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J. Biol. Chem., Vol. 280, Issue 14, 13895-13901, April 8, 2005

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Intersubunit and Domain Interactions of the Meprin B Metalloproteinase

DISULFIDE BONDS AND PROTEIN-PROTEIN INTERACTIONS IN THE MAM AND TRAF DOMAINS^*

Faoud T. Ishmael, Vincent K. Shier, Susan S. Ishmael, and Judith S. Bond¶

From the Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Received for publication, December 17, 2004 , and in revised form, February 3, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Meprins, multimeric metalloproteases expressed in kidney and intestinal epithelial cells as well as in certain leukocytes and cancer cells, have the ability to hydrolyze a variety of growth factors, vasoactive peptides, cytokines, and extracellular matrix proteins. The meprin B isoform exists primarily as a cell-surface homooligomer composed of disulfide-linked, multidomain -subunits. To gain insight into how the tertiary and quaternary structure of meprin B affects function, the disulfide-bonding pattern and sites of domain-domain interactions were investigated using sedimentation equilibrium ultracentrifugation, cross-linking, and mass spectrometry techniques. Three symmetrical intersubunit disulfide bonds were identified in the noncatalytic interaction domains; two in the MAM (meprin, A-5 protein, protein-tyrosine phosphatase µ) domain and one in the TRAF (tumor necrosis factor receptor-associated factor) domain. These disulfide bridges are unique for the known homophilic interactions of these domains. Mutation of any of the intersubunit cysteine residues resulted in the inability of meprin B to form disulfide-linked dimers. The four cysteines of the protease domain formed intradomain disulfide bonds. The MAM domain also had one intradomain disulfide bond and one free cysteine. Cross-linking studies of the meprin B dimer with the amine-reactive cross-linker disuccinimidyl suberate revealed inter- and intradomain contacts within the protein, including prosequence-prosequence, protease-TRAF, protease-epidermal growth factor, and TRAF-TRAF interactions. From these observations, a model of the meprin B dimer structure is proposed that provides insight into the relationship between structure and function of this isoform.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Meprins are oligomeric, zinc endopeptidases of the astacin family of metalloproteinases and the metzincin superfamily (1, 2). They are either secreted from or localized to the brush border membranes of kidney and intestinal epithelial cells of humans and rodents and are expressed by leukocytes and cancer cells under some conditions (3, 4). Meprins cleave cytokines, growth factors, bioactive peptides, hormones, and extracellular matrix proteins and have been implicated in inflammatory intestinal disease and in cancer metastases (3–5).

Meprins are unique among mammalian metalloproteinases in that they are composed of multidomain, evolutionarily related and subunits that form disulfide-linked homo- or heterooligomeric dimers (6, 7). The heterodimers tend to form tetramers (₂,₂; ₃,₁), whereas the meprin homodimers form decameric 900-kDa complexes to very higher molecular mass multimers of up to 6 MDa, among the largest extracellular proteolytic complexes known. Only dimers of homooligomeric meprin have been observed thus far. However, the local concentrations of meprin B in kidney brush border membranes have been estimated to be in the high micromolar range, higher than conditions used for in vitro experiments (7, 8). Thus, it is possible that higher oligomers are formed under these conditions.

