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• W2079063162 abstract "The amino-terminal ectodomain of thyrotropin (TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked (N-linked) oligosaccharides. The present studies were designed to evaluate how acquisition and processing of N-linked oligosaccharides play a role in the functional maturation of human TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first step of N-linked glycosylation (acquisition ofN-linked oligosaccharides), and a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing were used. Inhibition of acquisition ofN-linked oligosaccharides by tunicamycin treatment in CHO cells stably expressing TSHR produced nonglycosylated TSHR, which was totally nonfunctional. In contrast, all of the TSHRs synthesized in mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient, and galactose-deficient oligosaccharides, respectively) bound TSH and produced cAMP in response to TSH with an affinity and an EC50 similar to those in TSHR expressed in parental CHO cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and Lec2-TSHR were not efficiently expressed on the cell surface, whereas the expression levels of Lec8-TSHR and CHO-TSHR were essentially identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two subunits. Finally, anti-TSHR autoantibodies from Graves' patients interacted with all of the TSHRs harboring different oligosaccharides to a similar extent. These data demonstrate that acquisition and processing of N-linked oligosaccharides of TSHR appear to be essential for correct folding in the endoplasmic reticulum and for cell surface targeting in the Golgi apparatus. We also show that complex type carbohydrates are not crucially involved in the interaction of TSHR with TSH and anti-TSHR autoantibodies. The amino-terminal ectodomain of thyrotropin (TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked (N-linked) oligosaccharides. The present studies were designed to evaluate how acquisition and processing of N-linked oligosaccharides play a role in the functional maturation of human TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first step of N-linked glycosylation (acquisition ofN-linked oligosaccharides), and a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing were used. Inhibition of acquisition ofN-linked oligosaccharides by tunicamycin treatment in CHO cells stably expressing TSHR produced nonglycosylated TSHR, which was totally nonfunctional. In contrast, all of the TSHRs synthesized in mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient, and galactose-deficient oligosaccharides, respectively) bound TSH and produced cAMP in response to TSH with an affinity and an EC50 similar to those in TSHR expressed in parental CHO cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and Lec2-TSHR were not efficiently expressed on the cell surface, whereas the expression levels of Lec8-TSHR and CHO-TSHR were essentially identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two subunits. Finally, anti-TSHR autoantibodies from Graves' patients interacted with all of the TSHRs harboring different oligosaccharides to a similar extent. These data demonstrate that acquisition and processing of N-linked oligosaccharides of TSHR appear to be essential for correct folding in the endoplasmic reticulum and for cell surface targeting in the Golgi apparatus. We also show that complex type carbohydrates are not crucially involved in the interaction of TSHR with TSH and anti-TSHR autoantibodies. The receptors for glycoprotein hormones (thyrotropin receptor (TSHR), 1The abbreviations used are: TSHR, thyrotropin receptor; LHR, lutropin receptor; FSH, follitropin; CHO, Chinese hamster ovary; endo H, endoglycosidase H; PEG, polyethylene glycol; TSAb, thyroid stimulatory autoantibody; TBIAb, TSH binding inhibitory autoantibody; TSH, thyrotropin. lutropin receptor (LHR), and follitropin receptor (FSH)) are members of a subfamily of G protein-coupled receptors, characterized by an extremely large amino-terminal extracellular domain, which is the high affinity binding site for the respective ligands (1Nagayama Y. Rapoport B. Mol. Endocrinol. 1992; 6: 145-156Crossref PubMed Scopus (151) Google Scholar, 2Segaloff D.L. Ascoli M. Endocr. Rev. 1993; 14: 324-347PubMed Google Scholar) and is, in the case of TSHR, the primary autoantigen in autoimmune thyroid disease, including Graves' disease (1Nagayama Y. Rapoport B. Mol. Endocrinol. 