Glycosylation Is Important for Cell Surface Expression of the Water Channel Aquaporin-2 but Is Not Essential for Tetramerization in the Endoplasmic Reticulum
2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês
10.1074/jbc.m310767200
ISSN1083-351X
AutoresGiel Hendriks, Marco J. Koudijs, Bas W. M. van Balkom, Viola Oorschot, Judith Klumperman, Peter M.T. Deen, Peter van der Sluijs,
Tópico(s)Pancreatic function and diabetes
ResumoAquaporin-2 (AQP2) is a pore-forming protein that is required for regulated reabsorption of water from urine. Mutations in AQP2 lead to nephrogenic diabetes insipidus, a disorder in which functional AQP2 is not expressed on the apical cell surface of kidney collecting duct principal cells. The mechanisms and pathways directing AQP2 from the endoplasmic reticulum to the Golgi complex and beyond have not been defined. We found that ∼25% of newly synthesized AQP2 is glycosylated. Nonglycosylated and complex-glycosylated wild-type AQP2 are stable proteins with a half-life of 6-12 h and are both detectable on the cell surface. We show that AQP2 forms tetramers in the endoplasmic reticulum during or very early after synthesis and reaches the Golgi complex in 1-1.5 h. We also report that glycosylation is neither essential for tetramerization nor for transport from the endoplasmic reticulum to the Golgi complex. Instead, the N-linked glycan is important for exit from the Golgi complex and sorting of AQP2 to the plasma membrane. These results are important for understanding the molecular mechanisms responsible for the intracellular retention of AQP2 in nephrogenic diabetes insipidus. Aquaporin-2 (AQP2) is a pore-forming protein that is required for regulated reabsorption of water from urine. Mutations in AQP2 lead to nephrogenic diabetes insipidus, a disorder in which functional AQP2 is not expressed on the apical cell surface of kidney collecting duct principal cells. The mechanisms and pathways directing AQP2 from the endoplasmic reticulum to the Golgi complex and beyond have not been defined. We found that ∼25% of newly synthesized AQP2 is glycosylated. Nonglycosylated and complex-glycosylated wild-type AQP2 are stable proteins with a half-life of 6-12 h and are both detectable on the cell surface. We show that AQP2 forms tetramers in the endoplasmic reticulum during or very early after synthesis and reaches the Golgi complex in 1-1.5 h. We also report that glycosylation is neither essential for tetramerization nor for transport from the endoplasmic reticulum to the Golgi complex. Instead, the N-linked glycan is important for exit from the Golgi complex and sorting of AQP2 to the plasma membrane. These results are important for understanding the molecular mechanisms responsible for the intracellular retention of AQP2 in nephrogenic diabetes insipidus. Aquaporins (AQPs) 1The abbreviations used are: AQP(s), aquaporin(s); BFA, brefeldin A; CHO cells, Chinese hamster ovary cells; EGFP, enhanced green fluorescent protein; Endo H, endoglycosidase H; ER, endoplasmic reticulum; GFP, green fluorescent protein; MDCK cells, Madin-Darby canine kidney cells; NDI, nephrogenic diabetes insipidus; PNGase F, peptide N-glycosidase F; SNARE, soluble NSF attachment protein receptors; TGN, trans-Golgi network; PDI, protein disulfide isomerase.1The abbreviations used are: AQP(s), aquaporin(s); BFA, brefeldin A; CHO cells, Chinese hamster ovary cells; EGFP, enhanced green fluorescent protein; Endo H, endoglycosidase H; ER, endoplasmic reticulum; GFP, green fluorescent protein; MDCK cells, Madin-Darby canine kidney cells; NDI, nephrogenic diabetes insipidus; PNGase F, peptide N-glycosidase F; SNARE, soluble NSF attachment protein receptors; TGN, trans-Golgi network; PDI, protein disulfide isomerase. are ubiquitously expressed pore-forming proteins that facilitate water transport across membranes along an osmotic gradient (see Ref. 1.Kozono D. Yasui M. King L.S. Agre P. J. Clin. Invest. 