comparison of cleavage site specificity of gelatinases a and b using collagenous peptides

ELSEVIER Biochimica et Biophysica Acta 1293 (1996) 259-266 et Biophysica A~ta

Comparison ,of cleavage site specificity of gelatinases A and B using collagenous peptides

Tian Xia 1, Kathryn Akers, Arthur Z. Eisen, Jo Louise Seltzer * Dirision of Dermatology, Washington Unirersity School of Medicine, 660 S. Euclid Acenue, Box 8123, St. Louis, MO 63110, USA

Received 20 September 1995; accepted 4 December 1995

Abstract

The gelatinases (type IV collagenases) are members of the matrix metalloproteinase family that not only have a high degree of structural homology but are known to be nearly identical in their digestion profile against macromolecular substrates. We have shown previously that the preferred cleavage sites in the hydrolysis of type 1 gelatin, catalyzed by gelatinase A (72 kDa type IV collagenase), are bracketed by hydroxyproline in the P5 and P5' positions. In this report, a kinetic investigation using a series of collagenous dodecylpeptides in which the P5 and P5' hydroxyprolines were systematically varied and used as substrates for recombinant human gelatinase A, we show that replacement with either proline or alanine "always resulted in increased K,,. In contrast, substitution of the hydroxylated amino acids tyrosine and serine at P5 and P5' reduced the K m significantly, indicating that the hydroxyl moiety of the hydroxyproline is the functional group responsible for favorable enzyme-substrate affinity. This was shown by the kc~t/K m ratio, which was doubled by the substitution of serine in that site. Cleavage of the same series of dodecylpeptides by recombinant human gelatinase B (92 kDa type IV collagenase) showed a very different kinetic profile for which no patterns were discernible. In subsequent comparisons of the two enzymes, it was found that gelatinase B cleaved the thiopeptolide substrate AcProLeuGly-S-LeuLeuGly-OC2H 5 at double the velocity of gelatinase A. In ,zontrast, gelatinase A digested type 1 gelatin about 2.5-times faster than gelatinase B. SDS-PAGE analysis of gelatin cleavage products showed different patterns of product peptides for each enzyme. Further comparisons of the proteinases using synthetic peptide substrates with variations in size and in substituents at the P2' site again showed marked kinetic differences. Although these two matrix metalloproteinases seem similar in that they are both gelatinolytic and can degrade a nearly identical battery of macromolecular matrix components including type IV collagen, it is clear from these results that they are very different enzymatically. Since the regulatory portions of gelatinases A and B differ markedly, it has been assumed that the enzymes serve the same function, but respond to different stimuli. The differences in substrate specificity described herein suggest that their proposed physiological roles may require reevaluation.

Keywords: Cleavage site; Collagen: Gelatin; Gelatinase; Kinetics; Peptide

I. Introduct ion

The matrix metalioproteinases are a group of extracellu- lar neutral proteinases that show a high degree of homology in domain structure and sequence. All contain a zinc binding catalytic domairt and require bound calcium for stabilization [1-4]. Within the family are enzymes capable of hydrolyzing all matrix proteins, including helical colla- gens [2]. Both gelatinase A (72 kDa type IV collagenase) and gelatinase B (92 kDa type 1V collagenase) are called

Corresponding author. Fax: +1 (314) 3628159; e-mail: seltzer @ visar.wustl.edu.

I Present address: Monsant:) Company, 7(KI Chesterfield Parkway, Chesterfield. MO 63198, USA.

0167-4838/96/$15.00 © 1996 Elsevier Scicnce B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 5 ) 0 0 2 5 9 - 6

gelatinases since denatured collagen is their greatly preferred substrate [5-9]. Both have a propeptide domain, an N-terminal domain, an = 175 amino-acid domain homologous to the F2 collagen (gelatin) binding domain of fibronectin, a zinc-binding active site, and a carboxyl-terminal residue domain homologous to hemopexin [6]. The catalytic site is nearly identical in both enzymes. The fibronectin-like domain allows the proenzyme to bind to gelatin [10], and the hemopexin domain binds tissue inhibitors of metalloproteinases (TIMPs) [11-14]. Gelatinase B is larger due to a 53-residue insertion after the catalytic domain homologous to the a 2(V) collagen chain. In addi- tion to gelatins, types IV and V collagen [6,7] and elastin are macro'aolecular substrates for both enzymes [15]. Fi- bronectin [7] and types VII [16] and X [18] collagen are

260 T. Xia et al. / Biochimica et Biophysica Acta 1293 (1996) 259-266

also substrates for gelatinase A. Types VII and X collagen have not been examined for susceptibility to gelatinase B cleavage.

