N-Linked Oligosaccharides on the Meprin A Metalloprotease Are Important for Secretion and Enzymatic Activity, but Not for Apical Targeting

Abstract

The α and β subunits of meprins, mammalian zinc metalloendopeptidases, are extensively glycosylated; ∼25% of the total molecular mass of the subunits is carbohydrate. The aim of this study was to investigate the roles of the N-linked oligosaccharides on the secreted form of mouse meprin A. Recombinant meprin α and mutants in which one of the 10 potential Asn glycosylation sites was mutated to Gln were all secreted and sorted exclusively into the apical medium of polarized Madin-Darby canine kidney cells, indicating that no specific N-linked oligosaccharide acts as a determinant for apical targeting of meprin α. Several of the mutant proteins had decreased enzymatic activity using a bradykinin analog as substrate, and deglycosylation of the wild-type protein resulted in loss of 75–100% activity. Some of the mutants were also more sensitive to heat inactivation. In studies with agents that inhibit glycosylation processes in vivo, tunicamycin markedly decreased secretion of meprin, whereas castanospermine and swainsonine had little effect on secretion, sorting, or enzymatic properties of meprin. When all the potential glycosylation sites on a truncated form of meprin α (α-(1–445)) were mutated, the protein was not secreted into the medium, but was retained within the cells even after 10 h. These results indicate that there is no one specific glycosylation site or type of oligosaccharide (high mannose- or complex-type) that determines apical sorting, but that core N-linked carbohydrates are required for optimal enzymatic activity and for secretion of meprin α. The α and β subunits of meprins, mammalian zinc metalloendopeptidases, are extensively glycosylated; ∼25% of the total molecular mass of the subunits is carbohydrate. The aim of this study was to investigate the roles of the N-linked oligosaccharides on the secreted form of mouse meprin A. Recombinant meprin α and mutants in which one of the 10 potential Asn glycosylation sites was mutated to Gln were all secreted and sorted exclusively into the apical medium of polarized Madin-Darby canine kidney cells, indicating that no specific N-linked oligosaccharide acts as a determinant for apical targeting of meprin α. Several of the mutant proteins had decreased enzymatic activity using a bradykinin analog as substrate, and deglycosylation of the wild-type protein resulted in loss of 75–100% activity. Some of the mutants were also more sensitive to heat inactivation. In studies with agents that inhibit glycosylation processes in vivo, tunicamycin markedly decreased secretion of meprin, whereas castanospermine and swainsonine had little effect on secretion, sorting, or enzymatic properties of meprin. When all the potential glycosylation sites on a truncated form of meprin α (α-(1–445)) were mutated, the protein was not secreted into the medium, but was retained within the cells even after 10 h. These results indicate that there is no one specific glycosylation site or type of oligosaccharide (high mannose- or complex-type) that determines apical sorting, but that core N-linked carbohydrates are required for optimal enzymatic activity and for secretion of meprin α. endoplasmic reticulum Madin-Darby canine kidney Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis endoglycosidase H endoglycosidase F/N-glycosidase F bradykinin analog, 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Lys(dintrophenyl)-Gly-OH brefeldin A Meprins belong to the astacin family of metalloendopeptidases and to the metzincin superfamily (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (353) 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 (634) Google Scholar). The prototype of the family is the crayfish astacin (EC 3.4.24.21), a 20-kDa monomer secreted from the hepatopancreas. The meprins are oligomers composed of α and/or β subunits that are evolutionarily related, but differ in function (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (353) Google Scholar,3Wolz R.L. Bond J.S. Methods Enzymol. 1995; 248: 325-345Crossref PubMed Scopus (57) Google Scholar). Meprin β subunits are integral membrane proteins; mature α subunits are secreted from cells unless associated with β subunits (4Johnson G.D. Hersh L.B. J. Biol. Chem. 1994; 269: 7682-7688Abstract Full Text PDF PubMed Google Scholar, 5Marchand P. Tang J. Bond J.S. J. Biol. Chem. 1994; 269: 15388-15393Abstract Full Text PDF PubMed Google Scholar). Meprin A (EC 3.4.24.18) is defined as those isoforms that contain the α subunit; this includes membrane-bound α/β hetero-oligomeric forms and a secreted α homo-oligomeric form (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (353) Google Scholar, 6Beynon R.J. Oliver S. Robertson D.H.L. Biochem. J. 1996; 315: 461-466Crossref PubMed Scopus (31) Google Scholar). Meprins are tissue-specific proteases that are implicated in developmental processes as well as in normal and pathological processes in adult tissues (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (353) Google Scholar, 7Matters G.L. Bond J.S. Mol. Carcinogen. 