Polypeptide GalNAc-transferase T3 and Familial Tumoral Calcinosis
2006; Elsevier BV; Volume: 281; Issue: 27 Linguagem: Inglês
10.1074/jbc.m602469200
ISSN1083-351X
AutoresKentaro Kato, Charlotte Jeanneau, Mads A. Tarp, Anna Benet‐Pagès, Bettina Lorenz‐Depiereux, Eric Bennett, Ulla Mandel, Tim M. Strom, Henrik Clausen,
Tópico(s)Skin and Cellular Biology Research
ResumoMutations in the gene encoding the glycosyltransferase polypeptide GalNAc-T3, which is involved in initiation of O-glycosylation, were recently identified as a cause of the rare autosomal recessive metabolic disorder familial tumoral calcinosis (OMIM 211900). Familial tumoral calcinosis is associated with hyperphosphatemia and massive ectopic calcifications. Here, we demonstrate that the secretion of the phosphaturic factor fibroblast growth factor 23 (FGF23) requires O-glycosylation, and that GalNAc-T3 selectively directs O-glycosylation in a subtilisin-like proprotein convertase recognition sequence motif, which blocks processing of FGF23. The study suggests a novel posttranslational regulatory model of FGF23 involving competing O-glycosylation and protease processing to produce intact FGF23. Mutations in the gene encoding the glycosyltransferase polypeptide GalNAc-T3, which is involved in initiation of O-glycosylation, were recently identified as a cause of the rare autosomal recessive metabolic disorder familial tumoral calcinosis (OMIM 211900). Familial tumoral calcinosis is associated with hyperphosphatemia and massive ectopic calcifications. Here, we demonstrate that the secretion of the phosphaturic factor fibroblast growth factor 23 (FGF23) requires O-glycosylation, and that GalNAc-T3 selectively directs O-glycosylation in a subtilisin-like proprotein convertase recognition sequence motif, which blocks processing of FGF23. The study suggests a novel posttranslational regulatory model of FGF23 involving competing O-glycosylation and protease processing to produce intact FGF23. The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) 2The abbreviations used are: GalNAc-transferase, UDP-N-acetyl-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase; FTC, familial tumoral calcinosis; FGF23, fibroblast growth factor 23; SPC, subtilisin-like proprotein convertase; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. isoform GalNAc-T3 was recently implicated in familial tumoral calcinosis (FTC) (1Topaz O. Shurman D.I. Bergman R. Indelman M. Ratajczak P. Mizrachi M. Khamaysi Z. Behar D. Petronius D. Friedman V. Zelikovic I. Raimer S. Metzker A. Richard G. Sprecher E. Nat. Gen. 2004; 36: 579-581Crossref PubMed Scopus (469) Google Scholar, 2Ichikawa S. Lyles K.W. Econs M.J. J. Clin. Endocrinol. Metab. 2005; 90: 2420-2423Crossref PubMed Scopus (123) Google Scholar). GalNAc-T3 is a member of the large polypeptide GalNAc transferase gene family containing 20 genes of which 15 have been shown to encode functional enzymes. The GalNAc-transferase gene family is the largest mammalian family of glycosyltransferases, which are involved in catalysis of a single glycosidic linkage, GalNAcα1-O-Ser/Thr, and collectively the GalNAc-transferase isoforms control the initiation of mucin-type O-glycosylation in the Golgi complex (3Hassan H. Bennett E.P. Mandel U. Hollingsworth M.A. Clausen H. Ernst B. Hart B.W. Sinay P. Carbohydrates in Chemistry and Biology, A Comprehension Handbook. 2000: 273-292Google Scholar, 4Ten Hagen K.G. Fritz T.A. Tabak L.A. Glycobiology. 