2002; 109: 1395-1399Crossref PubMed Scopus (216) Google Scholar). AQPs share a highly conserved domain organization with six transmembrane domains and cytoplasmically oriented N and C termini. AQPs form tetramers or sometimes higher order oligomers in membranes. Structural studies of AQP1 revealed that the functional unit is a tetramer with each monomer providing an independent water pore (2.Murata K. Mitsuoka K. Hirai T. Walz T. Agre P. Heymann J.B. Engel A. Fujiyoshi Y. Nature. 2000; 407: 599-605Crossref PubMed Scopus (1394) Google Scholar). Stability of the tetramer is thought to be conferred by the assembly of monomers as a tight fitting wedge within the tetramer. With the exception of AQP2, which is regulated by the antidiuretic hormone vasopressin, most of the AQPs are expressed constitutively at the plasma membrane (see Ref. 3.Nielsen S. Frokjaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1005) Google Scholar). AQP2 is localized in a tubulovesicular subapical storage compartment. The intracellular retention is thought to be caused by tethering of the storage vesicles to the actin cytoskeleton and is regulated by members of the rho subfamily of small GTPases (4.Klussmann E. Tamma G. Lorenz D. Wiesner B. Maric K. Hofmann F. Aktories K. Valenti G. Rosenthal W. J. Biol. Chem. 2001; 276: 20451-20457Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Binding of vasopressin to the basolateral V2 receptor increases cAMP concentration which activates protein kinase A. Protein kinase A phosphorylates AQP2-Ser256 which is required for fusion of AQP2 storage vesicles with the plasma membrane (5.Fushimi K. Sasaki S. Marumo F. J. Biol. Chem. 1997; 272: 14800-14804Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 6.van Balkom B.W.M. Savelkoul P.J.M. Markovich D. Hofman E. Nielsen S. van der Sluijs P. Deen P.M.T. J. Biol. Chem. 2002; 277: 41473-41479Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The precise mechanism responsible for the recruitment of tetrameric AQP2 to the cell surface is not known, but phosphorylation of at least three of the four monomers, reportedly, is required and sufficient for the localization of AQP2 on the plasma membrane (7.Kamsteeg E.J. Heijnen I. van Os C.H. Deen P.M.T. J. Cell Biol. 2000; 151: 919-929Crossref PubMed Scopus (147) Google Scholar). The regulated recruitment of plasma membrane proteins from intracellular storage vesicles provides an efficient means to control cell surface expression and was originally discovered for the insulin-regulated glucose transporter GLUT4 (see Ref. 8.Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 267-277Crossref PubMed Scopus (907) Google Scholar). GLUT4 and AQP2 translocation share some similarities such as the requirement for the v-SNARE synaptobrevin-2 in fusion with the plasma membrane (8.Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 267-277Crossref PubMed Scopus (907) Google Scholar, 9.Gouraud S. Laera A. Calmita G. Carmosini M. Procino G. Rosetto O. Mannucci R. Rosenthal W. Svelto M. Valenti G. J. Cell Sci. 2002; 115: 3667-3674Crossref PubMed Scopus (63) Google Scholar). Nevertheless, there are important differences. For instance, GLUT4 translocates from a basolateral storage compartment in polarized epithelial cells, and phosphorylation of GLUT4 inhibits glucose transport, whereas phosphorylation of AQP2 is essential for its recruitment from an apical storage compartment. Despite the progress that has been made in understanding the physiology of AQP-mediated water transport, important aspects such as biosynthetic maturation and intracellular transport of AQP water channels are incompletely understood. For instance, it has been shown for AQP1 that conformational changes and rearrangements take place during insertion in the endoplasmic reticulum (ER), but whether insertion occurs cotranslationally or post-translationally has become a controversial issue (10.Skach W.R. Shi L. Calayag M.C. Frigeri A. Lingapppa V.R. Verkman A.S. J. Cell Biol. 1994; 125: 803-815Crossref PubMed Scopus (68) Google Scholar, 11.Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In addition, with the exception of AQP4 (12.Madrid R. Le Maout S. Barrault M.B. Janvier K. Benichou S. Merot J. EMBO J. 2001; 20: 7008-7021Crossref PubMed Scopus (134) Google Scholar), the sorting signals required for transport of AQPs between intracellular compartments remain to be defined. The observations that AQP2 and AQP6 (13.Yasui M. Hazama A. Kwon T.H. Nielsen S. Guggino W.B. Agre P. Nature. 1999; 402: 184-187Crossref PubMed Scopus (395) Google Scholar) reside in distinct intracellular compartments and the other AQPs localize to the plasma membrane suggest that crucial differences exist in sorting mechanisms of the AQPs. The consensus N-linked glycosylation site in AQP1 and AQP2 is used inefficiently. Most of the AQP1 and AQP2 molecules on the cell surface remain unglycosylated (14.Smith B.L. Preston G.M. Spring F.A. Anstee D.J. Agre P. J. Clin. Invest. 1994; 94: 1043-1049Crossref PubMed Google Scholar, 15.Baumgarten R. van de Pol M.H.J. Wetzels J.F.M. van Os C.H. Deen P.M.T. J. Am. Soc. Nephrol. 1998; 9: 1553-1559Crossref PubMed Google Scholar). Intriguingly, AQP2-T125M, a mutant that causes recessive nephrogenic diabetes insipidus (NDI) (16.Goji K. Kuwahara M. Gu Y. Matsuo M. Marumo F. Sasaki S. J. Clin. Endocrinol. Metab. 1998; 83: 3205-3209PubMed Google Scholar, 17.Marr N. Bichet D.G. Hoefs S. Savelkoul P.J.M. Konings I.B.M. de Mattia F. Graat M.P.J. Arthus M.F. Lonergan M. Fujiwara T.M. Knoers N.V.A.M. Landau D. Balfe W.J. Oksche A. Rosenthal W. Müller D. van Os C.H. Deen P.M.T. J. Am. Soc. Nephrol. 2002; 13: 2267-2277Crossref PubMed Scopus (95) Google Scholar), is not glycosylated because the consensus N-linked glycosylation motif is disrupted. This mutant is absent from the plasma membrane (17.Marr N. Bichet D.G. Hoefs S. Savelkoul P.J.M. Konings I.B.M. de Mattia F. Graat M.P.J. Arthus M.F. Lonergan M. Fujiwara T.M. Knoers N.V.A.M. Landau D. Balfe W.J. Oksche A. Rosenthal W. Müller D. van Os C.H. Deen P.M.T. J. Am. Soc. Nephrol. 2002; 13: 2267-2277Crossref PubMed Scopus (95) Google Scholar), suggesting that it is misfolded and retained in the ER, or for some other reason does not reach the cell surface. The significance of this observation transcends well beyond the particular AQP2-T125M patient mutant because it translates directly into two general questions relating to mechanisms responsible for ER quality control of AQPs and how nonglycosylated wild-type AQP2 is normally targeted to the plasma membrane. Mutations in AQP2 result in NDI (see Ref. 18.Morello J.P. Bichet D.G. Annu. Rev. Physiol. 2001; 63: 607-630Crossref PubMed Scopus (254) Google Scholar) through two putative mechanisms. Recessive nonfunctional mutants are thought to be retained in the ER and fail to tetramerize, whereas dominant AQP2 mutants oligomerize with the product of the wild-type allele, forming tetramers that are improperly targeted and do not reach the apical cell surface (19.Kamsteeg E.J. Wormhoudt T.A.M. Rijss J.P.L. van Os C.H. Deen P.M.T. EMBO J. 1999; 18: 2394-2400Crossref PubMed Scopus (169) Google Scholar, 20.Marr N. Bichet D.G. Lonergan M. Arthus M.F. Jeck N. Seyberth H.W. Rosenthal W. van Os C.H. Oksche A. Deen P.M.T. Hum. Mol. Genet. 