Asparagine-linked Oligosaccharides Protect Lamp-1 and Lamp-2 from Intracellular Proteolysis
Lysosomes contain several integral membrane proteins (termed Lamps and Limps) that are extensively glycosylated with asparagine-linked oligosaccharides. It has been postulated that these glycans protect the underlying polypeptides from the proteolytic environment of the lysosome. Previous attempts to test this hypothesis have been inconclusive because they utilized approaches that prevent initial glycosylation and thereby impair protein folding. We have used endoglycosidase H to remove the Asn-linked glycans from fully folded lysosomal membrane proteins in living cells. Deglycosylation of Lamp-1 and Lamp-2 resulted in their rapid degradation, whereas Limp-2 was relatively stable in the lysosome in the absence of high mannose Asn-linked oligosaccharides. Depletion of Lamp-1 and Lamp-2 had no measurable effect on endosomal/lysosomal pH, osmotic stability, or density, and cell viability was maintained. Transport of endocytosed material to dense lysosomes was delayed in endoglycosidase H treated cells, but the rate of degradation of internalized bovine serum albumin was unchanged.These data provide direct evidence that Asn-linked oligosaccharides protect a subset of lysosomal membrane proteins from proteolytic digestion in intact cells. Lysosomes contain several integral membrane proteins (termed Lamps and Limps) that are extensively glycosylated with asparagine-linked oligosaccharides. It has been postulated that these glycans protect the underlying polypeptides from the proteolytic environment of the lysosome. Previous attempts to test this hypothesis have been inconclusive because they utilized approaches that prevent initial glycosylation and thereby impair protein folding. We have used endoglycosidase H to remove the Asn-linked glycans from fully folded lysosomal membrane proteins in living cells. Deglycosylation of Lamp-1 and Lamp-2 resulted in their rapid degradation, whereas Limp-2 was relatively stable in the lysosome in the absence of high mannose Asn-linked oligosaccharides. Depletion of Lamp-1 and Lamp-2 had no measurable effect on endosomal/lysosomal pH, osmotic stability, or density, and cell viability was maintained. Transport of endocytosed material to dense lysosomes was delayed in endoglycosidase H treated cells, but the rate of degradation of internalized bovine serum albumin was unchanged. These data provide direct evidence that Asn-linked oligosaccharides protect a subset of lysosomal membrane proteins from proteolytic digestion in intact cells. endoglycosidase H Chinese hamster ovary normal rat kidney 1-deoxymannojirimycin phosphate-buffered saline polyacrylamide gel electrophoresis mannose 6-phosphate/insulin-like growth factor II receptor fluorescein isothiocyanate One of the proposed functions of asparagine-linked oligosaccharides is to protect the underlying polypeptide from proteolysis (1Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4951) Google Scholar). The strongest evidence for this conclusion comes fromin vitro studies that utilize mature glycoproteins that are treated with various glycosidases to remove their carbohydrate units. By comparing glycosylated proteins with their unglycosylated counterparts, it has been shown that glycosylation increases thermal stability, solubility, dynamic stability, and resistance to protease digestion (2Stochaj U. Cramer M. Mannherz H.G. Biochim. Biophys. Acta. 1992; 1122: 327-332Crossref PubMed Scopus (5) Google Scholar, 3Rudd P.M. Joao H.C. Coghill E. Fiten P. Saunders M.R. Opdenakker G. Dwek R.A. Biochemistry. 