of neutrophils

The primary function of neutrophils is to engulf (phagocytose) and kill in-vading extra-cellular pathogens. The body may encounter a great variety of extracellular pathogens, and the killing mechanism used by neutrophils is therefore rather general. The main weapons of neutrophils are the generation of reactive oxygen intermediates (“respiratory burst”), and the secretion of antibiotic polypeptides and proteins (reviewed in 116, 198). Antibiotic poly-leptides and proteins are stored in the granules of neutrophils. However, neutrophil granules are very heterogenic. This is because the granules are formed continuously throughout neutrophil differentiation, whereas different proteins are synthesized only at certain developmental stages. The protein content of a granule thus reflects the myelopoietic stage during which it was packaged (15, 66, 196). The earliest granules are formed during the promye-locyte stage and are designated azurophil (primary) granules. In the order of formation, specific (secondary) granules, gelatinase granules and secretory vesicles follow. The subsequent section will focus on azurophil granules, because these store the only neutrophil-specific protease encoded from the mast cell chymase locus, Cts G.

Azurophil granules are characterized by a high content of myeloperoxidase (MPO) and are therefore also called peroxidase-positive granules. MPO re-acts with H2O2, which induces formation of reactive molecules, such as tyro-sine radicals and halide oxidates. These molecules attack the surface of mi-croorganisms (reviewed in 104). A second antimicrobial peptide in azurophil granules is bacterial permeability increasing protein (BPI) (225). BPI binds with its N-terminal half to lipopolysaccharide (LPS) on the bacterial surface. This initiates the hydrolysis of phospholipids in the bacteria, increases the permeability of the outer membrane, arrests cell division and finally leads to the bacterial death (230, 231, and reviewed in 39). In addition, the C-terminal domain of BPI promotes the attachment of bacteria to neutrophils, which can then easily be phagocytosed (90). The combined effect is so strong that BPI kills gram-negative bacteria at nanomolar concentration.

In human azurophil granules, 30 to 50 % of the protein content is supplied by members of the -defensin family. This family comprises four small ( 3,5 kDa), cationic peptides, i.e. neutrophil protein 1, -2, -3 and -4. Alpha-defensins have protective effects against a broad range of bacteria, fungi, enveloped viruses and protozoa (35, 54, 117). This is achieved by the forma-

33

tion of multimeric transmembrane pores in the target (226). Interestingly, -defensins are absent from mouse neutrophils (50, 119 and reviewed in 53).

In addition, four highly abundant proteases in azurophil granules contribute to the destruction of microorganisms (reviewed in 116, 198). These prote-ases are therefore called serprocidins (serine proteases with microbicidal activity). Human serprocidins include Cts G from the mast cell chymase locus, and azurocidin, neutrophil elastase and proteinase-3 from the Gzm M/elastase locus. Like T cell granzymes, the neutrophil proteases are prote-olytically trimmed upon arrival in the granule compartment (55, 67, 120). The storage of serprocidins does not seem to involve serglycin proteoglycan, although it is present in the granules. Rather, the main role of proteoglycan in neutrophils seems to be in granule formation (146).

Serprocidins from the mast cell chymase locus

The only neutrophil protease from the mast cell chymase locus is Cts G (Ta-ble 2). Human Cts G holds a negatively charged aa, Glu, in position 226, which confers dual chymotrypsin-like and trypsin-like specificity (82, 164). As mentioned above, Cts G has antimicrobial properties. Interestingly, these properties are retained even when the protease is inactivated or clostripain-digested (8). The proteolytic activity of Cts G is on the other hand necessary for cleavage of many targets. For example, Cts G digests components of the ECM, such as collagen, fibronectin and proteoglycans (133, 200, 219). This may clear the way for other inflammatory cells that need to access a site of infection. Moreover, Cts G cleaves and inactivates the neutrophil chemoat-tractants interleukin 8 (IL-8) and tumor necrosis factor (TNF- ) (150, 186, 217). Neutrophils that have reached a target region may thereby prevent over-recruitment of further neutrophils. Cts G has also targets in the blood. It has been shown to cleave and activate several clotting factors, and to activate platelets in vivo (reviewed in 130). In addition, Cts G converts the blood pressure-regulating peptide angiotensin I (Ang I) to angiotensin II (Ang II) (170). This array of molecular targets is very similar to that of mast cell chymases, which will be discussed in the next section. Interestingly, there are indications that Cts G may also be expressed by human mast cells (79, 181). The functions of human chymase and Cts G could therefore be par-tially redundant.

On the other hand, Cts G does not seem to be present in rodent mast cells (125). Furthermore, mouse, rat, dog and cattle Ctsg hold an uncharged Ala instead of Glu in position 226. It is therefore likely that Cts G in these spe-

34

cies has only chymotryptic specificity, and that the biological role for Cts G differs between species. Surprisingly however, neutrophils from Cts G-deficient mice have no obvious functional defects (130).

Serprocidins from the Gzm M/elastase locus

The three remaining serprocidins, azurocidin, neutrophil elastase and prote-inase-3, are encoded from the elastase locus. (This locus holds also the NK cell granzyme M.) Neutrophil elastase and proteinase-3 are functional elasta-ses with preference for Ala and Val in P1 position (105). Azurocidin (also known as Heparin-binding protein or CAP37), in contrast, is a proteolyti-cally inactive serine-protease homolog (21).

Neutrophil elastase and proteinase-3 are exocytosed at sites of neutrophil accumulation and degrade connective tissue proteins, such as collagen type IV, fibronectin, vitronectin, laminin and elastin (reviewed in 243). The two elastases have been implicated in a number of pathological conditions, in-cluding pulmonary emphysema, rheumatoid arthritis and glomerulonephritis (reviewed in 229). Proteinase-3 is also a known major target antigen of auto-reactive antibodies, anti-neutrophil cytoplasmic antibodies (ANCA) (re-viewed in 203). Azurocidin, like -defensins, is expressed in neutrophils in human (141), but not in mouse. In spite of its proteolytic inactivity, azuro-cidin has bactericidal effects (4, 21). In addition, azurocidin chemotactically attracts monocytes (155), recruits T lymphocytes (32) and increases vascular permeability during neutrophil extravasation (56).

Reflections

Both neutrophils and NK/T cells store granule proteases from the mast cell chymase locus and the Gzm M/elastase locus. From the mast cell chymase locus, the knock-out of T cell Gzm B and that of neutrophil Cts G have been reported to affect the expression of adjacent genes (Gzm C and Gzm F, and mMCP-2, respectively). This indicates that proteases encoded from this lo-cus may underlie common regulatory mechanisms, even though they are expressed in different cell types. Thus, the evolution of NK/T cells and neu-trophils as cell types seems to be closely interlinked with the evolution of the mast cell chymase locus and the Gzm M/elastase locus.

35

Table 2. Selected contents of azurophil granules in neutrophils.

+: expressed; -: not expressed.

Neutrophil serine proteases: Serprocidins

human mouse Function

mast cell chymase locus

Cts G + + m Cts G: Chy-mase? h Cts G: Tryptase and chymase Antimicrobial Chemotaxis ECM remodeling Blood clotting

Azurocidin + - Inactive enzyme Antibacterial

Neutrophil elastase + + Elastase Degrades connec-tive tissue

Gzm M/elastase locus

Proteinase-3 + + Elastase Degrades connec-tive tissue

Neutrophil protein 1

+ - Cytotoxic, antim-icrobial

Neutrophil protein 2

+ - Cytotoxic, antim-icrobial

Neutrophil protein 3

+ - Cytotoxic, antim-icrobial

Other antibacterial effector molecules

Defensins

Neutrophil protein 4

+ - Cytotoxic, antim-icrobial

Proteoglycan matrix

Serglycin + + Formation of granules

(Data from: (4, 8, 21, 35, 50, 53, 54, 105, 115, 117, 130, 133, 146, 150, 164, 186, 200, 217, 219, 243)

36

TAKE HOME:

• The main function of neutrophils is to kill microbes.

• From the mast cell chymase locus, neutrophils express Cts G.

• From the Gzm M/elastase locus, human neutrophils express azuro-cidin, neutrophil elastase and proteinase-3.

• The antibacterial properties of Cts G and azurocidin are independent of proteolytic activity.

• Cts G and azurocidin show different expression patterns in different species. Neutrophils may therefore not have exactly the same func-tions in these species.

Mast cell granules

Mast cells (MCs) are found close to many body surfaces, such as the connec-tive tissue of the skin, the mucosa of the airways and intestine, and in the peritoneum. They are also often positioned close to blood vessels and nerves. MCs are therefore among the first immune cells in the body to come in con-tact with bacteria, viruses or nematodes that intrude the body, and have been called the gatekeepers of the immune system. These cells seem therefore to have evolved to be receptive for a variety of stimuli. In response, MCs can release pre-formed mediators that are stored in their granules, and synthesise other mediators de novo (Fig. 7). As the functional studies in this thesis (study III, IV and V) focus on serine proteases that are expressed in MCs, these cells will be discussed in somewhat greater detail.