Previous studies of cleavage site specificity have shown that in collagenous (i.e., Gly-X-Y-GIy-X-Y...) sequences. gelatinasc A preferentially cleaved between glycine and hydrophobic amino acids. However, PI' could also be acidic, an acid amide, or hydroxylated [17,19]. Of the actual sequences surrounding the cleavage site, hydroxyproline was invariant at P5', and nearly invariant at subsite P5 [17]. Hypothesizing that the bracketing of the cleavage site by hydroxyproline might be a major factor in the marked specificity for gelatin substrates, we devised a series of peptides based upon AcGluHypGlyProAlaG- lyValArgGlyGluHypGlyNH 2 in which first proline, then alanine was systematically substituted for the hydroxyprolines.

In this paper we report that with gelatinase A, substitutions of proline and alanine for the P5 and P5' hydroxyproline consistently resulted in higher K m values. Kinetic analysis of the same series of peptides and other collagenous peptide substrates using gelatinase B showed variations in both K m and Vm, x of nearly an order of magni- tude. Further comparison of the kinetic properties of the two enzymes showed that, surprisingly, gelatinase B is a much less effective gelatinase than gelatinase A, and that the gelatin breakdown patterns as shown by SDS-PAGE are different. From this evidence we conclude that the physiological function of these two 'gelatinases' may be quite different.

2. Materials and methods

2.1. Production o f recombinant gelatinases (~.'pe IV collagenases)

Cell lines p2AHT7211A and p2AHT72298, derived from the p2AHT2a cell line [20], were cultured in RPMI with 5% fetal bovine serum. In these cell lines (a generous gift from Dr. Gregory Goldberg, Washington University), expression of all metalloproteinases by HTI080 cells has been suppressed by stable transfection with the adenovirus EIA gene. Subsequently, expression plasmids containing the complete gelatinase A cDNA (p2AHT7211A) and gelatinase B cDNA (p2AHT72298), under control of the RSV LTR promotor, which escapes repression by EIA, were engineered into hygromycin-resistant cassettes and transfected into p2AHT2a cells [21]. The resultant clones overproduced the gelatinase proenzymes, which are se- creted into the culture medium.

2.2. Proenzyme purification and actication

Proenzymes were purified from serum-free culture medium as previously described [7] using chromatography

on Red Agarose followed by affinity purification on gelatin agarose with elution by 10% DMSO. The purified gelatinases are TIMP-free.

Slightly different protocols were used for organomercurial activation of the two proteinases. Progelatinase A was stored with the chelator 1,10-phenanthroline to prevent autoactivation and autodegradation. For activation, 20-50 btg of enzyme was incubated for 2 hours at room temperature in 0.05 M borate (pH 7.5), containing 5 mM CaC12, 20% glycerol, 0.005% Brij, 0.01 mM ZnCl 2 and 0.16 mM aminophenyl mercuric acetate (APMA). To remove APMA, the mixture was passed through a G-25 column.

Progelatinase B was activated by incubation in 0.05 M borate (pH 9.0) containing 5 mM CaC12, and 0.5 mM APMA for either 2 h at 37°C or overnight at room temperature. APMA was removed as described above. The activated enzymes were stored in aliquots at -80°C and were stable for at least 5 months.

2.3. Synthesis o f peptides

All peptides were synthesized at the Washington Uni- versity School of Medicine Protein Chemistry Laboratory with an Applied Biosystems Model 430A automated peptide synthesizer, using t-Boc chemistry. Peptides were acetylated while on the resin, then deblocked and cleaved from the resin with anhydrous hydrofluoric acid. Carboxyl-terminal amidation was accomplished by synthesis on a methyl-benzhydrylamine resin.

All derivatized peptides were purified by reverse-phase HPLC, using macroporous C18 columns (Vydac). Se- quence, mass, and derivatization were verified using a Vestee Model 201 electrospray mass spectrometer at the Washington University School of Medicine Protein Chem- istry Center. Sequence of cleavage fragments was determined using a gas phase Applied Biosystems Model 470A sequenator, with phenythiohydantoin derivatives identified by high pressure liquid chromatography.