1994; 33: 17-22Crossref PubMed Scopus (281) Google Scholar, 4van Berkel P.H. Geerts M.E. van Veen H.A. Kooiman P.M. Pieper F.R. de Boer H.A. Nuijens J.H. Biochem. J. 1995; 312: 107-114Crossref PubMed Scopus (136) Google Scholar, 5Rudd P.M. Woods R.J. Wormald M.R. Opdenakker G. Downing A.K. Campbell I.B. Dwek R.A. Biochim. Biophys. Acta. 1995; 1248: 1-10Crossref PubMed Scopus (95) Google Scholar). In contrast, experiments designed to analyze the importance of glycosylation in protecting endogenous proteins from proteolysis in living cells have used approaches that prevent the initial glycosylation of the nascent protein. Most of these studies have used tunicamycin, an inhibitor of UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-phosphate transferase, to produce unglycosylated proteinsin vivo. However, the inhibition of initial glycosylation disrupts protein folding to such an extent that it induces the “unfolded protein response,” a complex stress response characterized by the up-regulation of several chaperones in the endoplasmic reticulum (6Brewer J.W. Cleveland J.L. Hendershot L.M. EMBO J. 1997; 16: 7207-7216Crossref PubMed Scopus (77) Google Scholar). Many of the misfolded proteins are rapidly degraded in the endoplasmic reticulum (7Sidrauski C. Chapman R. Walter P. Trends Cell Biol. 1998; 8: 245-249Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Therefore, a decrease in the half-life of a protein synthesized in the presence of tunicamycin usually reflects endoplasmic reticulum-mediated proteolysis of misfolded proteins. The use of site-directed mutagenesis to remove Asn-linked glycosylation signals has provided a means for exploring the role of individual glycosylation sites on the behavior of the protein, but potential problems with proper protein folding remain. To avoid these difficulties, we sought a method for removing Asn-linked glycans from mature proteins in vivo, thereby bypassing any effects on initial protein folding. In this paper, we describe such a method based on the ability of endoglycosidase H (endo H)1 to cleave high mannose oligosaccharides from fully folded proteins. Using this method, we have examined the role of glycosylation in protecting lysosomal membrane proteins from degradation. Of all intracellular proteins, the components of the lysosome exist in the most proteolytic environment. The integral membrane proteins that are major constituents of the lysosomal membrane are extensively glycosylated and include Lamp-1 and Lamp-2 (lysosome-associatedmembrane proteins) and Limp-1 and Limp-2 (lysosomal integral membraneproteins) (for a review, see Ref. 8Hunziker W. Geuze H.J. BioEssays. 1996; 18: 379-389Crossref PubMed Scopus (237) Google Scholar). Indeed, the majority of the weight of these molecules is derived from carbohydrates (9Lewis V. Green S.A. Marsh M. Vihko P. Helenius A. Mellman I. J. Cell Biol. 1985; 100: 1839-1847Crossref PubMed Scopus (194) Google Scholar, 10Barriocanal J.G. Bonifacino J.S. Yuan L. Sandoval I.V. J. Biol. Chem. 1986; 261: 16755-16763Abstract Full Text PDF PubMed Google Scholar, 11Mane S.M. Marzella L. Bainton D.F. Holt V.K. Cha Y. Hildreth J.E. August J.T. Arch. Biochem. Biophys. 1989; 268: 360-378Crossref PubMed Scopus (124) Google Scholar). As such, these proteins make excellent models to test the hypothesis that core glycosylation protects against proteolysis in vivo. Using endo H to deglycosylate the various lysosomal membrane proteins in intact cells, we have determined that Asn-linked high mannose glycans protect Lamp-1 and Lamp-2 from degradation but are not essential for the prolonged survival of Limp-2. All chemicals were analytical grade and obtained from Sigma except for Percoll (Amersham Pharmacia Biotech), ECL reagents including horseradish peroxidase-conjugated secondary antibodies and Na125I (Amersham Pharmacia Biotech), fluorophore-conjugated secondary antibodies (Jackson Immunoresearch), nitrocellulose (Schleicher and Schuell), 1-deoxymannojirimycin (Oxford Glycosystems), IODO-BEADs (Pierce), fluorescein isothiocyanate-labeled dextran (FITC-dextran) (Molecular Probes, Inc., Eugene, OR), and endo H and endo Hf (New England Biolabs). β-Glucuronidase was purified from the secretions of 13.2.1 mouse L cells and iodinated as described previously (12Jadot M. Canfield W.M. Gregory W. Kornfeld S. J. Biol. Chem. 1992; 267: 11069-11077Abstract Full Text PDF PubMed Google Scholar). This cell line, which has been engineered to secrete large amounts of β-glucuronidase, was provided by William Sly (St. Louis University). Mouse monoclonal antibodies UH1 (anti-CHO Lamp-1) and UH3 (anti-CHO Lamp-2) developed by Dr. Thomas August (Johns Hopkins) were from the Developmental Studies Hybridoma Bank (University of Iowa). Mouse monoclonal E10D10 (anti-CHO