Multimeric Structure of the Secreted Meprin A Metalloproteinase and Characterization of the Functional Protomer

Abstract

Meprin A secreted from kidney and intestinal epithelial cells is capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. The secreted form of meprin A is a homo-oligomer composed of α subunits, a multidomain protease of 582 amino acids coded for near the major histocompatibility complex of the mouse and human genome. Analyses of the recombinant homo-oligomeric form of mouse meprin A by gel filtration, nondenaturing gel electrophoresis, and cross-linking (with disuccinimidyl suberate orN-(4-azido-2,3,5,6-tetraflourobenzyl)-3-maleimidylpropionamide) indicate that the secreted enzyme forms high molecular weight multimers, with a predominance of decamers. The multimers are composed of disulfide-linked dimers attached noncovalently by interactions involving the meprin, A5 protein, receptor protein-tyrosine phosphatase μ (MAM) domain. The active protomer is the noncovalently linked dimer. Linkage of active protomers by disulfide-bonds results in an oligomer of ∼900 kDa, which is unique among proteases and distinguishes meprin A as the largest known secreted protease. Electron microscopy revealed that the protein was present in two states, a crescent-shaped structure and a closed ring. It is concluded from this and other data that the covalent attachment of the protomers enables noncovalent associations of the native enzyme to form higher oligomers that are critical for hydrolysis of protein substrates. Meprin A secreted from kidney and intestinal epithelial cells is capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. The secreted form of meprin A is a homo-oligomer composed of α subunits, a multidomain protease of 582 amino acids coded for near the major histocompatibility complex of the mouse and human genome. Analyses of the recombinant homo-oligomeric form of mouse meprin A by gel filtration, nondenaturing gel electrophoresis, and cross-linking (with disuccinimidyl suberate orN-(4-azido-2,3,5,6-tetraflourobenzyl)-3-maleimidylpropionamide) indicate that the secreted enzyme forms high molecular weight multimers, with a predominance of decamers. The multimers are composed of disulfide-linked dimers attached noncovalently by interactions involving the meprin, A5 protein, receptor protein-tyrosine phosphatase μ (MAM) domain. The active protomer is the noncovalently linked dimer. Linkage of active protomers by disulfide-bonds results in an oligomer of ∼900 kDa, which is unique among proteases and distinguishes meprin A as the largest known secreted protease. Electron microscopy revealed that the protein was present in two states, a crescent-shaped structure and a closed ring. It is concluded from this and other data that the covalent attachment of the protomers enables noncovalent associations of the native enzyme to form higher oligomers that are critical for hydrolysis of protein substrates. meprin, A-5 protein, receptor protein-tyrosine phosphatase μ matrix-assisted laser desorption/ionization Tris-(2-carboxyethyl)phosphine hydrochloride polyacrylamide gel electrophoresis high pressure liquid chromatography 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Lys(Dnp)-Gly-OH, where Dnp is dinitrophenyl Meprins are zinc endopeptidases that are secreted from or found in brush border membranes of kidney and intestinal epithelial cells of humans and rodents. They are members of the astacin family of metalloproteases and the metzincin superfamily (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (351) Google Scholar, 2Stöcker W. Grams F. Baumann U. Reinemer P. Gomis-Ruth F.X. McKay D.B. Bode W. Protein Sci. 1995; 4: 823-840Crossref PubMed Scopus (633) Google Scholar). The expression of meprins enables activation or degradation of bioactive peptides, growth factors, cytokines, hormones, and matrix proteins by limited proteolysis (3Bertenshaw G.B. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 4Chestukhin A. Litovchick L. Muradov K. Batkin M. Shaltiel S. J. Biol. Chem. 1997; 272: 3153-3160Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 5Johnson G.D. Bond J.S. Barrett A.J. Woessner F. Rawlings N. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego, CA1998: 1222-1229Google Scholar). Meprins are multidomain, oligomeric glycoproteins with subunit masses of 85,000–110,000 Da (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (351) Google Scholar). The subunits, α and β, may associate to form either homo- or hetero-oligomeric proteins (6Gorbea C.M. Flannery A.V. Bond J.S. Arch. Biochem. Biophys. 