Protease Domain Glycans Affect Oligomerization, Disulfide Bond Formation, and Stability of the Meprin A Metalloprotease Homo-oligomer

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; protein, protein-tyrosine and tumor necrosis factor receptor-associated factor are their is for the of a stable and oligomer Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The MAM domain in oligomerization through the of intersubunit disulfide in to of the noncovalent 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, P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, J. P. Biochem. J. 1996; PubMed Scopus Google Scholar). the of protein as glycosylation on the formation and of the homo-oligomer not for of the molecular mass of the meprin α subunit J. Bond J.S. Biochem. PubMed Scopus (31) Google Scholar). on the the secreted α subunit from mouse 10 potential N-linked glycosylation sites the of is Bond J.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the and of a protein in several in the endoplasmic protein folding is by the of the protein as and A. M. Biochem. 2004; PubMed Scopus Google Scholar). The of glycosylation sites within folding of the protein the to and determine of to the Zhang B. A. J. Biol. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google Scholar). the of a protein, as stability and protein 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. 1996; PubMed Scopus Google Scholar). glycans at the by the of the proteins and by as and the M. J.S. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). glycans are in the and of meprin to the mannan-binding protein the of meprin to be M. M. J. Immunol. 2005; PubMed Scopus (38) Google Scholar). Mutagenesis of glycosylation in mouse meprin A demonstrated that no one glycan was for meprin A to the of epithelial cells Bertenshaw G.P. Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). several of the glycosylation mutants decreased activity as well as the stability of meprin as by These may from affecting or an in the or quaternary structure. at meprin glycosylation sites by of glycans in the MAM and of the α subunit in meprin A Bond J.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This isoform of meprin A is a of α and subunits. the not for the glycosylation and is no the glycosylation of the α subunit within the of the homo-oligomer. into the role of the carbohydrates of meprin, the glycosylation sites of the mouse meprin A homo-oligomer were using a mass the of glycans on the protein, the and stability of a oligomer were the of glycans on the formation of the oligomer was using a of single and glycosylation and of Meprin meprin A was from the of human embryonic kidney 293 cells the meprin mouse α P. J. Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google or the mouse α protein at and at the J.P. Scholar). were in Dulbecco's modified Eagle's and and at The cells were in for The protein was by for the protein or by a for the protein, as (11Doll B.A. Villa J.P. Ishmael F.T. Bond J.S. Biol. Chem. 2002; 383: 1167-1173Crossref PubMed Scopus (7) Google Scholar, G.P. Villa J.P. Bond J.S. Biol. Chem. 2002; 383: PubMed Scopus Google Scholar). of and of the single N-linked glycosylation mutants of mouse meprin α were Bertenshaw G.P. Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The and mutants were in of mutagenesis using mutagenesis the as a to the The was the and The was the and were by at the of of and were in HEK-293 cells using of HEK-293 cells were to in without The meprin and were into to the and to the of HEK-293 The were at for and the were The from was and for protein by and oligomeric and activity the were using and the was The were and at further and were on or at in were at using a and were in in and an α to was in and were the or glycosylation a mass was at glycosylation using a chemical deglycosylation from Biochem. PubMed Scopus Google Scholar, P. Biochem. PubMed Scopus Google Scholar). of by electrospray ionization/mass a of meprin A was to in a and in of The deglycosylation for on and was the of The was for using molecular The meprin was to in of and for at bonds were to the of the was a to a mass The was and a was used to the were the under the conditions of A for the to The were under a to of the the conditions were for and the was ionization in was used for and were and were using the the to for the of the A of meprin A was in the or absence of the deglycosidase to determine the of the glycosylation A of a of mouse meprin α was by for at and the were in the of for at in was to the the of the to a of and the was for an at and of the was into and using a The was into and of the was in phosphate at for in the of the deglycosidase and the was under the conditions in the absence of in was to that the was and the was phosphate The meprin were at for of was using a a a to the A mass