The granule-stored mediators in mast cells include histamine, proteoglycans and neutral proteases. Proteoglycans mainly provide a storage matrix for the proteases (e.g. reference (1). The two families of proteases that are present in MC granules are discussed below. Histamine is a biogenic amine that can bind to the histamine receptors H1, H2, H3 and H4. These receptors are found on many cell types, e.g. on endothelial cells, nerve cells and on smooth muscle cells of the bronchiae and intestine. Binding of histamine to histamine receptors causes the bronchiae and the intestinal muscles to con-strict, increases vascular permeability and mediates neurotransmission (re-

37

viewed in 62 and 129). This leads to typical allergic symptoms, such as edema, itching and diarrhea. Histamine is therefore a target of many anti-allergic drugs. Like many mast cell proteases, histamine depends on heparin proteoglycan for storage in the mast cell granules (12, 227). MC proteases, in turn, appear to be stored more efficiently when histamine is present (re-viewed in 148). The release of the granule-stored mediators is a very rapid event, and its effects can be seen within minutes. The most striking example of this is an anaphylactic shock. This systemic reaction can be caused when a strongly allergic individual is exposed to allergen.

Fig. 7. Upon various stimuli, MCs release pre-stored and newly synthesized media-tors. Figure adapted freely from (5). Fc, Fc receptor; TL, Toll-like receptor: C, com-plement receptor.

De novo synthesized MC mediators comprise prostaglandins, leukotrienes, and cytokines. Prostaglandins and leukotrienes can be produced quickly (within minutes), whereas cytokines are mainly responsible for delayed reac-tions, which occur within hours. Prostaglandins and leukotrienes are lipid mediators that are derived from arachidonic acid. In mast cells, Prostaglan-din D2 (PGD2) and leukotriene C4 (LTC4) are most prominent. These media-tors cause similar symptoms as histamine, including vasodilation, bron-choconstriction and enhanced vascular permeability (98, and reviewed in 16).

The cytokines produced by MC are numerous and will not be discussed in detail. They include tumor necrosis factor (TNF- ), interleukin (IL)-4 and IL-13 (reviewed in 17). IL-4 and IL-13 promote the production of IgE by B cells. IgE can then bind to high-affinity receptors (Fc RI) on MC. This pro-longs the half-life of both IgE and Fc RI on the MC surface (reviewed in

Pre-stored mediators:

Histamine ProteoglycansProteases

De novo synthesized mediators:

Prostaglandins LeukotrienesCytokines

Fc

R

TL

R

C

R

C3aC5aLPS

Pep-tido- glycandsRNA

IgE IgG

+ antigen

Stimuli MC response

38

51). TNF- is a multifunctional cytokine that is important in the defence against bacteria. It is not only de novo synthesized, but also stored in the MC granules (reviewed in 131).

The most thoroughly studied way of MC activation is the cross-linkage of IgE that is bound to Fc RI on the MC surface. IgE can be cross-linked by a specific antigen, or (experimentally) by anti-IgE antibodies, which can result in MC degranulation and synthesis of leukotrienes, prostaglandins and cyto-kines. MCs with higher levels of Fc RI on their surface cannot only bind more IgE, but can also be activated by lower concentrations of allergen or anti-IgE, and secrete higher amounts of preformed mediators, lipid mediators and cytokines (reviewed in 14, 51). MCs are also susceptible to stimulation with IgG. In human, this stimulation probably acts via the high-affinity re-ceptor Fc RI, whereas mouse MC apparently only express the low-affinity receptor Fc RIII (76, 102, 144 and reviewed in 213).

MCs can also be activated via pathways that belong to the “innate” immune system. For example, they express receptors for the complement components C3a and C5a, and binding of C3a and C5a induces the release of histamin (49, 94). In addition, MCs express toll-like receptors (TLR), a receptor fam-ily that binds to conserved structures of microbes such as peptidoglycan, lipopolysaccharide (LPS) and double-stranded RNA (reviewed in 207). En-gagement of TLR-2 and –4 on MCs can induce cytokine secretion and de-granulation, depending on the stimulating agent (137, 204, 205).

MCs are mainly known for their role in allergic reactions. However, the fact that MCs have been conserved throughout evolution (30, 178) strongly indi-cates that they do have important functions. MC-deficient mice have been used as a tool to investigate these functions. The well-studied KitW/W-v

mice lack MC, because they are deficient in c-kit, the receptor for a mast cell growth factor called stem cell factor (SCF). However, KitW/W-v mice also suffer from melanocyte deficiency, macrocytic anemia and sterility, and they almost completely lack interstitial cells of Cajal, which are essential for normal gastrointestinal motility (reviewed in 52). By reconstituting control KitW/W-v mice with bone-marrow derived MCs, MC-dependent effects can still be isolated in studies using this strain. Moreover, an additional strain with a different mutation in c-kit has recently been developed, W-sh/W-sh

(63). This strain is neither sterile nor anemic, which is promising for future studies.

KitW/W-v are more sensitive than wild-type mice to a number bacterial and parasitic infections. For example, when infected with Klebsiella pneumo-

niae, KitW/W-v mice have a 20-fold reduced rate of bacterial clearance as compared to wild-type or MC-reconstituted mice (132). Moreover, KitW/W-

39

v mice expel the nematodes Trichinella spiralis and Strongyloides ratti

slower than their MC-reconstituted littermates (144, 149). Impaired bacterial clearance has also been shown in W-sh/W-shmice against Mycoplasma pneu-

moniae (235). MCs thus appear to play an important part in the defence against both bacteria and parasites. It is however not completely clear how these findings apply to humans, because rodent MCs and human MCs may not be entirely functionally equivalent. This is in part reflected by the fact that different subtypes of MCs exist in human and rodents.

Human MCs are classified in two subtypes with regard to their storage of granule-associated serine proteases. The MC serine proteases belong to two families, tryptases and chymases (Table 3). MC chymases are encoded from the mast cell chymase locus, while MC tryptases are encoded from the tryp-tase locus on human chromosome 16 (151) (mouse chromosome 17 (231)). Virtually all human MC express tryptase, but only a subset also contains chymase. MCs that store both tryptase and chymase are called MCTC,whereas MCT contain only tryptase. In addition, MCTC store carboxypepti-dase A (CPA), a metalloprotease. The granules of both human MC types contain the proteoglycans heparin and chondroitin sulphate (34). MCT and MCTC seem to reside in most human tissues, but in varying ratios. MCTC are prevailing in the skin, connective tissue, and in the submucosa of the esophagus. MCT are the dominant MC type in the lung (224). Also in dog, MCT and MCTC have been described. In addition, some dog mast cells seem to be single-positive for chymase, and these are called MCC (147). Dog MCs display a similar tissue distribution as human MCs.

In rodents, in contrast, the two MC types are found in separate tissues and are also named with regard to their tissue localisation. Connective tissue mast cells (CTMC) are mainly present in the skin and in the peritoneal cav-ity, whereas mucosal mast cells (MMC) reside beneath the mucosal surfaces of the respiratory and intestinal tract (40). CTMC contain high amounts of heparin proteoglycan. MMC contain instead chondroitin sulphate, which is less negatively charged (reviewed in 107). The two rodent MC types can therefore easily be distinguished with basic dyes staining heparin. CTMC resemble MCTC, in that they also store tryp-tase, chymase and CPA. MMC store chymase, but not tryptase, similar to dog MCC (reviewed in 26). The number of MMC increases considerably during parasitic infections (reviewed in 139).

40

Serine proteases from the mast cell chymase locus (chymases)

MC chymases are stored in the granules as monomers that are complexed with the highly negatively charged heparin (81). Similar to T cell granzymes, the chymases are activated by DPPI (230), which removes the acidic acticva-tion dipeptide in the course of granule formation. In contrast to Gzm B, chymase remains tightly bound to heparin proteoglycan after its release from the granule. This protects the chymase from protease inhibitors (152) and increases its enzymatic activity (153).

Only one chymase is expressed in the mast cells of human and dog. This chymase, Cma1, belongs phylogenetically to the group of -chymases. Also rodents hold a single -chymase, which is expressed in CTMC. The mouse and rat -chymases are called mMCP-5 and rMCP-5, respectively. However, mMCP-5 and rMCP-5 are functionally not chymases, but have secondarily acquired elastase-like activity (98, 113). This is largely due to a Val in posi-tion 216 (194), where chymases generally hold a Gly. Rodent MCs express however additional proteases that belong to the group of -chymases. Mouse CTMC mainly express the -chymase mMCP-4 (145) and rat CTMC ex-press its homolog, rMCP-1 (58). These -chymases are functionally rather similar to the human chymase (28, 177, 211), although they are not as closely related. Rodent MMC express no -chymase, but several -chymases. The -chymases in mouse MMC are and mMCP-1 and mMCP-2 (86, 123, 124), where mMCP-2 appears to be catalytically inactive (154). The homolog of mMCP-1 in rat MMC is rMCP-2 (11). Rat MMC also ex-press lower levels of rMCP-3 and -4 (125).

