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• W2012702020 abstract "The meprin A homo-oligomer is a highly glycosylated, secreted zinc metalloprotease of the astacin family and metzincin superfamily. This isoform of meprin is composed of disulfide-bonded dimers of α subunits that further associate to form large, secreted megadalton complexes of 10 or more subunits. The aim of this study was to determine the sites of glycan attachment and to assess their ability to affect the formation and stability of the homo-oligomer. Nine of the ten potential N-linked glycosylation sites (Asn-41, Asn-152, Asn-234, Asn-270, Asn-330, Asn-426, Asn-452, Asn-546, and Asn-553) were found to be glycosylated in recombinant mouse meprin A using chemical and enzymatic deglycosylation methods and electrospray ionization mass spectrometry. Chemical cross-linking demonstrated that carbohydrates are at or near the noncovalent subunit interface. The removal of two glycans in the protease domain at Asn-234 and Asn-270, as well as one in the tumor necrosis factor receptor-associated factor domain at Asn-452, by a deglycosidase under nondenaturing conditions decreased the chemical and thermal stability of the homo-oligomer without affecting quaternary structure. Site-directed mutagenesis demonstrated that no single glycan was essential for oligomer formation; however, the combined absence of the glycans at Asn-152 and Asn-270 in the protease domain hindered intersubunit disulfide bond formation, prevented noncovalent associations, and abolished enzymatic activity. These studies provide insights into the role of glycans in the biosynthesis, activity, and stability of this extracellular protease. The meprin A homo-oligomer is a highly glycosylated, secreted zinc metalloprotease of the astacin family and metzincin superfamily. This isoform of meprin is composed of disulfide-bonded dimers of α subunits that further associate to form large, secreted megadalton complexes of 10 or more subunits. The aim of this study was to determine the sites of glycan attachment and to assess their ability to affect the formation and stability of the homo-oligomer. Nine of the ten potential N-linked glycosylation sites (Asn-41, Asn-152, Asn-234, Asn-270, Asn-330, Asn-426, Asn-452, Asn-546, and Asn-553) were found to be glycosylated in recombinant mouse meprin A using chemical and enzymatic deglycosylation methods and electrospray ionization mass spectrometry. Chemical cross-linking demonstrated that carbohydrates are at or near the noncovalent subunit interface. The removal of two glycans in the protease domain at Asn-234 and Asn-270, as well as one in the tumor necrosis factor receptor-associated factor domain at Asn-452, by a deglycosidase under nondenaturing conditions decreased the chemical and thermal stability of the homo-oligomer without affecting quaternary structure. Site-directed mutagenesis demonstrated that no single glycan was essential for oligomer formation; however, the combined absence of the glycans at Asn-152 and Asn-270 in the protease domain hindered intersubunit disulfide bond formation, prevented noncovalent associations, and abolished enzymatic activity. These studies provide insights into the role of glycans in the biosynthesis, activity, and stability of this extracellular protease. With a molecular mass of 1–8 MDa, the meprin A homooligomer is the largest known secreted protease (1Bertenshaw G.P. Norcum M.T. Bond J.S. J. Biol. Chem. 2003; 278: 2522-2532Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 2Villa J.P. Bertenshaw G.P. Bylander J.E. Bond J.S. Biochem. Soc. Symp. 2003; 70: 53-63Crossref PubMed Scopus (31) Google Scholar). It forms unique crescent, ring, barrel, and spiral structures through both disulfide bonds and noncovalent associations, and it is composed of α subunits that have a monomeric molecular mass of 78–85 kDa depending on the species (1Bertenshaw G.P. Norcum M.T. Bond J.S. J. Biol. Chem. 2003; 278: 2522-2532Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 3Ishmael F.T. Norcum M.T. Benkovic S.J. Bond J.S. J. Biol. Chem. 2001; 276: 23207-23211Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Meprin A is secreted as an oligomer from the brush border epithelial cells of the kidneys and the intestine, as well as certain leukocytes and cancer cells (4Norman L.P. Matters G.L. Crisman J.M. Bond J.S. Curr. Top. Dev. Biol. 2003; 54: 145-166Crossref PubMed Google Scholar, 8Matters G.L. Bond J.S. Mol. Carcinog. 