2002; 11: 779-789Crossref PubMed Scopus (114) Google Scholar). Given the consequences of mutant AQP2 alleles for the health of affected individuals, surprisingly little is known about biosynthetic maturation and sorting of AQP2. To develop rationally therapeutic protocols for NDI, we need to understand the pathways of wild-type AQP2 to the cell surface. Earlier studies with tunicamycin were interpreted to indicate that glycosylation was not essential for AQP2 shuttling (15.Baumgarten R. van de Pol M.H.J. Wetzels J.F.M. van Os C.H. Deen P.M.T. J. Am. Soc. Nephrol. 1998; 9: 1553-1559Crossref PubMed Google Scholar). Given the recent findings that this glycosylation inhibitor severely affects multiple ER and secretory pathway functions (21.Ye J. Rawson R.B. Komuro R. Chen X. Dave U.P. Prywes R. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 1355-1364Abstract Full Text Full Text PDF PubMed Scopus (1311) Google Scholar, 22.Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1562) Google Scholar), a reevaluation of the significance of glycosylation in AQP2 function became necessary. Using biochemical and morphological methods, we show that tetramerization of AQP2 occurs in the ER and that N-linked glycosylation is important for its transport from the Golgi complex to post-Golgi compartments. Reagents—AQP2-N123Q was constructed by overlap extension PCR and ligated in pcDNA3 (Invitrogen). Synthetic cDNAs were verified by restriction analysis and dye termination sequencing. Furin-pEGFP was generously provided by Gary Thomas. Antibodies against the C terminus of AQP2 were raised in rabbits and guinea pigs as described (23.Deen P.M.T. van Aubel R.A. van Lieburg A.F. van Os C.H. J. Am. Soc. Nephrol. 1996; 7: 836-841PubMed Google Scholar). The following antibodies were used: rabbit anti-protein disulfide isomerase and rabbit anti-calnexin (Ineke Braakman, University of Utrecht), mouse anti-gp114 (Kai Simons, Max Planck Institute, Dresden), mouse anti-CTR433 (Michel Bornens, Curie Institute, Paris), and mouse anti-α-tubulin Thomas Kreis, University of Geneva). The mouse antibodies against γ-adaptin and GM130 were from Transduction Labs, and conjugated secondary antibodies were purchased from Jackson Immunoresearch Laboratories and Molecular Probes. Cell Culture and Transfection—Madin-Darby canine kidney type I (MDCK-I) cells expressing human wild-type AQP2, or vesicular stomatitis virus G-tagged AQP2-R187C were maintained as described (24.Deen P.M.T. Rijss J.P.L. Mulders S.M. Errington R.J. van Baal J. van Os C.H. J. Am. Soc. Nephrol. 1997; 8: 1493-1501Crossref PubMed Google Scholar). MDCK-I cells were transfected with AQP2-N123QpcDNA3 using the calcium phosphate method. CHO and CHO-15B cells were transfected with wild-type AQP2pcDNA3 and AQP2-N123QpcDNA3 in the same way. Stable transfectants were selected in medium containing 0.6 mg/ml G418 and screened by immunofluorescence microscopy and Western blot. Expression of cytomegalovirus-driven constructs was induced by culturing the cells for 14-17 h in the presence of 5 mm sodium butyrate. Pulse-Chase and Immunoprecipitation—Cells were grown in 6-cm tissue culture dishes, washed with phosphate-buffered saline, and incubated for 30 min with methionine- and cysteine-free minimum Eagle's medium (Sigma). The cells were labeled for 30 min at 37 °C with 0.2 mCi/ml [35S]methionine/cysteine (Redivue Promix, Amersham Biosciences) and chased for different periods of time in Dulbecco's modified Eagle's medium, 10% fetal calf serum, 1 mm methionine, 1 mm cysteine. Cells were lysed in 1 ml of 1% Triton X-100, 50 mm Tris, pH 7.4, 1 mm EDTA, and a protease inhibitor mix (Complete mini, Roche Applied Science) on ice. Detergent lysates were centrifuged at 14,000 rpm for 5 min to remove insoluble debris. Lysates were next