The physiologic functions of each single MC chymase and the functional relationships between the chymases in rodents and the human chymase are not yet entirely clear. In general, many of the actions attributed to chymases involve a modulation of the local MC environment, which may promote an immune response. For example, chymases seem to remodel the extra-cellular matrix (ECM) by degrading fibronectin, (211) and by the activation of ma-trix metalloproteases (209). This may render sites of infection accessible for inflammatory cells. Chymases have also been reported to recruit inflamma-tory cells (76, 208), in part probably by the cleavage of cytokines (reviewed in 113). mMCP-1 has been shown to be involved in the expulsion of nema-todes (e.g. 185, 216, 222). However, the action of chymases also contributes to allergic symptoms. For example, they induce microvascular leakage and bronchial gland secretion, and increase the mucosal permeability in the gas-trointestinal tract (reviewed in 113). Chymases have also been implicated in the activation of MCs and in the wheal reaction (reviewed in 25).

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Similar to neutrophil Cts G, chymases have additional targets in the blood. These include thrombin, plasmin and serum albumin (169, 210, 211). A well-studied chymase substrate from the blood is the blood-pressure regulat-ing peptide angiotensin I (Ang I). Cleavage of the Phe8-His9 bond in the decapeptide Ang I generates the biologically active product Ang II. How-ever, Ang I contains a second potential cleavage site for chymases, the Tyr4-Ile5 bond. If this bond is cleaved, Ang is degraded rather than converted. The human chymase effectively generates Ang II without further degradation (170, 228). Molecular modelling predicts that positions 41, 143, 153, 173, 174, 176 and 193 (Chymotrypsin numbering) are of importance for this dis-tinctive specificity (236). A reconstructed ancestral chymase also forms Ang II from Ang I (31), indicating that this function may be ancient, although it may not be the most important in vivo role of extant chymases. The rodent chymases mMCP-1, mMCP-4 and rMCP-1 have also been shown to cleave Ang I. Of these proteases, the MMC protease mMCP-1 is the most efficient at generating Ang II, whereas the CTMC proteases mMCP-4, and especially rMCP-1, degrade Ang II to a considerable degree (28, 114). Rodent CTMC and human MCTC thus seem to differ in this respect. The conversion of Ang I by rat chymases is further discussed in study III.

In addition to - and -chymases, rat MMC also express rMCP-8, -9 and -10 (125). These three proteases belong to the Mcpt8-family, which to date has members only in rat and mouse. No proteolytic specificity or any other physiologic role is yet known for this family. I discovered four novel mem-bers of the Mcpt8 family in the rat genome, Mcpt8-rs1, -rs2, -rs3 and -rs4 (study I). Three of these genes appeared to be functional, and their expres-sion profile is addressed in study III. These multiple family members in rat may have redundant functions. On the contrary, only one Mcpt8 gene is pre-sent in the mouse, and this gene is expressed in basophils (124, 165).

Serine proteases from the tryptase locus (tryptases)

Tryptases constitute approximately a quarter of the cellular protein in mast cells (184). Human tryptases comprise the subforms I, I, II, III, and .The -tryptases are the most abundant and catalytically active of these. They are stored in the mast cell granules as active tetramers that are stabilized by interaction with proteoglycans (70, 183). The genes for all MC tryptases are clustered in the tryptase locus (see above) together with an inactive mastin gene. Like the mast cell chymase locus, this cluster probably evolved from a primordial gene that duplicated a number of times to generate these diverse and functionally distinct proteases (85). However, this functional diversifica-tion has progressed beyond that of the mast cell chymase locus and probably

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started out earlier. Tryptases have a number of reported activities that are associated with asthmatic symptoms. For example, they degrade the bron-chodilatory vasoactive intestinal peptide (VIP) (27) and stimulate prolifera-tion of fibroblasts (175), smooth muscle cells (19) and epithelial cells (20). This may lead to increased airway responsiveness in asthmatic patients.

The beneficial roles of tryptases appear to be similar to those of the chy-mases. For example, tryptases have been suggested to degrade targets in the ECM, such as fibronectin, fibrinogen and collagen type VI (reviewed in 135). However, the relevance of these substrates has been questioned (195), because these molecules are too large to fit into the pore shaped by tryptase tetramers. Interestingly, evidence is emerging that even active tryptase monomers may be present (42, 71). Moreover, tryptases can recruit inflam-matory cells (75, 84). They also increase vascular permeability by activation of prekallikrin and by production of bradykinin from kininogens (89).

The -tryptases I, II and III are very similar to each other. III is the allelic variant of II (reviewed in 25). Because -tryptases build tetrameric complexes, they are shielded from serine protease inhibitors (serpins). They remain therefore active after prolonged incubation in plasma, in contrast to many other proteases (156). In mouse, the tryptase mMCP-6 is most similar to the human -tryptases (70). In contrast, to the -tryptases, 1-tryptase is catalytically inactive. This is probably caused by a subsite distortion in posi-tions 214 to 219 (135). Alpha1-tryptase is not stored in granules, but is con-stitutively secreted as a monomer. Surprisingly, 1 is the allelic variant of the I-tryptase. Therefore, not all individuals inherit the gene, and 21% of the human population is 1-deficient (197). Gamma-tryptase is the only membrane-anchored tryptase. The -tryptase gene therefore seems to have diverged early from other tryptase genes. It was probably present already before the divergence of rodents and primates. (reviewed in 26). Even after secretion, -tryptase remains attached to the plasma membrane. It is suscep-tible to inactivation by serpins. However, the physiological function and importance of -tryptase is to date unknown. Delta-tryptase is also enzymati-cally inactive. Exon 5 displays similarity to the mouse tryptase mMCP-7, which otherwise has no direct human homologue.

Other proteases in mast cells

Carboxypeptidase A (CPA) is expressed only in the CTMC-subset of rodent MCs and in the human equivalent, MCTC. This protease belongs to the Zn2+-dependent metalloproteases (41, 60). In contrast to the serine proteases, CPA

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is an exopeptidase, i.e. it cleaves peptide bonds at the ends of peptides. CPA prefers targets with C-terminal aromatic or aliphatic residues (59, 220). This is interesting, because peptides with C-terminal aromatic aa are produced when chymases cleave a target. It has therefore been suggested that CPA uses substrates that have been generated by MC chymases (106). In support of this idea, CPA and chymase seem both to be involved in the degradation of endothelin-1 (ET-1) (138). This may limit inflammatory reactions medi-ated by ET-1. CPA and chymase also depend on each other for storage in-side the mast cell granule (81).

Table 3. Selected stored mediators in mast cell granules.

Protease names indicate expression of the respective protease. Conflicting data exist regarding the expression of mMCP-2 in mouse CTMC. +: expressed; -: not ex-pressed.

CTMC MMC MC serine proteases: chymases and tryptases human

MCTC

mouse rat human MCT

mouse rat

-chymases

Cma1 mMCP-5 rMCP-5 - - -

- - - - mMCP-1 rMCP-2 - (mMCP-2) - - mMCP-2 - - mMCP-4 rMCP-1 - - - - - - - - rMCP-3

-chymases

- - - - - rMCP-4

Others - - - - - rMCP-8 - - - - - rMCP-9

MC chy-mase locus

- - - - - rMCP-10

tryptase mMCP-6 rMCP-6 tryptase - - Tryptase locus - mMCP-7 rMCP-7 - - -

Other prote-ases

CPA + + + - - -

Heparin + + + + - -

(Data from: (1, 26, 58, 70, 81, 86, 87, 98, 113, 125, 145, 154, 183, 201)

Reflections

Interestingly, the functions of MC proteases resemble those of neutrophil proteases, since Ctsg, as well as chymases and tryptases, seem to be involved

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in ECM remodeling, chemotaxis towards cytokines and blood clotting. Dif-ferent sets of neutrophil and MC proteases are expressed in human and ro-dents. For example, human neutrophils are equipped with more proteases than their rodent counterpart, whereas the contrary is true for mast cells. The question is therefore whether human neutrophils may in part hold functions that in rodents are carried out by mast cells, and vice versea.

TAKE HOME:

• Mast cells are involved in the defense against bacteria and helminths, and in allergies. They can be activated in many different ways.

• Mast cells express chymases (from the mast cell chymase locus) and tryptases (from the tryptase locus). Some mast cells express onlytryptase, others only chymase, and some both tryptase and chymase.

• Different subtypes of mast cells exist in different species. It is there-fore not easy to understand how the mast cells from different species are functionally related.

IV) MAMMALIAN EVOLUTION – A SHORT OVERVIEW

Novel species can arise from a common ancestor in the course of evolution. We can learn in what order and approximately when different species di-verged by comparing their genes and molecules, and by studying fossil re-cords. A set of species that are studied thus covers a certain evolutionary time frame. In studies I and II of this thesis, I have analysed homologous genes from human, rat, mouse, dog, sheep, cattle, opossum and platypus. This set of species was chosen, because DNA or mRNA sequence informa-tion was available that could be studied with bioinformatics tools. I could thereby map the chymase loci of these species. To understand how the chy-mase locus evolved, I compared my results to the reported order and time of divergence for the concerned species. (If novel reports refine the order of divergence, our picture of the evolution of the mast cell chymase locus can be adjusted accordingly.)

