Interaction of human rheumatoid synovial collagenase (matrix ...

Vol. 264, No. 15, Issue of May 25, pp. 8779-8785, 1989 Prrnted in U . S . A .

THEJOURNAL OF BIOLOGICAL CHEMISTRY 01989 hv The American Society for Biochemistry and Molecular Biology, Inc

Interaction of Human Rheumatoid Synovial Collagenase(Matrix Metalloproteinase 1) and Stromelysin (MatrixMetalloproteinase 3) with Human a2-Macroglobulinand Chicken Ovostatin BINDING KINETICSANDIDENTIFICATION

OF MATRIXMETALLOPROTEINASE

CLEAVAGE SITES*

(Received for publication, November 21, 1988)

J a n J. Enghild, Guy Salvesen, Keith Brew$,and Hideaki Nagasetll From the Departmentof Pathology, Duke University Medical Center, Durham, North Carolina 27710, the $Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, and the §Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103

form gelatin, collagenase has The homologous proteinaseinhibitors,human a2- substrates are heat-denatured to macroglobulin (a2M) and chicken ovostatin, have been little or no action on them, indicating that thehelical struccompared with respect to their “bait” region sequences ture of collagen molecules is important for enzyme recogniand interactions with two human matrix metallopro- tion. On theotherhand, stromelysin (matrix metalloproteinases, collagenase and stromelysin. A stretch of 34 teinase 3), another metalloproteinasesecreted from the conamino acid residues of the ovostatin bait region se- nective tissue cells, has a wide spectrum of action on extraquence was determined and the matrix metalloprocellular matrix macromolecules. It digests proteoglycans, fia bronectin, laminin, type teinase cleavage sites identified. Collagenase cleaved IV collagen, and gelatins and removes X-Leu bond where X was unidentified, whereas the the NH,-terminal propeptides of type I procollagen (3, 7, 8). major cleavagesite by stromelysin wasat theGly-Phe Stromelysin also digests casein and reduced carboxymethylbond, 4 residues on the COOH-terminal side of the collagenase cleavage site. Collagenase cleavedthe a2M ated transferrin (3,7). Collagenase and stromelysin are about bait region at the Glye79-Le~680 bond, and stromelysin 55% identical in sequence (9-12), but stromelysin does not a t Glye7s-Le~s80andPhe684-Tyres5bonds. Sequence digest interstitial collagen types I and I1 althoughithas of members of the Q- limited activity on type I11 collagen.’ The enzymic activities similarity in the bait region of these matrix metalloproteinases are thoughtbetoregulated macroglobulin family is strikingly low. The kinetic or body fluid, in particular a2M is a 150-fold better substrate by inhibitors present in the tissue studies indicate that for collagenase than typeI collagen. Structural predic- by a tissue inhibitor of metalloproteinases (TIMP)3which is a also secreted from most of the connective tissue cells (13-15). tions based onthe bait region sequences suggest that collagen-like triple helical structure is not a prerequi- However, the level of TIMP in body fluids such as plasma site for the efficient bindingof tissue collagenase to a and rheumatoid synovial fluid is very low, and the predomisubstrate. The bindingof stromelysin toa2M is slower nant inhibitory activity for collagenase resides in a,M (16, than that of collagenase. Stromelysin reacts with ovo- 17). Thus, a,M appears to be one of the majorregulatory statin even more slowly.Despite the preference of factors for the activity of matrix metalloproteinases in the chicken ovostatin for metalloproteinases, human a 2 M , extracellular space. a far less selective inhibitor, reacts more rapidly with a,M is amajor plasmaproteinaseinhibitor with M , = collagenase and stromelysin. These results suggest that 725,000 (18, 19). The inhibitorymechanism of a,M is unique. a 2 M may play a n important role in regulating the ac- Unlike many other proteinase inhibitors, it inhibits almost tivities of matrix metalloproteinases in the extracelall endopeptidases from all four catalytic classes of proteinlular space. (20). The reaction of a

Connective tissue cells produce a group of related metalloproteinases that digest various extracellular matrix macromolecules (1-3). One of them, collagenase (matrix metalloproteinase l),’specifically digests collagen types I, 11, and I11 at a Gly-(Ile or Leu) bond located in the triplehelical region approximately three-fourths of the distance from the NH, terminus to theCOOH terminus (4-6). However, when these