in was used to the and was used to for the of the Meprin through the of the meprin A homo-oligomer were in the of 10 in for at in the was a the cross-linking was to the at a of and of the intersubunit disulfide bond to the noncovalent was in the of the and was to a of 10 in The cross-linking for at The were by and as of the Meprin meprin A was in the of in phosphate in the absence of and and nondenaturing were at for and used for further A deglycosylation at of deglycosidase to of by determine glycans were in the protein, of meprin was of under nondenaturing and conditions and for as of the was in The from the was using the of the for the mass from the of to by and of the and proteins in the absence of were using a a were from to at a of at using a of are an of of meprin A were as to and at for were a and were to 10 in and were to a of and at were at for of were and the was at was and was was to protein and and the were from to oligomeric of and meprin A were by on a in A was at a of and were A of was by and the meprin to determine the of the of the Meprin A The of the meprin oligomer and was by the in were to 10 and of and at for were an of The and were and The at the of of the protein was and to an activity of meprin A was using the is as (10Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). were of the by for or for for at in to the was a of for at activity in in the of 10 The was at and using a the of protein were by and The of the protein in the was from activity the of the and stability of the meprin glycosylation were of and for at was for at by a of were and The of was for an by and the stability of meprin were in the and absence of under as The were at for and a of the protein was to into and for activity in at The of activity was for of meprin A the were as at for and and as for protein that the were into and to at for at The were to and activity was at this of the Meprin A deglycosylation the from the of the This a mass of mass on that be by mass spectrometry. certain the of on the protein Biochem. PubMed Scopus Google Scholar, P. Biochem. PubMed Scopus Google Scholar). was no in a of meprin and the protein was not by a the protein was by a deglycosidase that of N-linked is no glycosylation on meprin A not The in the were and a of study is to and determine the of the in the of the glycosylation was used to from a of meprin A. The from the was for the of at their glycosylation at Asn-152, Asn-234, Asn-270, Asn-452, Asn-546, and were were no potential proteolytic sites and two glycosylation sites were in the both one and two were in the in this The not glycosylated was near the were no to the or glycosylated for and the of sites not be from this on mutagenesis and studies of and protein-tyrosine MAM are highly disulfide-bonded F.T. Ishmael Bond J.S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 25: PubMed Scopus Google Scholar). of the protein in this of the disulfide bonds may be one the MAM domain were not it is that at is glycosylated, this was to be glycosylated in meprin A Bond J.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). an deglycosylation was used to determine the and glycosylation sites were by a glycosylation of mouse meprin A using chemical in the of the to a of to a of not the glycosylated were not the glycosylated were in the of the to a of not the glycosylated were in a of the deglycosidase the from N-linked to a mass on the of the protein in the MAM domain, the disulfide bonds in meprin A were and With this of the glycosylation sites by chemical deglycosylation was and to the glycosylated forms of the and were In a that was in the absence that to the were for glycosylation a of in of not of the ten potential glycosylation sites in mouse meprin A are glycosylated, the of at the glycosylation of mouse meprin A using enzymatic in the of the the of in the the of in the the of in the the of in the the of in the the of in the in the of the the of in the in a at or the intersubunit disulfide bonds of the meprin A homo-oligomer be to a that is the of the protease 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). determine glycans were in of the noncovalent the of the the noncovalent of meprin A was the and and by and the intersubunit disulfide bonds were to form a noncovalent this was into a during in the absence of the noncovalent is on a is that carbohydrates are at or near the noncovalent and a of this The a of is to the of meprin intersubunit disulfide bonds the and forms of the noncovalent be not The of glycans in this to the ability of the α subunit to noncovalent by of the protein or and of in a determine the of glycans on the of the protein, glycans were from the homo-oligomer under nondenaturing Full deglycosylation of meprin A and the protein; under nondenaturing a of the glycans are In the oligomer no activity, in studies the protein was at for to Bertenshaw G.P. Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the protein was for at an of of the activity the of was a further were using a deglycosylation under nondenaturing the the of the to a mass of mass that be by of a of the homooligomer that glycans are from the oligomer under nondenaturing to Asn-234 and Asn-270 in the protease domain and in the domain were of the glycosylation sites by in meprin in the of the in the of the in a and of the the removal of glycans from the protein not affect the activity of the it is that the of the subunits is The of deglycosylation on the and of meprin A was by and were in the of and meprin A in the of and was no in the of the protein A in the of was in the of the protein from for wild to for the not affect the quaternary of the The of the protein in in of a the formation of an The species in and 10 for the protein were less in the protein, may be of or of the was no of the oligomeric species in the absence of the protease and domain on of the the activity were in the of both the and was no in the oligomeric of the protein, glycans decreased the stability of the as by and stability The protein at a than the wild The was not and was a not be to the in This was more in the protein; or no was for the protein, under the conditions the protein was at for of the protein enzymatic activity than the protein glycans in the protease and are the protein Oligomerization of The of N-linked glycan to the formation of both the and noncovalent was using a of single N-linked glycosylation mutants in the of the was to have to the of disulfide bond formation in intestinal Biochem. J. 2003; PubMed Scopus (39) Google Scholar). In the meprin α is 10 from an intersubunit disulfide and the of of the single mutants into HEK-293 cells, the the secreted proteins were by of the mutants was of the disulfide-bonded to the as the protein, the the mutants were by and for their ability to form the molecular mass of the mutants to wild the and mutants in the protease domain in of a formation of an The of the in the this is of a of the on the of was of the wild A more of of was protein and may be to the of the the in activity and stability of the single glycosylation mutants by Bertenshaw G.P. Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google are not of in the quaternary of the Oligomerization of no one glycosylation the oligomeric of the meprin A the of was of the protease domain glycans are in the meprin α subunits of and It was that the absence of glycosylation in this domain the domain to the was in ability to form the disulfide-bonded In this no species was on the of was of the on This was the of by the protein The in formation for the may of of of the protein into the of the was to noncovalent of the α subunits. of the that the of the protein in to a The of protein in to a on the of protein was to be monomeric the were under conditions not In more of the disulfide-bonded species was by more was in as species that disulfide-bonded species for or less of the species may be to and of the protein, or may a of the oligomer to a or determine the of the on the oligomerization was of a and of the domain or a more of a of mutants were and by and The and mutants the disulfide-bonded to the as the wild and were of the molecular mass the the was in ability to form the disulfide-bonded the of in was of the and the protein as a a of species These that is a the glycans at Asn-152 and Asn-270 that oligomerization through both the and noncovalent and the on oligomerization is not of the absence of more than one the glycan this and the of oligomerization is not of the to This was demonstrated by the or in the of the A to the in of formation and oligomer formation, the single mutants not have an on the quaternary not and of the glycosylation mutants were further for their ability to be by their of activity, and their stability in to the wild and the single glycosylation of the mutants be by removal of the at the monomeric mutants and were at of These mutants not have activity These that is in the protein of The and the were to degradation both the single and protease domain mutants activity In to the single protease domain glycosylation mutants activity in the study Bertenshaw G.P. Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This may conditions that affect the of meprin A The may be of at a activity of the glycosylation not not in a The single and glycosylation mutants of as by a The and single mutants activity than the wild type, the was The of the mutants was the was the activity 10 These the study of the protein, the of protease domain N-linked glycans in the stability of the homo-oligomer. In this N-linked glycans were as factors that to both the oligomerization and stability of the mouse meprin A homo-oligomer. A unique the glycans at Asn-152 and Asn-270 in the protease domain was glycans to formation of a oligomer that form intersubunit disulfide bonds in the MAM In addition, several glycans were to to the stability of the The role of the protease domain glycans in a of the protease domain in the and activity of the are the oligomeric of the astacin of a and a protease domain, is monomeric and not glycosylation sites PubMed Scopus Google Scholar). is a protease and is not single protease domain glycosylation not have a on activity and stability of the protein J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). two of the protease domain glycans of the meprin α subunit are to form an stable This is not of a of the protease domain in the absence of is a more on the folding and that the subunits. the folding of a protein by A. M. Biochem. 2004; PubMed Scopus Google Scholar). the of meprin A is highly on a protease domain affect the of the protein in the MAM and Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). by the absence of the Asn-152 and Asn-270 glycans intersubunit disulfide bond formation in the MAM domain of the disulfide bonds during folding of the In this disulfide form and are by an C.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). are as in the of meprin the process is in and intersubunit disulfide bonds not form (10Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, J. P. Biochem. J. 1996; PubMed Scopus Google Scholar, F.T. Ishmael Bond J.S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the to form the two disulfide bonds in the protease domain the protein in a that not in the protease MAM to the or to their on a of the protease domain, the of the Asn-152 and Asn-270 glycans to the two disulfide bonds to disulfide bond formation, a that was in a from the J. Biochem. PubMed Scopus Google Scholar, B. Chem. Biol. Full Text PDF PubMed Scopus Google Scholar). The to form the noncovalent is a of intersubunit disulfide bond formation, as well as of the The protease domain glycans are in the of the oligomer chemical and thermal The of the glycan by for the the of the protein are decreased D. Full Text Full Text PDF PubMed Scopus Google Scholar). the Asn-234 glycan in the meprin α protease domain within a in the of this domain, on it the of this J. Biochem. PubMed Scopus Google Scholar). the Asn-234 and Asn-270 glycans not affect the of meprin A. These are a study of that that glycans not affect the and of proteins M. A. 1996; PubMed Scopus Google Scholar). of the was to the of to the for the meprin A glycosylation mutants and The role of the meprin A protease domain glycans may be in protease activity in the of the kidney and intestinal on the of the glycans to the of the protein, the glycans may of the oligomer from in the extracellular as was demonstrated for the 2001; PubMed Scopus Google Scholar). The glycans of the MAM and were to be the to the oligomeric of meprin that oligomerization in proteins and that meprin A oligomerization (10Marchand P. Volkmann M. Bond J.S. J. Biol. Chem. 1996; 271: 24236-24241Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. P. Biochem. J. 1996; PubMed Scopus Google Scholar, F.T. Ishmael Bond J.S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the glycans in were not by to form the molecular mass a MAM no on disulfide or activity. The glycans of the domain, not to the of the protein, may to be in as MBP, the of meprin that to M. M. J. Immunol. 2005; PubMed Scopus (38) Google Scholar). The at and in the domain be an factor in the of meprin A or extracellular proteins by of meprin the glycan was by Asn-234 and Asn-270, it may not as much to oligomer stability as the two glycosylation sites the was to have stability Bertenshaw G.P. Bond J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of at the of the protein, it may The absence of a glycan at near the may be to of the protein in this or the of the protein may the from a glycan to this J. 2005; PubMed Scopus Google Scholar). The of the of meprin and the of the glycans of the protein in to the of the to this of the glycans or by and using or cells that the glycans in form P. Mol. Biol. PubMed Scopus Google Scholar). This study demonstrated that the be more the MAM domain glycans were by the protease domain glycans are for and a deglycosidase to Asn-234 and Asn-270 from the protein two more of the In addition, of the intersubunit disulfide bond to a noncovalent the protein much more to The glycans of meprin A have to be factors in meprin A by meprin subunits glycans may meprin and and secreted proteins glycans are by be in this of to meprin activity. the of the glycans of meprin may a role in as the of glycans be in as a in the glycan may the of and secreted for of and the of in the of meprin glycosylation sites by electrospray mass spectrometry. for of the and as well as for