1992; 6: 145-156Crossref PubMed Scopus (151) Google Scholar). Like many other secreted and cell surface proteins, these receptors undergo a series of posttranslational modifications such as disulfide bonding (1Nagayama Y. Rapoport B. Mol. Endocrinol. 1992; 6: 145-156Crossref PubMed Scopus (151) Google Scholar), glycosylation (3Misrahi M. Ghinea N. Sar S. Saunier B. Jolivet A. Loosfelt H. Cerutti M. Devauchelle G. Milgrom E. Eur. J. Biochem. 1994; 222: 711-719Crossref PubMed Scopus (109) Google Scholar, 4Tanaka K. Nagayama Y. Yamasaki H. Hayashi H. Namba H. Yamashita S. Niwa M. Biochem. Biophys. Res. Commun. 1996; 228: 21-28Crossref PubMed Scopus (10) Google Scholar, 5Dattatreyamurty B. Reichert Jr., L.E. Endocrinology. 1992; 131: 2437-2445Crossref PubMed Scopus (17) Google Scholar, 6Petaja-Repo U.E. Merz W.E. Rajaniemi H.J. Biochem. J. 1993; 292: 839-844Crossref PubMed Scopus (15) Google Scholar, 7Davies D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170PubMed Google Scholar, 8Zhang R. Cai H. Fatima N. Buczko E. Dufau M.L. J. Biol. Chem. 1995; 270: 21722-21728Crossref PubMed Scopus (79) Google Scholar, 9Davis D.P. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1997; 11: 550-562Crossref PubMed Scopus (76) Google Scholar), palmitoylation (10Zhu H. Wang H. Ascoli M. Mol. Endocrinol. 1995; 9: 141-150Crossref PubMed Google Scholar, 11Tanaka K. Nagayama Y. Nishihara E. Namba H. Yamashita S. Niwa M. Endocrinology. 1998; 139: 803-806Crossref PubMed Scopus (53) Google Scholar), and proteolytic cleavage (subunit formation in TSHR) (12Rees Smith B. McLachlan M. Furmaniak J. Endocr. Rev. 1988; 9: 106-121Crossref PubMed Scopus (529) Google Scholar, 13Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Seto P. Rapoport B. Mol. Endocrinol. 1991; 5: 1607-1612Crossref PubMed Scopus (76) Google Scholar, 14Loosfelt H. Pinchon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (176) Google Scholar), many of which appear to play roles in protein maturation and/or intracellular trafficking. In human TSHR, the receptor ectodomain has asparagine-linked (N-linked) oligosaccharides, which represent 30∼40% of its molecular weight (15Rapoport B. McLachlan S.M. Kakinuma A. Chazenbalk G.D. J. Clin. Endocrinol. Metab. 1996; 81: 2525-2533Crossref PubMed Scopus (61) Google Scholar). Earlier studies with in vitrosite-directed mutagenesis suggest that, among six potentialN-linked glycosylation sites, the first (amino acid 77) and third (amino acid 113) glycosylation sites are essential for the cell surface expression and function of the full-length TSHR (16Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Rapoport B. Mol. Endocrinol. 1991; 5: 29-33Crossref PubMed Scopus (93) Google Scholar). The possibility, however, cannot be excluded in this paper that the amino acid substitutions introduced per se affected receptor function (9Davis D.P. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1997; 11: 550-562Crossref PubMed Scopus (76) Google Scholar). Subsequent studies on the functional role ofN-linked oligosaccharides of TSHR have been performed with the truncated form of TSHR ectodomain expressed in mammalian cells and insect cells, yielding controversial data (15Rapoport B. McLachlan S.M. Kakinuma A. Chazenbalk G.D. J. Clin. Endocrinol. Metab. 1996; 81: 2525-2533Crossref PubMed Scopus (61) Google Scholar, 17Seetharamaiah G.S. Dallas J.S. Patibandla S.A. Thotakura N.R. Prabhakar B.S. J. Immunol. 1997; 158: 2798-2804PubMed Google Scholar, 18Vlase H. Matsuoka N. Graves P.N. Magnusson R.P. Davies T.F. Endocrinology. 1997; 138: 1658-1666Crossref PubMed Scopus (23) Google Scholar). However, it is difficult to interpret these data, because conformational integrity of TSHR protein has not been verified in these studies. Conformationally intact TSHR protein showing high affinity TSH binding has so far been expressed only in mammalian cells as the full-length receptor or the ectodomain fused to a membrane-anchoring peptide (19Shi Y. Zou M. Parhar R.S. Farid N.R. Thyroid. 1993; 3: 129-133Crossref PubMed Scopus (31) Google Scholar, 20Da Costa C.R. Johnstone A.P. J. Biol. Chem. 1998; 273: 11874-11880Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 21Osuga Y. Liang S.-G. Dallas J.S. Wang C. Hsueh A.J.W. Endocrinology. 