* This work was supported by National Institutes of Health Grants AR 39189, CA29589, and GM 21363. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll To whom correspondence should he addressed Dept. of Biochemistry, Universityof Kansas Medical Center, 39th and Rainbow Blvd., Kansas City, KS 66103. The numerical names formatrix metalloproteinasessecreted from connective tissue cells followthe proposal by Okada et al. (3).

ases regardless of their specificities proteinase and a,Mrequires proteolytic attack of the enzyme on a particular locus located near the middle of the quarter subunit, theso-called “bait” region. The cleavage of a peptide bond in this region triggers a conformational change in the a,M molecule that,inturn,entrapsthe enzyme without blocking the active site (21). Thus, the enzyme within the complex is able to catalyze the hydrolysis oflow molecular weight substrates but is restricted from reaction with large protein substrates. Although the specificity of collagenase is limited to native interstitial collagens, the ability of a,M to inhibit collagenase indicates that a,Mserves as a collagenase substrate. Earlier work of Werb et al. (22) indicates that this reactiontakes place very slowly. On theotherhand, our

* Gunja-Smith, Z., Nagase, H., andWoessner, J. F., Jr. (1989) Biochem. J.258, 115-119. The abbreviationsused are:TIMP, tissue inhibitorof metalloproteinases; a 2 M , a2-macroglohulin;APMA,4-aminophenylmercuric acetate; SDS-PAGE,sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