2003; 13: 1R-16Crossref PubMed Scopus (413) Google Scholar). Mucin-type O-glycosylation is one of the most abundant forms of glycosylation of proteins, and is found on a large variety of cell membrane and secreted glycopeptides and glycoproteins. O-Glycosylation imparts unique physicochemical features to glycoproteins and O-glycans have been shown to play important functions in almost all known biological processes including intracellular sorting, cell-cell adhesion, and microbial adhesion events (5Jentoft N. Trends Biochem. Sci. 1990; 15: 291-294Abstract Full Text PDF PubMed Scopus (627) Google Scholar). Inactivating mutations in GALNT3 were originally discovered in FTC (1Topaz O. Shurman D.I. Bergman R. Indelman M. Ratajczak P. Mizrachi M. Khamaysi Z. Behar D. Petronius D. Friedman V. Zelikovic I. Raimer S. Metzker A. Richard G. Sprecher E. Nat. Gen. 2004; 36: 579-581Crossref PubMed Scopus (469) Google Scholar, 2Ichikawa S. Lyles K.W. Econs M.J. J. Clin. Endocrinol. Metab. 2005; 90: 2420-2423Crossref PubMed Scopus (123) Google Scholar, 6Specktor P. Cooper J.G. Indelman M. Sprecher E. J. Hum. Genet.,. 2006; (in press)PubMed Google Scholar, 7Campagnoli M.F. Pucci A. Garelli E. Carando A. Defilippi C. Lala R. Ingrosso G. Dianzani I. Forni M. Ramenghi U. J. Clin. Pathol. 2006; 59: 440-442Crossref PubMed Scopus (53) Google Scholar), but more recently mutations in the gene encoding the phosphaturic factor FGF23 have also been identified in FTC (8Benet-Pages A. Orlik P. Strom T.M. Lorenz-Depiereux B. Hum. Mol. Genet. 2005; 14: 385-390Crossref PubMed Scopus (426) Google Scholar, 9Larsson T. Davis S.I. Garringer H.J. Mooney S.D. Draman M.S. Cullen M.J. White K.E. Endocrinology. 2005; 146: 3883-3891Crossref PubMed Scopus (119) Google Scholar, 10Araya K. Fukumoto S. Backenroth R. Takeuchi Y. Nakayama K. Ito N. Yoshii N. Yamazaki Y. Yamashita T. Silver J. Igarashi T. Fujita T. J. Clin. Endocrinol. Metab. 2005; 90: 5523-5527Crossref PubMed Scopus (183) Google Scholar). Thus, FGF23 mutations affecting folding and secretion were identified in FTC patients without mutations in GalNAc-T3. Furthermore, ablation of FGF23 in mice leads to hyperphosphatemia resembling FTC (11Shimada T. Kakitani M. Yamazaki Y. Hasegawa H. Takeuchi Y. Fujita T. Fukumoto S. Tomizuka K. Yamashita T. J. Clin. Investig. 2004; 113: 561-568Crossref PubMed Scopus (1287) Google Scholar). FTC patients with mutations in GalNAc-T3 or FGF23 exhibit hyperphosphatemia and have reduced serum intact FGF23 levels, which may suggest that GalNAc-T3 and FGF23 act in a common pathway. GalNAc-T3 and FGF23 were found to be co-expressed in a number of tissues (2Ichikawa S. Lyles K.W. Econs M.J. J. Clin. Endocrinol. Metab. 2005; 90: 2420-2423Crossref PubMed Scopus (123) Google Scholar). FGF23 is a key regulator of phosphate homeostasis (12White K.E. Evans W.E. O'Riordan J.L.H. Speer M.C. Econs M.J. Lorenz-Depiereux B. Grabowski M. Meitinger T. Strom T.M. Nat. Genet. 2000; 26: 345-348Crossref PubMed Scopus (1297) Google Scholar, 13Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1231) Google Scholar), and is an O-glycosylated glycoprotein of ∼32 kDa (14Shimada T. Muto T. Urakawa I. Yoneya T. Yamazaki Y. Okawa K. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Endocrinolology. 2002; 143: 3179-3182Crossref PubMed Scopus (387) Google Scholar). FGF23 is partially processed intracellularly by subtilisin-like proprotein convertases (SPC) at the consensus sequence RXXR↓ (RHTR179) between Arg179 and Ser180 (14Shimada T. Muto T. Urakawa I. Yoneya T. Yamazaki Y. Okawa K. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Endocrinolology. 