Cysteine Mutations in the MAM Domain Result in Monomeric Meprin and Alter Stability and Activity of the Proteinase*

Meprins are oligomeric, glycosylated cell surface or secreted metalloendopeptidases that are composed of multidomain disulfide-linked subunits. To investigate whether subunit oligomerization is critical for intracellular transport or for the enzymatic and/or physical properties of the proteinase, specific cysteine residues were mutated, and the mutants were expressed in 293 cells. Mutation of mouse meprin (cid:97) Cys-320 to Ala in the MAM domain (an extracellular domain found in meprin, A-5 protein, and receptor protein-tyrosine phosphatase (cid:109) ) resulted in expression of a monomeric form of meprin, as determined by SDS-polyacrylamide gel electro- phoresis and nondenaturing gel electrophoresis. The monomeric subunits were considerably more vulnera- ble to proteolytic degradation and heat inactivation in vitro compared with the oligomeric form of the enzyme. Proteolytic activity of the monomeric meprin using a bradykinin analog or aminobenzoyl-Ala-Ala-Phe- p -ni- troanilide as substrate was similar to that of disulfide-linked oligomeric meprin; however, activity against azo- casein was markedly decreased. Mutation of another cysteine residue in the MAM domain (C289A), predicted to be involved in intrasubunit disulfide bridging, resulted in disulfide-linked oligomers and monomers. These results indicated that this mutant was capable of forming intersubunit disulfide bonds but less efficiently than wild-type meprin subunits. Mutant C289A also re-tained activity toward peptides but not the protein sub- strate

Meprins are cell surface and secreted metalloendopeptidases that have been characterized in renal and intestinal brush border membranes of mice, rats, and humans (1)(2)(3)(4)(5)(6)(7). Active and latent forms of meprins have been found in mammalian tissues, and azocasein has generally been used as a good protein substrate for these enzymes (8). Meprin A (EC 3.4.24.18) from mouse and rat kidneys is the most thoroughly investigated isoform; it hydrolyzes a variety of biologically active peptides such as bradykinin, angiotensins, glucagon, luteinizing hormone-releasing hormone, parathyroid and melanocyte-stimulating hormone and is implicated in the processing and degradation of membrane-bound and matrix proteins (7)(8)(9)(10)(11)(12).
Meprins are proteases that are unique in their oligomeric structure. They are dimers of disulfide-linked dimers that are highly glycosylated and can exist as homo-or heterooligomers (13,14). The oligomers are composed of ␣ and/or ␤ subunits where the ␤ subunits are type I integral membrane proteins, and the ␣ subunits are associated with ␣ or ␤ subunits through disulfide bridges or noncovalent associations (14,15). The fulllength cDNA sequences of the ␣ and ␤ subunits are about 45% identical. They predict similar domains for ␣ and ␤ including, from the NH 2 terminus, signal and prosequences and protease, MAM, X, EGF 1 -like, transmembrane, and cytoplasmic domains (2, 3) (see Fig. 1, top, for diagram of ␣ subunit domains). The MAM domain consists of 170 amino acids; it was identified by computer analyses of protein sequences and termed MAM (an acronym for meprin, A-5 protein and receptor protein-tyrosine phosphatase ) for the proteins originally found to contain this sequence (16). The ␣ subunit contains an I (inserted) domain, between the X and EGF-like domain, not present in the ␤ subunit. In spite of the similarities, ␣ and ␤ have different substrate specificities; they undergo different posttranslational modifications during biosynthesis, and their tissue-specific expression is regulated independently. For example, meprin A (isoforms containing the ␣ subunit) has activity against peptides such as bradykinin and synthetic arylamides, whereas meprin B (the homooligomer of ␤ subunits; EC 3.4.24.63) has no activity against these substrates (1,8). Mouse kidney meprin ␤ subunits retain their COOH-terminal transmembrane domain, whereas ␣ subunits are totally extracellular as a consequence of proteolytic processing during biosynthesis at or near the I domain (15). Many mouse strains express both ␣ and ␤ subunits in kidney proximal tubule cells, whereas some inbred strains express only ␤ in adult kidney (2,3). Thus meprin isoforms are regulated at both the transcriptional and post-translational levels differentially.
