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• W2051606980 abstract "The hinge region of human immunoglobulin A1 (*IgA1) possesses multiple O-glycans, of which synthesis is initiated by the addition of GalNAc to serine or threonine through the activity of UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferases (pp-GalNAc-Ts). We found that six pp-GalNAc-Ts, pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9, were expressed in B cells, IgA-bearing B cells, and NCI-H929 IgA myeloma cells. pp-GalNAc-T activities of these six enzymes for a synthetic IgA hinge peptide, which has nine possible O-glycosylation sites, were examined using a reversed phase-high performance liquid chromatography, a matrix-assisted laser desorption ionization time of flight mass spectrometry, and peptide sequencing analysis. pp-GalNAc-T2 showed the strongest activity transferring GalNAc to a maximum of eight positions. Other pp-GalNAc-Ts exhibited different substrate specificities from pp-GalNAc-T2; however, their activities were extremely weak. It was reported that the IgA1 hinge region possesses a maximum of five O-glycans, and their amino acid positions have been determined. We found that pp-GalNAc-T2 selectively transferred GalNAc residues to the same five positions. These results strongly suggested that pp-GalNAc-T2 is an essential enzyme for initiation of O-linked glycosylation of the IgA1 hinge region. The hinge region of human immunoglobulin A1 (*IgA1) possesses multiple O-glycans, of which synthesis is initiated by the addition of GalNAc to serine or threonine through the activity of UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferases (pp-GalNAc-Ts). We found that six pp-GalNAc-Ts, pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9, were expressed in B cells, IgA-bearing B cells, and NCI-H929 IgA myeloma cells. pp-GalNAc-T activities of these six enzymes for a synthetic IgA hinge peptide, which has nine possible O-glycosylation sites, were examined using a reversed phase-high performance liquid chromatography, a matrix-assisted laser desorption ionization time of flight mass spectrometry, and peptide sequencing analysis. pp-GalNAc-T2 showed the strongest activity transferring GalNAc to a maximum of eight positions. Other pp-GalNAc-Ts exhibited different substrate specificities from pp-GalNAc-T2; however, their activities were extremely weak. It was reported that the IgA1 hinge region possesses a maximum of five O-glycans, and their amino acid positions have been determined. We found that pp-GalNAc-T2 selectively transferred GalNAc residues to the same five positions. These results strongly suggested that pp-GalNAc-T2 is an essential enzyme for initiation of O-linked glycosylation of the IgA1 hinge region. O-Glycan is a general term for one of the carbohydrate chains, of which synthesis is initiated by a transfer of GalNAc, xylose, mannose, or fucose to a serine or threonine residue in proteins. In mammalian cells, O-glycans are mostly produced by a transfer of GalNAc through the activity of UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase (pp-GalNAc-T). 1The abbreviations used are: pp-GalNAc-T, UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase; IgA1, immunoglobulin A1; IgAN, IgA nephropathy; RT, reverse transcriptase; PBMC, peripheral blood mononuclear cell; 5-FAM, 5-carboxyfluorescein; HPLC, high performance liquid chromatography; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; m/z , molecular size; ACTH, adrenocorticoid hormone; PTH, phenylthiohydantoin; HRP, VPSTPPTPSPSTPPTPSPSK-FAM; C1Gal-T, UDP-gal:GalNAc-α-peptide β1,3-galactosyltransferase; ST, sialyltransferase 1The abbreviations used are: pp-GalNAc-T, UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase; IgA1, immunoglobulin A1; IgAN, IgA nephropathy; RT, reverse transcriptase; PBMC, peripheral blood mononuclear cell; 5-FAM, 5-carboxyfluorescein; HPLC, high performance liquid chromatography; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; m/z , molecular