2.4. Thiopeptolide cleacage assay

The thioester substrate AcProLeuGly-S-LeuLeuGly- OC2H 5 (Bachem) was first dissolved in 50% acetic acid, and then added to an equal amount of 0.2 M Hepes (pH 7.6) containing 0.05 M CaCI 2 to make a 20 mM stock solution. Aliquots of the stock solution were lyophilized in microtubes, and reconstituted in dimethylformamide just before use in an assay.

Enzyme activity was assayed in 0.2 M Hepes (pH 9.0) containing 0.01 M CaCI 2, 1 mM thioester peptide, and 0.9 mM Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid)) (Sigma). As the thioester bond was cleaved, forming free -SH, Ellman's reagent traps the sulfhydryl forming a col- ored anion whose formation can be monitored spectropho- tometrically at 412 nm. The time-course of the increase in optical density was monitored on a Gilson recording spec-

T. Xia et al. / Biochimica et Biophysica Acta 1293 (1996) 259-266 261

trometer, and the initial velocity determined from the slope of the linear portion of the curve. Blanks consisted of the assay mixture without enzyme; non-enzymatic increase in optical density was less ~an 3% of the total increase per assay. Under the conditions described, the initial velocity was linear with enzyme concentration, and both thioester peptide and Ellman's reagent were present in saturating amounts. Therefore, the initial velocity is proportional to the amount of activated enzyme.

2.5. Assay of gelatin cleaz age

Gelatinase activity was assayed using ~4C-labeled collagen as described previously [5]. Briefly, 25 /zl of the collagen was denatured tc gelatin by heating in a boiling water bath for 10 min. S.eventy-five /.tl of enzyme was added, and after incubation at 37°C, the reaction was stopped with 25 p~l of 10:3% trichloroacetic acid. Product peptides soluble in 20% acetic acid were counted in a liquid scintillation spectrometer. 14C-labeled type I collagen of 4500 c p m / m g (a gift from Dr. John Jeffrey, SUNY, Albany) was purified as described [22].

When cleavage pattern~ of gelatin degradation were to be compared by examining substrate breakdown on SDS- polyacrylamide gels, type I collagen purified from rat tail tendons [23] was used. Collagen was denatured to gelatin by boiling in 0.1 M borate (pH 7.5) containing 5 mM CaCI 2, and incubated with proteinase at an enzyme/substrate ratio of 1:6500. At timed intervals aliquots were withdrawn into 25 mM EDTA to stop the reaction.

SDS-polyacrylamide gel electrophoresis was carried out according to the method of Laemmli [24]. Gels were stained with Coomassie blue G-250 and destained with 10% methanol, 5% acetic acid.

2.6. Kinetic analysis of peptide clea~,ages

Kinetic analyses were carried out fluorometrically, using fluorescamine to label the amino-terminal groups cre- ated by peptide bond cleavage [25,26]. Peptides were dissolved in 0.05 M borate buffer (pH 7.5) containing 10% ethanol. Varying concentrations of the peptides were incubated with 0.03 to 0.2 /zg of the enzymes at 37°C in 0.05 M borate (pH 7.5) containing 5 mM CaCI 2. At intervals, aliquots were withdrawn into 0.2 M borate (pH 6.8) containing 0.01 M EDTA. A solution of 0.1 m g / m l fluorescamine in acetone was added with continuous mixing to an equal volume of the borate-EDTA mixture. Fluores- cence was read in a Farrand fluorometer, using leucylleucine as a standard. Excitation was at 390 nm and emission measured at 475 nm. Three to six complete data sets were collected for each substrate. Standard deviations were _< 15%.

To assure measurement of initial velocity, all calculations are based upon conversion of less than 5% of the substrate to product. Kinetic constants were determined from double-reciprocal plots using the computer graphics program 'Cricket Graph'. Lines were drawn using linear least-squares fit.