1999; 25: 169-178Crossref PubMed Scopus (38) Google Scholar). Oligomerization of meprin A serves to concentrate the proteolytic activity of the α subunit in the extracellular space for potential delivery to downstream targets in the kidney and intestinal lumen or for degradation of extracellular matrix proteins during leukocyte migration and cancer metastasis (2Villa J.P. Bertenshaw G.P. Bylander J.E. Bond J.S. Biochem. Soc. Symp. 2003; 70: 53-63Crossref PubMed Scopus (31) Google Scholar, 6Crisman J.M. Zhang B. Norman L.P. Bond J.S. J. Immunol. 2004; 172: 4510-4519Crossref PubMed Scopus (68) Google Scholar, 7Lottaz D. Maurer C.A. Hahn D. Buchler M.W. Sterchi E.E. Cancer Res. 1999; 59: 1127-1133PubMed Google Scholar, 9Matters G.L. Manni A. Bond J.S. Clin. Exp. Metastasis. 2005; 22: 331-339Crossref PubMed Scopus (38) Google Scholar). In addition, mutants that are not oligomeric are much less stable than the wild type, and this may serve to protect the protein from the harsh extracellular milieu (10Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 11Doll B.A. Villa J.P. Ishmael F.T. Bond J.S. Biol. Chem. 2002; 383: 1167-1173Crossref PubMed Scopus (7) Google Scholar). The folding and oligomerization process of the meprin A homo-oligomer is influenced by several factors within the α subunit. Mutagenesis of His-167, a zinc ligand in the protease domain, leads to a monomeric, inactive protein (11Doll B.A. Villa J.P. Ishmael F.T. Bond J.S. Biol. Chem. 2002; 383: 1167-1173Crossref PubMed Scopus (7) Google Scholar). The MAM 4The abbreviations used are: MAM, meprin, A-5 protein, protein-tyrosine phosphate μ; TRAF, tumor necrosis factor receptor-associated factor; ER, endoplasmic reticulum; MBP, mannan-binding protein; HEK-293 cells, human embryonic kidney 293 cells; DMEM, Dulbecco's modified Eagle's media; ESI/MS, electrospray ionization/mass spectrometry; TFMS, trifluoromethanesulfonic acid; TCEP, Tris-(2-carboxyethyl)phosphine HCl; BMPH, N-[β-maleimidopropionic acid] hydrazide·trifluoroacetic acid; PNGaseF, peptide:N-glycanase F; BME, β-mercaptoethanol; BK+, fluorogenic bradykinin analog 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Lys(Dnp)-Gly-OH; NEM, N-ethylmaleimide; HPLC, high pressure liquid chromatography; DTT, dithiothreitol. (meprin, A5 protein, protein-tyrosine phosphatase μ) and tumor necrosis factor receptor-associated factor (TRAF) domains are noncatalytic, but their presence is required for the secretion of a stable and active oligomer (12Tsukuba T. Bond J.S. J. Biol. Chem. 1998; 273: 35260-35267Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The MAM domain has also been implicated in oligomerization through the presence of intersubunit disulfide bonds, in addition to forming part of the noncovalent interface (3Ishmael F.T. Norcum M.T. Benkovic S.J. Bond J.S. J. Biol. Chem. 2001; 276: 23207-23211Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 10Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 13Chevallier S. Ahn J. Boileau G. Crine P. Biochem. J. 1996; 317: 731-738Crossref PubMed Scopus (20) Google Scholar). However, the influence of protein modifications such as glycosylation on the formation and structure of the homo-oligomer has not been investigated. Glycosylation accounts for ∼18–20% of the molecular mass of the meprin α subunit (14Tang J. Bond J.S. Arch. Biochem. Biophys. 1998; 349: 192-200Crossref PubMed Scopus (31) Google Scholar). Based on the cDNA-deduced amino acid sequence, the secreted α subunit from mouse contains 10 potential N-linked glycosylation sites with the consensus sequence of NX(S/T), where X is any amino acid except proline (Fig. 1) (15Jiang W. Gorbea C.M. Flannery A.V. Beynon R.J. Grant G.A. Bond J.S. J. Biol. Chem. 1992; 267: 9185-9193Abstract Full Text PDF PubMed Google Scholar). Glycosylation can influence the structure and function of a protein in several ways. During biosynthesis in the endoplasmic reticulum (ER), protein folding is monitored by the glycan-mediated interaction of the protein with ER-resident chaperones such as calnexin and calreticulin (16Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1629) Google Scholar). The placement of glycosylation sites within critical folding regions of the protein can recruit the chaperones to these regions and determine which set of ER chaperones will bind to the molecule (17Hebert D.N. Zhang J.X. Chen W. Foellmer B. Helenius A. J. Cell Biol. 1997; 139: 613-623Crossref PubMed Scopus (221) Google Scholar, 20Molinari M. Helenius A. Science. 