1998; 139: 671-676Crossref PubMed Scopus (31) Google Scholar). The process of N-linked glycosylation of proteins has been well documented (see Fig. 1) (22Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3776) Google Scholar). Briefly, a dolichol pyrophosphate precursor (Glc3Man9GlcNAc2) is at first transferred to Asn side chain of Asn-X-Ser/Thr consensus sequence for N-linked oligosaccharides in a nascent polypeptide in the endoplasmic reticulum. Processing is initiated by the removal of the three terminal glucose residues and at least one mannose residue in the endoplasmic reticulum, followed by transportation to Golgi apparatus, where mannose residues are further trimmed, and N-acetylglucosamine, galactose, and sialic acid residues are sequentially added. The newly synthesized glycoproteins then exit the Golgi and are transported to their final destination. In the present studies, to further clarify the functional roles ofN-linked oligosaccharides in human TSHR, we evaluated the functional properties of nonglycosylated TSHR and TSHRs with different types of N-linked oligosaccharides. Nonglycosylated TSHR was produced by treatment of TSHR expressed in Chinese hamster ovary (CHO) cells with tunicamycin, and TSHRs with different oligosaccharides were expressed in a series of mutant CHO-Lec cell lines harboring mutations in the distinct steps in carbohydrate processing (see Fig. 1) (23Stanley P. Siminovitch L. Somatic Cell. Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar). Mutant CHO cell lines, CHO-Lec1, 2, and 8, originally established by Dr. Pamela Stanley (23Stanley P. Siminovitch L. Somatic Cell. Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar), were obtained from the American Type Culture Collection (Rockville, MD). CHO-Lec1 cells (ATCC CRL-1735) have no detectableN-acetylglucosaminyl-transferase I activity, and proteins expressed carry oligosaccharides bearing mannose-rich intermediates (Man5GlcNAc2) at sites normally occupied by complex carbohydrates (Sia2Gal2GlcNAc2Man3GlcNAc2) in parental cell line CHO cells (Fig. 1). CHO-Lec2 (ATCC CRL-1736) and -Lec8 (ATCC CRL-1737) cells lack CMP-sialic acid and UDP-galactose translocases and are incapable of transporting CMP-sialic acid and UDP-galactose from the cytosol to the Golgi, thereby producing sialic acid-deficient (Gal2GlcNAc2Man3GlcNAc2) and sialic acid- and galactose-deficient (GlcNAc2Man3GlcNAc2) oligosaccharides, respectively. These cells were maintained at 5% CO2 in minimal essential medium α supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). Parental CHO cells were grown in Ham's F-12 medium with 5% fetal calf serum and antibiotics described above. The human full-length TSHR cDNA (24Nagayama Y. Kaufman K.D. Seto P. Rapoport B. Biochem. Biophys. Res. Commun. 1989; 165: 1184-1190Crossref PubMed Scopus (514) Google Scholar) was ligated into the expression vector pCAGGS (25Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-200Crossref PubMed Scopus (4597) Google Scholar) to yield pCAG-TSHR. The plasmid was transfected into the cells described above with Lipofectin reagent (Life Technologies, Inc.). The cells were selected with 500 μg/ml G418 (Geneticin; WAKO, Osaka, Japan) and pooled. Surviving clones (12 clones for CHO cells and 24 for CHO-Lec cells) were also isolated with cloning cylinders, and the clonal cell lines expressing the highest levels of the receptor, determined by [125I]TSH binding (see below), were selected. The cells were treated with 5 μg/ml tunicamycin (WAKO) for 3 days. The medium was replaced daily to purge the cells of wild-type TSHR. Tunicamycin inhibits transfer of dolichol pyrophosphate precursor to Asn in the consensus sequence forN-linked glycosylation (Asn-X-Ser/Thr). Metabolic labeling with [35S]methionine/cysteine (Trans 35S Label; ICN, Irvine, CA), immunoprecipitation of TSHR with mouse anti-TSHR monoclonal antibodies A10 and A11 (26Nicholson L.B. Vlase H. Graves P. Nilsson M. Molne J. Huang G.C. Morgenthaler N.G. Davies T.F. McGregor A.M. Banga J.P. J. Mol. Endocrinol. 1996; 16: 159-170Crossref PubMed Scopus (68) Google Scholar), deglycosylation of TSHR with endoglycosydase H (endo H) and N-glycosidase F, and SDS-polyacrylamide gel electrophoresis were performed as described previously (4Tanaka K. Nagayama Y. Yamasaki H. Hayashi H. Namba H. Yamashita S. Niwa M. Biochem. Biophys. Res. Commun. 1996; 228: 21-28Crossref PubMed Scopus (10) Google Scholar, 11Tanaka K. Nagayama Y. Nishihara E. Namba H. Yamashita S. Niwa M. Endocrinology. 1998; 139: 803-806Crossref PubMed Scopus (53) Google Scholar, 27Chazenbalk G.D. Tanaka K. Nagayama Y. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1997; 138: 2893-2899Crossref PubMed Scopus (92) Google Scholar). Signals were obtained with a Fujix Bioimaging Analyzer Bas-5000 (Fuji, Tokyo, Japan). Extraction of the crude cell membrane and immunoblotting were performed as described previously (28Tanaka K. Nagayama Y. Takamura N. Namba H. Fukada S. Kuma K. Yamashita S. Niwa M. Endocrinology. 