2.7. Protein measurement

Protein concentration of enzyme solutions was determined using Quantigold (Diversified Biotech), an assay based upon the binding of colloidal gold to proteins [27,28]. Optical density is read at 595 nm, and readings interpo- lated into a standard curve of increasing BSA concentrations. This assay is linear over a range of 40-200 ng protein. The accuracy of the Quantigold assay was tested

Table 1 Determination of thiopeptolide t'ydrolysis per pmol enzyme

Preparation p.g protein/ Average JA412/h per/xg Average AA4t2/pmol assay AA412/h total protein active enzyme

Gelatinase A

#1, n = 3 0.15.3 1.85 12.33 1.01 #2, n = 3 0.15.5 1.85 11.91 1.06 #3, n = 3 0.144 1.64 11.37 1.02 #4, n = 24 0.153 1.56 10.40 0.93

Weighted average I 0.80 0.96

Gelatinase B #1, n = 17 0.144 1.77 12.29 2.08 #2, n = 5 0.119 1.17 9.81 1.67 #3, n = 12 0.123 1.56 12.66 2.14

Weighted average 12.1 2.05

Aliquots from different preparat ons were taken from storage at - 80°C and assayed over several months' storage. As measured by SDS-PAGE, each gg gelatinase A enzyme protein yielded 0.7/~g active enzyme of molecular weight 62 kDa, or I 1.2 pmol. Likewise, l /,tg total gelatinase B protein yielded 0.5 /zg active enzyme of molecular weight 84 kDa. or 5.9 pmol. In calculating k,a , values, the J A / h per pmol enzyme were rounded to 1.0 for gelatinase A and 2.0 for gelatinase B.

262 72 Xia et al . / Biochimica et Biophysica Acta 1293 (1996) 259-266

against the Lowry protein method [29] and the results were nearly identical.

3. R e s u l t s

3.1. Quantitation o f enzyme used per assay

In our previous studies dealing with cleavage site specificity of gelatinase A, the enzyme used had been purified in a completely active form from culture medium of human skin explants [17]. The studies reported here use recombinant proenzyme that requires reaction with organomercurials to form the enzymatically competent, lower molecular weight (62 kDa) active proteinase. Proen- zyme activated with APMA must be passed through a desalting column of Sephadex G-25, since thc organomercurial will interfere with the fluorometric assays. Previ- ously we included a gelatinolytic assay as an internal standard to quantitate the very dilute enzyme used in these kinetic studies, using a predetermined value of 80 /xg of gelatin degraded/ / . tg of e n z y m e / h o u r [17]. Although quite reproducible, this assay greatly underestimates the actual number of cleavages [5]. We have previously shown that the thiopeptolide substrate Ac-ProLeuGly-S-LeuLeuGly- OCzH.s [30] is cleaved by gelatinase A [26] and found that it is also a substrate for gelatinase B. The commercial availability of the thiopeptolide substrate makes it possible to use the rate of cleavage of a single thioester bond as an internal standard to quantitate precisely the amount of active enzyme.

To obtain a reliable measure of optical density change per mole of active 62 kDa gelatinase A, we assayed multiple samples from each organomercurial-activated preparation. Samples were stored as aliquots at - 8 0 ° C ; assays were performed using freshly thawed aliquots. The extent of conversion to active enzyme was measured from stained SDS-polyacrylamide gels. Table 1 shows that the specific activity of thiopeptolide hydrolysis was quite re-

producible from preparation to preparation. Under the conditions used, conversion of gelatinase A from the 72 kDa proenzyme to the 62 kDa enzymatically competent species was always approx. 70% complete, while activation of gelatinase B (to the 84 kDa active enzyme) was 50% complete. Activation was terminated at these points to prevent autodegradation to active fragments of lower molecular weight. Therefore, to calculate the true turnover number of the active species, each /,~g of gelatinase A proenzymc was considered to yield 0.7 /zg of active enzyme, or 11.2 pmol. Similarly, each /xg of gelatinase B yields 0.5 /xg active enzyme after organomercuriai treat- ment, or 5.9 pmoi. Thus the rate of thiopeptolide hydrolysis, expressed as AA412 /h , could be calculated as AA412/h per pmol active enzyme, and could be used to measure enzyme concentration in the dilute G-25 eluates used for the kinetic assays. This value for gclatinase A, 0.96 A A 4 ~ 2 / h per pmol active enzyme, was rounded to 1 for calculations of kcat; for gelatinase B, the value of 2.05 was rounded to 2 for kca t calculations. As shown in Table 1, activity remained constant through multiple assays over a period of months, and was consistent from preparation to preparation.

Because the organomercurial activating agent APMA was removed after the initial incubation, the ratio of proenzyme to active enzyme remained constant during storage up to at least five months. SDS-PAGE analysis indicated no autodegradation of freshly thawed aliquots (not shown). In the assay conditions used. the thiopeptolide concentration was saturating for both enzymes, so that the rate of hydrolysis was an accurate reflection of the amount of active proteinase.