8779

8780

Binding of Matrix Metalloproteinases Macroglobulins to

kinetic studies of the binding of rabbit synovial collagenase generated by stromelysin were conducted. The major cleavage to chicken ovostatin, ana,M-like protein fromegg white (23), site of stromelysin in ovostatin was found at the Gly-Phe demonstratedthatovostatinisan excellent substratefor bond 4 residueson the COOH-terminalside of the collagenase collagenase(24)in spite of the fact that the specificity of cleavage site (Fig. 3). Inthe second experiment a minor mammalian collagenase is believed to be limited to helical sequence starting at the same site as collagenase was observed, regions in collagen typesI, 11, and 111. This observation but the major sequence started with the phenylalanine resisuggested that the triple helices of collagen might not be an due. absolute requirement for thebinding of collagenase to a Thermolysin (23) and Serratia metalloproteinase(45) have substrate, since it is very unlikely that ovostatin contains a beenshown to be stoichiometrically inhibited by chicken collagen-type helix in its “bait” region. Thus,information ovostatin. The resultsof sequence analyses of the similarM, regarding the aminoacid sequence of the collagenase-suscep- = 88,000 fragmentsgenerated by these enzymes are also tible bait region of ovostatin and a,M may be important in summarized (Fig. 3). Although chymotrypsin is not inhibited developing an understandingof the interactionof collagenase by chicken ovostatin (241, the initial reaction of chymotrypsin with its substrates. with ovostatin generates the M, = 88,000 and M , = 78,000 We report here the amino acid sequence of a &residue fragments (24). Sequence analyses of the M , = 88,000 fragbait region of chicken ovostatin and the locations of collagen- ment are also shown (Fig. 3). From alignment of the NH,ase cleavage sites in both chicken ovostatin and human aZMterminal sequences of the proteolytic fragments generatedby determined using humanrheumatoid synovial collagenase. five different enzymes, a stretch of 34 amino acid residues in The sites of cleavage in the two inhibitors by stromelysin, a the proteinase-susceptible “bait”region of chicken ovostatin metalloproteinase homologous to collagenase, have also been was assembled (Fig. 3). Although thereis somesequence determined. Detailed kinetic studies of the bindingof human similarity in the bait region of azM and ovostatin, the level is synovialcollagenase with a2M and ovostatin indicate that quite low (Fig. 4). Out of 34 residues in the bait region of azM is the best substrate for collagenase that has been char- ovostatin, only 8 residues (24%) arefound to be identical with acterized to date. human a2M, 11 residues (32%) with rat aZM, and6 residues (18%)with themajor isoform of a,-inhibitor 3.5 EXPERIMENTAL PROCEDURES4 Binding of Collagenase to azM and Ovostatin-The rate of binding of human rheumatoid synovial collagenase to azM or ovostatin was determined using ‘251-labeledcollagenase. The RESULTS inhibitor was incubated with ‘251-collagenase (8 rg/ml) at Cleavage Sites of a2M by Collagenase and Stromelysin-The 25 “C, and the reaction was terminated by the addition of “bait” region of human a,M has been characterized with 1,lO-phenanthroline and EDTA to give final concentrations various proteinases. However, the sites of azM cleavage by of 17 and8.3 mM, respectively. ‘251-Collagenasebound to a2M connective tissuemetalloproteinases have notbeendeteror ovostatin was quantified by measuring the radioactivity mined, although a collagenasecleavage site has been predictedincorporated into the inhibitor band after electrophoresis of at theGly679-Leu660 bond (40)since the sequence around this the sample on 5% polyacrylamide gels. A typical analysis is bond, Gly-Pro-Glu-Gly-Leu-Arg-Val-Gly, is somewhat simi- shown in Fig. 5. The addition of ‘z51-collagenaseto apM in lar to the collagenase cleavage site in interstitial collagens. the presence of the chelating agentsat the above concentraCleavage of aZM by either collagenase or stromelysin gener- tions completely prevented bindingof the enzyme, indicating ated fragments of M, = 98,000 and M, = 80,000 (Fig. 1).Two that the enzyme reaction was instantaneouslyterminated separate sequence analyses of the upper fragments of M, = (Fig. 5). 98,000 reveal a single sequence of Leu-Arg-Val-Gly-Phe-TyrThe bindingof collagenase t o a2M was very rapid, and the Glu-Ser-Asp-Val, indicatingthat collagenasecleaved the data were analyzed to give an apparentpseudo first-order rate Gly679-Le~680 bond predicted. as On the other hand, the NH2-constant, kaPp,using Equation 3 (Miniprint Supplement). A terminal sequence of the fragment generated by stromelysin plot of the data is shown in Fig. 5. The values of k,, obtained showed two sequences in an equal amount, Leu-Arg-Val-Gly- with various concentrations of a2M inthismanner were Phe-Tyr-Glu-Ser-Asp-Val and Tyr-Glu-Ser-Asp-Val-Met- plotted according to Equation4 (seeplot in Fig. 6A). The line Gly-Arg-Gly, indicating cleavage sites at Gly679-Le~680 and was drawn according to the mediumvalues of K, and kz Phe684-Tyr685 (Fig. 2).Both the collagenase and stromelysin obtained by the method of Eisenthal and Cornish-Bowden cleavage sites in azM are located in the region cleaved by (38). The dissociation constant, K,, and the first-order rate other proteinases (40). constant for the irreversible formation of the a,M-collagenase Sequence Analyses of the Ovostatin "Bait I'Region-Reaction complex, k,, were 171 nM and 0.48 s-’, respectively. The kz/ of chicken ovostatin with human collagenase resulted in the K, value of 2.8 X lo6 M-’ s-’ indicates that azM is approxicleavage of the M, = 165,000 subunit into two fragments of mately 150-fold better a substrate for collagenase compared M , = 88,000 and M , = 78,000 (Fig. 1).Electrophoretic elution with human type I collagen (kcat/Km = 1.8 X lo4 M” s” (see of both bands and subsequent sequence analyses of the frag- Table I)). ments indicated that the fragment of M, = 88,000 was the The rate of binding of human collagenase with chicken COOH-terminal half of the ovostatin subunit and the frag- ovostatin was slower compared with that toa Z M ;the K , and ment of M, = 78,000 the NHZ-terminal half. Two separate the k, values were 323 nM and 5.8 X s-’, respectively sequence analyses of the M, = 88,000 fragment generated by (Fig. 6B). However, the k2/K;= 1.8 X lo3”’ s-’ is comparable collagenase gave a single sequence as shown in Fig. 3, indi- to thek J K , values reported for various types of interstitial cating thatcollagenase cleaves ovostatin subunits at a single collagens (see Table I). site. Similar sequence analyses of the M , = 88,000 fragment Binding of Strornelysin to a2M and Ovostatin-The kinetic constants for the formation of a,M-stromelysin were deterPortions of this paper (including “ExperimentalProcedures,” for collagenase. Although Figs. 1, 3, and 5-7, and Equations 1-4) are presented in miniprint at mined by thesamemethodas the end of this paper. Miniprint is easily read with the aid of a stromelysin has proteolytic activity against various protein standard magnifying glass. Full size photocopies are included in the J. J. Enghild, unpublished data. microfilm edition o f the Journal that is available from Waverly Press.