1998; 139: 852-858Crossref PubMed Scopus (31) Google Scholar) with mouse anti-TSHR monoclonal antibodies A10 and A11 (26Nicholson L.B. Vlase H. Graves P. Nilsson M. Molne J. Huang G.C. Morgenthaler N.G. Davies T.F. McGregor A.M. Banga J.P. J. Mol. Endocrinol. 1996; 16: 159-170Crossref PubMed Scopus (68) Google Scholar). 125I-labeled bovine TSH was from the TRAb kit (Cosmic, Tokyo, Japan). [125I]TSH binding to intact cells and intracellular cAMP measurement with a cAMP radioimmunoassay kit (Yamasa, Tokyo, Japan) were performed as described previously (11Tanaka K. Nagayama Y. Nishihara E. Namba H. Yamashita S. Niwa M. Endocrinology. 1998; 139: 803-806Crossref PubMed Scopus (53) Google Scholar). Unlabeled TSH used in TSH binding studies was of bovine origin (Sigma). [125I]TSH binding to the detergent-solubilized extracts was performed with polyethylene glycol (PEG) precipitation previously described (29Ginsberg J. Shewring G. Rees Smith B. Clin. Endocrinol. 1983; 19: 305-311Crossref PubMed Scopus (25) Google Scholar). Briefly, the detergent-solubilized extract was prepared by solubilization of the crude membrane (11Tanaka K. Nagayama Y. Nishihara E. Namba H. Yamashita S. Niwa M. Endocrinology. 1998; 139: 803-806Crossref PubMed Scopus (53) Google Scholar) with 1% Triton X-100 at 4 °C for 1 h. One-hundred μl of detergent-solubilized extract from 100 μg of crude membrane was incubated with 100 μl of [125I]TSH (∼15,000 cpm) for 1 h at 37 C, and solubilized extract and [125I]TSH complexes were precipitated with PEG. Two Graves' sera positive for TSAb activity, another two Graves' sera positive for TBIAb, and a mixture of three normal sera were used. Preparation of crude IgG fraction with PEG and measurements of TSAb activity were performed as described previously (30Kasagi K. Konishi J. Arai K. Misaki T. Iida Y. Endo K. Torizuka K. J. Clin. Endocrinol. Metab. 1986; 62: 855-862Crossref PubMed Scopus (106) Google Scholar). For the TBIAb assay, detergent-solubilized extract (100 μl) from 100 μg of crude membrane was incubated with 100 μl of serum and 100 μl of [125I]TSH (∼15,000 cpm) for 1 h at 37 C, and solubilized extract and [125I]TSH complexes were precipitated with PEG as described above. TBIAb activity was calculated as follows: [1 − (radioactivity with test IgG/radioactivity with normal IgG)] × 100 (%). Using clonal cell lines stably expressing the highest levels of receptors, we determined the molecular masses and glycosylation patterns of TSHR expressed in CHO cells treated with and without tunicamycin and in a series of CHO-Lec cells. In immunoprecipitation experiments (2-h pulse followed by 2-h chase), TSHR protein expressed in parental CHO cells (CHO-TSHR) was visualized as an ∼95-kDa precursor and an ∼120-kDa mature protein (Fig.2 A, lane 1) as previously reported (27Chazenbalk G.D. Tanaka K. Nagayama Y. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1997; 138: 2893-2899Crossref PubMed Scopus (92) Google Scholar). Both decreased in size to ∼85 kDa onN-glycosidase F treatment (Fig. 2 A, lane 3), a size compatible with that of the core polypeptide chain of TSHR (24Nagayama Y. Kaufman K.D. Seto P. Rapoport B. Biochem. Biophys. Res. Commun. 1989; 165: 1184-1190Crossref PubMed Scopus (514) Google Scholar) and also identical to that of nonglycosylated TSHR (Fig. 2 A, compare with lane 4; see below). The ∼95-kDa protein was sensitive to endo H, indicating that this has high mannose type oligosaccharides, whereas the ∼120-kDa species possesses complex type oligosaccharides because of its resistance to endo H (Fig. 2 A, lane 2). Tunicamycin treatment completely inhibited addition of oligosaccharides to TSHR (Fig. 2 A, lanes 4–6). Thus, TSHR was detected as an ∼85-kDa single species in CHO cells treated with 5 μg/ml tunicamycin. With respect to mutant cell lines, CHO-Lec1 cells produced a TSHR that migrated as an ∼90-kDa band, slightly smaller than a TSHR with high mannose oligosaccharides (Fig. 2 A, compare lanes 1 and 7) and was sensitive to endo H (Fig. 2 A, lane 8). This finding is consistent with the fact that proteins synthesized in CHO-Lec1 cells have oligosaccharides of mannose-rich intermediates (Man5GlcNAc2), and high mannose type oligosaccharides in the ∼95-kDa TSHR in parental CHO cells are presumably Man8–9GlcNAc2. CHO-Lec8 cells produced a TSHR migrating as a doublet with molecular masses of ∼90 and ∼95 kDa (Fig. 2 A, lane 10); the ∼90-kDa species is likely a TSHR with galactose- and sialic acid-deficient oligosaccharides (GlcNAc2Man3GlcNAc2) because of its resistance to endo H (Fig. 2 A, lane 11). In CHO-Lec2 cells, TSHR was visualized as a doublet of ∼105 and ∼95 kDa (Fig.2 A, lane 13). The ∼105-kDa species is likely a TSHR with sialic acid-deficient oligosaccharides (Gal2GlcNAc2Man3GlcNAc2). Although it is well known that TSHR cleaves into two subunits (12Rees Smith B. McLachlan M. Furmaniak J. Endocr. Rev. 1988; 9: 106-121Crossref PubMed Scopus (529) Google Scholar, 13Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Seto P. Rapoport B. Mol. Endocrinol. 