3.2. Effect o f substitutions.fi)r P5 and P5' hydroxyprolines on gelatinase A

The dodecylpeptide AcGluHypGlyProAlaGlyValArg- GIyGIuHypGlyNH, was chosen as a basis for variation because it closely resembles an actual cleavage site, whose

Table 2 Effect of variations in the P5 and P5' position on kinetics of dodecylpeptide hydryolysis by gelatinasc A

Sequence of substrate K m Vm~ ~ (nmol / A A k~.~t k ~.~,/ K ,, (mM) per hour) (X 10 5 ) (× 10 5)

Ac-Glu-Hyp-Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Hyp-Gly-NH 2 0.3 136 Ac-Glu-Hyp-Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Pro "-GIy-NH 2 0.32 133 Ac-Glu-Pro ' -Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Hyp-(}ly-NH, 0.35 147 Ac-Glu-Ala ' -Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Ala "-GIy-NH 2 0.42 146 Ac-Glu-Pro "-Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Pro "-GIy-NH 2 0.36 150 Ac-Glu-Pro "-Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Ala "-Gly-NH 2 0.49 129 Ac-Glu-Lys "-Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Lys "-GIy-NH 2 0.46 76 Ac-Glu-Phe "-Gly-Pro-Ala-Gly-VaI-Arg-Gly-Glu-Phe "-Gly-NH 2 0.3 103 Ac-Glu-Tyr "-Gly-Pro-Ala-Gly-Va]-Arg-Gly-Glu-Tyr "-Gly-NH 2 0.21 I 1 I Ac-Glu-Ser "-Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Ser "-GIy-NH 2 0.14 125

1.36 4.52 1.33 4.15 1.47 4.19 1.46 3.48 1.49 4.13 1.29 2.64 0.76 1.65 1.03 3.44 1.06 5.31 1.25 8.90

Assays were performed as described in Section 2. Quantitation of enzyme used in calculations of kca t is based upon thiopeptolide substrate hydrolysis, using the value of I absorbance unit per hour per pmol of enzyme, as shown in Table 1.


0.010,

0 . ~ '

0 .0tS '

/ 0.0¢6.

0.0~, J o ; ; ; ;

lt$ (m~

Fig. 1. Comparison of the hydrolysis of peptide substrates by gelatinase A with either hydroxyproline or serine in the P5 and P5' position. V is in nmol substrate c l eaved /AOD4t " , per hour. ( Q ) AcGluHyp- G l y P r o A l a G l y V a l A r g G l y G l a H y p G l y - N H , , ( / , ) A c G l u S e r - GlyProAlaGlyValArgGlyGluSerGly-NH 2.

corresponding hexapeptide is cleaved with a high turnover number and moderately low K m [17]. Our previous obser- vations showed that cleavage sites of type I gelatin by gelatinase A were almost invariably bracketed by hydroxyproline in the P5 and P5' positions. To investigate the role of these residues, we used the series of peptide substrates shown in Table 2. Systerr.atic replacements of the P5 and P5' hydroxyprolines with proline had little effect on kca t. Specificity, as measured by the ratio k c a t / K m w a s slightly lowered (from 4.5 to 4.1] because of a small increase in K m . Surprisingly, replazement of either proline or hydroxyproline with alanine did not affect turnover number. Consistently, however, replacement of the hydroxy-

proline resulted in a decrease in affinity for the substrate, as shown by the increase of K m values.

The effect of hydroxyproline on substrate binding seemed rather small to account for the consistent require- ment for that amino acid in the P5 and P5' positions around the cleavage sites in the natural substrate. There- fore, we devised peptide substrates with substitutions in these positions. A bulky hydrophobic residue (phenyi- alanine), a positively charged residue (lysine), and two other hydroxylated residues (serine and tyrosine) were substituted at P5 and P5'. Phenylalanine did not affect affinity, but slightly reduced kca r Substitution of the basic lysine for hydroxyproline reduced kca t 50%, from 1.36. 105 to 0.76 • 10 s, and raised the K m from 0.3 mM to 0.46 mM, so that the specificity ratio was reduced about two- thirds. Substitution of tyrosine lowered the K m from 0.3 to 0.21 mM and serine in those positions lowered the K m to

0.14 mM (Fig. 1). Turnover number was not greatly affected, so that, for serine, the k c a t / K m specificity ratio became double that for the base substrate. These results suggest that the hydroxyl moiety of hydroxyproline is the functional group which has the most profound effect on the kinetic behavior of gelatinase A.