Lamp-2) (Affinity Bioreagents), E9D9 (anti-CHO Lamp-2), and affinity-purified rabbit anti-canine Rab7 were generous gifts of S. Pfeffer (Stanford) (13Lombardi D. Soldati T. Riederer M.A. Goda Y. Zerial M. Pfeffer S.R. EMBO J. 1993; 12: 677-682Crossref PubMed Scopus (434) Google Scholar, 14Soldati T. Rancano C. Geissler H. Pfeffer S.R. J. Biol. Chem. 1995; 270: 25541-25548Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 15Riederer M.A. Soldati T. Shapiro A.D. Lin J. Pfeffer S.R. J. Cell Biol. 1994; 125: 573-582Crossref PubMed Scopus (243) Google Scholar). Affinity-purified rabbit anti-human Lamp-1 and mouse monoclonal 10D10 (anti-rat Lamp-2) were generous gifts of M. Fukuda (Burnham Institute) (16Carlsson S.R. Roth J. Piller F. Fukuda M. J. Biol. Chem. 1988; 263: 18911-18919Abstract Full Text PDF PubMed Google Scholar) and M. Jadot (Facultes Universitaires Notre Dame De la Paix, Belgium) (17Jadot M. Dubois F. Wattiaux-De Coninck S. R. J. Biochem. 1997; PubMed Scopus Google Mouse monoclonal antibodies against rat Limp-2 were provided by I. Sandoval (University of J.G. Bonifacino J.S. Yuan L. Sandoval I.V. J. Biol. Chem. 1986; 261: 16755-16763Abstract Full Text PDF PubMed Google Scholar) and A. M. H. H. J. Biochem. 1994; Google Scholar). Affinity-purified anti-human and affinity-purified rabbit receptor antibodies were provided by W. Sly and L. cells in have been previously described C. Kornfeld S. U. S. A. PubMed Scopus Google Scholar). rat kidney cells were obtained from The cells were in essential serum in a with The cells were treated with 1-deoxymannojirimycin for to use in the The was or cells were in to in endo H for the of were in and by and the cell was in were to and with various antibodies as described in the In the protease and were with endo H. To the effect of the endo H on a intracellular protein, cells were to purified for and for to the of the and cell were with in and were with or of endo H in and for The was by the of and the were for and the were for Limp-2. and protein nitrocellulose were the II and to the were of and PubMed Scopus Google Scholar, H. T. J. U. S. A. PubMed Scopus Google Scholar). with proteins were in in for and with the as in the for with All antibodies were in the with the were with the horseradish peroxidase-conjugated secondary for and in The ECL was for as by the and the signals were on were a (Molecular Inc., with The of the endosomal/lysosomal was determined as described by and with S. U. S. A. PubMed Scopus Google Scholar). The method of Percoll has been described previously J. A. Kornfeld S. J. Cell Biol. 1995; PubMed Scopus Google Scholar). cells were in to and in in the presence or absence of endo H for were with and in of for was by with and the cells were for or in were and were on Percoll as described J. A. Kornfeld S. J. Cell Biol. 1995; PubMed Scopus Google Scholar). were from the of the was for and the was in a cells were on in and treated with endo H for were with for in in in bovine serum and for with and with antibodies as described in the antibodies were and were examined by a were on and treated with endo H as described was to the for and the cells were We have the role of glycosylation of lysosomal membrane proteins the of the endosomal/lysosomal by removing the Asn-linked oligosaccharides from mature proteins in living cells a method based on the of endo H. This the core of Asn-linked high mannose and a to this as endo H is of we to prevent the of such in the cells In the we utilized a cell in C. Kornfeld S. U. S. A. PubMed Scopus Google Scholar, C. J. Kornfeld S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This is for and the Asn-linked oligosaccharides in the cell as high mannose which are endo H R. Kornfeld S. Biochem. 1985; PubMed Scopus Google Scholar). The the inhibitor J. Kornfeld R. Biochem. Biophys. 