1991; 5: 1607-1612Crossref PubMed Scopus (76) Google Scholar, 14Loosfelt H. Pinchon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (176) Google Scholar), TSHR subunits were detectable only after longer chase andN-glycosidase F digestion in our immunoprecipitation (27Chazenbalk G.D. Tanaka K. Nagayama Y. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1997; 138: 2893-2899Crossref PubMed Scopus (92) Google Scholar). Therefore, the subunit structures of TSHRs with different oligosaccharides were analyzed on immunoblotting. On immunoblotting, the single polypeptide of TSHR, for example, the ∼95- and ∼120-kDa species in immunoprecipitation of CHO-TSHR, were not visualized. Instead, several nonspecific reactions were observed between 70 and 120 kDa (Fig. 2 B). However, the diffuse “A subunit” with the mean molecular mass of ∼55 kDa could be clearly detected in CHO-TSHR. The majority of TSHRs expressed on the cell surface probably cleave into two subunits at the steady state levels. A similar diffuse band, slightly smaller than that in CHO-TSHR, was also present in Lec2-TSHR (Fig. 2 B, lane 5). The diffuse banding pattern of TSHR A subunit in CHO and CHO-Lec2 cells may be attributable to different degrees of glycosylation of the core polypeptides. These oligosaccharides may contain various outer chain sequences including different sizes of polylactosamine (22Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3776) Google Scholar). In contrast, TSHR A subunit was observed as a doublet in Lec1-TSHR and particularly in Lec8-TSHR (Fig. 2 B, lanes 3 and 4), in which oligosaccharides must be homogeneous. This finding agrees with our recent hypothesis of “two cleavage sites in TSHR” (27Chazenbalk G.D. Tanaka K. Nagayama Y. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1997; 138: 2893-2899Crossref PubMed Scopus (92) Google Scholar). Thus, these data suggest that all of the TSHRs expressed in CHO-Lec cells can cleave into two subunits. Cleavage seems to occur at the cell surface and to be mediated by matrix metalloprotease(s) (31Couet J. Sar S. Jolivet A. Hai M.-T.V. Milgrom E. Misrahi M. J. Biol. Chem. 1996; 271: 4545-4552Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). No cleaved product was observed in tunicamycin-treated, nonglycosylated TSHR (Fig.2 B, lane 2), suggesting that nonglycosylated TSHR may not be expressed on the cell surface. These data are in agreement with the expected consequences of the mutations represented by the cell lines used. Pooled clones of CHO cells stably expressing TSHR were treated with 5 μg/ml tunicamycin for 3 days and subjected to [125I]TSH binding and TSH-induced cAMP production experiments. As shown in Fig. 2 A, 5 μg/ml tunicamycin completely inhibited transfer of dolichol precursor to Asn in the nascent TSHR. Although effect of 3-day treatment with tunicamycin should be evaluated with immunoblot, not immunoprecipitation, immunoblot showed no specific signal as mentioned above. However, this period of treatment has been previously demonstrated to be enough to purge the cells of wild-type recombinant LHR (9Davis D.P. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1997; 11: 550-562Crossref PubMed Scopus (76) Google Scholar). As shown in Fig. 3 A, [125I]TSH binding determined with intact cells was of high affinity in CHO-TSHR. Untransfected CHO cells also showed nonspecific TSH binding of low affinity. This specific, high affinity TSH binding was largely abolished in CHO-TSHR treated with tunicamycin. Consistent with TSH binding data, little or no TSH-induced cAMP production was observed in tunicamycin-treated CHO-TSHR (Fig.3 C). In addition, as shown in Fig. 3 B, the solubilized extract from tunicamycin-treated CHO-TSHR also showed no TSH binding, although the solubilized extract from CHO-TSHR cultured in the absence of tunicamycin demonstrated significantly higher TSH binding than untransfected CHO cells. Thus, together with the immunoblot