2002; 143: 3179-3182Crossref PubMed Scopus (387) Google Scholar, 15Benet-Pages A. Lorenz-Depiereux B. Zischka H. White K.E. Econs M.J. Strom T.M. Bone. 2004; 35: 455-462Crossref PubMed Scopus (213) Google Scholar, 16White K.E. Carn G. Lorenz-Depiereux B. Benet-Pages A. Strom T.M. Econs M.J. Kidney Int. 2001; 60: 2079-2086Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). This processing step appears to be essential in the regulation of phosphate homeostasis, because mutations in the SPC cleavage sequence prevent processing and result in autosomal dominant hypophosphatemic rickets (12White K.E. Evans W.E. O'Riordan J.L.H. Speer M.C. Econs M.J. Lorenz-Depiereux B. Grabowski M. Meitinger T. Strom T.M. Nat. Genet. 2000; 26: 345-348Crossref PubMed Scopus (1297) Google Scholar). Many secreted proteins and peptides essential for regulation of biological activities are synthesized as inactive proproteins, which are subsequently activated by site-specific proteolysis mainly by SPCs to form the intact active forms (17Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). Examples of inactivation of proteins by SPCs have emerged more recently including FGF23 (14Shimada T. Muto T. Urakawa I. Yoneya T. Yamazaki Y. Okawa K. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Endocrinolology. 2002; 143: 3179-3182Crossref PubMed Scopus (387) Google Scholar, 15Benet-Pages A. Lorenz-Depiereux B. Zischka H. White K.E. Econs M.J. Strom T.M. Bone. 2004; 35: 455-462Crossref PubMed Scopus (213) Google Scholar, 16White K.E. Carn G. Lorenz-Depiereux B. Benet-Pages A. Strom T.M. Econs M.J. Kidney Int. 2001; 60: 2079-2086Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar), endothelial lipase (18Jin W. Fuki I.V. Seidah N.G. Benjannet S. Glick J.M. Rader D.J. J. Biol. Chem. 2005; 280: 36551-36559Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and matrix metalloproteinase-2 (19Cao J. Rehemtulla A. Pavlaki M. Kozarekar P. Chiarelli C. J. Biol. Chem. 2005; 280: 10974-10980Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Interestingly, the SPC site in FGF23 contains the sequence RHTR179↓ with a potential O-glycosylation site at Thr178. We hypothesized that O-glycosylation and in particular O-glycosylation directed by GalNAc-T3 could play a role for processing and secretion of FGF23. The results presented in this study demonstrate that secretion of FGF23 is dependent on O-glycosylation in CHO ldlD cells and that co-expression of GalNAc-T3 in these cells results in a marked increase of secretion. Furthermore, in vitro studies demonstrate that GalNAc-T3 can glycosylate the SPC signal sequence of FGF23 at Thr178, which suggests that the underlying mechanism for GalNAc-T3 involvement in FTC is at least in part a role in O-glycosylation of the furin protease consensus sequence in FGF23. Expression of FGF23 and Glycosyltransferases in CHO ldlD Cells—An expression construct of the entire coding sequence of human FGF23 cloned into pcDNA3.1/myc-His (C-terminal tags) was prepared as previously reported (15Benet-Pages A. Lorenz-Depiereux B. Zischka H. White K.E. Econs M.J. Strom T.M. Bone. 2004; 35: 455-462Crossref PubMed Scopus (213) Google Scholar). Full coding expression constructs of human GalNAc-T3 (20Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) and human core 3 β3GlcNAc-transferase T6 (21Iwai T. Inaba N. Naundorf A. Zhang Y. Gotoh M. Iwasaki H. Kudo T. Togayachi A. Ishizuka Y. Nakanishi H. Narimatsu H. J. Biol. Chem. 2002; 277: 12802-12809Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) were prepared as follows. The 5′-end of GalNAc-T3 (nucleotide 1–1032) was amplified by PCR using primer pair EHBC238 (5′-GCGGGATCCGTCAGAATGGCTCACCTAAAGCGA-3′)/EBHC222 (5′-ATCAGGAAGCGACTCCCAGCC-3′) with salivary gland mRNA (Clontech). An internal EcoRI site was used together with the BamHI site contained in EBHC238 to directionally clone the entire 5′-end of GalNAc-T3 