1999; 25: 169-178Crossref PubMed Scopus (38) Google Scholar, 8Lottaz D. Maurer C.A. Hahn D. Buchler M.W. Sterchi E.E. Cancer Res. 1999; 59: 1127-1133PubMed Google Scholar). They are secreted from or localized in mammalian brush-border membranes of the intestine and kidney epithelial cells (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (353) Google Scholar, 9Jiang W. Sadler P.M. Jenkins N.A. Gilbert D.J. Copeland N.G. Bond J.S. J. Biol. Chem. 1993; 268: 10380-10385Abstract Full Text PDF PubMed Google Scholar, 10Gorbea C.M. Marchand P. Jiang W. Copeland N.G. Gilbert D.J. Jenkins N.A. Bond J.S. J. Biol. Chem. 1993; 268: 21035-21043Abstract Full Text PDF PubMed Google Scholar). Secreted meprin A is found in mouse urine and in the lumen of the colon (6Beynon R.J. Oliver S. Robertson D.H.L. Biochem. J. 1996; 315: 461-466Crossref PubMed Scopus (31) Google Scholar, 8Lottaz D. Maurer C.A. Hahn D. Buchler M.W. Sterchi E.E. Cancer Res. 1999; 59: 1127-1133PubMed Google Scholar). Meprins are capable of degrading proteins such as collagens and gelatin; hormones such as parathyroid hormone, luteinizing hormone-releasing hormone, and melanocyte-stimulating hormone; and small peptides such as bradykinin, angiotensins, and gastrin (3Wolz R.L. Bond J.S. Methods Enzymol. 1995; 248: 325-345Crossref PubMed Scopus (57) Google Scholar, 11Chestukhin 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). The meprins may therefore be involved in activation or inactivation of important extracellular proteins and peptides and are highly regulated at transcriptional and post-transcriptional levels themselves. The deduced amino acid sequences of meprin α subunits from mouse, rat, and human cDNAs have been determined (12Johnson G.D. Bond J.S. Barrett A.J. Woessner F. Rawlings N. Handbook of Proteolytic Enzymes. Academic Press, Inc., San Diego, CA1999: 1222-1227Google Scholar). The subunit encodes an amino-terminal signal sequence (see Fig. 1, S), a prosequence (Pro), a protease (astacin-like) domain, a MAM (meprin, A5 protein, protein-tyrosine phosphatase μ) domain, a MATH (meprinand tumor necrosis factor receptor-associated factor homology) domain, an AM (afterMATH) domain, an I (inserted) domain, an epidermal growth factor (EGF)-like domain, a putative transmembrane-spanning domain (T), and a cytoplasmic domain (C) (13Tsukuba T. Bond J.S. J. Biol. Chem. 1998; 273: 35260-35267Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The mouse meprin α subunit is extensively glycosylated, containing ∼25% carbohydrate (14Tang J. Bond J.S. Arch. Biochem. Biophys. 1998; 349: 192-200Crossref PubMed Scopus (31) Google Scholar). It contains 10 potential N-linked glycosylation sites (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). The functions of the oligosaccharides of meprins, however, have not been investigated. Appropriate glycosylation of a number of proteins is important for proper expression and function. For example, nonglycosylated rabies virus glycoprotein is not expressed at the cell surface, nor is it effective as a vaccine (16Shakin-Eshleman S.H. Remaley A.T. Eshleman J.R. Wunner W.H. Spitalnik S.L. J. Biol. Chem. 1992; 267: 10690-10698Abstract Full Text PDF PubMed Google Scholar); N-glycosylation of GD3 synthase is necessary for folding, catalytic activity, and trafficking to the Golgi complex (17Martina J.A. Daniotti J.L. Maccioni H.J.F. J. Biol. Chem. 1998; 273: 3725-3731Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar); and glycosylation of the human high affinity immunoglobulin E receptor is necessary for efficient folding in the endoplasmic reticulum (18Letourneur O. Sechi S. Willette-Brown J. Robertson M.W. Kinet J.-P. J. Biol. Chem. 1995; 270: 8249-8256Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The features of meprins that direct the enzymes to apical membranes have not been characterized; however, the final destination of these proteases is critical for normal tissue physiology and for pathological processes. Basolateral targeting of transmembrane proteins generally is mediated by discrete amino acid sequence motifs in the cytoplasmic domain (19Kirchhausen T. Bonifacino J.S. Riezman H. Curr. Opin. Cell Biol. 1997; 9: 488-495Crossref PubMed Scopus (352) Google Scholar). For apical targeting, some proteins, such as erythropoietin, require specific N-linked oligosaccharides (20Kitagawa Y. Sano Y. Ueda M. Higashino K. Narita H. Okano M. Matsumoto S.