1991; 290: 549-553Crossref PubMed Scopus (28) Google Scholar). Multimers containing α subunits (both homo- and hetero-oligomers) are designated meprin A (EC 3.4.24.18), whereas homo-oligomers of β are designated meprin B (EC 3.4.24.63). The mouse α and β subunits are ∼42% identical at the amino acid level, and their cDNA-deduced primary sequences predict a similar arrangement of functional domains (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (351) Google Scholar, 5Johnson G.D. Bond J.S. Barrett A.J. Woessner F. Rawlings N. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego, CA1998: 1222-1229Google Scholar). Meprin α cDNA (Structure FTI) encodes a 760-amino acid protein with an NH2-terminal signal sequence followed by a prosequence, the protease domain (containing the zinc active site), a MAM1domain, a MATH (meprin and TRAF homology; TRAF, tumor necrosis factor receptor-associated factor) domain, followed by an AM (after MATH) domain, an EGF (epidermal growth factor)-like domain, a putative COOH-terminal transmembrane domain (TM), and a cytoplasmic (C) tail. The α subunit contains a 56-amino acid domain (I) inserted between the AM and EGF-like domains that is not present in the β subunit. The I domain has been shown to direct COOH-terminal proteolytic processing of the α subunit during biosynthesis (7Marchand P. Tang J. Johnson G.D. Bond J.S. J. Biol. Chem. 1995; 270: 5449-5456Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The mature α subunit lacks the domains COOH-terminal to the AM domain, including the transmembrane domain. As a result, meprin α homo-oligomers are secreted, and meprin α is only found membrane-bound via interactions with the β subunit (which does not undergo COOH-terminal proteolysis and is a type I integral membrane protein). Studies of membrane-bound meprins indicated that the subunits are linked via disulfide bonds to form dimers (6Gorbea C.M. Flannery A.V. Bond J.S. Arch. Biochem. Biophys. 1991; 290: 549-553Crossref PubMed Scopus (28) Google Scholar, 7Marchand P. Tang J. Johnson G.D. Bond J.S. J. Biol. Chem. 1995; 270: 5449-5456Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Marchand P. Tang J. Bond J.S. J. Biol. Chem. 1994; 269: 15388-15393Abstract Full Text PDF PubMed Google Scholar). These dimers can further associate via noncovalent interactions to form tetramers (8Marchand P. Tang J. Bond J.S. J. Biol. Chem. 1994; 269: 15388-15393Abstract Full Text PDF PubMed Google Scholar). Meprins are unique among proteases in their oligomeric structure. Because hetero-oligomer formation requires interactions between the α subunits and membrane-bound β subunits, it was unclear whether the secreted homo-oligomer, which contains only α subunits, could form multimers of the same oligomeric state or even larger complexes. Native gel electrophoresis of unpurified secreted meprin A followed by Western blot detection of the enzyme demonstrated that it migrated with a mobility larger than 700 kDa (9Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Mutation of Cys-320, the cysteine residue in the MAM domain involved in an intersubunit disulfide bond, to an alanine (C320A) resulted in expression of a monomeric form of meprin. The mutant had little or no ability to hydrolyze proteins, although it retained activity against peptide substrates and showed decreased thermostability and an increased susceptibility to tryptic degradation (9Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This work indicated that the oligomeric state is critical for activity and stability of the enzyme but could not determine whether the properties exhibited by the monomeric mutant were due to lack of formation of the disulfide-linked dimer or the inability of the subunits to associate noncovalently. Moreover, the oligomeric state of meprin could not be accurately determined by native gel electrophoresis of the unpurified protein because the band observed could have been due to meprin subunits in association with other proteins. Thus, the oligomeric structure of the purified meprin protein was investigated, and the importance of this structure to function was examined. Recombinant mouse meprin A was purified from the media of human embryonic kidney 293 cells (American Type Culture Collection) stably transfected with full-length mouse meprin α cDNA (10Kadowaki T. Tsukuba T. Bertenshaw G.P. Bond J.S. J. Biol. Chem. 2000; 275: 25577-25584Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Briefly, the purification involved mono-Q and superose-12 fast protein liquid chromatography column chromatography; the protein was activated by trypsin after mono-Q chromatography (5Johnson G.D. Bond J.S. Barrett A.J. Woessner F. Rawlings N. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego, CA1998: 1222-1229Google Scholar, 10Kadowaki T. Tsukuba T. Bertenshaw G.P. Bond J.S. J. Biol. Chem. 2000; 275: 25577-25584Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). An 8 μm meprin solution was labeled with 10 molar equivalents ofN-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidylpropionamide (Molecular Probes) for 12 h at 4 °C in the dark. Excess label was removed by passing the mixture through a Sephadex G-25 column. Cross-linking was initiated by subjecting the protein to ultraviolet light for 7 min, as described elsewhere (11Alley S.C. Ishmael F.T. Jones A.D. Benkovic S.J. J. Am. Chem. Soc. 2000; 122: 6126-6127Crossref Scopus (65) Google Scholar), and quenched with 5 μl of gel loading buffer. Fifty μl of an 800 μm N-biotinyl-N′-[6-maleimidiohexanoyl]-hydrazide (biotin-maleimide) solution dissolved in dimethylformamide was added to 500 μl of an 8 μm meprin solution. The protein was incubated for 10 h at 4 °C, and excess label was removed with a Sephadex G-25 column. The labeled protein was subsequently deglycosylated with peptide-N-glycosidase F (PNGase F; New England BioLabs), and proteins were separated from carbohydrates by gel filtration. Deglycosylated, labeled protein was incubated with 50 μg of trypsin at pH 8.5, 37 °C for 14 h. The resulting peptides were separated by a reverse phase C-18 HPLC column (11Alley S.C. Ishmael F.T. Jones A.D. Benkovic S.J. J. Am. Chem. Soc. 2000; 122: 6126-6127Crossref Scopus (65) Google Scholar), and fractions were analyzed by reflector mode MALDI mass spectrometry using an α-cyano-4-hydroxycinnamic acid matrix. Samples from different preparations of meprin were diluted with deionized water to ∼25 μg/ml and then negatively stained with 1% uranyl acetate using a double carbon method (12Norcum M.T. J. Biol. Chem. 1989; 264: 15043-15051Abstract Full Text PDF PubMed Google Scholar). Electron micrographs were obtained with a LEO 912AB microscope operated at 100 kV at an absolute magnification of 31,500. The spectrometer slit of the energy filter was used to remove inelastically scattered electrons. For preparation of figures, negatives were digitized at an optical resolution corresponding to 6.4 Å/pixel on an Agfa Duoscan flatbed scanner, and then composites were prepared using Adobe Photoshop. Statistics of particle measurements were compiled from enlarged prints on a digitizing tablet with SigmaScan (Jandel). Eight μm meprin was reduced with 5 mm TCEP (Molecular Probes), pH 8.0, for 30 min at 4 °C. Excess reducing agent was removed by a Sephadex G-25 column, and the enzyme was subjected to native gel electrophoretic analysis to determine the dissociation of the oligomer and nonreducing SDS-PAGE to assess the amount of disulfide bond reformation. Proteolytic activity was determined with an internally quenched fluorescent bradykinin substrate analog, BK+ (2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Lys(Dnp)-Gly-OH, where Dnp is dinitrophenyl), and the protein substrate azocasein as described previously (5Johnson G.D. Bond J.S. Barrett A.J. Woessner F. Rawlings N. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego, CA1998: 1222-1229Google Scholar). An 8 μm solution of native or reduced meprin was incubated at 47 °C for 5–35 min at 5-min intervals. The protein was cooled to 25 °C, and the activity toward BK+ was determined. Susceptibility to trypsin digestion was assessed by mixing 2 μl of an 800 nm solution of either the native protein or the reduced protein with trypsin at final concentrations of 20–100 ng/ml. After 15 min at 25 °C, the reaction was stopped by the addition of a 3-fold excess of soybean trypsin inhibitor, and the protein was subjected to SDS-PAGE followed by Western blotting to determine the extent of proteolysis. When purified recombinant mouse meprin A homo-oligomer was subjected to nondenaturing, nonreducing gel electrophoresis, the native protein migrated as a species larger than the 669,000 molecular weight marker (Fig.1 A). The monomeric molecular weight of a meprin α subunit is ∼90,000, indicating that the multimeric protein is capable of forming at least octamers. Cross-linking the protein with disuccinimidyl followed by SDS-PAGE in the presence of 2-mercaptoethanol resulted in dimers and higher order oligomers (Fig. 1 B). The largest cross-linked