into an existing secreted GalNAc-T3 construct (20Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The full coding core 3 β3GnT6 construct was amplified using primer pair MTHC2307 (5′-GCGGATCCACCATGGCTTTTCCCTGCCGCAGG-3′)/MTHC2302 (5′-GCGAATTCTCAGGAGACCCGGTGTCCCCG-3′) with human genomic DNA, and the product was cloned into the BamHI/EcoRI site of pBluescript (Stratagene, La Jolla, CA). Both constructs were cloned into pcDNA3.1. Chinese hamster ovary mutant cells, CHO ldlD (22Kingsley D.M. Kozarsky K.F. Segal M. Krieger M. J. Cell Biol. 1986; 102: 1576-1585Crossref PubMed Scopus (125) Google Scholar), with defective UDP-Gal/UDP-GalNAc 4-epimerase were grown in Ham's F-12/Dulbecco's modified Eagle's medium (1:1, v/v) supplemented with 3% fetal bovine serum and 0.03% glutamine. Cells were transfected with pcDNA3.1 constructs and stable transfectants were selected in 0.4 mg/ml G418 and/or 0.4 mg/ml Zeocin (Invitrogen) using immunostaining for expression of FGF23 (monoclonal anti-myc antibody (Invitrogen)) and GalNAc-T3 (monoclonal antibody UH5) (23Bennett E.P. Hassan H. Mandel U. Hollingsworth M.A. Akisawa N. Ikematsu Y. Merkx G. van Kessel A.G. Olofsson S. Clausen H. J. Biol. Chem. 1999; 274: 25362-25370Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In the case of core 3 β3GnT6, stable transfectants were selected by loss of reactivity with an anti-Tn (GalNAcα1-O-Ser/Thr) monoclonal antibody (5F4) when cells were grown in medium with 1 mm GalNAc (24Sorensen A.L. Reis C.A. Tarp M.A. Mandel U. Ramachandran K. Sankaranarayanan V. Schwientek T. Graham R. Taylor-Papadimitriou J. Hollingsworth M.A. Burchell J. Clausen H. Glycobiology. 2006; 16: 96-107Crossref PubMed Scopus (210) Google Scholar). Double transfectants were prepared sequentially with FGF23 followed by glycosyltransferases. To evaluate the effects of O-glycosylation CHO ldlD transfectant cells were grown in medium supplemented with 0.1 mm Gal, 1 mm GalNAc, or a combination. Immunocytology—Immunostaining of CHO ldlD cells was performed with washed air-dried cells on multiwell slides using fixation with 3% paraformaldehyde and permeabilization with 0.5% Triton X-100. Slides were incubated with monoclonal antibodies overnight at 4 °C, followed by fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulins (F261, Dako) and examination on a Zeiss fluorescence microscope using epi-illumination. ELISA for Quantification of FGF23—A commercial ELISA kit for quantification of intact FGF23 was used with culture medium from CHO ldlD transfectant cells according to the manufacturer's protocol (Kainos Laboratories International). Culture medium from cells was diluted to 1:500 and the assay run with an internal standard. Glycosyltransferase Assays and Chemoenzymatic Synthesis of Glycopeptides—All glycosyltransferases were expressed as soluble secreted truncated proteins in insect cells as previously described (24Sorensen A.L. Reis C.A. Tarp M.A. Mandel U. Ramachandran K. Sankaranarayanan V. Schwientek T. Graham R. Taylor-Papadimitriou J. Hollingsworth M.A. Burchell J. Clausen H. Glycobiology. 2006; 16: 96-107Crossref PubMed Scopus (210) Google Scholar, 25Schwientek T. Bennett E.P. Flores C. Thacker J. Hollmann M. Reis C.A. Behrens J. Mandel U. Keck B. Schafer M.A. Haselmann K. Zubarev R. Roepstorff P. Burchell J.M. Taylor-Papadimitriou J. Hollingsworth M.A. Clausen H. J. Biol. Chem. 2002; 277: 22623-22638Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Screening assays for GalNAc-transferases with peptides were performed with UDP-[14C]GalNAc as previously described (25Schwientek T. Bennett E.P. Flores C. Thacker J. Hollmann M. Reis C.A. Behrens J. Mandel U. Keck