-I. Sasaki R. Exp. Cell Res. 1994; 213: 449-457Crossref PubMed Scopus (77) Google Scholar); other proteins require juxtamembrane segments with clusteredO-linked oligosaccharides or interactions of transmembrane domains or glycosylphosphatidylinositol anchors with the lipid bilayer (21Yeaman C. Le Gall A.H. Baldwin A.N. Monlauzeur L. Le Bivic A. Rodriguez-Boulan E. J. Cell Biol. 1997; 139: 929-940Crossref PubMed Scopus (246) Google Scholar, 22Scheiffele P. Roth M.G. Simins K. EMBO J. 1997; 16: 5501-5508Crossref PubMed Scopus (570) Google Scholar, 23Brown D.A. Crise B. Rose J.K. Science. 1989; 245: PubMed Scopus Google Scholar). meprin α not contain and the transmembrane and cytoplasmic domains are in the endoplasmic reticulum those features be however, N-linked oligosaccharides are targeting Madin-Darby canine kidney cells, a well cell for study of of epithelial cell Y. Sano Y. Ueda M. Higashino K. Narita H. Okano M. Matsumoto S.-I. Sasaki R. Exp. Cell Res. 1994; 213: 449-457Crossref PubMed Scopus (77) Google Scholar, C. Le Gall A.H. Baldwin A.N. Monlauzeur L. Le Bivic A. Rodriguez-Boulan E. J. Cell Biol. 1997; 139: 929-940Crossref PubMed Scopus (246) Google Scholar, 22Scheiffele P. Roth M.G. Simins K. EMBO J. 1997; 16: 5501-5508Crossref PubMed Scopus (570) Google Scholar, 23Brown D.A. Crise B. Rose J.K. Science. 1989; 245: PubMed Scopus Google were in this study to oligosaccharides are required for the apical targeting of meprin α or secretion, folding, and activity of the meprin α in which potential glycosylation sites were (see Fig. and of and of N-linked oligosaccharides were for this The containing wild-type mouse meprin α subunit the and the MATH were (see Fig. (13Tsukuba T. Bond J.S. J. Biol. Chem. 1998; 273: 35260-35267Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). These wild-type and mutant cDNAs in were into at site to expression The mutant was by the using a that the of the wild-type at to a was for to was using the For the Asn in the sequence was to Gln using The was to site in the site of to a site for were as by sequence cells and human kidney cells were in Dulbecco's modified Eagle's medium with and in a with The expression containing wild-type or mutant meprin α were into cells by the 10 of were to cells on tissue after the cells were with and for in to For expression of wild-type and mutant meprin the expression were into human kidney cells by the using 10 of expression and of tissue P. Tang J. G.D. Bond J.S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). were to by in The medium was with I and in the for an h. The tissue of cells were after of and to at for The were to using and The cells were with from the with a and to at The cells were in containing for at and to at for the is to as the cell cell were in containing of secretion of wild-type and mutant meprin the cells were on on a was were with an of and cells were and as The and cell were to electrophoresis using M. J. Mol. Biol. PubMed Scopus Google Scholar). was as P. M. Bond J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The proteins were with meprin α in Meprin subunits were using the The cells were for at in and with The cells were for with and in the the cells were from the with in containing and and and for The were to at 10 The cells and were with of for at to of protein A and at for The were with of meprin α at for 10 and at for h. The were with of protein gel for at with The were with containing and 10 with the containing and and with The were for at with of containing and with For total the or secreted proteins were by for at with containing The were to a final of containing and protease and The were with 10 of endoglycosidase H or of endoglycosidase F/N-glycosidase F and at for h. were by the in investigate the of deglycosylation on enzymatic activity, containing meprin were with as that no or were in the was to the at a final of in to meprin at for was at a were at for and at A bradykinin analog from was as a P. M. Bond J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (38) Google Scholar). For specific activity the of meprin protein in was determined by and P. M. Bond J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (38) Google Scholar). For all of activity, at were and are as of cells wild-type mouse meprin α were on tissue a was were with or tunicamycin for and to was in all the the of other cells wild-type mouse meprin α were on on a was as determined by cells were with or 10 or for or with or castanospermine or swainsonine for in the were with I with or at the and for h. The cells and were as and to cells with mouse meprin α subunit were on and apical and were for meprin protein after meprin A was exclusively secreted into the apical medium with a molecular mass of When cells were by with such as which or which apical of meprin was markedly In the of of the secreted meprin was found in the apical and in the of was to the apical was no that these cell or from the of cells, and the was not in the apical or medium not These results indicate that meprin A is secreted into the apical medium by a that