Although the meprin and subunits are 42% identical at the amino acid level, they differ markedly in their ability to self-associate, in proteolytic processing during biosynthesis in the endoplasmic reticulum, and in substrate specificity (1, 7, 9, 10). For example, meprin has a preference for acidic residues in the P1 and P1' sites of the substrate, whereas meprin selects for small or hydrophobic residues at these sites (10, 11). Multimers containing the subunit (homooligomers or heterooligomers) are designated meprin A (EC 3.4.24.18 [EC] ), whereas homooligomers of the subunit are designated meprin B (EC 3.4.24.63 [EC] ). The cDNA-deduced primary sequences of both subunits predict a similar arrangement of functional domains beginning with an NH₂-terminal signal sequence, a prosequence followed by a protease domain containing the active site, a meprin, A-5 protein, receptor protein-tyrosine phosphatase µ domain (MAM),¹ and a tumor necrosis factor receptor-associated factor (TRAF) domain (see Fig. 1). The MAM domain is present in many cell surface proteins and is thought to be involved in cell-cell adhesion, protein-protein interactions, and signal transduction (12). The TRAF domain is found in a diverse population of intracellular proteins and is also involved in protein associations and signal transduction; meprins are the only plasma membrane and extracellular proteins known to contain TRAF domains (13). Distal to the TRAF domain is an epidermal growth factor (EGF)-like domain, a putative COOH-terminal transmembrane region, and a cytoplasmic tail. Meprin retains the EGF, transmembrane region, and cytoplasmic tail during biosynthesis and moves to the cell surface as a type I membrane-bound protein. The meprin subunit is primarily found at the plasma membrane; however, it can be shed into the extracellular milieu (14, 15). The subunit contains a 56-amino acid inserted (I) domain between the TRAF- and EGF-like domains, not present in the subunit, that directs COOH-terminal proteolytic processing of this subunit during biosynthesis, resulting in removal of the region proximal to the TRAF domain (16). Consequently, homooligomers of are secreted, and meprin is only found membrane-bound via interactions with the meprin subunit.

View larger version (7K): [in this window] [in a new window]   FIG. 1. Cysteine residues and domain structure of the secreted rat meprin subunit. For the meprin cDNA-deduced domain structure lines above the domains indicate cysteine residues, and numbers below indicate the first amino acid of the domain. The domains are, prosequence (Pro) and catalytic domain (Protease).

Previous studies have demonstrated the importance of one cysteine residue in each of the MAM domains for intersubunit disulfide bridging of the subunits (21, 22). Chevallier et al. (21) demonstrated that mutation of rat Cys-309 in the MAM domain resulted in the expression of a monomeric enzyme and that mutations of rat Cys-560 and Cys-562 in the TRAF domain do not affect dimerization. In addition, mutation of the equivalent residue in the MAM domain of rat meprin , Cys-306, resulted in no dimers or heterodimers. On the basis of these studies it was concluded that there was a unique Cys residue in the MAM domain of each meprin subunit that formed a single intersubunit disulfide link. However, no systematic study has been performed on the disulfide bonding pattern of either subunit.

For the studies herein a number of techniques were used to define the oligomeric structure of meprin B and the effect that it may have on function. Self-association properties and the molecular mass of rat meprin B were determined by analytical ultracentrifugation using sedimentation equilibrium studies in which protein concentrations in the micromolar range are achieved. The intra- and intersubunit disulfide bonding pattern was elucidated using mass spectroscopy analyses to gain insight into the tertiary and quaternary structure of the protein. Point mutations of selected cysteine residues were created at positions based on the disulfide mapping experiments to define the role of intersubunit disulfide bond formation on oligomerization of the protein. In addition, cross-linking experiments were conducted to reveal additional points of inter- and intrasubunit contact between and within domains.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Analytical Ultracentrifugation—Recombinant rat meprin B was purified as described previously and dialyzed into ultracentrifugation buffer containing 50 mM potassium phosphate, pH 7.5, 150 mM potassium chloride (7). Sedimentation equilibrium measurements of meprin B were conducted using a Beckman XL-I analytical ultracentrifuge in absorbance mode. The supplied XL-I acquisition software was used to acquire data. Simultaneous acquisition at a range of protein concentrations (500 nM to 5.5 µM) was accomplished using a six-channel center-piece while monitoring at wavelengths appropriate for the protein concentration (220–300 nm). Data were acquired at rotor speeds of 6,000, 9,000, and 12,000 rpm. Equilibrium was achieved when 2 consecutive sets of data taken 2 h apart were completely superimposable. After collection of these data, the rotor speed was increased to 36,000 rpm to deplete the meniscus, allowing determination of base-line absorbance. The partial specific volume of meprin B was determined to be 0.72 by performing parallel sedimentation equilibrium experiments in solutions of H₂O and D₂O as described elsewhere (23, 24).

Data were edited using the Microcal Origin software package (Version 3.78, Microcal Software). Local and global nonlinear least squares analyses were employed using the program WinNonlinLR (version 1.05) to extract solution molecular masses.