B. Schafer M.A. Haselmann K. Zubarev R. Roepstorff P. Burchell J.M. Taylor-Papadimitriou J. Hollingsworth M.A. Clausen H. J. Biol. Chem. 2002; 277: 22623-22638Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) or as product development assays in 25 μl of 25 mm cacodylic acid sodium, pH 7.4, 10 mm MnCl2, 0.25% Triton X-100, 1.5 mm UDP-GalNAc (Sigma), 8.5 μg of acceptor peptides, and 0.4 μg of purified recombinant GalNAc-transferases. Peptides were custom synthesized by Schafer-N (Copenhagen, Denmark). Microsomal fractions derived from 200 μl of packed cell pellets were resuspended in 200 μl of 10 mm Hepes, 150 mm NaCl, 1% Triton X-100 and incubated on ice for 1 h, followed by centrifugation at 100,000 × g for 1 h, and the supernatant used for assays with or without immuno-clearance of human GalNAc-T3. Immuno-clearance was performed by incubating the supernatant overnight with anti-GalNAc-T3 monoclonal antibody UH5 (23Bennett E.P. Hassan H. Mandel U. Hollingsworth M.A. Akisawa N. Ikematsu Y. Merkx G. van Kessel A.G. Olofsson S. Clausen H. J. Biol. Chem. 1999; 274: 25362-25370Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), followed by incubation with protein G-Sepharose (Amersham Biosciences) for 1 h and centrifugation. The resulting supernatants were used for GalNAc-transferase assays and incorporation of GalNAc was quantified by scintillation counting after Dowex-1 chromatography. Synthesis of glycopeptides was performed sequentially with HPLC purification between each step. GalNAc-O-glycosylation was performed in 25 mm cacodylic acid sodium, pH 7.4, 10 mm MnCl2, 0.25% Triton X-100, 1.5 mm UDP-GalNAc (Sigma), 0.4 mg/ml of acceptor peptides, and 1.6 μg/100 μl of GalNAc-T3 (24Sorensen A.L. Reis C.A. Tarp M.A. Mandel U. Ramachandran K. Sankaranarayanan V. Schwientek T. Graham R. Taylor-Papadimitriou J. Hollingsworth M.A. Burchell J. Clausen H. Glycobiology. 2006; 16: 96-107Crossref PubMed Scopus (210) Google Scholar). Core 1 galactosylation was performed in 100 mm MES, pH 6.0, 20 mm MnCl2, 0.1% Triton X-100, 2.6 mm UDP-Gal (Sigma), 0.4 mg/ml of GalNAc-glycosylated FGF23b peptide, and dC1Gal-T1 (24Sorensen A.L. Reis C.A. Tarp M.A. Mandel U. Ramachandran K. Sankaranarayanan V. Schwientek T. Graham R. Taylor-Papadimitriou J. Hollingsworth M.A. Burchell J. Clausen H. Glycobiology. 2006; 16: 96-107Crossref PubMed Scopus (210) Google Scholar, 26Ju T. Brewer K. D'Souza A. Cummings R.D. Canfield W.M. J. Biol. Chem. 2002; 277: 178-186Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Sialylation with ST3Gal-I was performed in 25 mm Tris-HCl, pH 6.5, 0.1% Triton X-100, 2 mm CMP-NANA (Sigma), 0.4 mg/ml of Galβ1–3GalNAcα1-O-glycosylated FGF23b peptide, and human ST3Gal-I (27Tsuji S. Datta A.K. Paulson J.C. Glycobiology. 1996; 6: R5-R7Crossref Google Scholar, 28George S.K. Schwientek T. Holm B. Reis C.A. Clausen H. Kihlberg J. J. Am. Chem. Soc. 2001; 123: 11117-11125Crossref PubMed Scopus (68) Google Scholar). Sialylation with ST6GalNAc-I was performed in 50 mm MES, pH 6.0, 20 mm EDTA, 2 mm dithiothreitol, 2 mm CMP-NANA, 0.4 mg/ml of GalNAc-glycosylated or Sialα2–3Galβ1–3GalNAcα-O-glycosylated FGF23b, and human ST6GalNAc-I (29Ikehara Y. Kojima N. Kurosawa N. Kudo T. Kono M. Nishihara S. Issiki S. Morozumi K. Itzkowitz S. Tsuda T. Nishimura S. Tsuji S. Narimatsu H. Glycobiology. 1999; 9: 1213-1224Crossref PubMed Scopus (112) Google Scholar). All glycopeptides were purified by HPLC on a C18 column (ZORBAX 300 SB C-18, 9.4 × 250 mm) and analyzed by MALDI-TOF. MALDI-TOF MS Analysis—Evaluation of the number of GalNAc residues and subsequently Gal and NeuAc residues incorporated into peptides was done by MALDI-TOF MS as previously described (24Sorensen A.L. Reis C.A. Tarp M.A. Mandel U. Ramachandran K. Sankaranarayanan V. Schwientek T. Graham R. Taylor-Papadimitriou J. Hollingsworth M.A. Burchell J. Clausen H. Glycobiology. 