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It is estimated that the tetrapod species (vertebrates with four limbs) have separated from fish approximately 410 million years (Myr) ago. The lineage leading to birds and reptiles and the mammalian lineage then diverged ap-proximately 310 Myr ago. Two different subclasses of mammals emerged around 215 Myr ago, and these split into three subclasses around 185 Myr ago. All three subclasses have extant (presently existing) representatives. These three subclasses are called Prototheria (monotremes), Metatheria (marsupials) and Eutheria (placental mammals) (Fig. 8). Eutheria and Metatheria are together called Theria. The Theria subclasses comprise vi-viparous mammals, whereas Prototherian species are egg-laying mammals (223, 238 and reviewed in 61).

Only a few extant Prototherian species exist, and these include the Platypus (Ornithorhynchus anatinus) and two Echidna species (Tachyglossus aculea-

tus and Zaglossus bruijnii). The Platypus is a duck-billed semi-aquatic mammal that is confined to eastern Australia and Tasmania. Echidnas are covered with coarse hair and spines, and superficially resemble hedgehogs and porcupines. The Short-beaked echidna (Tachyglossus aculeatus) is the most widespread native mammal of Australia, while the Western long-beaked echidna (Zaglossus bruijnii) is present only in certain regions of New Guinea.

Metatheria (marsupial mammals) comprise approximately 270 extant spe-cies, 70 of which are South American and 200 Australian. Many, but not all species of the Methatheria subclass have a pouch (marsupium). A widely known representative of the Metatheria is the Red Kangaroo (Macropus

rufus). Two additional examples are the Gray short-tailed opossum (Monodelphis domestica) and the Fat-tailed dunnart (Sminthopsis crassicau-

data). The Gray short-tailed opossum is a rat-like marsupial that is found mainly in South America. The Fat-tailed dunnart lives in diverse habitats in South-western Australia. It is a mouse-like mammal that weighs only 10 to 20 grams, and is thereby one of the smallest carnivorous marsupials.

The subclass Eutheria is represented by the majority of all living mammals. Eutherian mammals, e.g. humans, are found on all continents. One of the earliest known eutherian species is the extinct Eomaia scansoria. A fossil of this species was rather recently discovered in China (92). Eomaia was also very little with an estimated weight of 20 to 25 grams.

It is to date not entirely clear in which order the three mammalian subclasses diverged. Most recent reports state that Prototheria first diverged from The-ria, about 215 Myr ago. The Theria then separated into Eutheria and Metath-eria about 185 Myr ago (110, 218, 232). This scenario, the so-called Theria hypothesis, is supported by data from nuclear genes, and by fossil and anat-

46

omic evidence (10, 100). However, some mitochondrial data place Protothe-ria and Metatheria closer to each other (Marsupionta hypothesis) (91).

The early mammalian evolution coincided with major geographical changes. About 140 Myr ago, the primordial super-continent Pangaea divided into a northern half, Laurasia (comprising North America, Greenland and Eurasia) and a southern half, Gondwana (comprising Antarctica, Australia and South America). Gondwana split into two parts approximately 84 to 38 Myr ago, and this separated American and Australian mammals (reviewed in 143). Another strong influence on the evolution of mammals was the abrupt ex-tinction of dinosaurs in the late Cretaceous (more than 65 Myr ago), which freed ecological niches and apparently allowed numerous mammalian spe-cies to emerge.

Fig. 8. Schematic time scale for the evolution of selected mammalian species.

The dendrogram in Fig. 8 summarizes the current view of how the mammal-ian species included in our studies evolved. It is largely based on a study by Springer et al., which analysed the largest available data set for placental mammals (2003) (199). The mouse and rat are at present estimated to have diverged between 12 and 24 Myr ago (2, 33, 57, 199, 237), and the average of 18 Myr is presented here. The sheep and cattle lineages diverged between 20 and 40 Myr ago (6, 83, 140, 194). Interestingly, primates appear to be more closely related with rodents than with carnivores (102, 134, 142). The

Ruminants

Carnivores

Cetartiodactyls

Monotremes

1885 8095 30 0time (- Myr)

Marsupials

185215

Rodents

Primates

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mast cell chymase locus, on the other hand, is much more similar between human and dog and than between human and rodents (study I).

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PRESENT INVESTIGATIONS

AIMS

The presented studies were conducted to clarify how serine proteases from the mast cell chymase loci of selected mammals are related in evolution and function. This information can improve our understanding of the functions of mast cells, neutrophils, NK cells and T cells in these species. Beta-chymases and the Mcpt8-family are two gene families from the mast cell chymase lo-cus that have been described in rodents only. To track the evolutionary ori-gin of these gene families, I mapped the chymase loci from species where sequence material was available for bioinformatics studies, i.e. human, mouse, rat, dog, cattle, sheep, platypus and opossum. I identified several novel -chymase- and Mcpt8-like genes in the rat chymase locus and then analyzed their expression. I also studied the proteolytical properties of one member of the Mcpt8-family, mMCP-8. Beta-chymases and the Mcpt8-family are mainly expressed in rodent mast cells. In human and dog mast cells, the only protease from the mast cell chymase locus is -chymase. As a further step to elucidate the functional relations between mast cells in hu-man, dog and rodents, the extended cleavage specificities of human and dog

-chymase were determined.

RESULTS AND DISCUSSION

Repeated duplications have remarkably expanded the mast cell chymase locus of the rat (study I)

The mast cell chymase locus in human (Homo sapiens) spans over 129 kb and contains only four genes: the -chymase Cma1, the neutrophil protease

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Ctsg, and the T/NK cell granzymes Gzmh and Gzmb. The chymase locus in mouse (Mus musculus) holds a number of additional genes that belong to the

-chymase family, the Mcpt8-family and the family of granzymes. The mouse mast cell chymase locus is with 324 kB also almost three times larger than its human counterpart. Previous studies suggest that the -chymase and Mcpt8-family are rodent-specific.

We wanted to clarify the evolutionary origin of the genes that are present in mouse, but not in human, and to understand the mechanisms that have shaped the chymase locus. With this purpose, we mapped the chymase loci of human (H. sapiens), mouse (M. musculus), rat (Rattus norvegicus) and dog (Canis familiaris) in detail, and compared them to each other. Compari-sons between the dog locus and the loci of human, mouse and rat shed light on evolutionary events approximately 95 Myr ago, the time when the ro-dent/primate and carnivore/ruminant lineage are believed to have diverged (Fig. 8). No additional mammalian species could be included in this study, because their genome sequences were not available at satisfactory quality at the time.

The four studied loci display the same basic structure, with the Cma1 and Gzmb genes at the borders, and the Ctsg gene in a central position. The mast cell chymase locus in dog is with 73 kb somewhat smaller than the human locus. Our analysis here retrieved the homologs to human Cma1, Ctsg, Gzmh

and Gzmb, and an additional novel chymase gene, which was termed Cma2.This gene is very interesting, because phylogenetic analyses based on nu-cleotide sequences classify it as -chymase, whereas amino acid compari-sons place it with the -chymases. Cma2 is thereby the most closely -chymase-related gene that has been described in a non-rodent species. Due to a frame-shift mutation, Cma2 does most likely not give rise to a functional protein. The presence of this gene in the dog chymase locus nevertheless indicates that an ancestral -chymase gene might have been present before the divergence of rodents and carnivores.

The mast cell chymase locus in the rat (R. norvegicus) is with 1,1 Mb more than nine times larger than in human. Apart from previously described ho-mologs to the chymase locus genes of human and mouse, we identified eight novel genes in this locus. Four of these genes belong to the Mcpt8-family and are designated Mcpt8-rs1, Mcpt8-rs2, Mcpt8-rs3 and Mcpt8-rs4. The four other genes are related to the -chymase Mcpt2 and are called Mcpt2-

rs1, Mcpt2-rs2a, Mcpt2-rs2b and Mcpt2-rs2c. The novel Mcpt2-rs2a,Mcpt2-rs2c, Mcpt8-rs1 and Mcpt8-rs4 genes hold intact open reading frames without mutations in the catalytical triad. Thus, the -chymase- and Mcpt8-family are even more extensively expanded in the rat than previously known.

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In addition, we found a number of Mcpt2-related sequences that lack exon 1 and can therefore not be transcribed, s2a, s2b, s2c and s2d. These are ar-ranged with the complete Mcpt2-related sequences and with Mcpt8-like se-quences in repetitive units (Fig. 9). The members of the Mcpt2-family in these units display a similar degree of identity with each other as the mem-bers of the Mcpt8-family, respectively. It is therefore likely that the Mcpt2-and the Mcpt8-family were expanded by duplications of a unit that contained a member of each family. The duplication process may have been mediated by L1_RN repeats also found in the region (Fig. 9). To further establish whether the Mcpt2- and Mcpt8-related genes were co-duplicated, we sepa-rately calculated the approximate time of duplication for each family. Spe-cifically, we calculated the evolutionary distance of each family relative to the divergence of the mouse and rat lineages. We used intron sequences for this analysis, which are assumed to evolve neutrally. Our examination con-firmed that the concerned Mcpt2- and Mcpt8-related genes diverged around the same time, approximately 5 Myr ago. Interestingly, when we subjected exon sequences to the same type of analysis, we found a considerably higher substitution frequency in the Mcpt8- than in the Mcpt2-related genes. This indicates that evolutionary selection favours diversification in the members of the Mcpt8-family.