3.3. Effect of substitutions for P5 and P5' hydroxyprolines on gelatinase B

When the same set of peptides was subjected to hydrolysis by gelatinase B, the results were strikingly different (Table 3). Kca , values for all peptides were significantly lower (2.5- to 25-fold) than corresponding kca t values with gelatinase A, showing a sevenfold range, from 0.6. 104 to 4.5. 104, as contrasted to the twofold range for gelatinase A. There was also an eightfold range in K m values, from 0.3 to 2.4 mM. With this set of peptides, there was no discernible trend in the variations. The contrasts between kinetic constants for the two enzymes are striking. For example, substitution of two alanines for two hydroxyprolines did not affect kca t for gelatinase A, but reduced kca t

Table 3 Effect of variations in the P5 an,:l P5' position on kinetics of dodecyl peptide hydrolysis by gelatinase B

Sequence of substrate K m V,,~ ( n m o l / ~ A k~, k~,t/K,~ (mM) per hour) ( × 10 ~ ) ( x 10 ~)

Ac-Glu-Hyp-Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Hyp-Gly-NH 2 0.6 49.9 Ac-Glu-Hyp-Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Pro "-Gly-NH 2 0.86 72.9 Ac-Glu-Pro "-Gly-Pro-Ala-Gly- VaI-Arg-Gly-Glu-Hyp-Gly-NH 2 0.72 89.9 Ac-Glu-Ala ° -Gly-Pro-Ala-Gly- Val-Arg-Gly-Glu-Ala "-Gly-NH 2 0.42 12.3 Ac-Glu-Pro "-Gly-Pro-Ala-Gly- Val-Arg-Gly-Glu-Pro "-GIy-NH 2 0.67 36.5 Ac-Glu-Pro ' -Gly-Pro-Ala-Gly- VaI-Arg-Gly-Glu-Ala "-GIy-NH 2 2.39 79.0 Ac-Glu-Lys "-Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Lys "-GIy-NH 2 0.79 59.2 Ac-Glu-Phe "-Gly-Pro-Ala-Gly-Val-Arg-Gly-Glu-Phe "-GIy-NH 2 0.31 19.0 Ac-Glu-Tyr "-Gly-Pro-Ala-Gly-VaI-Arg-Gly-Glu-Tyr "-GIy-NH 2 0.32 24.0 Ac-Glu-Ser "-Gly-Pro-Ala-Gly- VaI-Arg-Gly-Glu-Ser "-GIy-NH 2 1.71 77.9

2.49 4.15 3.65 4.24 4.49 6.24 0.61 1.46 1.83 2.73 3.94 1.65 2.95 3.74 0.95 3.07 1.20 3.75 3.90 2.28

Assays were performed as described in Section 2. Quantitation of enzyme used in calculations of kca , is based upon thiopeptolide susbstrate hydrolysis, using the value of 2 units absorbance change per hour per pmol of enzyme, as shown in Table 1.

264 T. Xia et al. / Biochimica et Biophysica Acta 1293 (1996) 259-266

for gelatinase B sevenfold from 2.49.104 to 0.61 • 104. 0.2" Substitution of two serines for two hydroxyprolines reduced the K m of gelatinase A by three, as described above, but more than doubled the K m for gelatinase B, ~ o.Js- from 0.6 to 1.71. Cleavage sites were confirmed as Gly-Val by random sequence analysis of product peptides, o

3.4. Comparison of gelatinase A and gelatinase B with ~ ~ 0.1-~

different peptides ~

Several other synthetic peptides were used for a kinetic comparison between the two gelatinases. The hexapeptide ~ 005. AcProAlaGlyValArgGlyNH 2 was compared with its nonamidated form. Also, since we had previously found that arginine in position P2' was favorable to the cleavage rate for gelatinase A, we made dodecylpeptides with glutamic 0

0 acid and isoleucine substituted into the P2' position. The results, given in Table 4, show striking differences between the enzymes. The negatively charged, nonamidated peptide is a good substrate for gelatinase A, but is essen- o l25. tially not a substrate for gelatinase B. The twofold reduc- tion in kca t for the charged peptide is the same for the recombinant enzyme as for the explant enzyme [17]. As 0.1-