2000; 288: 331-333Crossref PubMed Scopus (288) Google Scholar). Glycosylation also influences the physicochemical properties of a protein, such as conformational stability and protein solubility (21Wormald M.R. Dwek R.A. Structure. 1999; 7: R155-R160Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 22Wang C. Eufemi M. Turano C. Giartosio A. Biochemistry. 1996; 35: 7299-7307Crossref PubMed Scopus (333) Google Scholar). Some glycans mediate protein-protein interactions at the cell surface by influencing the orientation of the proteins and by serving as recognition and binding determinants between the molecules (23Rudd P.M. Wormald M.R. Stanfield R.L. Huang M. Mattsson N. Speir J.A. DiGennaro J.A. Fetrow J.S. Dwek R.A. Wilson I.A. J. Mol. Biol. 1999; 293: 351-366Crossref PubMed Scopus (211) Google Scholar). For instance, glycans are involved in the recognition and binding of meprin to the C-type serum lectin mannan-binding protein (MBP), the first endogenous inhibitor of meprin to be identified (24Hirano M. Ma B.Y. Kawasaki N. Okimura K. Baba M. Nakagawa T. Miwa K. Oka S. Kawasaki T. J. Immunol. 2005; 175: 3177-3185Crossref PubMed Scopus (38) Google Scholar). Mutagenesis of each individual glycosylation site in mouse meprin A demonstrated that no one particular glycan was responsible for targeting homo-oligomeric meprin A to the apical membrane of polarized epithelial cells (25Kadowaki 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). However, several of the glycosylation site mutants decreased peptidase activity as well as the stability of meprin A, as assessed by heat inactivation assays. These changes may result from subtle differences affecting catalysis or an overall change in the tertiary or quaternary structure. Previous attempts at mapping meprin glycosylation sites by lectin blotting of cyanogen bromide fragments detected 2–3 glycans in the MAM and TRAF domains of the α subunit in kidney-purified meprin A (15Jiang W. Gorbea C.M. Flannery A.V. Beynon R.J. Grant G.A. Bond J.S. J. Biol. Chem. 1992; 267: 9185-9193Abstract Full Text PDF PubMed Google Scholar). This isoform of meprin A is a membrane-bound hetero-oligomer of α and β subunits. However, the lectin experiments did not account for the other glycosylation sites, and there is no information regarding the glycosylation of the α subunit within the context of the homo-oligomer. To gain insight into the role of the carbohydrates of meprin, the glycosylation sites of the mouse meprin A homo-oligomer were first mapped using a mass spectrometry approach. To elucidate the effect of glycans on the native, folded protein, the structure and stability of a partially deglycosylated oligomer were then investigated. Finally, the effect of glycans on the formation of the oligomer was assessed using a series of single and multiple glycosylation site mutants. Expression and Purification of Wild-type Meprin A—Homooligomeric meprin A was purified from the media of human embryonic kidney 293 cells (HEK-293; ATCC 1573 CRL) stably expressing the wild-type, full-length meprin mouse α cDNA (26Marchand P. Tang J. Johnson G.D. Bond J.S. J. Biol. Chem. 1995; 270: 5449-5456Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) or the wild-type mouse α protein truncated at Arg-615 and His-tagged at the C terminus (27Villa J.P. Integrative Biosciences. Ph.D. thesis, Pennsylvania State University2003Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum and antibiotics/antimycotics (100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B; Invitrogen) at 37 °C with 5% CO2 until confluent. The cells were then maintained in serum-free media for 3–4 days. The protein was purified by anion exchange chromatography for the full-length protein or by a nickel chelating column for the His-tagged