Mapping of Disulfide Bonds—A dithio-bis-nitrobenzoic acid assay was performed to determine the number of free thiols in meprin as described previously (25). Meprin (1 ml of an 8 µM solution, in 20 mM HEPES, pH 7.8, 150 mM NaCl) was then denatured by the addition of 480 mg of urea (Sigma). A 100 mM solution of N-ethylmaleimide (Sigma) was prepared using dimethylformamide (Fisher) and was immediately added to the above solution (10 mM final concentration) to alkylate any free thiols. The mixture was allowed to sit at 25 °C for 1 h before dialyzing into 100 mM NH₄HCO₃ (Sigma), pH 8.5. Endoproteinase Glu-C was prepared (Sigma, 1 mg/ml in 100 mM NH₄HCO₃, pH 8.5), added to the meprin solution at a final concentration of 50 µg/ml, and incubated at 37 °C for 18 h. The resulting peptide mixture was passed through a 10-kDa cutoff concentrator (Amicon) to remove the endoproteinase Glu-C. One-half of this sample was analyzed without further digestion, whereas chymotrypsin (Sigma, 1 mg/ml in 100 mM NH₄HCO₃, pH 8.5) was then added to the other half of the mixture at a final concentration of 50 µg/ml and incubated for 18 h at 37 °C. The digested mixtures were then separated by HPLC using a peptide separation program described previously (26). Samples were directly analyzed by a Mariner electrospray time of flight mass spectrometer (PerSeptive Biosystems) as they eluted from the column.

In addition, the proteins were alkylated then digested with endoproteinase Glu-C alone or in combination with chymotrypsin as described above. The samples were subjected to HPLC, and one-ml fractions were collected. These fractions were dried in a speed-vac and resuspended in 5 µl of a solution containing 5 mg/ml of -cyano-4-hydroxycinnamic acid (Sigma) dissolved in 60% acetonitrile, 0.1% trifluoroacetic acid. 0.5 microliter of each fraction was spotted on a target plate and analyzed with a Voyager matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer (PerSeptive Biosystems) as described previously (6, 27). MALDI data were analyzed using the Automated Spectrum Assignment Program website (roswell.ca.sandia.gov/~mmyoung/index.html).

Cross-linking of Meprin B and Mapping the Points of Contact— Meprin B (0.5 ml of an 8 µM solution in 20 mM HEPES, pH 7.8, 150 mM NaCl) was mixed with 50 µl of a 4 mM solution of disuccinimidyl suberate (DSS, Pierce) dissolved in dimethylformamide (Fisher). Crosslinking was allowed to proceed for 3 h at 25 °C and quenched with 20 mM Tris, pH 7.5 (final concentration). The protein solution was then dialyzed into 100 mM NH₄HCO₃, pH 8.5, and digested with endoproteinase Glu-C as described above. The protease was then removed, and trypsin (Sigma) was added to a final concentration of 50 µg/ml. The mixture was incubated at 37 °C for 18 h, separated by HPLC, and analyzed by MALDI mass spectrometry as described above. The peak list from each fraction was exported to the Automated Spectrum Assignment Program website, and the software was configured to search for potential cross-links between lysine residues and/or the amino terminus of meprin (28). Parameters for detection included an error of no more than 100 parts per million and an allowance of one missed cleavage site for endoproteinase Glu-C and two missed cleavage sites for trypsin. Only peptides with unique masses were considered.

Generation of Cysteine Mutants—Single cysteine mutations were created using the QuikChange kit (Stratagene). Mutation of meprin Cys-492 to an alanine was accomplished using the primers 5'-TCAATTACAGTGGCCGGCTCCTTGGCAAGC-3' and 5'-GCTTGCTGCCAAGGAGCCGGCCACTGTAATTGA-3'. Polymerase chain reaction established the mutation in the rat meprin cDNA, and transformation of the construct was conducted as previously described (7, 29). This DNA was then stably transfected into human embryonic kidney 293 cells as described previously (30). The meprin C274A mutation was generated as described above using the primers 5'-TTTTGAGCTGGAGAATATCGCTGGCATGATCCAAAGTTCAC-3' and 5'-GTGAACTTTGGATCATGCCAGCGATATTCTCCAGCTCAAAA-3'.