The related crayfish protease astacin (EC 3.4.24.21), which in its mature form is comprised of only the protease domain, is a monomeric enzyme (17). In contrast, all mature secreted and membrane-associated forms of the meprin ␣ subunit contain the protease, MAM, and X domains. The functions of the latter two domains are unknown, and no matching sequences for the X domain have been identified in the data banks. MAM domains, however, are found in otherwise unrelated proteins, such as enteropeptidase, tyrosine phosphatase and , and A5 protein and have been suggested to serve as "adhesion" domains (16, 18 -20). There is some evidence that indicates that MAM domains can mediate specific noncovalent interactions between the tyrosine phosphatase and receptors (21). Comparisons of MAM domains of the different proteins indicate that they contain four conserved cysteine residues. Meprin MAM domains contain a fifth cysteine residue not present in the MAM domains of the other proteins. On the basis of these comparisons and other considerations, it seemed reasonable to hypothesize that the fifth cysteine of the MAM domain is involved in covalent interactions between the meprin subunits; this proposition is tested by mutational analyses herein.
The unusual oligomeric structure of meprins evokes questions of the necessity of oligomers and disulfide bridges for the function and structure of the enzymes. Does the oligomeric structure affect activity? What is the role of the disulfide bonds? Is covalent or noncovalent oligomerization a prerequisite for intracellular transport? Do the interactions of the COOH-terminal domains of the mosaic proteins affect the activity or stability of the enzyme? The present work indicates that oligomerization (covalent or noncovalent) does not affect the secretion or specific activity of meprin ␣ subunits against peptides but greatly affects vulnerability of the subunits to proteolytic degradation and heat inactivation and activity against protein substrates.

EXPERIMENTAL PROCEDURES
Plasmid Construction and Mutagenesis-The pcDNA I/Amp (Invitrogen) plasmid expressing full-length wild-type mouse meprin ␣ subunit cDNA was described previously (15). The COOH-terminal truncation mutant was generated by the polymerase chain reaction using a mutagenic antisense primer that changed the codon for Cys-571 to a stop codon. Meprin ␣ cysteine mutants were constructed according to the method of Deng and Nickoloff (22) using the Transformer site-directed mutagenesis kit (Clontech). Two separate mutagenic primers were designed to target either the Cys-289 or the Cys-320 codon. The selection primer was designed to change a XhoI site in the multiple cloning site of pcDNAI/Amp to a SalI site. The same selection primer was used together with either one of the two mutagenic primers in the mutagenesis reactions. The presence of an Ala codon at position 289 or 320 was confirmed by DNA sequencing.
Tissue Culture and Transfection-Human 293 cells (ATCC 1573 CRL) from an adenovirus-transformed kidney cell line were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 50 units/ml penicillin and 50 g/ml streptomycin (complete Dulbecco's modified Eagle's medium) in a 37°C incubator with 5% CO 2 . The recombinant wild-type or mutant meprin ␣ subunits were expressed in human 293 cells after transfection by the calcium phosphate precipitation method using 10 g of expression plasmid and 1 g of helper plasmid pVA1/100-mm tissue culture plate (23,24). Cells were grown to approximately 90% confluency by overnight incubation in complete Dulbecco's modified Eagle's medium. Then the medium was replaced with serum-free Opti-MEM (5 ml/plate) and returned to the incubator for an additional 48 h. The tissue culture media were collected, cleared of cell debris by centrifugation at 16,000 ϫ g for 20 min, and concentrated 10-fold to 500 l/plate using Centriprep-30 and Microcon-30 concentrators (Amicon, Inc.). The samples were stored at Ϫ70°C until analysis.
SDS-PAGE and Immunoblotting-Electrophoresis was carried out according to the method of Laemmli and Favre (25) on 7.5, 9, or 12% polyacrylamide gels. Immunoblotting was performed as described previously (15). The proteins were probed with one of two different anti-bodies, which had been produced by injection of meprin antigen into rabbits. In one instance, the purified mouse meprin had been deglycosylated prior to injection (referred to as anti-deglycosylated ␣ antibody); the second antibody was produced in response to native meprin antigen (anti-␣ antibody). Detection was accomplished using the enhanced chemiluminescence detection method (Pierce).