We also identified a large number of pseudogenes with similarity to Gzmb

and Gzmc in the rat locus. These pseudogenes are located between Ctsg and Gzmc. In the mouse chymase locus, Gzmd, e, f, g, h, and n (the orphan gran-zymes) are found in the corresponding region. Thus, similar duplications of granzyme genes have apparently occurred in the mouse and rat, but these yielded far less functional genes in the rat. It is possible that functions of the orphan granzymes in the mouse are in the rat performed by members of the Mcpt8-family. Mouse granzymes are however expressed in NK/T cells, whereas the previously characterized members of the rat Mcpt8-family are MC-specific. The functions of both gene families remain to be resolved.

Taken together, the findings from study I emphasize the importance of gene duplications in the evolution of mammalian gene families. The chymase loci in rat and mouse provide remarkable examples. In both loci, -chymase and granzyme genes duplicated repeatedly, which generated different numbers of functional genes. The -chymases might be derived from an ancestor that existed before the divergence of the rodent and carnivore lineages. The Mcpt8-family was expanded in the rat lineage, but not in the mouse, from one to seven genes. No members of the Mcpt8-family were found in human or dog. The Mcpt8-family thereby still appears to be rodent-specific.

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Fig. 9. Mcpt2-and Mcpt8-related genes interleaved in the rat mast cell chymase lcous. Exons are depicted as larger black boxes and are emphasized by gray back-ground panels. Horizontally aligned parts share >80% sequence identity. I, Ib and II represent suggested duplication units.

The mammalian mast cell chymase locus was probably founded by a single ancestral gene over 215 million years ago (study II)

The evolution of the mast cell chymase locus could be tracked in two addi-tional mammalian lineages, cetartiodactyls and marsupials, when the ge-nome sequences for cattle (Bos taurus) and opossum (Monodelphis domes-

tica) became available. Additional information was provided by a previously published granzyme sequence from the platypus (Ornithorhynchus

anatinus), a monotreme. This set of species allowed us to follow evolution-ary events about 215 Myr back in time.

The cattle chymase locus shares the basic structure of the loci in mouse, rat, dog and human. In all of these species, Ctsg is found near the middle of the locus, while the chymase- and Gzmb genes are located at the borders. We identified a second, novel granzyme gene in cattle, which clusters with hu-

52

man Gzmh. Moreover, the cattle chymase locus is the first where two -chymase genes (Cma1a and Cma1b) and two cathepsin G genes (Ctsg1 and Ctsg2) were found. These chymase genes and Ctsg genes are highly similar. To track the duplication of these genes, we screened the available expressed sequence tags (ESTs) from sheep and could retrieve two -chymases. With the same method as in study I, we calculated the divergence time of the cat-tle and sheep -chymases relative to the divergence from the rodent lineage. The result indicates that the -chymase gene could have duplicated shortly before the cattle and sheep lineages diverged, about 30 Myr ago.

Interestingly, the cattle chymase locus also holds genes from the duodenase-family, which appears to be present only in ruminants. Duodenases are most likely involved in food digestion rather than immune function. By phyloge-netic classification, we could determine that the duodenases are not only co-localized but also closely related with the other genes from the chymase locus. This suggests that duodenases were derived from a protease with im-mune functions in a process of neofuctionalization. In addition to the known duodenase BDMD1, we found three novel duodenase genes in the cattle chymase locus, and one in the sequence material that has not yet been as-signed a chromosomal location. The novel genes were named BDMD2,BDMD3, BDMD4 and BDMD5. All four genes appear to encode functional serine proteases. In the chymase loci of mouse and rat, the members of the

-chymase, Mcpt8- and granzyme families are positioned in clusters. In con-trast, the duodenase genes are separated by the Ctsg1, Ctsg2 and Gzmh

genes. In addition, the most closely related duodenases lie on different sides of these three genes. The duplication process for the duodenaess appears therefore to have been rather complicated.

Our analysis of the opossum genome yielded only two genes with clear rela-tion to genes from other chymase loci (graspases). The first gene groups in phylogenetic analyses firmly with the -chymases. This suggests that an ancestral -chymase existed already before the divergence of marsupials from placental mammals, approximately 185 Myr ago. The opossum chy-mase holds the same specificity-conferring residues 189, 216 and 226 as most other mammalian chymases (S-G-A). It is thus likely that also the an-cestral chymase already had this triplet and displayed chymotryptic activity. The second opossum gene was named grathepsodenase, as it is approxi-mately equally related to granzymes, cathepsin G, duodenases and the Mcpt8-family. Thus, a second ancestral chymase locus gene was thus proba-bly present before the divergence of marsupials and placental mammals, but this gene was not as strongly conserved. The grathepsodenase holds A-G-R in the specificity-conferring triplet, and this triplet is also found in rat and mouse Gzm B, which have aspartase activity. The second ancestral gene could therefore also have been an aspartase. The importance of the aspar-

53

tase-function is further emphasized by the fact that Gzmb, together with -chymase, borders the chymase loci of all previously investigated mammals.

In contrast to the graspases of all other investigated species, the two opos-sum genes are not arranged in one locus. Their neighbour genes are however homologs to the neighbour genes of the human and dog chymase loci. In addition, the opossum sequences lack the Cys191-Cys220 disulphide bridge, like all other graspases. We therefore suggest that a chromosomal rear-rangement in the marsupial lineage separated these two genes. If this oc-curred early in the marsupial lineage, the separation of the genes could have hampered an expansion of the chymase locus in marsupials. This possibility can be investigated when additional marsupial genomes become available.

The previously reported platypus “granzyme” sequence appears in fact ap-proximately equally related to all extant chymase locus gene families. This sequence also lacks the Cys191-Cys220 disulphide bridge. The platypus ge-nome sequence is not yet available at satisfactory quality, but experimen-tally, no other graspase-related sequences could be retrieved in platypus despite intense trials (Poorafshar and Hellman, unpublished results). It is therefore likely that a single graspase progenitor has existed before the di-vergence of Prototheria and Theria, about 215 Myr ago, and that all known chymase locus genes were derived from this ancestor.

In summary, a single founder gene of the mammalian chymase locus was probably present more than 215 Myr ago. This progenitor duplicated more than 185 Myr ago, and the copies evolved into an ancestral -chymase and a possibly aspartase-like second ancestor. In marsupials, these ancestors were probably subsequently separated by a chromosomal rearrangement, and did apparently not expand further. In placental mammals, the aspartase-ancestor founded the granzyme-, cathepsin G-, Mcpt8- and duodenase families. These families were expanded or deleted differentially in the various mammalian lineages over the past 100 Myr. At some point, and possibly before the di-vergence of rodents and carnivores, the -chymase gene was also duplicated and gave rise to the -chymases. In addition, the -chymase gene was probably duplicated more recently in the ruminant lineage.

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Novel members of the Mcpt2- and Mcpt8-families are expressed in rat MMC, and modelling suggests that mMCP-8 is proteolytically active (study III)

Mcpt2- and Mcpt8-related genes have expanded in the rat mast cell chymase locus by duplications of a common unit about 5 Myr ago (study I). A number of previously uncharacterized genes were identified within the duplicated units. In this study, we analyzed the expression of the novel genes that ap-peared to be functional, i.e. Mcpt2-rs2a, Mcpt2-rs2c, Mcpt8-rs1 and Mcpt8-

rs4. We also included Mcpt8-rs3, which holds an intact open reading frame, but encodes a mutation in the catalytical triad (H57Q). The previously known members of the studied gene families, Mcpt2, Mcpt8, Mcpt9 and Mcpt10, are expressed in rat MMC. Mcpt2 is the most prominent MMC protease and provides a marker for MMC in our analyses. The expression of the novel genes and Mcpt2 was studied by RT-PCR with carefully designed specific primers. We analyzed 14 rat tissues and the rat cell line RBL-1, which displays the characteristics of rat MMC (188).

Interestingly, the mouse MMC protease mMCP-1 has been shown to convert angiotensin I (Ang I), like the human chymase (177, 228), but no protease in rat MMC has yet been ascribed this activity. The Ang-converting properties of rat Mcpt2 have to our knowledge not yet been addressed. However, an-other -chymase from the mast cell chymase locus, rat vascular chymase (rVch), has been shown to efficiently convert Ang I (68). RVch is with 89% amino acid sequence identity closely related to Mcpt2. The expression of rVch has only been studied in a restricted number of tissues, and mainly in hypertensive rats. However, apart from vascular muscle cells, expression of rVch has been shown in the cell lines RBL-1 and RBL-2H3 (68). This is an indication that rVch may be expressed in rat MMC, equipping them with Ang I-converting activity similar to mouse MMC. To examine this possibil-ity, we included rVch in our analyses of tissue expression.