125: Scopus Google Scholar). cells in the presence of for contain glycoproteins with high mannose oligosaccharides that are by endo H. In the initial endo H was to the of cells or cells. various of cell were and the of the lysosomal membrane proteins was by By of endo H a decrease in the weight of the lysosomal glycoproteins and Limp-2 was with the of Asn-linked high mannose oligosaccharides in cells and cells the in the weight of Lamp-1 and Lamp-2 was with a of on the This such that all of the Lamp-1 was by and the Lamp-2 by The rate of of Lamp-2 was by the of the lysosomal protease and to the with the to most in the lysosome However, the protease were protecting the weight of This the of the Lamp-2 protein core to proteolysis in the absence of a of Lamp-1 was in the presence of and not These the importance of the Asn-linked glycans of Lamp-1 and Lamp-2 for protecting the protein core from digestion in the proteolytic environment of the lysosome. The weight of Limp-2 was cells were in endo H However, in to the with Lamp-1 and Limp-2 was relatively stable in the lysosomal environment in the absence of high mannose oligosaccharides as by the in Limp-2 protein with of endo H To that all of the Asn-linked glycans of Limp-2 are to endo cells that had been in the presence of endo H for were in and the proteins were by in by with endo H. As shown in the Limp-2 the on as the material from the endo cells. This that endo H all the high mannose from Limp-2. the endo H was to the of living cells, it the various lysosomal membrane proteins they to the cell J. D. Full Text PDF PubMed Scopus Google Scholar). the endo H internalized by and the of the endosomal/lysosomal To these we of and cells with various of endo H for and determined the extent of of Lamp-1 and Limp-2 It is that proteins are by of endo H and fully by the living cells a of the endo H from the the of the intracellular endo H to deglycosylate the lysosomal membrane that the endo H in the proteolytic environment of the endosomal/lysosomal To test for intracellular endoglycosidase we examined the effect of endo H on an intracellular of β-glucuronidase, a lysosomal cells were to for and were with endo H for various As in the of internalized was of endo H endo H was cells were with the and in thereby the that of the of the in the have to the cell endo H have the the mannose and these that a of the endo H was internalized by the cells. of endo H of Lamp-1 and Lamp-2 was of the was in treated cells we the that prolonged to the endoglycosidase resulted in In an to this cells were in the presence of endo H for to Lamps and to for the of the endo H from the As shown by the in the of Lamp-1 protein from endo H to normal the in the absence of endo H. This that the cells are and that the Lamp-1 is intact the prolonged endo H The rate which Lamp-1 was is with the half-life of this protein (9Lewis V. Green S.A. Marsh M. Vihko P. Helenius A. Mellman I. J. Cell Biol. 1985; 100: 1839-1847Crossref PubMed Scopus (194) Google Scholar, 10Barriocanal J.G. Bonifacino J.S. Yuan L. Sandoval I.V. J. Biol. Chem. 1986; 261: 16755-16763Abstract Full Text PDF PubMed Google Scholar). were with Lamp-2 not the of endo H to Lamps not cell we used this method to lysosomal functions of Lamp-1 and Lamp-1 and Lamp-2 have been to of the proteins in the lysosomal membrane M. J. J. S.R. J. Biol. Chem. 1988; 263: Full Text PDF PubMed Google Scholar). This of in with their has been to a carbohydrate to the lysosomal that protect proteins in the lysosomal membrane components of the as as the membrane W. Geuze H.J. BioEssays. 1996; 18: 379-389Crossref PubMed Scopus (237) Google Scholar, V. Green S.A. Marsh M. Vihko P. Helenius A. Mellman I. J. Cell Biol. 1985; 100: 1839-1847Crossref PubMed Scopus (194) Google Scholar). To test the that Lamps prevent of the we the of this complex of The and lysosomal in living cells was based on the signals from S. U. S. A. PubMed Scopus Google Scholar). In these cells were to for and were in the absence or presence of endo H to the these the endosomal/lysosomal was determined to for cells and for endo cells These that the and of Lamps have no effect on the endosomal/lysosomal The of the was by the by the of and the rate and of this from the the endosomal/lysosomal with to initial in endo and cells, which the presence of an Therefore, we that glycosylated Lamp-1 and Lamp-2 are not for To test the that the Lamps a role in the lysosomal membrane or as a to protect the membrane from we the ability of lysosomes to and lysosomes were to with of the in not in with