protein, as described previously (11Doll B.A. Villa J.P. Ishmael F.T. Bond J.S. Biol. Chem. 2002; 383: 1167-1173Crossref PubMed Scopus (7) Google Scholar, 28Bertenshaw G.P. Villa J.P. Hengst J.A. Bond J.S. Biol. Chem. 2002; 383: 1175-1183Crossref PubMed Scopus (29) Google Scholar). Expression of Single and Multiple Glycosylation Site Mutants— Full-length cDNAs of the single N-linked glycosylation site mutants of mouse meprin α were made previously (25Kadowaki 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). The double and triple mutants were generated in successive rounds of mutagenesis using QuikChange site-directed mutagenesis kit (Stratagene) with the N234Q mutant plasmid as a template, according to the manufacturer's instructions. The N152Q mutation was introduced with the following primers: 5′-GAT CAA CAG GTG GGA CAG CAA ATT TCC ATT GGT GAG GGA TG-3′ and 5′-CAT CCC TCA CCA ATG GAA ATT TGC TGT CCC ACC TGT TGA TC-3′. The N270Q mutation was introduced with the following primers: 5′-ATA AGA CTG AAT CGA ATG TAC CAG TGC ACC GCA ACA CAT ACT CTG-3′ and 5′-CAG AGT ATG TGT TGC GGT GCA CTG GTA CAT TCG ATT CAG TCT TAT-3′. All plasmids were sequenced by Joe Bednarczyk at the Molecular Genetics Core Facility of Pennsylvania State College of Medicine. Wild-type and mutant plasmids were transiently expressed in HEK-293 cells using Lipofectamine 2000 transfection reagent (Invitrogen). Briefly, 60 × 15-mm plates of HEK-293 cells were grown to 90% confluency in DMEM with 10% FBS but without antibiotics/antimycotics. The meprin plasmids (8 μg) and Lipofectamine 2000 reagent were each diluted into Opti-MEM reduced serum medium (Invitrogen), mixed according to the manufacturer's instructions, and added to the plates of HEK-293 cells. The plates were incubated at 37 °C for 4–6 h and then the media were replaced with DMEM lacking FBS. The medium from each plate was collected 24 h post-transfection and analyzed for protein expression by SDS-PAGE and Western blotting. For oligomeric size, stability, and activity assays, the 24-h post-transfection media samples were concentrated 4-fold using YM-50 Centricon concentrators (Amicon), and the EDTA-free Complete Mini inhibitor mixture (Roche Applied Science) was added after concentrating. The media samples were aliquoted and stored at –80 °C until further use. Gel Electrophoresis and Western Blotting—For SDS-PAGE, samples were separated on 7.5% Tris-HCl Ready gels (Bio-Rad) or 8% polyacrylamide gels at 40–45 mA in 25 mm Tris, 192 mm glycine, pH 8.5, with 0.1% SDS. Gels were transferred onto nitrocellulose membrane (Bio-Rad) at 16 V using a Bio-Rad semi-dry blotting apparatus with transfer buffer containing 25 mm Tris, 192 mm glycine, 0.04% SDS, and 20% methanol. Blots were blocked in 10% nonfat dry milk in 20 mm Tris, 138 mm NaCl, with 0.1% Tween 20 (TBS-T) and then incubated with an appropriate anti-meprin α antibody. Anti-rabbit secondary antibody coupled to horseradish peroxidase (Amersham Biosciences) was added after washing in TBS-T, and blots were developed with the appropriate chemiluminescent substrate (SuperSignal West Pico or West Dura, Pierce). N-Linked Glycosylation Site Mapping—To facilitate glycosylation site mapping, a mass marker was created at each glycosylation site using a chemical deglycosylation method adapted from previously published protocols (29Sojar H.T. Bahl O.P. Arch. Biochem. Biophys. 1987; 259: 52-57Crossref PubMed Scopus (79) Google Scholar, 30Edge A.S. Faltynek C.R. Hof L. Reichert Jr., L.E. Weber P. Anal. Biochem. 1981; 118: 131-137Crossref PubMed Scopus (537) Google Scholar). For detection of peptides by electrospray ionization/mass spectrometry (ESI/MS), a 500-μg aliquot of purified meprin A was concentrated to dryness in a Speedvac and then reconstituted in 75 μl of trifluoromethanesulfonic acid (TFMS; Sigma). The deglycosylation reaction proceeded for 2 h on ice and was quenched with the slow addition of pre-chilled 60% pyridine (200 μl, aqueous). The sample was dialyzed against Nanopure water for 4 h, using SpectraPor dialysis tubing (10 mm, 3500 molecular weight cutoff). The deglycosylated meprin was again concentrated to dryness, resuspended in 100 μl of 100 mm ammonium bicarbonate, pH 8.0, and digested 1:20 with trypsin (Sigma) for 