2006; 16: 96-107Crossref PubMed Scopus (210) Google Scholar). Briefly, 0.5 μl of enzyme reaction mixtures were diluted with 3.5 μl of 0.1% trifluoroacetic acid, H2O, and 1 μl was applied and mixed with 1 μl of 25 mg/ml 2,5-dihydroxybenzoic acid dissolved in H2O/CH3CN (2:1) solution. Mass spectra were obtained on a Voyager-DE™ instrument (Applied Biosystems) operating at an accelerating voltage of 20 kV (grid voltage 96.5%, guide wire voltage 0.05%) in the linear mode with the delayed extraction setting. Recorded data were processed using GRAMS software. In Vitro Furin Cleavage Assay—An in vitro SPC cleavage assay with a human furin protease (Sigma) was developed with FGF23-related peptides covering the RHTR cleavage site. Assays were performed in 100 mm Hepes, pH 7.5, 1 mm CaCl2, 0.5% Triton X-100, 1 mm 2-mercaptoethanol using 10 μg of (glyco)peptides substrate and 1 unit of enzyme in a total volume of 50 μl with incubations at 37 °C for 1, 4, and 24 h. Product development was evaluated by MALDI-TOF. The effects of O-glycosylation of FGF23 were studied using the CHO ldlD mutant cell line. This cell line was originally developed and used to demonstrate that O-glycosylation of the low density lipoprotein receptor was required for stable cell-surface expression by preventing proteolytic cleavage of the extracellular domain (30Kozarsky K.F. Call S.M. Dower S.K. Krieger M. Mol. Cell. Biol. 1988; 8: 3357-3363Crossref PubMed Scopus (37) Google Scholar). CHO ldlD cells are deficient in the UDP-Gal/UDP-GalNAc 4-epimerase, which catalyzes the reaction from UDP-glucose to UDP-galactose. The defect can be selectively restored by addition of GalNAc and/or Gal. Interestingly, addition of GalNAc alone results in O-glycosylation limited to GalNAc with no apparent elongation or sialylation (24Sorensen A.L. Reis C.A. Tarp M.A. Mandel U. Ramachandran K. Sankaranarayanan V. Schwientek T. Graham R. Taylor-Papadimitriou J. Hollingsworth M.A. Burchell J. Clausen H. Glycobiology. 2006; 16: 96-107Crossref PubMed Scopus (210) Google Scholar), whereas the substitution of GalNAc and Gal leads to a complete restoration of O-glycosylation. Secretion of FGF23 Is Dependent on GalNAc O-Glycosylation—FGF23 has previously been expressed in CHO cells and both intact 32 kDa and processed fragments are secreted suggesting that CHO cells process FGF23 partially (31Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Bone. 2001; 28: S89Google Scholar). As shown in Fig. 1 secretion of FGF23 in CHO ldlD cells requires O-glycosylation with at least GalNAc, and the level of secretion is dramatically increased when GalNAc-T3 is co-expressed. All cells were characterized for expression of respective genes by immunocytology (Fig. 1A) and SDS-PAGE Western blot analysis (Fig. 1B, top panel). The stable transfectants of CHO ldlD cells with FGF23 alone or subsequent introduced GalNAc-T3 or control β3GnT6 expressed similar levels of FGF23 evaluated by anti-Myc reactivity. The β3GnT6 construct encodes the human β3GlcNAc-transferase that forms the core 3 mucin-type O-linked structure (GlcNAcβ1–3GalNAcα1-O-Ser/Thr), and is expected to elongate and mask the Tn structures expressed in CHO ldlD cells grown in only GalNAc (21Iwai T. Inaba N. Naundorf A. Zhang Y. Gotoh M. Iwasaki H. Kudo T. Togayachi A. Ishizuka Y. Nakanishi H. Narimatsu H. J. Biol. Chem. 2002; 277: 12802-12809Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The GalNAc-T3 transfectant cells