8781

Binding of Matrix Metalloproteinases to Macroglobulins T . SGT . PA

%"

T.PL,TH.TL.S=l TL CS

CS

collagenaseStromelysin stromelysin

FIG. 2. Collagenase and stromelysin cleavage sites in azM bait region. Human aaM (1 nmol) was incubated with 0.8nmol of collagenase for 2 min at 37 "C, and the reaction was terminated by 1 mM 1,lOphenanthroline. Proteolytic fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described under "Experimental Procedures" and subjected to NH,-terminal sequence analysis. To identify the stromelysin cleavage sites, a2M (300 pmol) was reacted with 206 pmol of stromelysin for 10 min at 37 'C, and the reaction was terminated by 1 mM 1,lO-phenanthroline. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteins were transferred to polyvinylidene difluoride-Millipore Immobilon transfer membrane (35) and subjected to NHa-terminal analysis. The cleavage sites by other proteinases are shown: papain (PA),Streptomyces griseus trypsin (SGT), calf chymosin (CS), bovine chymotrypsin (CT), human plasmin ( P L ) ,bovine thrombin (TH), thermolysin (TL), and subtilisin ( S ) are after Mortensen et al. (40); bovine trypsin (T)and Staphylococcus aureus V8 proteinase ( S P ) after Mortensen et al. (40) and Hall et al. (42); porcine pancreatic elastase ( E ) after Sottrup-Jensen et al. (43); and human neutrophil cathepsin G (CG) and elastase (HLE) after Virca et al. (44).

** * ** Chicken Ovostatin

** * *

* * *

****

LNAGFTASI--HTVALSAEVAREERGKRHILETIRL

Rat ap

KPKVCERLRD----- NKGIPAAY--HLVSQSHMDAFLESSESP-TETRRSY

Human a p

KPKMCPQLQQYEMHGPEGLRVGFYESDVM-GRGHARLVHVEEPHTETVRKY

Ill I

I

I

R a t a, I n h i b i t o r 3

I

I

I I

IIIII

LPWAVKSP----LPQEP-PRKDPPPKDPVIETIRN

FIG. 4. Alignment of ovostatin bait region with human and rat azM and rat al-inhibitor 3. The bait region sequences of human aaM, rat a2M, and rata,-inhibitor 3 (clone pRLA113/2J) are after Sottrup-Jensen et al. (19), Gehring et al. (46), and Braciak et al. (47), respectively. The gaps (-) are introduced to optimize the alignment; * indicates residues where ovostatin is identical with one or more of the other sequences; 1 indicates identical residues in the two a,Ms.

TABLEI Comparison of kinetic parameters for hydrolysis of macroglobulins, collagens, and octapapeptides by human tissue collagenase and stromelysin All the values were obtained at 25 "C exceut for octaueutides (49) at 30 "C. K, or K ,

Substrate Enzyme

@

(A)Tissue collagenase (human) Tissue collagenase (rabbit) Stromelysin (human) (B) Tissue collagenase (human)

a,M (human) Ovostatin (chicken) Ovostatin (chicken) a,M (human) Type I collagen Human Calf Guinea pig Rat Type I1 collagen Human Calf Rat Type 111 collagen Human Guinea pig

0.17 0.32 0.57 0.10

kZ or kcak s" x

la

k,/K, or LtlKrn M' s - ~x 10"