Our results indicate that the studied genes are expressed mainly by MMC, although at different levels. One indication for this is that all analysed genes are expressed in RBL-1 cells. Moreover, expression of the analysed genes in rat tissues was confined to tissues that also expressed Mcpt2. Apart from Mcpt2, Mcpt8-rs1 and Mcpt8-rs4 were the most expressed of the analysed genes, with mRNA detected in all studied parts of the intestine, and in spleen and testis. Noteworthy, the mutated Mcpt8-rs3 is not transcribed at detect-able levels in any tissue, although mRNA was amplified from RBL-1 cells. Proteolytic activity thus appears to be of importance for sustained transcrip-tion of the Mcpt8-family members. Transcripts of rVch and Mcpt2-rs2a/2c

were amplified from stomach and large intestine, but not from small intes-

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tine, spleen or testis. The level of transcription may tentatively be up-regulated under pathological conditions. It is thus possible that rVch contrib-utes Ang I-converting activity to rat MMC. This role could be shared with Mcpt2, which remains to be analyzed. To obtain a prediction for the Ang I-converting properties of Mcpt2, we made an aa alignment of positions that are thought to be important for efficient conversion of Ang I (236). Judged from this analysis, rMCP-2 may have intermediate Ang-I converting activity.

Our findings indicate that the variety of proteases in rat MMC is even greater than previously known. To understand the functions of this cell type, the proteolytic activities of these proteases need to be characterized. This is es-pecially interesting for the Mcpt8-family, as no function for any member of this family has yet been described. Because many closely related members of this family are present in the rat, it is likely that their functions are par-tially redundant, and it is difficult to understand their main role. In the mouse, in contrast, only one family member is present, mMCP-8. The func-tion of this protease may therefore be more conserved. Although mMCP-8 is expressed in basophils rather than MCs, we chose to study the proteolytic activity of this protease as a first representative of the Mcpt8-family.

Members of the Mcpt8-family have previously been suggested to display aspartase activity, because they share similarities with GzmB (173). A mo-lecular model that was obtained from SWISS-MODEL confirms that the S1-site in mMCP-8 is similar to that in human Gzmb. In addition, a number of residues that are specifically conserved within the Mcpt8-family line the substrate-binding cleft of mMCP-8. Substrate binding should therefore be an important feature in the evolution of this family. We produced recombinant mMCP-8 and analysed its proteolytic activity with a number of chromogenic substrates. However, mMCP-8 did not cleave any of the substrates in an array representing all known enzymatic specificities of chymase locus prote-ases. We thus proceeded to an unbiased method to determine enzymatic specificitiy, substrate phage display (Fig. 10).

This method employs a library of T7 phages, where the N-terminus of a cap-sid protein is modified to contain a peptide of nine random amino acids (ran-dom nonapeptide). Enough phages are present in the library to represent all possible permutations. The random nonapeptide is followed by a His-tag. This His-tag serves to anchor the phages to Ni-NTA beads. Bound phages can thus be pelleted by centrifugation. When the studied protease is added, phages with a cleavage-susceptible peptide are released from the beads. These phages are collected in the supernatant and amplified in bacteria. Am-plified phages then enter the next round of selection (biopanning), and phages with cleavage-susceptible peptides are enriched over subsequent biopannings. A sufficient enrichment is usually achieved after five rounds.

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The DNA from the obtained phages is sequenced, and the consensus of the cleavage-susceptible inserts is determined.

Protease

Ni2+

Agarose

Cleavesusceptiblepeptide

Variable region His6 -tag

XXXXXXXXXHHHHHH

C-terminal of the phagecapsid protein

Immobilizethe phages

Removeuncleavedphages

T7

T7

Ni2+

Agarose

Ni2+

Agarose

Amplifyphagesin bacteria

Agarose

After several rounds of selection,isolate individual phage clones andsequence randomised region

T7

T7

Agarose

T7

Agarose

T7

Produce phagelibrary containingvariable sequence

1.

Add Ni-agarose

Addprotease

3.

2.

Ni2+

Ni2+

Ni2+

Collect the Selectedphages

4.

Fig. 10. The substrate phage display methodology is depicted schematically. This illustration was obtained from (97).

Using this methodology, we have previously determined the extended cleav-age specificity of several other chymase locus proteases. However, numer-ous trials did not succeed in determining the proteolytic specificity of mMCP-8. Several explanations for this can be thought of. First, the mMCP-8 model predicts that two Cys residues that are conserved in the Mcpt8-family, but absent in other graspases, are localized at the surface of the enzyme in proximity to the substrate-binding cleft. These residues might build disul-phide bridges joining mMCP-8 molecules to form homodimers. The sub-strate-binding cleft would then be hard to access. Moreover, an intramolecu-lar disulphide bridge could be formed with a third Cys that is present in mMCP-8, and in one other member of the family. The substrate-binding cleft would be obstructed also in this case. We investigated these possibilities by analysing recombinant mMCP-8 on non-reducing gels, and by chromogenic assays in the presence of the reducing agent dithiothreitol (DTT). No dimers were observed, and mMCP-8 activity was not detected with varying concen-trations of DTT. As a further explanation, mMCP-8 might require a substrate

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conformation that cannot be provided by the short peptides used in chro-mogenic assays and in substrate phage display. The enzyme may also be very specific for a sequence that is represented by too few phages to be suc-cessfully amplified. In the future, mMCP-8-deficient mice could be gener-ated to unravel the role of this enzyme. Moreover, molecular studies of other family members could be initiated.

The human chymase displays a well-defined substrate recognition profile (study IV)

Where the rodent chymase loci encode a large variation of MC proteases, the human locus encodes only one: the human -chymase (HC). The role of the human chymase is therefore difficult to infer from data generated in the rat and in the mouse. However, the functions of this enzyme are probably very important, because -chymase is found in all investigated mammals and can be traced back in time at least 185 million years (study II). The HC is stored together with tryptase in the granules of human MCTC mast cells. The en-zyme has been subject to a number of molecular investigations, and various substrates have been identified. For example, the HC has several targets within the extra-cellular matrix, and activates a number of cytokines. The enzyme also converts Ang I to Ang II with higher efficiency than any other studied mast cell chymase. In vivo, the HC has been implicated in the induc-tion of endothelial permeability and in the recruitment of inflammatory cells (76, 77), although the molecular targets in these processes are to date uni-dentified. The question remains however whether the primordial target of the human chymase has yet been found.

One way to address this issue is to determine the cleavage specificity of the HC. The results from these studies can be compared to sequences present in previously described substrates, and can be used to predict novel substrates. The substrate specificity of the HC has previously been investigated e.g. with chromogenic substrates and peptide libraries. These methods yield in-formation either about the preferred aa N-terminal of the scissile bond (in the unprimed positions, i.e. P1, P2, …), or about the positions C-terminal of this bond (primed positions, i.e. P1´, P2´, …). Insight is however not gained about the preferred substrate sequence over the entire substrate-binding cleft, and synergistic or antagonistic effects of aa in the primed and unprimed po-sitions cannot be studied. These limitations are overcome with the substrate phage display methodology (see above), where primed and unprimed posi-tions are permutated simultaneously.

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We have for the first time determined the complete extended cleavage speci-ficity of recombinant HC using substrate phage display. Phages with cleav-age-susceptible nonapeptides were enriched about 70 times, compared to control with omitted HC, after five selection rounds. A well-defined consen-sus was obtained from 48 aligned phage sequences. Only aromatic residues were found in position P1, with about equal occurrence of Phe and Tyr. Ali-phatic aa were generally preferred in the other unprimed positions. Ala, Val and Leu were equally represented in position P2, Val was preferred in posi-tion P3, and Gly, Leu and Val were the most frequent aa in position P4. As to the primed positions, Ser and small aliphatic aa (Gly, Ala) were frequent in position P1´, whereas a strong preference for negatively charged residues (Asp, Glu) was revealed in position P2´. Position P3´was dominated by small aliphatic residues.

These results confirm previous reports of a preference for aliphatic aa in the unprimed positions (166). However, where a preference for Pro in position P2 has been suggested to be an important feature of the HC (101, 166, 169) we rather observed an exclusion of Pro in this position. The clearest feature in the observed consensus, apart from aromatic P1 residues, was the strong preference for negatively charged aa in position P2´. Structural studies have previously predicted this interaction (157, 236); and these predictions were here confirmed biochemically for the first time. The presented consensus sequence will hopefully support the identification of novel in vivo substrates, and contribute to the understanding of the functional homologies between rodent chymases and the human chymase.

Similarities and differences in the substrate recognition profiles of dog and human chymase (study V)

Like in human, only one functional chymase gene is present in the mast cell chymase locus in dog, and this dog chymase (DC) is an -chymase. Dogs hold a MC subtype where DC is expressed together with tryptase, similar to human MCTC. The human and dog mast cell subtypes also display similar tissue distributions. In addition, cultured bone marrow-derived mast cells from dog are comparable to human cultured MCs with respect to differentia-tion requirements and cell phenotype (119). With regard to these similarities, studies of the DC may yield information that can be applied to the human situation more easily than data gained from the various rodent chymases.