species observed migrated above the 669,000 molecular weight marker, confirming that meprin can form an octamer or even higher order complexes. When the protein was subjected to analytical size exclusion chromatography to obtain more precise molecular weight information, the elution volume of meprin corresponded to a molecular weight of 905,000, indicating that the protein is primarily decameric (Fig. 1 C). The width of the meprin peak was comparable to that of thyroglobulin (669 kDa), the protein standard closest in mass. However, the meprin peak was more asymmetric than any of the monodisperse standard proteins. As shown in the elution profile (Fig. 1 C), there appears to be some trailing on the right side of the peak, indicating the presence of smaller oligomeric forms. The large size of the complex distinguishes the meprin A homoligomer as the largest known secreted protease. The ability of the secreted protein to form decameric complexes, compared with the tetrameric structure of hetero-oligomeric meprins (8Marchand P. Tang J. Bond J.S. J. Biol. Chem. 1994; 269: 15388-15393Abstract Full Text PDF PubMed Google Scholar), may result from the lack of the potential conformational barriers imposed by being membrane-bound. The electrophoretic mobility of the protein under both reducing and denaturing conditions corresponds to the monomeric molecular weight (Fig.2 A). Under denaturing (in the presence of SDS) but nonreducing conditions (no 2-mercaptoethanol), the protein migrates as a dimer (Fig. 2 B). When the enzyme was reduced with 2-mercaptoethanol and then subjected to native gel electrophoresis (no SDS), the protein migrated as a dimer of noncovalently linked subunits (Fig. 2 C). These results indicate that the oligomer is composed of disulfide-linked dimers that associate noncovalently to form the native structure. The interaction between the covalently linked dimers does not appear to be strong because only dimers, not higher order oligomers, are observed after reduction of the S-S bond. The secreted form of meprin A contains 11 cysteine residues per subunit (StructureFTII). Previous work indicated that Cys-320 in the MAM domain is responsible for the covalent interaction of subunits (9Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Mutation of Cys-320 to alanine (C320A) resulted in a meprin α monomer that exhibited altered proteolytic activity and stability (9Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This mutant was incapable of forming not only disulfide-linked dimers but noncovalently linked oligomers as well. To determine whether the changes seen in the mutant were due to the loss of the disulfide-linked dimer or instead to loss of interactions between the noncovalently linked subunits, we reduced wild type meprin with the phosphine TCEP and measured the activity and stability of the protein. TCEP was selected as a reducing agent because it does not contain a thiol group. Thiol-containing reagents such as 2-mecaptoethanol or have been shown to meprin by either of the active zinc or reduction of disulfide bonds in the or AM domains R.J. Bond J.S. Biochem. J. PubMed Scopus Google Scholar). with TCEP resulted in dissociation of the oligomer noncovalently linked dimers, as was the with 2-mercaptoethanol (Fig. 2 the disulfide bond not TCEP was even after a of not To determine the of that are meprin protein was incubated with 2 with which can be G.L. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). The indicated that there was thiol per subunit reduction and per subunit after reduction with disulfide bond was reduced in addition to the intersubunit disulfide When meprin was reduced with subjected to Sephadex G-25 column chromatography to remove the reducing and for little or no activity loss was The of hydrolysis of BK+ by the reduced enzyme was that of the form for the native and reduced This demonstrated that the reduction and formation of noncovalently linked dimers had not altered the protease domain of the which contains two disulfide and that the disulfide-linked dimer is not for activity toward peptides. the activity of the reduced enzyme toward the protein substrate azocasein was that of the protein, indicating that only the noncovalently dimer was for hydrolysis of proteins. The stability of the form of meprin was similar to that of the native as assessed by and to trypsin The thermostability was by the proteins at °C, at and subsequently the ability of the protease to hydrolyze the monomeric mutant (9Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), the