24 h at 37 °C. Disulfide bonds were reduced with 50 mm dithiothreitol (Sigma) immediately prior to injection onto the HPLC column. One-half of the tryptic digest was injected onto a BetaBasic C18 column (5 μm, 150 × 1 mm) coupled to a Mariner mass spectrometer (PerSeptive Biosystems). The split flow rate was 0.05 ml/min, and a gradient program was used to partially separate the peptides. Peptides were loaded onto the column under the starting conditions of 100% solvent A (0.1% formic acid/H2O) for 6 min, diverting the flow to prevent salt interference. The peptides were eluted under a gradient to 95% solvent B (0.1% formic acid/acetonitrile) over 44 min. Following elution of the peptides, the conditions were held for 5 min, and then the column was flushed with 90% solvent C (0.1% formic acid/isopropyl alcohol). Electrospray ionization in positive mode was used for detection, and data were collected between m/z 200 and 2500. MS data were analyzed using the accompanying DataExplorer version 4.0.0.1 software (Applied Biosystems), utilizing the extracted ion chromatogram feature to search for the predicted m/z values of the glycopeptides. A separate sample of meprin A was also incubated in the presence or absence of the deglycosidase PNGaseF (New England Biolabs) to determine the occupancy of the glycosylation sites. A 500-μg sample (500 μl of a 11.8 μm solution) of mouse meprin α was denatured by boiling for 5 min at 100 °C and then the disulfides were reduced in the presence of 20 mm DTT (Sigma) for 30 min at 37 °C. N-Ethylmaleimide (Sigma) dissolved in 100% ethanol (HPLC grade; Sigma) was added to the sample (in the presence of the DTT) to a final concentration of 60 mm, and the sample was incubated for an additional 30 min at 37 °C. After denaturation, reduction, and capping of cysteines, the sample was exhaustively dialyzed into Nanopure water (2 × 2 liters) and then concentrated ∼5.5-fold using a Speedvac. The sample was then split into two, and one-half of the sample was incubated in 50 mm sodium phosphate buffer, pH 7.5, at 37 °C for 5 h in the presence of the deglycosidase PNGaseF (glycerol-free; New England Biolabs), and the other half was incubated under the same conditions in the absence of PNGaseF. After deglycosylation, trypsin (Promega Gold in 50 mm acetic acid; Sigma) was added to each sample so that the final trypsin/meprin ratio was 1:20, and the pH was 7.5 (in 50 mm phosphate buffer). The meprin samples were digested with trypsin at 37 °C for 39 h. One-tenth of each sample was analyzed using a Waters 2695 HPLC system with a Discovery Bio C5 column (Supelco, 5 μm,5cm × 2.1 mm) with a gradient similar to the chemically deglycosylated samples. A Waters LCT Premier time-of-flight mass spectrometer in positive ion mode was used to detect the peptide ions, and Waters MassLynx 4.0 software was used to search for the expected peptide ions. Cross-linking of the Meprin Homo-oligomer Noncovalent Dimer through the Carbohydrate Moieties—The carbohydrate moieties of the meprin A homo-oligomer were oxidized in the presence of 10 mm sodium periodate (Sigma) in 100 mm sodium acetate, pH 5.5, for 2 h at 23 °C in the dark. Excess sodium periodate was removed with a 1-ml desalting column packed with G-25 Sephadex resin (Sigma) equilibrated with water. For the cross-linking reaction, triethanolamine, pH 7.5, was added to the sample at a final concentration of 40 mm, and reduction of the intersubunit disulfide bond to yield the noncovalent dimer was achieved in the presence of 5 mm Tris-(2-carboxyethyl)phosphine HCl (TCEP), pH 8.0. After ∼15 min, the carbohydrate and cysteine-reactive cross-linker N-[β-maleimidopropionic acid] hydrazide·trifluoroacetic acid (BMPH; Pierce) was added to a final concentration of 10 mm in 10% dimethylformamide (Fisher). The cross-linking reaction proceeded for 2 h at 23 °C. The samples were analyzed by nonreducing SDS-PAGE and Western blotting as described above. Partial Deglycosylation of the Meprin Homo-oligomer— For partially deglycosylated samples, meprin A was incubated in the presence of PNGaseF (New England Biolabs), in 50 mm sodium phosphate buffer, pH 7.5, in the absence of β-mercaptoethanol (BME) and SDS (nonreducing and nondenaturing conditions). Samples were