an and The mouse meprin α subunit contains 10 oligosaccharide in the and in the protease and in the MAM in the MATH and and in the AM domain A of mouse meprin α mutants were in which Gln was for Asn at the 10 potential N-glycosylation of the mutants was into cells, and apical and were by and mutants were secreted into the apical medium the wild-type meprin In a a small of meprin α was in the however, it was found to be of the total secreted meprin. These results indicate that oligosaccharide are not critical of apical The enzymatic of the secreted mutant proteins a of to with the wild-type activity at one mutant had a specific activity and the wild-type in the MATH of the other mutants had of the wild-type for a in the AM domain, the activity was the as that of the wild-type The decreased were not to of the subunit and no were in of the mutants to by of to as determined by not These results indicate that the N-linked oligosaccharides are not for activity, and the and AM of the meprin α subunit to optimal enzymatic activity. deglycosylation of secreted wild-type meprin A also resulted in loss of enzymatic activity In the of Fig. meprin were by and In the the were not with endoglycosidase or gel however, with after to enzymatic activity. the and were to oligosaccharides after of meprin but the subunits The activity after whereas the with had no activity. deglycosylation had on enzymatic of meprin A with at potential N-glycosylation to the wild-type protein to the wild-type protein to the wild-type protein to the wild-type protein to the wild-type protein to the wild-type protein to the wild-type protein to the wild-type protein to wild-type and mutant were by as The are of using as were as G.D. Bond J.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google to the wild-type protein in a to wild-type and mutant were by as The are of using as were as G.D. Bond J.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). in to heat inactivation were the wild-type protein and some meprin α site mutants meprin A retained activity after at for results were for of the Asn and mutants and were after of at The mutant was the mutant and activity in at These results indicate that the at and in the protease domain and at in the AM domain of the the of N-linked oligosaccharides of meprin α in with or tunicamycin were the of the N-glycosylation the of from to to form J.S. Biochem. Biophys. Res. PubMed Scopus Google Scholar). The wild-type meprin α subunit was as an protein in cells, to a protein, and secreted into the When with the meprin α proteins associated with cells had a molecular mass of however, the expression was markedly and the protein was not in the markedly decreased the and secretion of meprin α into the an of I and in the and an of in the Golgi had little effect on and secretion of meprin α in cells In the of the wild-type meprin α subunit was exclusively secreted into the apical with of the protein was secreted into the apical The wild-type meprin α subunit was to but sensitive to indicating that it was secreted as a protein containing oligosaccharide The decreased the molecular mass of the wild-type protein from to as When with and no with were not that the meprin α subunits not or with the secreted protein was sensitive to and indicating that the subunit contains high When with the protein with on that with indicating that it oligosaccharides The high proteins in the of and proteins in the of had enzymatic the to the wild-type protein not the apical and enzymatic properties of the meprin α subunit are of the type of N-linked oligosaccharide oligosaccharides are required for secretion and of meprin a mutant in which all of the N-linked glycosylation sites were from Asn to Gln was (see Fig. It been that the and mutants and mutants (see are secreted are expressed in human kidney cells (13Tsukuba T. Bond J.S. J. Biol. Chem. 1998; 273: 35260-35267Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). these mutants are sorted as the type to the apical were into cells, and the of secretion was The and mutants and mutants were all secreted into the apical medium When the and Asn mutants were into human kidney cells, the mutant was found in the however, there was no secretion of the Asn mutant not the of the mutant were The mutant was in the cell and the protein had a molecular mass of and was secreted into the medium within The mutant N-linked oligosaccharides was as a protein of the protein was retained in the cell a of with amino no secreted protein was in the of the mutant with not the molecular whereas was from to that the mutant had no N-linked These results indicate that N-linked oligosaccharides are required for secretion of the meprin α subunit into the The results with the Asn mutant and the tunicamycin that some core glycosylation is required for secretion of meprin α that carbohydrate are for the folding of the is required