Activity and Stability Assays—Activity of wild-type and mutant meprin B against the fluorogenic peptide substrate 2-aminobenzoic acid-MGWM-DEID-2,4-dinitrophenyl-SG-OH (OCK+) and the protein substrate azocasein were performed as previously described (10, 29). Tryptic stability experiments were carried out by incubating activated wild-type or mutant meprin B (each at a concentration of 33 ng/µl) with 0, 12.5, 25, 40, or 80 ng/µl trypsin (final concentration) for 1 h at 37 °C. Soybean trypsin inhibitor was added at 2-fold excess over trypsin to stop the reaction. These samples were then subjected to SDS-PAGE and analyzed by Western blot to determine the extent of meprin degradation by trypsin. Susceptibility to heat inactivation was assessed by incubation of activated wild-type meprin protein, C274A, and C492A mutants at 45 °C for 0, 5, 10, 15, 20, 30, and 40 min. The samples were allowed to cool to 25 °C, and then activity was measured against OCK+.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Oligomeric State of Meprin B—To determine whether meprin B forms higher order oligomers at high protein concentrations, the enzyme was subjected to analytical ultracentrifugation using sedimentation equilibrium. A representative plot of an equilibrium experiment is shown in Fig. 2. A global fit of the data using the program WinNonlin yielded a molecular mass of 177 kDa. The monomeric molecular mass of meprin was previously determined to be 85 kDa by electrophoretic and mass spectrometry techniques. Thus, the obtained molecular mass of 177 kDa is consistent with dimer formation and previous experimental determinations of the meprin B molecular mass using light scattering, size exclusion chromatography, and electron microscopy (7). The data fit best to a single ideal species, indicating that no oligomers higher than dimer formed even at the millimolar concentrations achieved during centrifugation.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Representative sedimentation equilibrium data set for the determination of the meprin B molecular mass. Meprin B samples were subjected to analytical ultracentrifugation as described under "Experimental Procedures." A global fit to nine data sets from three different protein concentrations and three rotor speeds is shown in the bottom graph as a solid line. The residuals due to deviation of the data from this line are shown above the curve.

Meprin B was exposed to NEM under denaturing conditions to alkylate free thiols, digested with endoproteinase Glu-C alone or with chymotrypsin, and analyzed by liquid chromatography/electrospray ionization mass spectrometry. A mass was identified as Cys-341 modified with NEM, indicating that this residue is the free cysteine (Table I). Masses were identified corresponding to disulfide bond formation between Cys-104 and Cys-256, Cys-274 and Cys-274, and Cys-306 and Cys-306.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Representative MALDI-MS data of disulfide-linked meprin B peptides. A disulfide-linked fragment corresponding to the linkage between Cys-104 and Cys-256, both in the protease domain, was isolated by HPLC and analyzed by MALDI-TOF. The expected mass of this fragment was 2307.66; a mass of 2307.55 was observed.

Mutation of Cys Residues Involved in Disulfide Bond Formation— Previous experiments by Chevallier et al. (21) demonstrated that rat meprin B was secreted as a monomer when Cys-306 was mutated to a serine residue (21). Because the results of the disulfide mapping experiment above showed that there were at least three intersubunit disulfide bonds, the question arose as to why this single mutation resulted in failure to form any disulfide linkages. Therefore, Cys-492 and Cys-274 were mutated to alanine residues in separate experiments to assess whether loss of these intersubunit disulfide bonds would also result in the inability to form the covalently linked dimer.

The meprin C274A mutant migrated predominantly as a monomer when subjected to SDS-PAGE in the absence of reducing agent (Fig. 4, panel A). A small amount of dimer is noted, less than 10% by densitometry. It is unclear whether this is due to disulfide formation inside the cell or some oxidation of cysteine residues after the protein was secreted. However, the protein migrated primarily as a dimer under native conditions (no SDS or reducing agent), revealing that the subunits are noncovalently linked (Fig, 4, panel A). Activity of the mutant was comparable with wild type against both the peptide and protein substrates, OCK+, and azocasein (Table III). However, the C274A mutant showed increased vulnerability to degradation by trypsin (Fig. 5, panel A). Wild-type meprin B retains 80% of its activity after 40 min at 45 °C, whereas the mutant lost most activity after 20 min and retained less than 10% activity after 40 min (Fig. 5, panel B). These data indicate that mutation of C274 results in no covalent linkages between the subunits, whereas the protein retains proteolytic activity. Meprin B can dimerize in the absence of covalent intersubunit linkages; however, it is less stable to heat and proteolytic degradation than its disulfide-linked counterpart.