cells, we no in the of by stress or in the the initial of lysosomes from the not these we that the of the lysosomal is not by Lysosomes are characterized not by the presence of but by the high We the that Lamp-1 and Lamp-2 for the of these dense To we cell of and endo cells to on Percoll these dense lysosomes are in the of the whereas including are in the of the As shown in the of Limp-2 in the was in the and cells. This that Lamp-1 and Lamp-2 are not for the of dense However, the dense lysosome of the cells had a decrease in the of in the decrease in the of in the dense lysosome was in endo cells not In and cells, with endo H not in a decrease in the of these the was to The that the of the from that of Limp-2 in the is with an in the of the in their to dense evidence for this was obtained by the of in dense In this and cells were to for and a of or the cells were and to on Percoll The of the was and the in the dense lysosome was It is that the of to the dense lysosomes is in the endo cells, it that This is with a in the of material and dense lysosomes and the in the of the We the ability of and endo to bovine serum albumin that had been internalized the The rate of proteolysis was in cell that the cells the proteolytic of the endosomal/lysosomal not In of the in to dense lysosomes in the endo cells, it that a of the proteolysis is in to the dense This is with the conclusion of A. G. T. J. Cell 1996; Google Scholar) that are major sites of protein degradation. of cells that had been treated with endo H for the presence of These an and The for which is in and lysosomes and but were for the a of G. Mellman I. Kornfeld S. 1988; Full Text PDF PubMed Scopus Google Scholar). However, the were for the a of P. Zerial M. Full Text PDF PubMed Scopus Google Scholar). The Limp-2 and Rab7 was in the and and not in the cells We that the dense the a receptor and The data in this that endo H used to the Asn-linked glycans from mature proteins in intact cells, thereby the of the of these The major of this approaches that prevent initial glycosylation is that the nascent protein is to and the to the of units. This is glycoproteins and are degraded Asn-linked glycosylation is the are several to the endo H The is that it used on cells that are to high mannose oligosaccharides to We used of cells to this The was a cell that is in C. Kornfeld S. U. S. A. PubMed Scopus Google Scholar). This cell is to the to As a all of Asn-linked oligosaccharides to the of endo H. The cell was cells that had been treated for prolonged with an inhibitor of I. This inhibitor the of to the and thereby the Asn-linked glycans in an endo This used with any cell that in is that the endo H the Asn-linked glycans from all of the glycoproteins of the cell and the endosomal/lysosomal this is not a functions intact endo H as most it proteins from in that The we have is Asn-linked glycans protect the underlying protein from in intact cells. We to the lysosomal membrane glycoproteins because they are glycosylated and exist in a proteolytic environment. These membrane proteins have large of sites for Asn-linked most of which are used (9Lewis V. Green S.A. Marsh M. Vihko P. Helenius A. Mellman I. J. Cell Biol. 1985; 100: 1839-1847Crossref PubMed Scopus (194) Google Scholar, 10Barriocanal J.G. Bonifacino J.S. Yuan L. Sandoval I.V. J. Biol. Chem. 1986; 261: 16755-16763Abstract Full Text PDF PubMed Google Scholar, 11Mane S.M. Marzella L. Bainton D.F. Holt V.K. Cha Y. Hildreth J.E. August J.T. Arch. Biochem. Biophys. 1989; 268: 360-378Crossref PubMed Scopus (124) Google Scholar). the majority of the oligosaccharides are to that contain and a high of (16Carlsson S.R. Roth J. Piller F. Fukuda M. J. Biol. Chem. 1988; 263: 18911-18919Abstract Full Text PDF PubMed Google Scholar). However, the absence of has no effect on the half-life of Lamps or lysosomal in cells S. R. M. Helenius A. Mellman I. J. Cell Biol. 1989; PubMed Scopus Google Scholar). it had been that the high of carbohydrates on these molecules protects from this had not been to We that the Asn-linked oligosaccharides of and Limp-2 are by cells in the presence of endo H for the high mannose of are it most that the endo H is the endosomal/lysosomal