483 0.58 6.0 5.8

280 0.18 1.1 5.6

0.8 0.8 0.9 0.9

0.15 9.5 6.0 5.5

1.8 1.2 0.70 0.60

0.013 2.1 0.047 1.6 0.111.1

0.28 0.75 1.3

1.4 0.7 Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln 0.00603300

160 5.0 200

Ref.

~~~

This study This study 24 This study 48 48 48 48 48 48 48

11 0.72

48 48 49

(le)

Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln 3600 0.0093 (5") Gly-Pro-Gln-Gly-Leu-Ala-Gly-Gln 0.0097 2800

330

49

270

49

270

49

(10")

Gly-Pro-Gln-Gly-Ile-Ala-Gly-Thr0.0112400 ( 1 1")

Gly-Pro-Asp-Gly-Ile-Ala-Gly-Gln

0.0018

49

(8')

The numbers for octapeptides are after Fields et al. (49).

substrates, the rate of binding with a2M was considerably slower compared with thatof collagenase; the K; and k2 values obtained were 104 nM and 5.8 X s-' (k2/Ki= 5.6 X lo4 M-' s-I), respectively (Fig. 6C). The reaction of stromelysin

with chicken ovostatin was even slower. The cleavage of the M , = 88,000 and M , = 78,000 ovostatin subunit into the fragments was not completed even after a 2-h incubation at 37 "C (Fig. 7). The complex formation was not clearly detected

Binding of Matrix Metalloproteinases to Macroglobulins by the gel electrophoresis methodindicatingthat chicken ovostatin is not an effective inhibitor of stromelysin.

8782

Fasman but was predicted to form a coil structure by the DELPHI program. This was also the case for the corresponding sequence in the rat protein. Based on these results, we DISCUSSION propose that the bait region, although perhaps having a loop structure and being flexible and accessible to solvent and The kinetic data presented here demonstrate that the binding of mammalian tissue collagenase to aZM is very rapid. proteinases, does not have any dominant secondary structure of a conventional type. In fact, thehigh level of susceptibility Since proteolytic cleavage of the a2M quartersubunitis prerequisite for a proteinase to bind to a2M, the kz/K,values of a large number of the peptide bonds in this region (Figs. 2 obtained in our study can be compared with kJKm values and 4) would be inconsistent with their involvement in extenreported for human skin collagenase with various types of sive H-bonding arrangementsof the typeproposed by Gettins collagen (48) and synthetic substrates (49). As summarized in et al. (52). The high susceptibility of the Glys79-Le~680 bond Table I, a Z Mserves as a far better substrate than any other in human a2M tocollagenase cannot be readily explained at substrate so far examined with tissue collagenase. The se- present. Similarly, the lower susceptibility of chicken ovoquence analysis of the COOH-terminal fragmentof a2M gen- statin to collagenase, although the kz/Ki values are compaerated by collagenase indicated that this enzyme cleaves the rable to kcat/Kmvalues for type I collagen, must reflect the combination of the sequencein this region and the local predicted Gly679-Le~G80 bond furthest upstream in the bait region sequence. The sequence around the collagenase cleav- conformation of the baitregion. It may be speculated that the is similar toGly- flexible nature of this region allows it to existin a multiplicity age site, Gly-Pro-Glu-Gly-Leu-Arg-Val-Gly, Pro-Gln-Gly-(Ile or Leu)-(Ala or Leu)-Gly-X incollagen, but of conformational stateswhich could fit specificities of various the azM bait region possesses an acidic and a basic amino proteinases. The kinetic parameters for the reactionof stromelysin with acid residue at the P, and P'z sites, respectively (subsites are according to Schechter and Berger (50)). Recent studies by azM and ovostatin indicate that stromelysin binds to these Fields et al. (49) on the sequence specificity of human skin macromolecules at a much slower rate thancollagenase. These fibroblast collagenase using a series of synthetic octapeptides results were unexpected, as stromelysin has a broader speciwith sequences based on potential collagenase sites indicate ficity on various protein substrates (3, 7, 8, 11, 27-29, 54) that aminoacid substitutions influenced the rateof hydrolysis whereas the action of collagenase is limited to interstitial of peptides. Interestingly, the substitution at the Ppsite with collagens. Analyses of the cleavage sites in azM and ovostatin or