.z=

shown in Fig. 2a, for gelatinase B the substitution of the ~ _ negatively charged glutamic residue for the positive arginine in the P2' position had little effect on Vm~ × and raised o • ,~ 0.075-

the K m from 0.6 to 2.83 mM, a difference no more striking than for many of the other peptides tested. How- ~ ever, for gelatinase A, this same substitution lowered the ~ 0.05. kca t almost five times, while increasing the K m almost ~ threefold, as shown in Fig. 2b. -e

E 0.025- 3.5. Comparison of gelatin hydrolysis by gelatinase A and = gelatinase B

Gelatin degradation was measured first by production of trichloroacetic acid soluble fragments of [~4C]gelatin, and then by examination of SDS-polyacrylamide gels showing the course of substrate cleavage. In the radioactive gelatin assay, gelatinase A was shown to degrade the labeled gelatin substrate more than twice as fast as gelatinase B, with a turnover number of 410 p.g gelatin a -cha in /~g active enzyme/hour (255 mol gelatin a-chain/mol active

I/S mM

(b)

I/S mM

Fig. 2. (a) Comparison of the hydrolysis of peptide substrates by gelatinase B: ( [] ) AcGluHypGlyProAlaGlyVal ArgGlyGluHypGly-NH 2, ( 0 ) AcGluHypGlyProAlaGlyValGluGlyGluHypGly-NH z. (b) Comparison of the hydrolysis of the same peptide substrates shown in Fig. 2a by gelatinase A: ( [] ) AcGluHypGlyProAlaGlyVal ArgGlyGluHypGly-NH 2 , ( 0 ) AcGIuHypGIyProAIaGIyValGIuGIyGluHypGIy-NH 2 .

Table 4 Kinetic comparisons between gelatinase A and gelatinase B when charge is changed at P2' and PY position in peptide substrates

Sequence of substrate Gelatinase A Gelatinase B

rm Vma, kc~, k . , / r , rm Vma, k., k.#K,. (raM) (nmol /AA (X105 ) (×105 ) (raM) (nmol /AA (×104 ) (×1 0 4 )

per hour) per hour)

AcGluHypGlyProAlaGlyValArgGlyGluHypGlyNH 2 0.3 136 1.36 4.52 0.6 50 2.49 4.15 AcProAlaGlyValArgGlyNH 2 0.33 144 1.43 4.34 0.71 38 2.04 2.87 AcProAlaGlyValArgGlyOH 0.47 78 0.78 1.65 1.24 2 0.09 0.07 AcGluHypGlyProAlaGlyValGlu ° GIyGIuHypGIyNH 2 1.01 35 0.35 0.35 2.83 39 1.95 0.69 AcGluHypGlyProAlaGlyVallle ° GIyGIuHypGlyNH 2 0.30 83 0.83 2.77 0.48 19 0.97 2.02

Assays were performed as described in Section 2. Quantitation of enzyme used in calculations of kca t is based upon thiopeptolide substrate hydrolysis, as shown in Table 1.


(A) E

"o

Gelatinase A Gelotinase B

E /..= I0' 20' 30' 40' 50' I0':' b I0' 20' 30' 40' 50' 105'

Fig. 3 also show some of the more obvious points where the cleavage patterns of type I collagen by gelatinase A and gelatinase B differ. Other differences can be detected upon inspection of these patterns.

(B) Gelatinase Gelatinase A B

• , , j

, z i , :

50' 105' Fig. 3. (a) Proteolytic degradation of type I gelatin catalyzed by gelatinases A and B. Approximately equal amounts of gelatinases A and B (0.16 ~g, and 0.13 /xg. respectively) were incubated at 37°C with one mg of type 1 collagen which had Ix:en denatured to gelatin by heating in a boiling water bath for 10 min. The reaction mixture also contained 0.05 M Tris (pH 7.5), 0.01 M CaCI 2, 10% glycerol and 0.005% Brij-35. Aliquots of 25 /.H were withdrawn from the reaction mixtures at the indicated times and subjected to electrophoresis on 12% SDS-polyacrylamide gels. Gels were stained with Coomassie brilliant blue G-250. (b) Indication of different sites of type I gelatin cleavages by gelatinases A and B. Lanes from Fig. 3a at approximately the same degree of degradation are compared directly. A:ToWS indicate some product cleavage peptides from gelatinase B dige.';tion which are not seen with gelatinase A.