Trypsin and Arg-C Treatment-Trypsin (Life Technologies, Inc.) was added to the expressed protein at a final concentration of 5, 10, 20, or 40 ng/l in 20 mM Tris-HCl, pH 7.5. After incubation at 37°C for 30 min, soybean trypsin inhibitor (Sigma, Type II-S) was added at a 2-fold excess of trypsin. Samples were incubated at 25°C for 15 min and then kept at 4°C until analysis. For meprin activity determinations, a trypsin concentration of 10 ng/l was used to activate the protease.
Endoproteinase Arg-C (Boehringer Mannheim) was used to activate meprin subunits at a concentration of 40 ng/l. After 30 min at 37°C, the reaction was stopped with 0.3 mM 3,4-dichloroisocoumarin.
Nondenaturing Gradient Gel Electrophoresis-Meprin A, purified from ICR mouse kidneys, expressed wild-type ␣, and the two ␣ cysteine mutant proteins were subjected to electrophoresis on nondenaturing gradient (3-20%) gels following the method of Cotton and Jennings (26). Electrophoresis was carried out at 200 V for 6 h.
Activity Determinations-Azocaseinase activity was measured as described previously (1,8). Activity toward the fluorogenic bradykinin analog 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Lys(Dnp)-Gly-OH, where Dnp is dinitrophenyl (BKϩ), obtained from Dr. Graham Knight of Strangeways Research Laboratory, Cambridge, UK, was determined using a Hitachi F2000 fluorescence spectrometer. The fluorometric assays were performed at 30°C in 50 mM ethanolamine HCl buffer, pH 8.7, in the presence of 4% dimethyl sulfoxide. The assay volume was 300 l, and the final concentration of the BKϩ substrate was 10 M. Excitation and emission wavelengths were 320 and 417 nm, respectively. Activity against aminobenzoyl-Ala-Ala-Phep-nitroanilide (ABz-AAF-pNA; Bachem Bioscience) was measured as for BKϩ except that the substrate was solubilized in buffer containing N,N-dimethylformamide (2% in the final reaction mixture), the substrate concentration was 84 M, and the excitation and emission wavelengths were 340 and 420 nm, respectively.

RESULTS
Rationale for Mutagenesis Studies-There are 19 cysteine residues in the meprin ␣ subunit (Fig. 1, top). Of those, four cysteines are in the protease domain, and they are highly conserved in all members of the astacin family of metalloendopeptidases (27). In astacin, they are known to be involved in intradomain disulfide bridges, and this is most probably true and is important for the proteolytic activity of all eukaryotic members of the family (17,27). There are five Cys residues in the MAM domain; four of them are conserved in all other MAM domains, and those cysteines are most likely in intradomain bridges because they are part of extracellular proteins that do not have covalent interactions with other proteins (16). The fifth Cys in the meprin MAM domain, Cys-320 in ␣, is unique to meprins and is conserved among the six meprin subunits cloned to date (␣ and ␤ subunits of mice, rats, and humans). There are two cysteines in the X domain of ␣ subunits, Cys-571 and Cys-573; these two are not conserved in meprin ␤ subunits. There are six Cys in the EGF-like domain; by analogy with other EGF-like units, all are intradomain bridged (28). There is one Cys in the signal sequence that is cleaved off during biosynthesis and one in the COOH-terminal transmembrane domain of meprin ␣ that is removed during maturation (3,14). Therefore, the Cys residues that are the most promising candidates for formation of disulfide bridges between mature subunits are Cys-320 of the MAM domain and Cys-571 and Cys-573 of the X domain.
Cys-571 and Cys-573 Are Not Required for Disulfide Linkage of ␣ Subunits-Previous studies had shown that transcripts truncated just after the X domain were secreted as disulfidelinked dimers (15). The two cysteine residues of the X domain that were candidates for intersubunit disulfide bridges are present near the COOH terminus of the X domain. Truncated subunits that were terminated immediately NH 2 -terminal to Cys-571 and Cys-573 were expressed in 293 cells (Fig. 1). The secreted truncated meprin ␣ protein, like the wild type, was predominantly a disulfide-linked oligomer as assessed by mobility after SDS-PAGE. Therefore, Cys-571 and Cys-573 are not essential for covalent interactions between subunits. The truncated mutant was expressed at high levels and, when activated, had a specific activity for the substrate azocasein that was comparable with wild type (data not shown). The level of expression of wild-type and truncated mutants varied from one experiment to another; the higher level of mutant compared with wild type observed in Fig. 1 was not consistently found in other experiments.