revealed thatstromelysin hydrolyzes theGlyG79-Le~680 an acidicresidue, aspartic acid (peptide 8 in Table I), debond in a2M at an equal rate and primarily the creased the susceptibility about 3-fold (49). Nevertheless, the Phe684-TyrG85 sequence specificityalone could not accountfor the hydrolysis Gly-Phe bond in ovostatin. A Gly-Phe bond is also present of native interstitialcollagens at a single site. Fieldset al. (49) on the NH,-terminal side of the stromelysin cleavage site in of re-emphasized the view that thelocal conformational features azM,but this bond was not attacked during the formation of the collagen molecule at the cleavage site govern the thestromelysin-a2M complex. Theseresultsindicatethat specificity of collagenase. Indeed, the k,,,/K,,, values for all of stromelysin favors hydrophobic amino acid residues at both the synthetic peptideswere considerably lower than those for the P, and P', sites and that the substrate specificities of collagen types I, 11, and 111 althoughtheir kcat values are collagenase and stromelysin are clearly distinguishable. The higher than those forcollagens (Table I). azM is a better preferential digestion of a bond between the hydrophobic specificity of stromelysin is similar substrate than type I collagen for tissue collagenase by a residues indicates that the factor of about 150. The high susceptibility of a,M to colla- to that of an acid metalloproteinase purified from human genase is also likely t o be controlledby the local conformation articular cartilage, which has been shown to cleave the B of the apM bait region. Gettins and Cunningham (51) have chain of insulin either at theAla-Leu or Tyr-Leu bond (55). Ovostatins belong to the family of a-macroglobulinsas concluded from 'H NMR studies of native and methylaminetreated human apM that the bait region is a flexible loop with indicated by their similar NH2-terminal sequences, quaterhigh mobility. This was extended by Gettins et al. (52), with nary structure, and proteinase inhibition mechanism (24,56so far also presentinallovostatins secondary structure predictionsfrom the aminoacid sequence, 59).A thiolesteris to provide a model for the bait region as a loop composed of characterized (56, 60, 61) with the notable exception of the two antiparallel strands of p structure (residues680-685 and one from chickenegg white (24). Complete primary structures azM (46), and 695-700) separated by a turn, with themajority of proteinase have been reported for human a2M (19,62), rat cleavage sites being located within thetwo strands of p sheet. rat al-inhibitor3 (47), and extensive overall sequenceidentity We have carried out similar secondary structure predictions has been demonstrated, e.g. human and rat azM share 73% 3 are 58% identity (46) and rat a2M and rat a,-inhibitor for the sequences around the bait region in both rat and human a,Ms, as well as for the ovostatin baitregion sequence identical (47). Partialsequence analysis of human pregnancy reported here using both the Chou and Fasman procedure zone protein has alsoshown 68% identityto human a,M (63). regions of these (53) and the DELPHI program (both were utilized on-line However, the sequences aroundthebait through the Protein Identification Resource of the National proteins are highly divergent in comparison to the restof the sequence, having only 10% identical residues (19, 46, 47, 61), Biomedical Research Foundation: programs PRPLOT and and show no significantsequence correlationinthisarea DELPHI). Neither prediction program supported the proposed two-stranded p structure of Gettins et al. (52) for any when compared usingthe programALIGN (64). This diversity of the bait region sequences appears to be a general feature of thethree sequences, although a turncenteredaround residue 678 was predicted for both human and rat a2Ms. A of the proteins in the a-macroglobulinfamily. The stretch of weak p structure prediction was obtained for residues 680- 34 residues in the chicken ovostatin bait region is also very 685 by the Chou and Fasman procedure, but this was not different from other known bait region sequences (Fig. 4 ) . It confirmed by the DELPHI program, while the region corre- is perhaps relevant to this observation that the amino acid sponding to the second strand of Gettins et al. (52), residues residues in the ovomucoid third domain whichform close 695-700 in human azM,showed a higher probability for helix contacts with serine proteinases also show high variability formation than for p structure by the method of Chou and (65). Sequence variations in the bait region may reflect ad-