for secretion of proteins A. F. N. K. EMBO J. 1998; PubMed Scopus Google Scholar); however, there are proteins secreted by such as the brush-border M. R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the glycosylation for secretion, it is more the effect of glycosylation on the of a specific protein that is for the It is of that the meprin Asn mutant is retained in the cell in the had that meprin the MAM domain were in the by a indicating that those were as proteins and were for into the (13Tsukuba T. Bond J.S. J. Biol. Chem. 1998; 273: 35260-35267Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The of as in the not in but carbohydrates as in into the as well as of meprin to the cell The results indicate that no one specific N-linked oligosaccharide is required for secretion or apical of the homo-oligomeric form of mouse meprin A. 10 of the potential Asn glycosylation sites were mutated for this study it is not of the sites are using of meprin peptides that at of the potential glycosylation sites are to high or complex oligosaccharides in mouse kidney (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). site is in the MAM this site is to complex The other sites are in the AM domain and at of these sites contain high and/or complex of those Asn and the of oligosaccharide however, had no effect on The potential glycosylation sites in the MAM and AM domains are not highly is in and mouse meprin and the not sorting, enzymatic activity, or of the subunit to inactivation. In mutants that are truncated the AM and MATH domains are secreted by cells in Fig. that glycosylation and other in these domains not an in targeting the protein to the apical of meprin A require and as by of cells with and The secreted form of meprin in the of indicating some in The glycosylation that in the Golgi for meprin secreted from kidney and intestine and for meprin secreted from or cells, is not for targeting or folding of the is on the results of the in which meprin was and secreted in the of castanospermine and These the of and from the core N-linked complex These also of with such as and S.H. J. P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). been to with proteins and may be an important in the folding of forms of the meprin Y. Y. D.B. Mol. 1999; 4: Full Text Full Text PDF PubMed Scopus Google Scholar). The that from this and (13Tsukuba T. Bond J.S. J. Biol. Chem. 1998; 273: 35260-35267Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, P. Tang J. G.D. Bond J.S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, P. M. Bond J.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (38) Google is that core the MAM domain, and the epidermal growth and cytoplasmic domains and meprin α from the are all required for of the meprin α protein from the into the In the of one of these the meprin protein is retained in the or to the for The also indicate that complex and or forms of meprin are not for The protease domain Asn mutants and the mutant in the AM domain were more to heat inactivation the wild-type protein or other For the protease domain it is that is to the on as there is no that meprins, or other of the astacin family contain oligosaccharides in the catalytic In of the Asn are in the astacin family The meprin protease domain on the of one to the of the within the protease domain The mutated Asn to the site is from the in a was the indicating that the of this mutant is to the of the from the the It is that and by zinc at the is to be on the of the and of this also the of inactivation. is with those that from the site the and enzymatic activity of meprin G.D. Bond J.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The inactivation of the AM domain mutant is of in that this is the of the secreted mature meprin α for meprin A from mouse kidney (14Tang J. Bond J.S. Arch. Biochem. Biophys. 1998; 349: 192-200Crossref PubMed Scopus (31) Google Scholar). in glycosylation or of a Gln at this site may the mature of the mutants with Asn had enzymatic activity For the protease domain the in activity was for the is the that the of The of a may the site to of the catalytic activity. The other protease domain not be to with but may the The in the prosequence domain also decreased enzymatic activity, indicating that of the prosequence folding of the or activation of the The Asn in the MAM and AM domains that decreased meprin activity that in the domains the catalytic The was the one that the specific activity and the catalytic may be to that in or of however, that the the and of the of protein in In the and on the deglycosylation of the mature by indicate that oligosaccharides an important in the catalytic properties of meprin. may from of the site or from on the subunit or that the of and The of the domains for folding, activity, and been with domain and these indicate that oligosaccharides an important in the of the the function of the