View larger version (27K): [in this window] [in a new window]   FIG. 4. Effects of cysteine mutations on the oligomerization of meprin B. The latent forms of recombinant wild-type (WT) meprin B and cysteine mutants were subjected to electrophoresis under nonreducing conditions on 7.5% SDS-PAGE gels or under nonreducing and nondenaturing conditions (native PAGE). Panel A, meprin C274A mutant and wild-type meprin ; panel B, meprin C492A mutant and wild-type meprin .

View this table: [in this window] [in a new window]   TABLE III Specific activity of meprin C274A and C492A mutants The activities of wild-type meprin or cysteine mutants were measured against a peptide substrate (OCK+) or a protein substrate (azocasein). Specific activity toward OCK+ is in fluorescent units s-1µg-1. Azocasein specific activity is in absorbance units min-1 µg-1. The amounts of wild-type and mutant meprins were determined by Western blot and densitometry. The data represent averages ± S.E. for three determinations.

View larger version (47K): [in this window] [in a new window]   FIG. 5. The meprin C274A and C492A mutants demonstrate increased susceptibility to trypsin digestion and heat inactivation. Panel A, wild-type and mutant meprin B were incubated with varying amounts of trypsin for 1 h at 37 °C, the reaction was stopped with soybean trypsin inhibitor, and then meprin protein was analyzed by Western blotting after reducing SDS-PAGE. Panel B, samples were incubated at 45 °C for the times indicated, cooled to 25 °C, and then measured for their activity against a fluorogenic peptide substrate, OCK+. (), wild-type; (), C274A; (), C492A.

Cross-linking and Mapping of Sites of Meprin B Interaction—To gain additional information about the tertiary and quaternary structure of meprin B, a cross-linking approach was employed. DSS, a homobifunctional, amine-reactive crosslinker that covalently links lysine residues, was used to probe sites of contact. The addition of 50 mol eq of DSS for 3 h at 24 °C yielded 50% cross-linking to dimer (Fig. 6, inset). The cross-linked products were then digested, separated by HPLC, and analyzed by MALDI mass spectrometry (Fig. 6). Peak lists generated from MALDI-MS of each purified fraction were analyzed using the Automated Spectrum Assignment Program to find putative cross-links.

View larger version (12K): [in this window] [in a new window]   FIG. 6. Representative MALDI-MS data from meprin B cross-linking. Meprin B was incubated in the presence or absence of the cross-linker DSS. Inset, Coomassie-stained gels. Lane 1, SDS-PAGE of meprin B without DSS in the presence of reducing agent. Lane 2, meprin B after incubation with DSS in the presence of reducing agent. The spectrum contains a proteolytic fragment corresponding to a lysine-lysine cross-link between the protease domain (Lys-185) and the TRAF domain (Lys-575).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The experiments described in these studies have elucidated the disulfide bonding pattern of the rat meprin B protease and characterized interactions between functional domains of the protein, thus providing insight into the multimeric structure of the metalloproteinase. The work shows that three disulfide bonds link the subunits rather than the one unique bond proposed from mutational studies and that the enzyme is a dimer under a wide range of concentrations. The intersubunit disulfide linkages in the MAM and TRAF domains serve to stabilize the dimer against heat inactivation and proteolytic degradation, and this may be particularly important in extracellular environments such as the lumen of the intestine or at sites of inflammation where meprins are found in abundance. The proposed disulfide pattern of the meprin B structure is reminiscent of the macrophage colony stimulating factor protein, which also contains three intersubunit disulfide bonds that have been shown to stabilize its structure (32, 33).

The overall pattern of disulfide bridging shows two intrasubunit disulfide bridges in the protease domain, one intra- and two intersubunit disulfide bridges in the MAM domain and one intersubunit disulfide bridge in the TRAF domain (Fig. 7). The presence of two intradomain disulfide bonds in the protease domain of meprin B is consistent with those found in the crayfish astacin, the only member of this evolutionary family for which there is a crystal structure (17). The protease domain cysteines are conserved in all members of the astacin family, except for flavastacin, the bacterial form of the protease that has no cysteine residues (34). The disulfide linkages and lysinelysine cross-links identified in this study are structurally consistent with a homology model of the meprin B protease domain (Fig. 8). As shown herein for meprin B, the protease domain acts catalytically as a monomer, although the protein forms a dimer. There is no indication of allosteric kinetics for meprins, and all other members of the astacin family are monomeric (1).