Mutagenesis of Cys-320 to Alanine Results in Monomeric Meprin ␣; Intersubunit Disulfide Bonds Are Not Required for Intracellular Transport-When the C320A mutant was transfected into 293 cells, the mutants were secreted as monomers (Fig. 2). The mutant protein had the same mobility when subjected to SDS-PAGE whether or not ␤-mercaptoethanol was present. Therefore, intersubunit S-S bridging was eliminated, and Cys-320 is likely responsible for intersubunit S-S bridging. The monomeric form of meprin was secreted at rates comparable with wild-type meprin, indicating covalent dimerization is not necessary for transport or secretion.
To assess the effect of eliminating an intramolecular disulfide bond in the MAM domain, Cys-289 was mutated to an alanine. The resulting mutant protein was also secreted from cells but yielded a mixture of monomers and covalently linked dimers as assessed by SDS-PAGE in the absence of ␤-mercaptoethanol (Fig. 2). One interpretation of these data is that the C289A mutation allowed covalent S-S bridging between subunits but that covalent dimerization was a less efficient process with the mutant compared with wild-type protein. The data lend further support to the conclusion that Cys-320 is likely responsible for intersubunit S-S bridging.
Cys Mutations in the MAM Domain Affect Glycosylation of Meprin ␣-The mobility of both cysteine mutants when subjected to SDS-PAGE appeared to be somewhat decreased compared with wild-type protein. To determine whether this mobility change was due to glycosylation differences, the wild-type and mutant proteins were deglycosylated with endoglycosidases F and H and subjected to SDS-PAGE as described previously (15). Treatment of the proteins with endoglycosidase F, which removes high mannose and most complex N-linked oligosaccharides, decreased the molecular masses of all the proteins to approximately 67 kDa and eliminated the mobility differences between the proteins (data not shown). All three proteins were resistant to endoglysidase H, indicating they were complex-glycosylated. These data indicate that both cysteine mutations altered the complex glycosylation patterns.
Cys Mutations in the MAM Domain Affect the Noncovalent Oligomerization of Meprins-Previous work has established that the covalently linked dimers of meprin ␣ associate noncovalently with other dimers to form tetramers and higher oligomeric complexes (14). In addition, the MAM domain has been implicated as an adhesion domain (16,21), and in spite of the fact that the cysteine mutants formed monomeric units in SDS gels, it is possible that they exist as oligomers under nondenaturing conditions. To determine the monomeric/oligomeric state of native cysteine mutant proteins, they were subjected to electrophoresis on nondenaturing gradient gels (Fig. 3). Under the conditions used, native proteins could be separated mainly on the basis of size (26). These data show that the C320A mutant protein is a monomer under nondenaturing conditions, with an apparent molecular mass of about 130 kDa. The observed subunit molecular mass in the nondenaturing gel was larger than that observed in SDS-PAGE (95 kDa); however, the value is consistent with previous results for monomeric meprin subunits in this gel system (14). The wild-type ␣ protein exists predominantly in a higher oligomeric state, and the C289A mutant protein is a mixture of monomers and oligomers (dimers) under these conditions. Meprin A, purified from

FIG. 2. Expression of meprin ␣ cysteine mutants in 293 cells.
Wild-type (wt) or mutant meprin ␣ subunits in which specific Cys residues were altered to Ala (C289A and C320A mutants) were transiently expressed in 293 cells. The tissue culture media containing the secreted expressed subunits were subjected to SDS-PAGE (7.5% gels) in the presence or absence of ␤-mercaptoethanol (␤-ME); immunoblots were performed using anti-meprin ␣ antibodies. mouse kidney (which contains ␣ and ␤ subunits), exists mainly as tetramers and higher oligomers. Thus, there is no evidence for noncovalent interactions of the mutant subunits that are not covalently linked.