One difference between the dog and human chymase locus is the presence of a pseudogene with similarity to -chymases in dog. This gene contains just a

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single deleterious mutation, which might have been introduced in evolution-ary terms rather recently (study I). Before its pseudogenization, the second dog chymase could thus have influenced the evolution of the DC, and this may have lead to a functional divergence of the DC and the human chymase (HC). However, the aa identity between mature DC and HC (82%) is even higher than between dog and human Cts G (67%) or Gzm B (71%). This indicates that the functions of the DC and HC are probably well conserved.

The DC has however not been studied extensively to date. The enzyme has mostly been implied in pathological conditions, such as adverse left ven-tricular remodelling (202) and haemodialysis vascular access dysfunction (93). Two common functions for the human and dog chymase are the effi-cient conversion of Ang I and the activation of matrix metalloproteases (43, 114, 176). These chymase functions may be important in other species as well. To further explore the functional properties of the DC, we analyzed the extended substrate specificity of recombinant DC by substrate phage display (see above).

Although phages with cleavage-susceptible peptide were enriched only eight times after five rounds of selection (to compare with about 70 times for the HC), a rather clear consensus was obtained from 51 phage sequences. As expected, only aromatic aa were present in position P1, with Phe and Tyr being the most frequent residues. In contrast to the HC, also Trp was present in about 20% of the sequences. The biggest difference to the HC was ob-served in position P2, where Arg was the single most frequently observed residue, followed by aliphatic aa. For the HC, Arg was very infrequently observed in this position. In positions P3 and P4, the profile of aa resembles that of the HC, with aliphatic aa in most sequences. Also in the primed posi-tions, both similarities and differences to the profile of the HC were re-vealed. In position P1´, the DC clearly prefers aliphatic aa, and Leu is the most frequent residue. In contrast, the HC prefers Ser in this position. Moreover, where the P2´position for the HC is dominated by the negatively charged aa Asp and Glu, the preference of the DC is much less defined in this position, and Asp and Glu are not found at higher frequency than ali-phatic aa or Ser. On the other hand, the profiles of DC and HC are very simi-lar in position P3´, which features mostly aliphatic residues.

In summary, the profiles of the DC and HC are similar in position P1, P3, P4 and P3´, whereas clear differences are observed in positions P2, P1´and P2´. The in vivo relevance of these observations remains to be investigated. Ten-tatively, the HC and DC may cleave a substrate where positions P1, P3, P4 and P3´are conserved between human and dog, whereas positions P2, P1´and P2´ differ. Bioinformatic screenings for such substrates are currently con-ducted.

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CONCLUDING REMARKS

In this thesis, more than 20 novel serine protease genes were identified in the mast cell chymase loci of different mammals. To date, four of these genes have been shown to be expressed, at least six are non-functional genes (pseudogenes), and the rest have yet to be analyzed. We have demonstrated that the gene content of the chymase locus varies greatly between mammal-ian species. Originating from one single ancestral gene more than 215 mil-lion years ago, the locus holds to date four genes in human, whereas at least ten functional genes are found in cattle, 14 in the mouse, and 17 in the rat. We have also found complex functional relationships between chymase lo-cus proteases among different species. This complicates inter-species com-parisons of immune cells, such as mast cells, neutrophils, NK cells and T cells. To have a better understanding of the immune system in mammals, further studies of the chymase locus proteases need to be conducted. In addi-tion, studies on this locus may even extend to non-immunologic terrain. A number of proteases from the chymase locus in cattle have apparently evolved their functions from immune proteases to digestive enzymes. The mast cell chymase locus continues to pose puzzling questions that remain to be solved.

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SVENSK SAMMANFATTNING

Maike, du har arbetat hårt i fem år och ska nu äntligen disputera. Vad har du kommit fram till, och vad kan det leda till?

Jag har undersökt en familj av enzymer som har funktioner i immunförsva-ret. Enzymerna är proteaser, som kan klyva sönder sekvenser i äggviteämnen (proteiner) som de känner igen. Proteaserna finns lagrade i små korn i olika immunceller, såsom T celler, NK celler (natural killer cells), mastceller och neutrofiler. När kroppen behöver försvara sig mot parasiter, bakterier och virus kan immuncellerna hjälpa till genom att släppa ut sina korn där protea-serna finns.

Generna som dessa proteaser kodas från finns hos de flesta däggdjur i ett genkluster som kallas ”mastcell kymas-lokus”. Jag har kartlagt detta kluster i olika arter och därigenom hittat ca 20 nya gener. Sex av dessa är ”döda” gener, så kallade pseudogener, som sannolikt inte uppfyller någon funktion. Men för fyra gener har jag redan kunnat visa att de uttrycks och sannolikt har en funktion i immunsystemet. De resterade generna återstår att undersö-ka. När vi jämför kartorna för mastcell kymas-lokus mellan olika arter ser vi likheter och skillnader. Vi kan då bättre förstå vilka vapen immuncellerna är utrustade med i de olika arterna. När vi vet vilka likheter och skillnader som finns kan vi också lättare bedöma om fynd som gjorts t ex i mus och råtta även kan gälla i människa.

Kan du ge några exempel på funktioner som proteaserna kan ha?

Ja, gärna. En grupp av proteaser, så kallade granzymer, finns i korn i NK celler och T celler. Dessa celler är specialister på att döda virusinfekterade celler och tumörceller, så att t ex virus inte kan sprida sig i kroppen. Avdöd-ningen sker genom att granzymerna sätter igång signaler i målcellen som säger åt cellen att förstöra sig själv. Ungefär som harakiri! En annan grupp av proteaserna heter kymaser. Kymaserna finns i mastceller, kända för att vara inblandade i allergier. Men man tror också att mastceller kan hjälpa kroppen att försvara sig mot bakterier och parasiter. Vävnaden som bakteri-erna har infekterat behöver luckras upp för att immuncellerna ska komma åt. Kymaserna är ansvariga för denna uppluckring. Dessutom kan kymaserna

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dra till sig immunceller och göra dem uppmärksamma på var i kroppen det finns en infektion. En ytterligare funktion av kymaser kan vara att reglera blodtrycket.

Vad gör de gener som du har undersökt då?

För de fem nya gener som jag upptäckt vet vi hittills bara att de är uttryckta i mastceller. Två av generna är släkt med ett proteas som verkar reglera blod-trycket, och de har kanske en liknande funktion. De tre andra generna hör till en familj som verkar hjälpa till i försvaret mot parasiter. Jag har också un-dersökt två proteaser som är kända sedan tidigare. Ett av dem är kymaset från människa. Vi har kommit fram till vilken proteinsekvens som kymaset helst vill klyva. Denna sekvens har vi hittat i en del bakterieproteiner. Det är t ex möjligt att kymaset klyver proteiner som olika bakterier behöver för att fästa sig vid kroppens celler, så att de kan infektera dem. Genom att klyva dessa proteiner skulle kymaset t ex kunna skydda oss mot lunginflammation. Detta behöver dock utforskas mer ingående innan vi vet om det verkligen är så. Det andra kända proteaset är kymaset i hund. Även här har vi hittat vil-ken proteinsekvens som hundkymaset föredrar att klyva. Vi förväntade oss att det skulle vara nästan samma sekvens som för det mänskliga kymaset, med så var inte fallet. I stället var sekvensen ganska lik den proteinsekvens som föredras av ett kymas från råtta! Vi har tidigare trott att detta råttkymas kanske hade fått förändrade funktioner som var viktiga för just råttan. Med våra nya fynd verkar det som om funktionerna är viktiga även för hunden och kanske för fler arter.

Titeln på din avhandling antyder att du har undersökt evolutionen av proteaser. Vilka evolutionära fynd har du gjort?

Jag har kommit fram till att alla gener som finns i ”mast cell chymase locus” hos däggdjur troligtvis har samma ursprung. De verkar härstamma från en stamfader-gen som har funnits för mer än 215 miljoner år sedan. De gener som finns idag hör huvudsakligen till två familjer. Dessa familjer verkar ha sitt ursprung i att stamfader-genen kopierades (”duplicerades”) för ungefär 185 miljoner år sedan. I de olika grenarna av däggdjursfamiljen, såsom hov-djur och gnagare, har det senare uppkommit underfamiljer som särskiljer dessa grenar. Det är mycket möjligt att dessa underfamiljer av proteaser har varit viktiga när nya arter utvecklades och erövrade nya biologiska nischer. I den nya biologiska nischen behövdes det kanske en annan sorts immunpro-teaser, eftersom det fanns andra typer av bakterier och parasiter.

Hur har du gjort för att komma fram till detta?