View larger version (10K): [in this window] [in a new window]   FIG. 7. Proposed pattern of disulfide bridging for meprin B. This pattern is based on the data herein for the Protease, MAM, and TRAF domains and on other EGF structures (e.g. 31).

View larger version (90K): [in this window] [in a new window]   FIG. 8. The rat meprin B protease domain modeled from the crystal structure of astacin. The cysteine residues are colored yellow and are arranged such that Cys-104 and Cys-256 are in close proximity, as are Cys-125 and Cys-145. This arrangement of cysteine residues is consistent with the disulfide mapping experiments. The lysine residues are colored dark blue. Lys-228 and Lys-185 are positioned 11.7 Å apart, and Lys-109 and Lys-89 are separated by 12.0 Å.

Meprin B has a complex disulfide arrangement in the MAM domain, which is unique to meprins. Although there is evidence that the intradomain disulfide bridges in MAM domains in proteins such as protein-tyrosine phosphatase µ are essential for MAM homophilic interactions, there is no evidence for interdomain disulfide bridging in MAM domains in proteins other than meprins (12). The finding herein that the C274A mutant has full enzymatic activity and results in the formation of a noncovalent meprin dimer indicates that the meprin subunit has a dimerization interface that is not dependent on intersubunit disulfide bridges. However, the disulfide bonding pattern of meprin B implies a different role for this protein compared with other members of the MAM domain family.

The identification of an intersubunit disulfide bridge in the meprin TRAF domain was unanticipated based on the structures of other TRAF domains and previous studies with meprins that had implicated only a unique intersubunit disulfide bridge in the MAM domain. Intracellular TRAF domains generally do contain cysteine residues (e.g. human TRAFs 1–6 contain at least 1 and up to 5 cysteine residues); however, they have not been reported to have intersubunit disulfide linkages. The intracellular TRAFs participate in signal transduction through noncovalent homo- and heterophilic interactions (36). In addition, the Cys-492 residue of the meprin TRAF domain (conserved in mouse, rat, and human ) is not present in meprin subunits. Meprin contains two cysteine residues in the TRAF domain (Cys-560 and Cys-562); however, mutation of these residues resulted in disulfide-linked dimers (21). This difference between meprin and in the TRAF disulfide linkages might account for some of the differences in the oligomeric properties of the two subunits, i.e. the inability of the meprin subunit to form the high molecular mass homomeric complexes that occur for meprin . Modeling of meprin TRAF domains based on the structure of TRAF 2 indicates that the meprin Cys-492 residue is in a loop region between sheets 4 and 5 of TRAF-C domains (37). This is a region identified in the crystal structure of TRAF 2 to be involved in homophilic interactions (38). It is intriguing that the mutation of Cys-492 to an alanine residue prevents covalent and noncovalent interactions of the meprin dimer, indicating it is essential for the dimeric structure although not essential for proteolytic activity. Thus, the TRAF domain plays a major role in the folding of meprin proteins into stable dimers and perhaps formation of higher order oligomers and is in close proximity to the protease domain in the native structure of meprin B.

In addition to stabilizing the structure of meprin B, the cysteine residues linked in intersubunit disulfides appear to play a role in the overall folding process leading to the native state. The single cysteine mutants demonstrated an "all-or-nothing" effect of these particular residues in forming covalently linked products; i.e. all of the cysteines need to be present for any of the intersubunit disulfide bonds to form. One interpretation of these results is that the cysteine residues in question play a role in the formation of cystines via a folding-coupled reshuffling pathway (39). In proteins that exhibit this type of folding, an initial disulfide bond forms which allows the protein to adopt a conformation that places a free cysteine residue in position to undergo a disulfide exchange with an existing disulfide bond, allowing a switch between the cysteine partners in the disulfide linkage (39). As such, mutation of any of the cysteines involved in this pathway precludes correct disulfide bond formation.