Cys Mutations in the MAM Domain Affect Activity against Azocasein but Not BKϩ and ABz-AAF-pNA and Decrease Resistance of the Subunit to Proteolysis and Heat Inactivation-To determine whether the mutants correctly fold into catalytically active conformation, proteolytic activity was measured for the wild-type and mutant meprins. Meprin subunits are secreted as inactive proproteases in cultured cells and have to be treated with trypsin-like proteinases to remove the prosequence and activate the protein (1,15). Trypsin activation decreases the molecular mass by approximately 5 kDa and results in a mobility shift on SDS-PAGE (Fig. 4). Under conditions that were usually used to activate wild-type meprin ␣ in the culture medium (40 ng of trypsin/l), the cysteine mutants were totally degraded. Further investigations of the susceptibility of the proteins to trypsin indicated that the mutants were more susceptible to proteolytic degradation than the wild type (Fig. 4). Conditions were found (5-10 ng of trypsin/l of sample) where the mutants were activated but not degraded. Alternatively, the protease Arg-C (40 ng/l) could be used to activate wild-type and mutant enzymes without degrading the bulk of the protein (data not shown).
The cysteine mutants and wild-type meprin had comparable specific activities against the bradykinin analog and ABz-AAF-pNA (Table I). Analysis of the catalytic efficiencies (k cat /K m values) using the BKϩ substrate also indicated that the mutant proteins were as active as wild-type proteins. Activity of the mutant subunits against azocasein, however, was markedly decreased. No activity of the mutants could be detected with azocasein as substrate at 30 or 37°C; at these temperatures at least 1,000 units/mg would have been measurable. The peptide-degrading activity of wild-type and mutant meprins was stable at 30°C over 30 min, the same conditions under which azocaseinase activity was measured. The results shown on Table I were typical for several different transfection preparations.
The recombinant meprins and purified kidney meprin A were also tested for activity against gelatin on zymgraphs (29). Purified kidney meprin and the recombinant wild type degraded gelatin, whereas no gelatinase activity was observed for the cysteine mutants (data not shown). Because the cysteine mutant proteins were considerably more vulnerable to proteolytic degradation than the wild-type protein, the vulnerability of the proteins to heat inactivation was further tested (Fig. 5). The cysteine mutant proteins were inactivated, as determined by activity against BKϩ, much faster than wild-type protein at temperatures of 40°C and above. Half the activity of the cysteine mutant proteins was lost in 2 min at 40°C, whereas the half-life for the wild-type protein Expressed wild-type (wt) or mutant meprin ␣ subunits were incubated for 30 min at 37°C with trypsin at concentrations of 5, 10, 20, or 40 ng of trypsin/l. The trypsin was then inactivated by incubation for 15 min at 25°C with a 2-fold excess of soybean trypsin inhibitor. The trypsintreated samples were subjected to SDS-PAGE (7.5% gels) in the presence of ␤-mercaptoethanol for 45 min. Then varying amounts of purified meprin A were added onto the same gels, and electrophoresis was continued for 30 min (shown in top gel only). Meprin standards in lanes 1-5 contained 12.5, 25, 50, 75, and 100 ng, respectively. The proteins were immunoblotted with the anti-meprin ␣ antibody, and the amounts of trypsin-treated meprin in each lane were quantitated by densitometry using the purified meprin A samples as standards. The results shown are typical of four separate experiments.

TABLE I Proteolytic activity of the wild-type and mutant meprins
Secreted wild-type (wt) cysteine mutants and meprin A, purified from ICR mouse kidney, were assayed for proteolytic activity after activation with Arg C using fluorogenic ABz-AAF-pNA, or azocasein as described under "Experimental Procedures." For specific activity calculations, the amount of meprin protein in each sample was determined by immunoblotting and densitometry. BKϩ and ABz-AAF-pNA specific activity units are mol substrate cleaved per min per mg of meprin; azocaseinase activities are in units/mg of meprin.  at this temperature was approximately 90 min. The protein concentrations of the meprin subunits, as determined by Western blotting, did not change during the course of incubations, therefore the observed inactivation was due to a loss of specific activity of the proteins. DISCUSSION This work demonstrates that the structure of the MAM domain and/or the oligomeric state of the meprin A is critical for stability of the meprin protein and for activity of the enzyme against proteins. The results support the hypothesis that Cys-320 of the MAM domain is essential for covalent interactions of the subunits; mutation of this residue to an alanine completely eliminated the formation of dimers. The fact that mutation of another cysteine (C289A) in the MAM domain, which is likely involved in the conserved structure of this domain, allowed formation of some dimers but also resulted in dramatic effects on oligomerization, activity, and stability of the subunit indicates that MAM domain structure is a critical determinant of the physical/chemical characteristics of the enzyme. It is not possible to determine at this point whether the MAM domain structure itself or the state of oligomerization of the protein affects the stability and activity of the protease domain because changes in one are coupled to the other.