incubated at 37 °C for 2 h and then used for further experiments. A typical deglycosylation reaction contained at least 300–500 units of deglycosidase to 20 μg of meprin. Mapping Glycans Removed by PNGaseF—To determine which glycans were removed in the partially deglycosylated protein, 900 μl of 4.6 μm meprin (330 μg total) was treated with 20 μl of glycerol-free PNGaseF (New England Biolabs) under nondenaturing and nonreducing conditions (–SDS, –BME, but +Nonidet P-40) and prepared for ESI/MS as described above. One-half of the sample was digested with trypsin (1:20 in 20 mm Tris, 150 mm NaCl, pH 7.5). The data set collected from the tryptic digest was searched using the extracted ion chromatogram tool of the DataExplorer software, looking for the predicted tryptic fragments plus 1 mass unit, which resulted from the conversion of Asn to Asp by PNGaseF. Circular Dichroism and Intrinsic Tryptophan Fluorescence Spectroscopy—The far-UV CD spectra of the wild-type and partially deglycosylated proteins in the absence of urea were obtained using a Jasco J-710 spectropolarimeter fitted with a xenon lamp. Samples were scanned from 190 to 250 nm at a scan rate of 50 nm/min at 25 °C using quartz cuvettes with a path length of 1 mm. Spectra are an average of three scans. Samples of latent meprin A were partially deglycosylated as described above, then diluted to 3.2 μm with Nanopure water (8 mm sodium phosphate, final), and incubated at 23 °C for 15 min before analysis. Fluorescence spectra were obtained with a PTI QuantaMaster luminescence spectrometer. Wild-type and partially deglycosylated samples were diluted to 10 μg/ml (118 nm) in Nanopure water. Tris and NaCl were added to a final concentration of 20 and 150 mm, respectively, at pH 7.5. Samples were incubated at 23 °C for 30 min before analysis. Quartz cuvettes with path lengths of 1 cm × 2 mm were used, and the cuvette holder was kept at 25 °C. Excitation slit width was 0.5 nm, and emission slit width was 2.0 nm. Excitation wavelength was 280 nm to measure total protein fluorescence (from tryptophan and tyrosine), and the emission spectra were recorded from 300 to 400 nm. Size Exclusion Chromatography—The oligomeric states of purified and crude meprin A samples were analyzed by size exclusion chromatography on a Superose 12 column (Amersham Biosciences) equilibrated in 20 mm Tris, 150 mm NaCl, pH 7.5. A 200-μl sample was injected at a flow rate of 0.25 ml/min, and 1-ml fractions were collected. A small portion of each fraction was analyzed by SDS-PAGE and Western blotting with the appropriate meprin antibody to determine the elution profile of the protein. Urea-induced Unfolding of the Meprin A Homo-oligomer— The urea-induced unfolding of the meprin oligomer (wild-type and partially deglycosylated) was followed by the change in intrinsic tryptophan fluorescence. Samples were diluted to 10 μg/ml with Nanopure water and increasing amounts of urea (0.6 mm phosphate, final) and then incubated at 25 °C for 30 min. Fluorescence spectra were acquired with an excitation wavelength of 280 nm. The excitation and emission slit widths were 0.25 and 1.75 nm, respectively. The fluorescence intensity at the wavelength of maximal emission (λmax) of the wild-type protein was recorded and plotted versus urea concentration to obtain an unfolding curve. Activity Assays—The peptidase activity of meprin A was measured using the fluorogenic substrate 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Lys(Dnp)-Gly-OH (BK+), where Dnp is dinitrophenyl, as described previously (10Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Assays were carried out after activation of the samples by limited trypsin digestion (1:20 trypsin/meprin for purified samples or 5 ng/μl trypsin for crude media samples) for 30–60 min at 37 °C in 20 mm Tris, 150 mm NaCl, pH 7.5, to remove the prosequence. Trypsin was inhibited with a 3–10-fold excess of soybean trypsin inhibitor (Sigma) for 15–20 min at 25 °C before assaying activity in 50 mm ethanolamine buffer, pH 8.7, in the presence of 10 μm BK+ (final). The fluorescence was monitored at 320 nm excitation and 417 nm emission using a Hitachi-F2000 fluorimeter. For crude media samples, the specific activities of each protein were calculated by