precleared by incubation with bovine serum albumin-coated protein A beads for 1 h at 4 °C. The supernatants were then transferred to a fresh tube and incubated with antibody-coated beads for 2 h at 4 °C. Beads were washed three times at room temperature for 5 min with 0.05% Triton X-100, 0.1% SDS, 0.3 m NaCl, 10 mm Tris-HCl, pH 8.6. Endoglycosidase H (Endo H) and peptide N-glycosidase F (PNGase F) digestions were performed on immune precipitates as described previously (25.Braakman I. Litty H.H. Wagner K.R. Helenius A. J. Cell Biol. 1991; 114: 401-411Crossref PubMed Scopus (248) Google Scholar). Samples were resuspended in 25 μl of Laemmli sample buffer containing 50 mm dithiothreitol, incubated for 30 min at 37 °C, and resolved by SDS-PAGE on 12.5% gels. Quantitation of immunoprecipitated AQP2 was done by phosphorimaging. Immunoelectron Microscopy—MDCK-AQP2 and MDCK-AQP2-N123Q cells were fixed for immunoelectron microscopy with a mixture of 2% freshly prepared formaldehyde and 0.2% glutaraldehyde in 0.1 m phosphate buffer, pH 7.4. After 6 h at room temperature the fixative was replaced, and cells were postfixed in 2% formaldehyde overnight at 4 °C. Cells were then prepared for ultrathin cryosectioning and immunogold labeled according to the protein A-gold method. Briefly, fixed cells were washed once in phosphate-buffered saline with 0.02 m glycine, after which cells were scraped in 1% gelatin in phosphate-buffered saline and embedded in a 12% gelatin solution. The cell-gelatin mixture was solidified on ice and cut into small blocks. After infiltration with 2.3 m sucrose at 4 °C, blocks were mounted on aluminum pins and frozen in liquid nitrogen. Ultrathin cryosections were picked up in a mixture of 50% sucrose and 50% methyl cellulose (26.Liou W. Geuze H.J. Geelen M.J.H. Slot J.W. J. Cell Biol. 1997; 136: 61-70Crossref PubMed Scopus (220) Google Scholar) and incubated with a rabbit antibody against AQP2, which was used previously for the ultrastructural localization of AQP2 in Xenopus oocytes (27.Mulders S.M. Bichet D.G. Rijss J.P.L. Kamsteeg E.J. Arthus M.F. Lonergan M. Fujiwara M. Morgan K. Leijendekker R. van der Sluijs P. van Os C.H. Deen P.M.T. J. Clin. Invest. 1998; 102: 57-66Crossref PubMed Scopus (220) Google Scholar). Labeling was done with protein A coated with 10 nm of gold. Miscellaneous Methods—Methods for cRNA injection in Xenopus oocytes, water permeability measurements, immunofluorescence microscopy, cell surface biotinylation, and velocity gradient centrifugation have been described previously (17.Marr N. Bichet D.G. Hoefs S. Savelkoul P.J.M. Konings I.B.M. de Mattia F. Graat M.P.J. Arthus M.F. Lonergan M. Fujiwara T.M. Knoers N.V.A.M. Landau D. Balfe W.J. Oksche A. Rosenthal W. Müller D. van Os C.H. Deen P.M.T. J. Am. Soc. Nephrol. 2002; 13: 2267-2277Crossref PubMed Scopus (95) Google Scholar, 19.Kamsteeg E.J. Wormhoudt T.A.M. Rijss J.P.L. van Os C.H. Deen P.M.T. EMBO J. 1999; 18: 2394-2400Crossref PubMed Scopus (169) Google Scholar, 28.Deen P.M.T. Verdijk M.A. Knoers N.V.A.M. Wieringa B. Monnens L.A.H. van Os C.H. van Oost B.A. Science. 1994; 264: 92-95Crossref PubMed Scopus (747) Google Scholar). Confocal laser scanning immunofluorescence microscopy of MDCK cells was done as described previously (29.Deneka M. Neeft M. Popa I. van Oort M. Sprong H. Oorschot V. Klumperman J. Schu P. van der Sluijs P. EMBO J. 2003; 22: 2645-2657Crossref PubMed Scopus (72) Google Scholar). Biosynthesis and Maturation of AQP2—To investigate the biosynthesis of AQP2 we used the MDCK cell line. These cells lack measurable levels of endogenous AQP2, whereas transfected AQP2 is subject to vasopressin-dependent translocation like AQP2 in the kidney (24.Deen P.M.T. Rijss J.P.L. Mulders S.M. Errington R.J. van Baal J. van Os C.H. J. Am. Soc. Nephrol. 