noncovalent reduced form of meprin exhibited the same of as the native both the wild type and forms of the enzyme were to proteolytic digestion by These results indicate that the changes exhibited by the mutant were not due to the inability of the protein to form the disulfide-linked dimer but to an inability to form the noncovalent the noncovalently dimer is the functional protomer of the 1 of the 11 cysteine residues present in the secreted form of the meprin subunit is a thiol that is to as determined by not This cysteine residue was labeled with the and the protein was subjected to ultraviolet light to cross-linking (Fig. A). the of and the protein migrated as a band corresponding to dimer (Fig. the of but in the presence of reducing the mobility of the protein was with monomer indicating that the intersubunit disulfide bond was reduced (Fig. the presence of and the of the reducing a band corresponding to a cross-linked was observed (Fig. When both and reducing agent were a cross-linked protein corresponding to the meprin dimer was observed (Fig. This that the have between the noncovalently linked subunits because cross-linking between the disulfide-linked dimers result in only dimers after of whether reducing agent was present or To determine the of the cysteine residue that was labeled and of the of interaction between the noncovalently linked subunits, meprin was labeled with the The of the cysteine residue was determined by subjecting the protein to tryptic digestion followed by MALDI mass spectrometry to the labeled peptide as described under A mass of Da was observed by reflector mode MALDI mass which is with the mass of the containing amino acids with the mass of (Fig. B). The labeled cysteine in the MAM domain, which has been described as an domain that For the MAM domains of receptor protein-tyrosine phosphatase μ and are for interactions in transfected cells J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The that the MAM domain an in both covalent and noncovalent interactions in the meprin of the MAM domain by or results in of the protein and degradation by the the importance of this domain in and of the native protein T. Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (45) Google Scholar). To further the high molecular weight oligomers, meprin was by negatively stained two of oligomers were seen As shown in a are that have the to a of and closed The which the of multimers, nm in The of the between 30 and some are as as nm or as large as of the are is in and is by a and a or The of the is of the and oligomers (Fig. 4 this both have and there are the The of meprin is composed of The of nm in (Fig. 4 4 This that are from the that is to interaction of the and of the The of the of is larger are seen (Fig. 4 These nm in and appear to be by interactions of two of the complex may subunits to form a such that the addition of subunits, the of the oligomer subunits are added to the ring. of a from a containing of subunits may be due to the of the structure. more the on the structure it than the the association of a critical of subunits for containing subunits than this be to form a and whereas containing more subunits A of the is in which the protein is from disulfide-linked dimers noncovalently to form either crescent-shaped or closed The subunit interactions are by the MAM domain, which is involved in both the covalent and noncovalent of the of the with TCEP results in dissociation the active the noncovalently linked dimer. reduction of the crescent-shaped only noncovalently linked As shown in reduction at the of the in the of the imposed by the are to associate noncovalently. Meprin may function to large of proteins in which could of the active protomer in The presence of subunits may to the of proteolysis. Linkage of in a could to proteins, in a of the J. P. W. 1995; PubMed Scopus Google Scholar). Meprin to other proteases such as and These are large multimeric composed of homo-oligomers that function to and in protein the Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Scopus Google Scholar, M. M. W. K. PubMed Scopus Google Scholar). I is a with a molecular mass of kDa K. Scopus Google Scholar). forms even larger complexes, between and kDa M. M. W. K. PubMed Scopus Google Scholar). It is composed of subunits of kDa that associate to form a structure 50 nm in and nm in M. M. W. K. PubMed Scopus Google Scholar). However, the meprin A homo-oligomer is secreted from the and extracellular proteins and peptides. A. Jones for with the MALDI mass spectrometry