Western blot and densitometry. The concentration of the wild-type protein in the media sample was measured from its BK+ activity compared with the fluorescence/μg of purified protein. Tryptic Stability Assays—To test the activation and stability of the meprin glycosylation mutants, crude media samples were incubated with increasing amounts of trypsin (0, 5, 40, and 80 ng/μl) for 45 min at 37 °C. Trypsin was inhibited for 15 min at 23 °C by a 3-fold excess of soybean trypsin inhibitor. Samples were mixed with SDS-PAGE sample buffer (+BME) and immediately boiled. The entire set of samples was then boiled for an additional 5 min before analysis by SDS-PAGE and Western blotting. Heat Stability Assays—To measure the heat stability of partially deglycosylated meprin A, activated samples were incubated in the presence and absence of PNGaseF under native conditions, as described above. The samples were then incubated at 55 °C for 0, 5, 10, 15, 30, and 45 min. At each time point, a small aliquot of the protein was removed, cooled to 23 °C, diluted into assay buffer, and assayed for BK+ activity in triplicate at 23 °C. The percent of initial activity over time was plotted for each treatment. For crude media samples of meprin A mutants, the samples were activated as indicated, then incubated at 55 °C for 0, 5, 15, 30, and 45 min, and processed as described for partially deglycosylated protein except that the samples were diluted into assay buffer and allowed to equilibrate at 23 °C for at least 15 min. The samples were then equilibrated to 30 °C, and BK+ activity was measured at this temperature. N-Linked Glycosylation Site Mapping of the Mouse Meprin A Homo-oligomer—Chemical deglycosylation with TFMS removes the carbohydrate moieties from glycoproteins with the exception of the innermost GlcNAc residue. This creates a mass marker of +203 mass units on each glycopeptide that can then be detected by mass spectrometry. Under certain conditions, the innermost GalNAc residue of O-linked sugars also remains on the protein (29Sojar H.T. Bahl O.P. Arch. Biochem. Biophys. 1987; 259: 52-57Crossref PubMed Scopus (79) Google Scholar, 30Edge A.S. Faltynek C.R. Hof L. Reichert Jr., L.E. Weber P. Anal. Biochem. 1981; 118: 131-137Crossref PubMed Scopus (537) Google Scholar). However, there was no detectable GalNAc in a total carbohydrate analysis of meprin A, and the protein was not detected by a carbohydrate-reactive label after the protein was fully deglycosylated by PNGaseF, a deglycosidase that removes all types of N-linked glycans; thus there is no significant O-linked glycosylation on meprin A (data not shown). The predominant monosaccharides in the carbohydrate analysis were GlcNAc, galactose, and mannose, with a small amount of fucose. Further study is needed to accurately quantify these monosaccharides and determine the location of the fucose in the context of the terminal glycosylation pattern. ESI/MS was used to detect glycopeptides from a tryptic digest of chemically deglycosylated meprin A. The total ion chromatogram from the digest was searched for the presence of glycopeptide ions at their calculated mass-to-charge (m/z) ratios. Peptide ions consistent with glycosylation at Asn-152, Asn-234, Asn-270, Asn-452, Asn-546, and Asn-553 were also detected (Table 1). Because there were no potential proteolytic cleavage sites between Asn-546 and Asn-553, these two glycosylation sites were contained in the same peptide. Masses consistent with both one and two GlcNAc residues were observed in the total ion chromatogram, indicating heterogeneity in this region (Table 1). The only site not glycosylated was Asn-614, near the putative C terminus. There were no ions corresponding to either the u" @default.
• W2012702020 created "2016-06-24" @default.
• W2012702020 creator A5006267438 @default.
• W2012702020 creator A5026441432 @default.
• W2012702020 creator A5078007274 @default.
• W2012702020 creator A5090568840 @default.
• W2012702020 date "2006-12-01" @default.
• W2012702020 modified "2023-09-27" @default.
• W2012702020 title "Protease Domain Glycans Affect Oligomerization, Disulfide Bond Formation, and Stability of the Meprin A Metalloprotease Homo-oligomer" @default.
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