1997; 8: 1493-1501Crossref PubMed Google Scholar). MDCK-AQP2 cells were pulse labeled with [35S]methionine/cysteine, and AQP2 was immunoprecipitated from detergent lysates after different periods of chase time and analyzed by SDS-PAGE on 12.5% reducing gels. As shown in Fig. 1A, two bands of 29 and 31-32 kDa (which we refer to as 31 kDa) can be discerned which are not present in immunoprecipitates prepared from nontransfected cells (not shown). Quantification of the 29- and 31-kDa bands at the end of the 30-min pulse by phosphorimaging showed that they are formed at a ratio of 3:1. Even after a short pulse of 2.5 min, about 25% of newly synthesized AQP2 was present in the 31-kDa band, suggesting that glycosylation occurs cotranslationally or very shortly after translation. Whereas the 29-kDa form remained present at all chase times, the 31-kDa form disappeared within 1-1.5 h of chase, and at the same time, a complex of proteins appeared as a faint smear of 40-46 kDa. We then explored the relationship between the 29-, 31-, and the 40-46-kDa bands using Endo H and PNGase F digestions of AQP2 immunoprecipitates. As shown in Fig. 1A, Endo H selectively removed the 31-kDa band but not the 29-kDa and 40-46-kDa bands, whereas after PNGase F only the 29-kDa protein remained detectable as a doublet. PNGase F treatment causes oxidation of the asparagine to an aspartate in the consensus N-linked glycosylation sequence. This modification adds extra negative charge to the protein which might be responsible for a small shift in mobility on SDS-polyacrylamide gels. Alternatively, the hydrophobic transmembrane domains of AQP2, which represent ∼50% of its mass, might fail to be completely denatured in SDS. This could limit the accessibility of the N-linked glycosylation site to PNGase F, preventing quantitative deglycosylation. Disappearance of the 31-kDa band within 1 h after translation and the appearance of the 40-46-kDa bands indicate transport from the ER to the Golgi complex. Although some of 40-46-kDa material is already present at the end of the pulse period, it is unlikely that complex glycosylation of AQP2 occurs cotranslationally. Assuming a translation rate of 5-10 amino acids/s (25.Braakman I. Litty H.H. Wagner K.R. Helenius A. J. Cell Biol. 1991; 114: 401-411Crossref PubMed Scopus (248) Google Scholar, 30.Horwitz M.S. Scharff M.D. Maizel J.V.J. Virology. 1969; 39: 682-694Crossref PubMed Scopus (36) Google Scholar), the pulse period is relatively long compared with the time it takes to translate the 270-amino acid protein AQP2. Likely the AQP2 molecules that were synthesized at the beginning of the pulse may have already left the ER. Indeed, when the pulse is reduced to 2.5 min, we did not observe the 40-46 kDa smear (Fig. 1A, lane indicated with *). The 29-kDa form of AQP2 is a very stable molecular species. After an initial decrease during the 1st h of the chase (likely because of degradation in the ER and transport to the Golgi complex), the half-life of this form is in the order of 6-12 h. The results of the pulse-chase experiments extend and confirm earlier steady-state measurements that document that the 29-kDa band represents nonglycosylated AQP2, whereas the 31- and 40-46-kDa bands are the high mannose and the complex glycosylated forms, respectively. Because the complex glycosylated forms of newly synthesized AQP2 ran as a faint smear between 40 and 46 kDa and because of a background signal along the entire length of the lane, an accurate quantification of the glycoforms was problematic and precluded the investigation of product precursor relationships between the various forms. To avoid this problem we expressed AQP2 in CHO-15B cells that lack functional GlcNAc transferase I (31.Schlesinger S. Gottlieb C. Feil P. Gelb N. Kornfeld S. J. Virol. 