enzyme per hour) compared to 135 txg gelatin a-chain/ /zg active enzyme per hour ( l l 0 moi gelatin a-chain/mol active enzyme per hour) for gelatinase B. It must be remembered that this assay greatly underestimates the number of cleavages, as :;hown in Fig. 3. The arrows in

4. Discussion

All matrix metalloproteinases share a considerable degree of homology, particularly in the zinc-binding, active- site region but are varied in their substrate specificities. It has been assumed, however, that those enzymes grouped under the same category, such as the gelatinases, are functionally interchangeable in that they are known to be nearly identical in their digestion profiles against macromolecular substrates, but responsive to different stimuli due to differences in the regulatory portions of the genes [31,32]. Both gelatinase A and gelatinase B greatly prefer denatured collagen as their protein substrate, and are known to hydrolyze the same battery of matrix components [2]. The present study shows that the preferred cleavage site sequence for gelatinase A is obviously not an optimal site for gelatinase B.

We previously found that hydroxyproline occurs as the fifth residue on either side of the cleavage site in type I gelatin hydrolysis catalyzed by gelatinase A. Interestingly, using synthetic peptides as substrates, systematic substitution first of proline, then alanine, for these hydroxyproline residues resulted in a small, but consistent, increase in K m values. Since substitution of serine and tyrosine in these P5 and P5' sites did serve to significantly lower the K m, it seems possible that the hydroxyl moiety of the hydroxyproline plays a favorable role in cleavage site specificity. Perhaps the reason that substrates with hydroxyproline in those positions do not bind better is because the bulky hydroxyproline causes steric hindrance that diminishes the effect of the hydroxyl group. Clearly a positive charge such as lysine in the P5 and P5' positions has a detrimental effect on both affinity and tumover, while a bulky hydrophobic group slightly lowers turnover rate.

An intriguing hypothesis is that the bracketed hydroxyprolines serve as recognition sites that allow the enzyme to attach to the end(s) of gelatin a-chains. As shown in Fig. 3, the molecule is not split randomly, but larger peptide products are gradually broken down to smaller ones. Perhaps bracketed hydroxyproline residues allow the enzyme to move down the chain from site to site, cleaving at favorable positions. Experiments to explore this hypothesis are in progress.

When the same set of substrates was tested using gelatinase B, the results were much more variable, with an approximate sevenfold range in both K m and Vma x. Al- though no pattern of substrate preference was apparent from these studies with systematic substitutions at P5 and P5', the differences between gelatinases A and B were clearly shown. For example, the only effect on gelatinase

266 T. Xia et al. / Biochirnica et Bioph3:~ica Acta 1293 (1996) 259-266

A kinetics with alanine substitutions at those sites was a 33% increase in Km. In contrast, for gelatinase B, the K m

was lowered 33% with an accompanying fourfold decrease in Vma x. Again, for gelatinase A the effect of carboxyl- terminal charge on the hexapeptide was to halve the Vma x. However, for gelatinase B. the presence of a negative charge in that position lowered the cleavage rate to the point where it could no longer be considered a substrate. While the use of synthetic peptides is a useful approach for designing both substrates and inhibitors, it seems reason- able to conclude from these results that a peptide useful tor probing cleavage site specificity for one metalloproteinase is of little use for another, even within a group with as much homology as the gelatinases. A similar conclusion was recently reached in a kinetic study comparing interstitial collagenase and gelatinase B using synthetic substrates developed for interstitial collagenase [33].

Using tht. assay that measures production of trichloroacetic acid soluble peptides from radiolabeled gelatin, gelatinase A was found to hydrolyze type I gelatin about 2.5-times as rapidly as gelatinase B. Inspection of Fig. 3 shows that even the pattern of gelatin cleavage differs greatly between the two gelatinases. It has been observed in this and other laboratories [34] that gelatinase B hydro- lyzes type IV collagen much more rapidly than gelatinase A. These differences in macromolecular substrate specificity taken together with the disparities observed using peptide substrates leads us to conclude that these enzymes may serve different functions in matrix remodeling. For detailed studies of cleavage site specificity, peptides based upon the cleavage sites in type IV collagen may be the most useful for gelatinase B. Such studies are currently in progress.

Acknowledgements

We are very grateful to Sharon Favors for her excellent technical assistance, and to Ginger Roberts for help in preparation of this manuscript. We also thank Dr. Gregory Grant for helpful suggestions. This work has been sup- ported by NIH grant AR12129 and AR07824.

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comparison of cleavage site specificity of gelatinases a and b using collagenous peptides

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