The work clearly shows that MAM domain cysteine mutations resulted in significant structural changes in the meprin protein. However, the fact that activity of the enzyme against the peptide substrate was not affected and that the amount of protein synthesized, COOH-terminally processed, transported, and secreted by cells was not significantly altered indicates that the protein was folded to some extent and transported as its wild-type counterpart. The protein was not degraded by endoplasmic reticulum or other subcellular compartment proteases as many unfolded or inappropriately assembled proteins are (30). This is consistent with the concept that domains of mosaic proteins form modules that fold independently. The contrast of the in vivo stability with in vitro instability may reflect different environments and interactions with other proteins. The folding of monomers in vivo is likely accompanied by interactions with chaperones and transport molecules. In vitro the protein seems to depend more on interactions with like molecules to form oligomers. It is of interest that wild-type meprin ␣ subunits form very high molecular weight complexes and that meprin A isolated from kidney forms tetramers and higher order complexes. The latter finding was observed previously in ultracentrifugation studies of purified preparations of meprin A where the protein behaved mainly as a tetramer but also associated into higher oligomeric forms (14). The cysteine mutants do not form higher oligomers, indicating that structural alterations of the MAM domain disrupt noncovalent subunit interactions as well as covalent bridges. It is possible that the covalent intersubunit disulfide bridges stabilize units to allow noncovalent interactions of the subunits or domain swapping (31). Whatever the mechanism, it is clear that the MAM domain acts as an adhesion domain and stabilizes subunit interactions, protein structure, and the function of the enzyme. The role of the disulfide interactions between subunits may be to stabilize subunit interactions so that higher oligomeric forms are favored.
MAM domains have been identified recently as distinct protein modules in several proteins, and one study has specifically addressed the role of this domain in protein structure and function. Zondag et al. (21) demonstrated that the MAM domains of receptor protein-tyrosine phosphatase and are essential for mediating specific homophilic cell-cell interactions in transfected nonadherent insect cells. The present study provides an example where the MAM domain mediates specific homooligomeric interactions of a secreted protein. In vivo, MAM domain interactions result in the concentration of meprin ␣ subunits at the cell surface in association with integral membrane ␤ proteins.
The effect of MAM domain cysteine mutations on the ability of meprin to hydrolyze azocasein implicates complex domain or subunit interactions for this activity. It is possible that there are interactions between subunit active sites for protein hydrolysis or that there is a different conformation of the active site in monomers and oligomers. There has been no indication of cooperative interactions between subunits in kinetic studies of meprin A (8). However, studies with inhibitors have yielded unexpected results that may indicate complex interactions at the active sites. For example, the inhibitor actinonin is a simple competitive inhibitor of astacin but shows complex inhibition patterns with meprin A (competitive/noncompetitive), and products of bradykinin hydrolysis that would be predicted to be simple competitive inhibitors show mixed inhibition patterns for meprin A.
The differences in the glycosylation of the mutants and wildtype meprin subunits could contribute to differences in resistance to proteases and oligomerization. There is at least one N-linked glycosylation site in the MAM domain near Cys-320; the Cys mutations could affect the interaction of that site with cellular glycosidases, which could in turn expose normally protected peptide bonds to proteases. Oligomerization to high molecular weight complexes may also confer increased proteolytic resistance by protecting areas of the protein that are vulnerable to proteases. Native forms of meprin are quite resistant to proteases; for example, functionally intact meprin can be solubilized from membranes by treatment with high concentrations of papain or trypsin/toluene. Many brush border enzymes exist as oligomers, and a standard treatment for solubilization of the bulk of these enzymes is to incubate membranes with proteases. Intracellular enzymes may also be stabilized by oligomerization, as in the case of cathepsin E. The latter aspartic proteinase is a homodimer in which the subunits are linked by a disulfide bond. Mutation of a specific cysteine residue resulted in a monomeric, active enzyme with decreased thermal stability (32). The function of the oligomerization for many FIG. 5. Heat inactivation of wild-type and mutant meprins. Recombinant wild-type (f) or mutant (q, C320A; å, C289A) meprins were activated by treatment with trypsin (10 ng of trypsin/l) as described under "Experimental Procedures." The samples were then incubated at 40°C for the indicated time periods. Samples were removed from the incubation mixture and assayed using BKϩ as substrate at 30°C.