1975; 17: 239-246Crossref PubMed Google Scholar) and do not express endogenous AQP2. This enzyme adds N-acetylglucosamine to mannose residues after trimming of the GlcNac2Man9Glc3 oligosaccharide by ER and Golgi mannosidases. In the absence of a functional enzyme, complex glycosylated proteins are not formed, and the resulting mannose-trimmed glycoproteins have a slightly higher mobility than the high mannose precursor. Results of pulse-chase experiments in CHO-15B-AQP2 cells are shown in Fig. 1B. As in MDCK cells, ∼75% of newly synthesized AQP2 was present in the 29-kDa band and was not glycosylated at the end of the pulse, whereas the remainder of radioactivity was present in the 31-kDa band. During the chase period this Endo H-sensitive band (not shown) was converted to a discrete band of 30 kDa. Quantification of the three bands (Fig. 1B) shows a clear precursor-product link between the high mannose and the 30-kDa glycosylated forms of AQP2. The kinetics of AQP2 glycosylation in MDCK and CHO-15B cells was similar, suggesting that the limited extent of glycosylation is not a property of the cells but instead is determined by intrinsic features contained within the AQP2 molecule. As in MDCK cells, the nonglycosylated and terminally glycosylated form of wild-type AQP2 are stable proteins, whose levels decrease with a half-life of 6-12 h after the 1st h of chase, indicating that glycosylation is not essential for the stability of the wild-type protein. The consensus site for N-linked glycosylation is located in the second extracellular loop, 7 amino acid residues upstream of the predicted fourth transmembrane domain. Efficient glycosylation requires a distance of at least 10 amino acids from a C-terminal transmembrane domain, possibly because space constraints combined with egress through the translocon limit a productive interaction with oligosaccharyltransferase (32.Nilsson I. von Heijne G. J. Biol. Chem. 2000; 275: 17338-17343Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We therefore hypothesized that a prolonged residence time of AQP2 in the ER would increase glycosylation. To address this issue, we performed pulse-chase experiments in MDCK cells expressing vesicular stomatitis virus G-tagged AQP2-R187C (Fig. 1C), a mutant that is retained in the ER in recessive NDI (33.Deen P.M.T. Croes H. van Aubel R.A. Ginsel L.A. van Os C.H. J. Clin. Invest. 1995; 95: 2291-2296Crossref PubMed Scopus (215) Google Scholar). As shown in Fig. 1C, the amount of the high mannose precursor of the mutant continued to increase during the first 2 h of chase. This is in sharp contrast to wild-type AQP2 for which the level of the high mannose form decreased continually after the pulse (Fig. 1A). Thus this result suggests that prolonged residence of AQP2 in the ER enhanced the efficiency of glycosylation in a post-translational manner. Glycosylation Is Required for Cell Surface Expression of AQP2—To analyze the function of N-linked glycosylation we substituted Asn123 for Gln and expressed this AQP2 mutant in MDCK cells. We first investigated the synthesis and stability of AQP2-N123Q in pulse-chase experiments of 35S-labeled cells followed by immunoprecipitation and SDS-PAGE. As expected and shown in Fig. 2A, removal of the N-linked glycosylation consensus site did not affect expression of the nonglycosylated 29-kDa precursor; however, neither the 31-kDa nor the mature 42-46-kDa were present. Quantitation of the 29-kDa band showed that its half-time was reduced from 6-12 h in MDCKAQP2 cells to 4 h in the MDCK-AQP2-N123Q cells. Many glycoproteins are O-glycosylated in the Golgi complex. It is not known, however, whether AQPs are