and is these intracellular However, we the that the Lamps are to the cell they the endo H J. D. Full Text PDF PubMed Scopus Google Scholar). of the of the is that Lamp-1 and Lamp-2 are rapidly degraded by in their environment. This that Asn-linked of the high mannose protect these membrane glycoproteins from the of that Lamp-1 synthesized in the presence of tunicamycin is rapidly but the of this is by the that the Lamp-1 have misfolded in the endoplasmic reticulum and been as an protein by the J.G. Bonifacino J.S. Yuan L. Sandoval I.V. J. Biol. Chem. 1986; 261: 16755-16763Abstract Full Text PDF PubMed Google Scholar). In to the rapid degradation of Lamp-1 and Limp-2 in the absence of Asn-linked high mannose This from the that Limp-2 synthesized in the presence of tunicamycin is rapidly degraded J.G. Bonifacino J.S. Yuan L. Sandoval I.V. J. Biol. Chem. 1986; 261: 16755-16763Abstract Full Text PDF PubMed Google Scholar). However, in that the to the Limp-2 in dense lysosomes in the presence of protease an of degradation. we that the Asn-linked glycans of Limp-2 are essential for the initial folding of the but are for the survival of the protein in the lysosome. The resistance of Limp-2 to degradation in the lysosome that not all lysosomal constituents glycosylation to avoid The of several lysosomal prolonged endo H that these proteins in the lysosome their of Asn-linked Lamp-1 and Lamp-2 were rapidly degraded in the presence of endo H the of cell we were to several of the Lamps in lysosomal Lamp-1 and Lamp-2 have been to make of the lysosomal membrane proteins (16Carlsson S.R. Roth J. Piller F. Fukuda M. J. Biol. Chem. 1988; 263: 18911-18919Abstract Full Text PDF PubMed Google Scholar). on this high with the dense it has been that Lamps protect the lysosomal membrane from by a the membrane and the W. Geuze H.J. BioEssays. 1996; 18: 379-389Crossref PubMed Scopus (237) Google Scholar, V. Green S.A. Marsh M. Vihko P. Helenius A. Mellman I. J. Cell Biol. 1985; 100: 1839-1847Crossref PubMed Scopus (194) Google Scholar). not this We that Lamp-1 and Lamp-2 are not for the pH, density, or membrane of the lysosomes or the of the endosomal/lysosomal we no evidence of of the lysosomal membrane and of lysosomal the which have cells fully from prolonged of glycosylated lysosomal membrane proteins for the of it is that the Asn-linked glycans of these proteins by intracellular endo H. The most is that the lysosomal membrane is to the in to the presence of the S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. W. J. Chem. PubMed Scopus Google Scholar, S. J. Biochem. PubMed Scopus (5) Google Scholar). the not to to the components of the from endo cells were to the of the endosomal/lysosomal One in these is that the endo cells a of the most the synthesized with the molecules a with endo H were in the of the Percoll the dense that contain the we the that a of molecules are to these lysosomal we the that a of Lamp-1 or Lamp-2 in the lysosomal membrane endo H but is because it by the antibodies We have to this by a of monoclonal and to for the proteins in the cell and have not proteolytic lysosomal intact in the presence of of Lamp-1 and a of were in the endo cells. Most was the of that for Limp-2 and Rab7 but were These have to the described of that by the of a of with dense lysosomes J. Cell 1996; Scholar, A. J. Cell Biol. 1996; PubMed Scopus Google Scholar, S.R. J. Cell Biol. 1998; PubMed Scopus Google Scholar, M. I. A. M. S. J. Cell PubMed Google Scholar). The complex is to the of and of and dense The of this in endo cells a in the and for the rate of of to dense lysosomes and the in the of the on Percoll However, to the of this is the of Lamp-1 and Lamp-2 or the of the of this was in G. Y. R. D. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) their with The were and and a in a of the including pH, osmotic stability, density, and were The that in several Lamp-2 was for the of Lamp-2 was not in the in derived from the which had a no These are with that Lamp-1 and Lamp-2 has effects on lysosomal The data that the of Lamp-1 and Lamp-2 in This a role of the in that is not for the viability of cells in We Dr. for in the experiments and and for the