8783

Binding of Matrix Metalloproteinases toMacroglobulins

aptational changes associated with theemergence of specialized a-macroglobulins that inhibit specific proteinases. Chicken ovostatin has been shown to bindrapidly and tightly to microbial metalloproteinases such as thermolysin(23) and Serratia proteinase (45). T h e binding of human tissue matrix metalloproteinases t o chicken ovostatin is not as efficient as for these microbial enzymes. Furthermore, chicken ovostatin does notinhibitserineandcysteineproteinases (24). In contrast, although aZM binds and forms stable complexes with almost all endopeptidases, Serratia proteinase-a,M complexes are not stable upon prolonged incubation, and about 90% of proteolytic activity can be restored (66). Such aneffect was not seen with ovostatin, as it forms a tight complex with the proteinase (46). Thus, ovostatin may function as an antibacterialagentin egg whitetoprotecttheembryo from infection.On the other hand, the rapid binding of tissue collagenase by a2M suggests that a2Mmay play an important role in regulating tissue breakdownby acting as a n inhibitor of matrix metalloproteinases. Connective tissue cells are capable of producing a tissue metalloproteinase inhibitorof M , = 27,000, T I M P (13-15), and the synthesis of T I M P by the cultured cells can be increasedby the agents or factors which induce the production of matrix metalloproteinases (67, 68). However, competitionexperimentsindicatethat a z M is a much moreeffective inhibitor of collagenase than TIMP(69), suggesting that a2Mmay be the major collagenase inhibitor, especially in inflamed lesions. For example, in rheumatoid synovial fluid, the level of a,M can be0.7-1.0 mg/ml, approximately one-third of the normal plasmallevel (70,71). Deposits of a2M in the rheumatoid synovial lining cells (72) and collagenase-a,Mcomplex in rheumatoid synovialfluid (70, 73) further suggest the importance of a2M in regulatingcollagenolysis. Acknowledgments-We thank Dr. Salvatore V. Pizzo for his encouragement of this work. We are also grateful to Rikako Suzuki and Ida Thdgersen for their expert technical assistance and L. Denise Byrd for typing the manuscript. REFERENCES 1. Vaes, G., Eeckhout, Y., Lenaers-Claeys, G., Francois-Gillet, C., and Douetz, J.-E. (1978) Biochern. J. 1 7 2 , 261-274 2. Murphy, G., Cawston, T. E., Galloway, W. A,, Barnes, M. J., Bunning, R. A. D., Mercer, E., Reynolds, J. J., and Burgeson, R. E. (1981) Biochem. J. 199,807-811 3. Okada, Y.,Nagase, H., and Harris, E.D., Jr. (1986) J. Biol. Chem. 261,14245-14255 4. Miller, E. J., Harris, E. D., Jr., Chung, E., Finch, J. E., Jr., McCroskery, P. A,, and Butler, W. T. (1976) Biochemistry 1 5 , 787-792 5. Hofmann, H., Fietzek, P. P., and Kuhn, K. (1978) J. Mol. Bid. 125,137-165 6. Dixit, S. N., Mainardi, C. L., Seyer, J. M., and Kang, A. H. (1979) Biochemistry 1 8 , 5416-5422 7. Galloway, W. A., Murphy, G., Sandy, J. D., Gavrilovic, J., Cawston, T. E., and Reynolds, J. J . (1983) Biochem. J. 209, 741752 8. Chin, J. R., Murphy, G., and Werb, 2. (1985) J . Biol. Chem. 260, 12367-12376 9. Goldberg, G. I., Wilhelm, S. M., Kronberger, A., Bauer, E. A,, Grant, G. A., and Eisen, A. Z. (1986) J. Biol. Chem. 261,66006605 10. Whitham, S. E., Murphy, G., Angel, P., Rahmsdorf, H.-J., Smith, B. J., Lyons, A,, Harris, T. J. R., Reynolds, J . J., Herrlick, P., and Docherty, A. J . P. (1986) Biochem. J . 240, 913-916 11. Wilhelm, S. M., Collier, I. E., Kronberger, A,, Eisen, A. Z., Marmer, B. L., Grant, G. A,, Bauer, E. A., and Goldberg, G. I. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 6725-6729 12. Saus, J., Quinones, S., Otani, Y., Nagase, H., Harris, E. D., Jr., and Kurkinen, M. (1988) J . Bid. Chem. 263, 6742-6745 13. Vater, C. A., Mainardi, C. L., and Harris, E. D., Jr. (1979) J . Eiol. Chem. 2 5 4 , 3045-3053

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