O-glycosylated. Removal of the N-linked glycosylation motif in AQP2 produced a protein that ran as single sharp band on SDS-polyacrylamide gels during the pulse and all chase times, suggesting that AQP2 is not O-glycosylated in these cells. Given the reduced stability of the glycosylation mutant, we next addressed whether or not this protein was misfolded, using water permeability in Xenopus oocytes as read-out system. Although oocytes lack the mechanisms for vasopressin-regulated translocation of AQP2, they nevertheless are very valuable for determining the biophysical properties of water channels (28.Deen P.M.T. Verdijk M.A. Knoers N.V.A.M. Wieringa B. Monnens L.A.H. van Os C.H. van Oost B.A. Science. 1994; 264: 92-95Crossref PubMed Scopus (747) Google Scholar). We injected wild-type AQP2 and AQP2-N123Q cRNAs in Xenopus oocytes and measured water permeability of the plasma membrane. As documented in Fig. 2B, both proteins were expressed at the same level at the plasma membrane and yielded similar water permeability. This showed that the absence of glycosylation did not affect the ability of AQP2 to form functional water channels and suggests that AQP2-N123Q is not misfolded. To investigate whether AQP2-N123Q reached the cell surface in a more physiological model, we performed cell surface biotinylation in the MDCK-AQP2-N123Q cells. Biotinylated proteins were then retrieved from detergent lysates by neutravidin-agarose precipitation, resolved by SDS-PAGE, and analyzed by Western blot. Although wild-type AQP2 was expressed at the plasma membrane, AQP2-N123Q was not detected on the cell surface (Fig. 2C). To rule out that the AQP2 signal on the plasma membrane was the result of cell leakage, causing biotinylation of intracellular proteins, we also analyzed the blots with a tubulin antibody. As expected, we did detect tubulin in total cell lysates; however, it was not retrieved on the neutravidin beads, showing that the plasma membrane remained intact during the experiment. Because plasma membrane expression of AQP2 can be induced by vasopressin or forskolin treatment, we added 10 μm forskolin to the cells. Even under this condition AQP2-N123Q was retained intracellularly. The observation that AQP2-N123Q is not delivered to the cell surface suggested that the mutant either cannot leave the ER or is allowed to exit the ER but retained in another compartment. To discriminate between these possibilities, we investigated the localization of AQP2-N123Q. Cells were treated with either indomethacin or forskolin, which diminishes or enhances cAMP-dependent AQP2 expression on the apical cell surface (24.Deen P.M.T. Rijss J.P.L. Mulders S.M. Errington R.J. van Baal J. van Os C.H. J. Am. Soc. Nephrol. 1997; 8: 1493-1501Crossref PubMed Google Scholar), respectively. As shown in Fig. 2D, AQP2-N123Q is predominantly localized in perinuclear structures but not on the plasma membrane. We confirmed with confocal immunofluorescence microscopy on filter-grown AQP2-N123Q MDCK cells, that the mutant was localized intracellularly (not shown). Even though the various kinase consensus sequences are retained in the mutant, it is clear that AQP2-N123Q localization is not affected by the drugs. In contrast wild-type AQP2 is translocated to the cell surface in the presence of forskolin, whereas the level of wild-type AQP2 at the cell surface is decreased by indomethacin as was reported before (Fig. 2D). Thus, glycosylation of AQP2 is required for cell surface expression, and the absence of glycosylation induces intracellular retention. Glycosylation of AQP2 Is Not Essential for Exit from the ER—To define the location where nonglycosylated AQP2 is retained, we performed double label immunofl
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