exhibited the expected supranuclear Golgi-like staining (23Bennett E.P. Hassan H. Mandel U. Hollingsworth M.A. Akisawa N. Ikematsu Y. Merkx G. van Kessel A.G. Olofsson S. Clausen H. J. Biol. Chem. 1999; 274: 25362-25370Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). CHO ldlD cells grown in GalNAc-substituted medium express very little Tn glycoproteins (Fig. 1A), but cells transfected with FGF23 as well as FGF23 and GalNAc-T3 produced the expected diffuse cytoplasmic staining with anti-Tn (GalNAcα1-O-Ser/Thr). This suggests that indeed FGF23 is GalNAc-glycosylated in cells grown in GalNAc (Fig. 1A). The control β3GnT6-transfected cells when grown in GalNAc exhibited marked reduction in reactivity with anti-Tn compared with the parent FGF23 cells, in agreement with the expected function of this enzyme in elongation of Tn to core 3 structures (GlcNAcβ1–3Gal-NAcα1-O-Ser/Thr). Analysis of lysates of all transfectant cells grown with or without addition of sugars by anti-myc Western blotting revealed comparable levels of intracellular FGF23, although there was a clear tendency that cells grown in GalNAc as well as GalNAc and Gal exhibited slightly reduced levels compared with cells grown without GalNAc (Fig. 1B, top panel). Cell lysates contained a major anti-myc reactive protein with a molecular mass of ∼30 kDa with a trace of a higher molecular mass band of 32 kDa. The upper band does not appear to represent an O-glycosylated glycoform because it does not vary with growth conditions of cells, i.e. presence or absence of added Gal or GalNAc. Analysis of FGF23 levels in medium of cells grown for 72 h by Western blot analysis as well as ELISA showed complete lack of FGF23 when cells were grown without GalNAc, whereas two distinct molecular species of ∼31 and 32 kDa were found in the medium when cells were grown in GalNAc or GalNAc and Gal, respectively (Fig. 1, B and C). CHO ldlD cells grown in both GalNAc and Gal produce sialylated core 1 structures, which should account for the apparent higher molecular weight. A faint band of ∼14 kDa was also observed in medium from all cells grown in GalNAc, which represents the C-terminal myc-tagged fragment (FGF23-(180–251)) (14Shimada T. Muto T. Urakawa I. Yoneya T. Yamazaki Y. Okawa K. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Endocrinolology. 2002; 143: 3179-3182Crossref PubMed Scopus (387) Google Scholar). ELISA analysis of levels of secreted FGF23 from CHO ldlD FGF23 and the double FGF23 and β3GnT6 transfectants showed low or undetectable levels, whereas the GalNAc-T3 transfectant cells showed a marked increase. This increase was especially significant with cells grown in both GalNAc and Gal, which suggests that GalNAc-glycosylation alone promotes secretion to a lesser degree than the more complex glycosylation achieved with addition of Gal and GalNAc. Interestingly, lack of GalNAc-T3 does not appear to result in significant intracellular accumulation of FGF23 or degradation products (Fig. 1B). The major anti-myc reactive species of ∼30 kDa in cell lysates did appear to exhibit a weak inverse correlation with levels of secreted FGF23. It may therefore be assumed that rapid degradation controls the intracellular pool similar to what was observed with a FGF23 mutant in HEK293 cells (8Benet-Pages A. Orlik P. Strom T.M. Lorenz-Depiereux B. Hum. Mol. Genet. 