data, nonglycosylated TSHR seems to accumulate intracellularly in an unfolded form, thereby suggesting an essential role for acquisition of oligosaccharides in correct folding and membrane expression of TSHR. As shown in Fig.4 A, [125I]TSH binding experiments with pooled clones of cells revealed that Lec8-TSHR bound to TSH with an affinity and a B max similar to those of CHO-TSHR. In contrast, specific TSH binding was significantly decreased in Lec1-TSHR and Lec2-TSHR. However, not only the detergent-solubilized extracts from CHO and CHO-Lec8 cells but also those from CHO-Lec1 and CHO-Lec2 cells demonstrated significantly higher [125I]TSH binding than that from untransfected CHO cells (Fig. 4 B). Consistent with the TSH binding data in intact cells, CHO-TSHR and Lec8-TSHR produced intracellular cAMP in response to TSH stimulation to a similar extent, and Lec1-TSHR and Lec2-TSHR showed blunted TSH-induced intracellular cAMP synthesis (∼10 and ∼30% of that in CHO-TSHR, respectively) (Fig.4 C). To further evaluate the function of TSHR expressed in CHO-Lec cells, particularly in Lec1 and Lec2, clonal cell lines expressing the highest levels of receptors were used. The clonal cells lines of Lec1-TSHR and Lec2-TSHR, as well as those of CHO-TSHR and Lec8-TSHR, demonstrated specific TSH binding, as shown in Fig.5 A. In these cells, the binding affinity for TSH was inversely correlated with the receptor number expressed. Thus, TSH binding affinity was highest in Lec1- and Lec2-TSHR and lowest in CHO-TSHR (TableI). In functional studies, however, the EC50 for TSH-induced cAMP production was very similar in all of the cells (Fig. 5 B and Table I), thereby suggesting that the apparently lower binding affinity for TSH in CHO-TSHR appears to be related to its higher expression levels as previously reported (32Chazenbalk G.D. Kakinuma A. Jaume J.C. McLachlan S.M. Rapoport B. Endocrinology. 1996; 137: 4586-4591Crossref PubMed Scopus (77) Google Scholar). Therefore, we interpret these data as that TSH binding is likely of high affinity in TSHR expressed in all of the cells.Table ISummary of functional properties of TSHR expressed in clonal cell lines of CHO and CHO-Lec cellsReceptor number/cellK DaDetermined by Scatchard analysis.EC50nmnmCHO110,400bThe mean of two separate experiments, each experiment determined in duplicate.0.852.0CHO-Lec198,7000.501.9CHO-Lec858,0000.323.8CHO-Lec251,8000.243.4a Determined by Scatchard analysis.b The mean of two separate experiments, each experiment determined in duplicate. Open table in a new tab These data indicate that Lec8-TSHR is correctly folded and fully expressed on the cell surface, and that Lec1-TSHR and Lec2-TSHR accomplish conformational maturation but are trapped intracellularly. However, some of these receptors can reach the cell surface and normally transduce a signal. The effect of different oligosaccharides on the ability of TSHR to interact with anti-TSHR autoantibodies was examined. In the TBIAb assay shown in Fig.6 A, two Graves' IgG, one with potent TBIAb activity (Graves 1) and the other with moderate activity (Graves 2) in CHO-TSHR, comparably inhibited [125I]TSH binding in all of the CHO-Lec cells. TSAb activity in two Graves' sera (Fig. 6 B, Graves 3 andGraves 4) was also similar in all of the cells. Therefore, receptor recognition by autoantibodies does not involve complex type oligosaccharides in TSHR. In the present studies, using a glycosylation inhibitor, tunicamycin, and a series of mutant CHO-Lec cells defective in the different steps of glycosylation process, we evaluated how acquisition and modification of oligosaccharides affect the functional maturation of TSHR. Our results indicate that nonglycosylated TSHR produced by inhibition of the first step of glycosylation with tunicamycin treatment is not folded correctly. In contrast, high affinity TSH binding to Lec1-TSHR clearly indicates that TSHR with mannose-rich intermediates (Man5GlcNAc2) is conformationally intact. Therefore, the acquisition of N-linked oligosaccharides appears to allow the nascent, highly unfolded TSHR protein to accomplish conformational maturation in the endoplasmic reticulum. Several roles of addition of N-linked oligosaccharides to nascent proteins have been demonstrated; for example,N-linked oligosaccharides attach to lectin-like molecular chaperons such as calnexin and calreticulin, facilitating correct protein folding, and also play a role in the “quality control” system of the endoplasmic reticulum that ensures selective transportation of the properly