A model of the meprin B dimer is proposed based on the disulfide mapping and cross-linking results (Fig. 9). The subunits are arranged with D₂ symmetry such that the TRAF domains, MAM domains, and prosequences comprise the interface, which is linked by three disulfide bonds. The protease domain and the TRAF domain are in close proximity, as are the protease and EGF-like domains, based on the cross-linking experiments. The cross-linking data provide a basic framework of inter- and intradomain contacts. It should be noted that two residues that are in contact but orientated away from each other may still cross-link if they are within the sphere delineated by the length of the spacer arm. Because the EGF-like domain is proximal to the transmembrane domain, the protease domain is positioned close to the cell membrane. However, meprin B has been shown to undergo a conformational change upon activation, and thus, the positioning of the protease domain relative to the membrane may be different in the active state. The positioning of the prosequence domain close to the cell membrane combined with the ability of this domain to dimerize may account for activation differences observed between the meprin and subunits. Proteolytic activation of homooligomeric meprin A is much more efficient than that of meprin B. Recent data indicated that meprin but not subunits can be activated by plasmin, which might also reflect differences in the accessibility of the prosequence cleavage sites of the subunits (40). The orientation of the protease domain close to the cell membrane has important implications for the physiologic role of the enzyme. For instance, the enzyme could be involved in releasing transmembrane proteins from the cell surface and could affect the activity and localization of growth factors and receptors at the cell surface. Initial experiments indicate that meprin B has the ability to shed proteins, including itself, from the cell surface (35). The complex interactions between the domains and subunits are clearly important for regulating the activation, stability, and protein-protein interactions of the meprins.

View larger version (26K): [in this window] [in a new window]   FIG. 9. Model of the meprin B dimer. A model of the dimeric structure is proposed based upon the disulfide and cross-linking mapping experiments. The dimer exhibits D2 symmetry and is arranged such that the subunit interface is formed between the MAM domains, the TRAF domains, and the prosequences. The protease domain interacts with the TRAF domain as well as the EGF domain, placing it near the COOH-terminal region of the protein close to the cell membrane.

	FOOTNOTES

Present address: Dept. of Medicine, The Pennsylvania State University College of Medicine, Hershey, PA 17033.

Present address: OSMcMN, 1940 Duke St., Arlington, VA 22314.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, H171, The Pennsylvania State University College of Medicine, Hershey, PA 17033. Tel.: 717-531-8586; Fax: 717-531-7072; E-mail: jbond{at}psu.edu.

¹ The abbreviations used are: MAM, meprin, A-5 protein, receptor protein-tyrosine phosphatase µ; TRAF, tumor necrosis factor receptor-associated factor; EGF, epidermal growth factor; NEM, N-ethylmaleimide; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; DSS, disuccinimidyl suberate; OCK+, 2-aminobenzoic acid-MGWM-DEID-2,4-dinitrophenyl-SG-OH; HPLC, high performance liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Bond, J. S., and Beynon, R. J. (1995) Protein Sci. 4, 1247-1261[Abstract]
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5. Bertenshaw, G. P., and Bond J.S. (2004) in Handbook of Proteolytic Enzymes, pp. 599-609, Elsevier Academic Press, London
6. Ishmael, F. T., Norcum, M. T., Benkovic, S. J., and Bond, J. S. (2001) J. Biol. Chem. 276, 23207-23211[Abstract/Free Full Text]
7. Bertenshaw, G. P., Norcum, M. T., and Bond, J. S. (2003) J. Biol. Chem. 278, 2522-2532[Abstract/Free Full Text]
8. Craig, S. S., Reckelhoff, J. F., and Bond, J. S. (1987) Am. J. Physiol. 253, C535-C540[Medline] [Order article via Infotrieve]
9. Hengst, J. A., and Bond, J. S. (2004) J. Biol. Chem. 279, 34856-34864[Abstract/Free Full Text]
10. Bertenshaw, G. P., Turk, B. E., Hubbard, S. J., Matters, G. L., Bylander, J. E., Crisman, J. M., Cantley, L. C., and Bond, J. S. (2001) J. Biol. Chem. 276, 13248-13255[Abstract/Free Full Text]
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