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Jag har haft stor nytta av projekten som sekvenserar arvsmassan (genom) av olika arter just nu. För en del däggdjur finns hela genomsekvensen redan upplagt på nätet. Det är som ett elektroniskt bibliotek där man kan leta efter gener. Jag har letat igenom alla däggdjursgenom som fanns tillgängliga vid tidpunkten för studien, vilka var människa, mus, råtta, hund, ko och opos-sum. Opossum är ett jätteroligt pungdjur som ser ut ungefär som en råtta med päls. Pungdjuren är avlägsna släktingar till andra däggdjur, och därför har opossum-genomet givit oss viktig information om vilka gener som har funnits långt tillbaka i evolutionen. När jag undersökte funktionen för hund-kymaset och det mänskliga kymaset använde jag mig av en klurig metod som heter ”substrate phage display”. Till grund för denna metod ligger en samling av bakteriofager. Bakteriofager är virus som enbart infekterar bakte-rier. Fagerna i samlingen har muterats så ett de visar upp en proteinbit på sin yta som är sammansatt av nio slumpmässigt valda olika aminosyror. Hela fagsamlingen innehåller så många som 50 miljoner fager med olika slump-sekvenser. Man låter det proteas man undersöker välja mellan dessa slump-sekvenser. Fager med sekvenser där proteaset har kluvit kan man sedan sam-la upp. Man förökar dessa fager i bakterier och erbjuder dem sedan till pro-teaset en gång till. Så gör man i fem omgångar och får därmed fram sekven-ser som klyvs bättre och bättre. Till slut kan man lista ut vilken sekvens proteaset föredrar mest.

Tack, nu har jag blivit lite klokare. Jag tänkte försöka sammanfatta det du har gjort för våra läsare i några korta meningar för att se om jag har förstått rätt.

Maike har alltså forskat om proteasgeners utveckling ur ett historiskt perpektiv, och hon är intresserat av vilken funktion proteaserna kan ha. Hon har undersökt när och hur olika gener har muterat genom att titta på generna i olika arter. Det har hon gjort genom att leta i ett elek-troniskt bibliotek där hela genom finns lagrade. Maike har på så sätt hittat olika uppsättningar av proteaser hos olika däggdjur. Mellan vissa arter är de mer överensstämmande, mellan andra mindre, men alla proteaser verkar härstamma från en och samma stamfader. När gen-klustret som Maike studerar har utvecklats i evolutionen verkar det ibland ha uppstått genkopior som inte har någon funktion. Andra gånger har det uppkommit gener som kan spela en stor roll för att just den art som de finns i ska ha ett bra immunförsvar. Kan du skriva un-der på detta, Maike?

Jajamen! Tack så mycket för denna roliga intervju!

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ACKNOWLEDGEMENTS

The last five years of PhD studies have been a very exciting time. I have moved to a new country, learned a new language, got married to my dearest Erik, and found good friends. I am very grateful for this adventurous time of my life! It all was much possible thanks to Lasse, who accepted me as his PhD student without ever having seen me work in the lab before.

Lasse, min handledare, tack för vår resa under de senaste åren. Tack för ditt stöd, din tid och ditt engagemang. Så roligt att även du tycker om att dryfta alla livets ämnen! Det har varit en utmaning och en hedersbetygelse att kän-na din tilltro.

Stor tacksamhet känner jag också för alla medarbetare från Lasses, Sandrasoch Pilis grupp. Ni har varit så mycket mer än kollegor! Jag kommer att sakna er alla! En person som lyser upp många dagar är Jenny. Tack för den glädje och tillförsikt som du sprider! Jag hoppas att jag kan ta efter dig i alla fall lite grand. Nu har jag äntligen mer tid att träffas igen! Mattias, tack för många goda samtal om kvällen, bade om forskningen och om livet. Nu när du har licat ska jag inte längre kalla dig för “Mattiaschen”… eller i varje fall lite mer sällan… Mattias E, jag kan ärligt säga att denna avhandling skulle inte har blivit det den är utan dig (det är bara att ta en titt på författarlistor-na). Du är helt enkelt toppen! Tack! Maria och Parvin, ni har varit mina “mammor”, bade i labangelägenheter och i allmänmänskliga ting. Många samtal med er har hjälpt mig att finna mig till rätta i det svenska samhället, och att känna mig varmt välkommen. Puss puss! Som tur är har jag även träffat många av Lasses föredetta doktorander och postdoks, ett glatt och härligt gäng! Anna, Camilla, Jeanette, Lotta, Maryam, Molly, Sara och Ulrika, det har varit rikt att dela vardagen med er. Anna, vilken bra idé att du grundade onsdaxölstraditionen! Camilla och Ulrika, ni har visat mig vä-gen genom den ibland invecklade doktorandtiden. Vad roligt det var när jag fick hjälpa till med immunologikursen för första gången! Sara, du är en förebild på många sätt. Jag tycker om din blandning av lugn och engage-mang. Tack för generositeten som du och din familj har visat mig! Ett stort tack vill jag också säga till alla examensarbetare som har varit med i grup-pen under de senaste fem åren. Vielen Dank für die Kuhsequenzen, Anna!Wie Du siehst, konnten wir sie gut gebrauchen. Viel Glück und viele Grüsse an Heidelberg! Per, tack för roligt och engagerat festande! Och för att du är

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så snäll. Maria och Lena, tack för gott samarbete, och lycka till i fortsätt-ningen! Good luck for your studies, Camilla, and thanks for many nice and summery lunches! To all people that have joined the group rather recently, Sayran, Niclas, Madeleine and Marie-Louise, it has been vey nice meetingyou. I wish you a good time in the lab and at the computer, hope you´ll have fun!

Det var mycket roligt att få bli granne med just din grupp, Sandra, när jag ju hade gjort mitt examensarbete hos dig. Jag tycker om att du äter lunch med oss, och att du ställer de tuffa frågorna! Sofia, så roligt att vi sjunger i sam-ma kör nu, det är en garanti för att vi kommer att träffas framöver. Skvallra gärna mycket! Jag är tacksam för din värme och omtänksamhet. Kajsa, nå-gon som dig har jag saknat när först jag kom till Sverige! Rak, orädd, cool! Det är väldigt roligt att känna dig. Maria Andrén, du är också en sån som gör en glad! Tack för alla fester du är med och ordnar! Sinnisky, you are one of the most generous persons I know. I wish you all the good you can get! Hope you will soon visit me at my place! So we can talk about all those Swedish habits, and probably more (Wanna have Sushi?). Jag har också fått med mig mycket från Pilis grupp. En av dina första kommentarer till mig, Pilis, var: “Det som är bra kan alltid bli bättre.” Jodå, det är ett väldigt an-vändbart motto! Tack för all hjälp med datorerna, Niklas, och för din torra humor. Sirje, eftersom du inte heller har bott i Sverige hela livet har jag alltid känt mig lite som dig. Synd att vi inte längre bor granne… Siv, tack för många varma samtal och trevliga fikastunder! Och för alla tips angående PCR…I am also very grateful to all colleagues and staff at ICM! I got so much help from you, in areas of life ranging from a residence permit for Sweden to the decision what to have for lunch… THANK YOU!

Den Weg zu meiner Doktorarbeit hast Du bereitet, Michael Reth. Ich bin froh, Dich zu kennen! Es ist mir ein Vorbild zu sehen, wie Du anderen ein Freund bist, wie Du Dich für Deine Mitmenschen einsetzt, und natürlich wie Du forschst. Hoffentlich sehen wir uns bald wieder! My friends around the world, thank you for being close to me. You make me feel at home in many places. It is much thanks to you that life feels so good! I love our talks, e-mails, computer conferences, travels, singing, work-outs, projects, lunches, family celebrations…. It is great to know you. Meiner erweiterten Familie, wenn ich Euch so nennen darf, Elke, Paul und Andreas, bin ich dankbar für die Grossherzigkeit und Wärme, die sie mir entgegengebracht hat. Elke, Du hast mir so vieles geschenkt und bist mir in vielem ein Vorbild! Paul und Andreas, Euch zu kennen macht mein Leben reicher. Meiner Familie bin ich dankbar, dass sie an mich und meinen Lebensweg glaubt. Danke für Eure Unterstützung, Euer Wohlwollen und Euer Interesse. Günther und Renate,Euch und Eurer Familie bin ich dankbar, dass Ihr meine Geschwister und mich so gut durch eine schwere Zeit gebracht habt. Danke für die Zukunft,

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die Ihr uns ermöglicht habt. Oma Ruth, es hat mich so gefreut, dass Du und Gerhard zu Eriks und meiner Hochzeit nach Schweden gekommen seid! Ich denke immer gern an unsere ”Lämmle”-Gespräche, die so einiges klarer werden liessen. Min nya svenska familj, främst Gunilla, Lennart och Svea,er vill jag tacka för ert tålamod med mina tyska egenheter! Tack för att ni har förståelse för det mesta! Jag hoppas att vi kommer att se mycket av varan-dra! Eva, Jens und Anne, ich bin froh, Eure Schwester zu sein! Danke für unsere guten Gespräche, für unseren Draht zueinander, für unsere geme-insamen Interessen und für Eure Eigenheiten. Euch zu treffen macht mich froh. Wie gut, dass Ihr gern nach Schweden kommt! Ohne Eure Unter-stützung wäre es unmöglich gewesen den Entschluss zu fassen, hier zu le-ben.

Erik, min man, älskade och vän, tack för värmen, närheten, vilan och stödet som du ger mig. Det har varit underbart att komma hem till Mickey Mouse, lyxmat och din famn! Jag älskar dig, knusket! Jag vill blanda mina gener med dina!

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