folded proteins for the Golgi complex (33Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (562) Google Scholar). Therefore, unfolded proteins such as nonglycosylated TSHR mentioned above may be trapped in the endoplasmic reticulum. Impaired cell surface expression in Lec1-TSHR suggests that processing of N-linked oligosaccharides from high mannose type to complex type in the Golgi apparatus appears to participate in cell surface targeting of TSHR. However, efficient cell surface expression of TSHR is not necessarily dependent on completion of complex oligosaccharides, because TSHR with sialic acid- and galactose-deficient oligosaccharides (Lec8-TSHR) is efficiently expressed on the cell surface. Contrarily, TSHR with sialic acid-deficient oligosaccharides (Lec2-TSHR) is not efficiently expressed on the cell surface. We cannot explain these data at present, although oligosaccharides terminating in galactose residues may somehow interfere with cell surface targeting of TSHR in the Golgi apparatus. Alternatively, lack of sialylation in the trans-Golgi network may affect a role of the trans-Golgi network as an important sorting site for different proteins destined for the apical or basolateral surfaces of cell membranes (34Simons K. Wandinger-Ness A. Cell. 1990; 62: 207-210Abstract Full Text PDF PubMed Scopus (422) Google Scholar). For any reason, these data are in agreement with the facts that N-linked oligosaccharides generally play critical roles in protein folding and intracellular trafficking in the endoplasmic reticulum and the Golgi apparatus, respectively (33Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (562) Google Scholar, 35Fiedler K. Simons K. Cell. 1995; 81: 309-312Abstract Full Text PDF PubMed Scopus (276) Google Scholar). However, our data on TSHR may not be generalized to other membrane proteins, because (i) the membrane receptor for Newcastle disease virus is reported not to be properly expressed in mouse mammary carcinoma Had-1 cells deficient in UDP-galactose transporter like Lec8 cells (36Hara T. Endo T. Furukawa K. Kawakita M. Kobata A. J. Biochem. 1989; 106: 236-247Crossref PubMed Scopus (39) Google Scholar), and (ii) the expression levels of rat LHR were decreased by 30–40% of that in CHO cells in all of the CHO-Lec cells. 2Y. Nagayama et al., unpublished data. Our data also indicate that complex type oligosaccharides do not appear to constitute TSH or autoantibody binding sites or to participate in signal transduction, although glycosylation is reported to be related to function in some proteins (37Thornhill W.B. Wu M.B. Jiang X. Wu X. Morgan P.T. Margiotta J.F. J. Biol. Chem. 1996; 271: 19093-19098Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 38Wright A. Morrison S.L. J. Immunol. 1998; 160: 3393-3402PubMed Google Scholar, 39Katoh S. Zheng Z. Oritani K. Shimozato T. Kincade P.W. J. Exp. Med. 1995; 182: 419-429Crossref PubMed Scopus (230) Google Scholar). It has recently been reported that the truncated type of TSHR ectodomain, which is not secreted, possesses high mannose type carbohydrates and is incapable of binding to TSH or anti-TSHR autoantibodies in CHO cells (15Rapoport B. McLachlan S.M. Kakinuma A. Chazenbalk G.D. J. Clin. Endocrinol. Metab. 1996; 81: 2525-2533Crossref PubMed Scopus (61) Google Scholar). However, whether this lack of binding was related to polypeptide misfolding or to the type of oligosaccharides on the protein was unknown in this study. From our results, it is now clear that misfolding is the primary event. Thus, TSHR ectodomain by itself may not be able to accomplish correct folding, thereby being trapped in the endoplasmic reticulum and unable to acquire complex oligosaccharides. Recent studies also suggest that only the TSHR ectodomain fused to a membrane-anchored peptide seems to be correctly folded (19Shi Y. Zou M. Parhar R.S. Farid N.R. Thyroid. 1993; 3: 129-133Crossref PubMed Scopus (31) Google Scholar, 20Da Costa C.R. Johnstone A.P. J. Biol. Chem. 1998; 273: 11874-11880Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 21Osuga Y. Liang S.-G. Dallas J.S. Wang C. Hsueh A.J.W. Endocrinology. 1998; 139: 671-676Crossref PubMed Scopus (31) Google Scholar). A nonessential role of galactose and sialic acid in function of TSHR shown in Lec8 cells is reminiscent of our previous finding (4Tanaka K. Nagayama Y. Yamasaki H. Hayashi H. Namba H. Yamashita S. Niwa M. Biochem. Biophys. Res. Commun. 1996; 228: 21-28Crossref PubMed Scopus (10) Google Scholar, 40Nagayama Y. Chazenbalk G.D. Takeshita A. Kimura H. Ashizawa K. Yokoyama N. Rapoport B. Nagataki S. Endocrinology. 