2005; 14: 385-390Crossref PubMed Scopus (426) Google Scholar). O-Glycosylation Capacity in CHO ldlD Cells—The remarkable induction of secretion of FGF23 by co-expression of GalNAc-T3 suggests that CHO ldlD cells do not express an endogenous hamster GalNAc-T3 ortholog or other GalNAc-transferase isoforms with the same specificity; however, the repertoire of GalNAc-transferases in CHO ldlD cells is unknown. To evaluate the presence of such activity, we tested detergent lysates of CHO ldlD cells for GalNAc-transferase activity with a peptide sequence derived from FGF23 (FGF23b) and a MUC1-derived peptide sequence as control (Table 1). CHO ldlD cell lysates contained significant activity with Muc1a peptide and essentially no detectable activity with the FGF23b peptide. The GalNAc-T3 CHO ldlD transfectant cell lysates contained almost 2-fold higher activity with Muc1a and significant activity with the FGF23 peptide. Clearing the lysates with anti-GalNAc-T3 immobilized antibodies resulted in selective removal of the activity with FGF23. We did test for activity in the immunoprecipitates, because our previous studies of immunoprecipitation of soluble recombinant GalNAc-T3 by UH5 was found to be active (32Mandel U. Hassan H. Therkildsen M.H. Rygaard J. Jakobsen M.H. Juhl B.R. Dabelsteen E. Clausen H. Glycobiology. 1999; 9: 43-52Crossref PubMed Scopus (107) Google Scholar), however, no activity above background could be demonstrated in precipitates of CHO ldlD GalNAc-T3 cells. This is likely due to the fact that the enzyme is the membrane-bound form, which is known to be very unstable. These experiments indicate that CHO ldlD cells do not express GalNAc-T3 or isoforms with FGF23 substrate specificities in a significant degree. We have previously identified unique in vitro substrate specificities for the GalNAc-T3 isoform and its close subfamily member GalNAc-T6 (23Bennett E.P. Hassan H. Mandel U. Hollingsworth M.A. Akisawa N. Ikematsu Y. Merkx G. van Kessel A.G. Olofsson S. Clausen H. J. Biol. Chem. 1999; 274: 25362-25370Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). One peptide substrate identified from the V3-loop of HIV gp120 has been confirmed as an exclusive in vivo substrate for GalNAc-T3 in COS7 cells, which is the only available correlation of in vitro and in vivo functions of GalNAc-transferases to date (33Nehrke K. Hagen F.K. Tabak L.A. Glycobiology. 1998; 8: 367-371Crossref PubMed Scopus (29) Google Scholar). Gal-NAc-T3 and -T6 have similar substrate specificities as revealed by in vitro analysis, however, the two isoforms have markedly different cell and tissue expression patterns (23Bennett E.P. Hassan H. Mandel U. Hollingsworth M.A. Akisawa N. Ikematsu Y. Merkx G. van Kessel A.G. Olofsson S. Clausen H. J. Biol. Chem. 1999; 274: 25362-25370Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar).TABLE 1Substrate specificities of cell lysates with or without immunoprecipitation with acceptorsAcceptor substrateMUC1a, AHGVTSAPDTRFGF23b, PIPRRHTRSAEDDSERDPcpm (pmol/min)aActivities expressed as measured counts in reactions. Eight μl of 200 μl of microsome detergent lysates derived from 200 μl of packed cell pellets were assayed as described under "Experimental Procedures" in a 25-μl reaction volume. Background of 70 cpm was substracted.Detergent-soluble cell lysates CHO ldlD FGF23 clone134(3.4)7(0.2) CHO ldlD FGF23/T3 clone270(6.9)110(2.8)Anti-GalNAc-T3 cleared lysates CHO ldlD FGF23 clone160(4.1)6(0.2) CHO ldlD FGF23/T3 clone160(4.1)2(0.1)a Activities expressed as measured counts in reactions. Eight μl of 200 μl of microsome detergent lysates derived from 200 μl of packed cell pellets were assayed as described under "Experimental Procedures" in a 25-μl reaction volume. Background of 70 cpm was substracted. Open table in a new tab Unique GalNAc-T3-mediated O-Glycosylation of Peptide Substrates from FGF23—To further explore the role of GalNAc-T3 we tested a
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