1994; 135: 1060-1065Crossref PubMed Scopus (12) Google Scholar) of high affinity TSH binding to TSHR expressed in 293 human embryonal kidney cells. These cells expressN-acetylgalactosaminyl-transferase andN-acetylgalactosaminyl-N-acetylglucosaminyl-mannosyl-sulfotransferase and produce N-linked oligosaccharides terminating in sulfate in >70% of the chains (41Smith P.L. Skelton T.P. Fiete D. Dharmesh S.M. Beranek M.C. MacPhail L. Broze Jr., G.J. Baenziger J.U. J. Biol. Chem. 1992; 267: 19140-19146Abstract Full Text PDF PubMed Google Scholar). Of interest is that some glycoproteins synthesized in the thyroid gland have been reported to be sulfated (42Edge A.S.B. Spiro R.G. J. Biol. Chem. 1984; 259: 4710-4713Abstract Full Text PDF PubMed Google Scholar,43Schneider A.B. McCurdy A. Chang T. Dudlak D. Magner J. Endocrinology. 1988; 122: 2428-2435Crossref PubMed Scopus (23) Google Scholar). TSHR expressed in the thyroid gland may also be, at least in part, sulfated. Furthermore, N-linked oligosaccharides in bovine pituitary TSH that we used in the present studies are exclusively sulfated, whereas human pituitary TSH terminates both in sulfation and sialylation (44Green E.D. Baenziger J.U. J. Biol. Chem. 1988; 263: 25-35Abstract Full Text PDF PubMed Google Scholar). Biological activity of sulfated TSH has been reported to be more potent than sialylated TSH (45Grossmann M. Szkudlinski M.W. Tropea J.E. Bishop L.A. Thotakura N.R. Schofield P.R. Weintraub B.D. J. Biol. Chem. 1995; 270: 29378-29385Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The carbohydrates, especially the terminal sialic acids, of TSH, have also been demonstrated to attenuate its biological activity (45Grossmann M. Szkudlinski M.W. Tropea J.E. Bishop L.A. Thotakura N.R. Schofield P.R. Weintraub B.D. J. Biol. Chem. 1995; 270: 29378-29385Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 46Grossmann M. Wong R. Teh N. Tropea J.E. East-Palmer J. Weintraub B.D. Szkudlinski M.W. Endocrinology. 1997; 138: 92-100Crossref PubMed Scopus (49) Google Scholar). In this regard, it will be interesting to compare the relative potency of TSH with different types of oligosaccharides to bind and stimulate TSHR with various oligosaccharides. In FSH receptor, although removal of N-linked oligosaccharide from the wild-type receptor does not affect FSH binding (5Dattatreyamurty B. Reichert Jr., L.E. Endocrinology. 1992; 131: 2437-2445Crossref PubMed Scopus (17) Google Scholar, 7Davies D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170PubMed Google Scholar), the deglycosylated receptor produced by in vitromutagenesis or tunicamycin treatment shows no FSH binding activity (7Davies D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170PubMed Google Scholar). Thus, the N-linked oligosaccharides seem to be necessary for the proper folding rather than hormone binding in FSH receptor. However, the role of N-linked oligosaccharides in LHR is controversial; although high affinity human chorionic gonadotropin binding to LHR does not appear to involve the carbohydrates on LHR, it remains unclear whether the oligosaccharides are necessary for the correct folding of the nascent LHR (6Petaja-Repo U.E. Merz W.E. Rajaniemi H.J. Biochem. J. 1993; 292: 839-844Crossref PubMed Scopus (15) Google Scholar, 8Zhang R. Cai H. Fatima N. Buczko E. Dufau M.L. J. Biol. Chem. 1995; 270: 21722-21728Crossref PubMed Scopus (79) Google Scholar, 9Davis D.P. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1997; 11: 550-562Crossref PubMed Scopus (76) Google Scholar). In conclusion, our data show that acquisition and processing ofN-linked oligosaccharides in TSHR are essential for correct folding in the endoplasmic reticulum and for intracellular trafficking in the Golgi apparatus. However, binding of TSH and anti-TSHR autoantibodies to TSHR does not appear to involve the oligosaccharides of the receptor. We thank Dr. J. P. Banga (Kings College School of Medicine, London, UK) for providing mouse anti-TSHR monoclonal antibodies and Dr. J. Miyazaki for the expression vector pCAGGS. We are also grateful to Dr. B. Rapoport (Cedars-Sinai Medical Center, Los Angeles, CA) for critical review of the manuscript." @default.
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• W2079063162 title "Role of Asparagine-linked Oligosaccharides in Protein Folding, Membrane Targeting, and Thyrotropin and Autoantibody Binding of the Human Thyrotropin Receptor" @default.
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