size; ACTH, adrenocorticoid hormone; PTH, phenylthiohydantoin; HRP, VPSTPPTPSPSTPPTPSPSK-FAM; C1Gal-T, UDP-gal:GalNAc-α-peptide β1,3-galactosyltransferase; ST, sialyltransferase O-Glycans were found in many glycoproteins, particularly in secretory glycoproteins such as mucins. Eight core structures, core 1–8, that are basal structures initiated by the GalNAc addition to peptides, are recognized in the mucin-type O-glycan. Each core structure is differentially expressed in conjunction with the differentiation and malignant transformation of various cells and tissues (1Brockhausen I. Yang J.M. Burchell J. Whitehouse C. Taylor-Papadimitriou J. Eur. J. Biochem. 1995; 233: 607-617Google Scholar, 2Piller F. Piller V. Fox R.I. Fukuda M. J. Biol. Chem. 1988; 263: 15146-15150Google Scholar, 3Fukuda M. Cancer Res. 1996; 56: 2237-2244Google Scholar, 4Yang J.M. Byrd J.C. Siddiki B.B. Chung Y.S. Okuno M. Sowa M. Kim Y.S. Matta K.L. Brockhausen I. Glycobiology. 1994; 4: 873-884Google Scholar, 5Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Google Scholar). pp-GalNAc-Ts are biologically important because they determine the number and position of mucin-type O-glycans in a protein. To date, at least 11 human pp-GalNAc-Ts, -T1, -T2, -T3, -T4, -T6, -T7, -T8, -T9, -T10, -T11, and -T12, have been identified (5–16). The proteins are 40–60% identical in their sequence and are therefore homologous. In particular, the predicted catalytic domains are highly conserved. Their substrate specificities have been examined using a variety of peptides, of which sequences are derived from native glycoproteins. They have shown different substrate specificities, different kinetic properties, and different tissue distributions, although some of them showed substantial overlaps in the assessed catalytic specificities and tissue distribution. The positions ofO-glycans in proteins are determined by a variety of pp-GalNAc-Ts expressed in the cells and their substrate specificities. Some pp-GalNAc-Ts exhibit strong “primary activity” toward peptides that have no GalNAc, whereas others, such as pp-GalNAc-T4 and -T7, prefer peptides having GalNAc residue(s) as acceptor substrates rather than the corresponding peptides with no GalNAc (11Bennett E.P. Hassan H. Hollingsworth M.A. Clausen H. FEBS Lett. 1999; 460: 226-230Google Scholar, 17Hassan H. Reis C.A. Bennett E.P. Mirgorodskaya E. Roepstorff P. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 2000; 275: 38197-38205Google Scholar, 18Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. Van Wuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Google Scholar). The “secondary activity,” i.e. GalNAc addition to a peptide already having GalNAc, of the latter may be directed by a lectin domain in their catalytic region. It is supposed that multiple GalNAc transfers to a single peptide are completed in the cells by a combination of multiple pp-GalNAc-Ts having primary activity and ones having secondary activity.Human serum IgA consists of two structurally and functionally distinct subclasses, IgA1 and IgA2, of which the ratio is about 85 and 15% of total IgA, respectively (19Mestecky J. Russell M.W. Monogr. Allergy. 1986; 19: 277-301Google Scholar, 20Mestecky J. Tomana M. Crowley-Nowick P.A. Moldoveanu Z. Julian B.A. Jackson S. Contrib. Nephrol. 1993; 104: 172-182Google Scholar, 21Iwase H. Trends Glycosci. Glycotechnol. 1999; 11: 113-118Google Scholar). Immunoglobulin A nephropathy (IgAN), which is the most common form of glomerulonephritis, is a serious disease. In southern Europe, Asia, and Australia, the ratio of IgAN is 20–40% among patients with primary glomerular disease (22Novak J. Julian B.A. Tomana M. Mesteck J. J. Clin. Immunol. 2001; 21: 310-327Google Scholar). Because the method to cure IgAN is still unknown, dialysis treatment or renal transplantation is necessary for IgAN patients in the final stage.IgAN is characterized by the selective deposition of IgA1 in the renal glomerular mesangium (23Conley M.E. Cooper M.D. Michael A.F. J. Clin. Invest. 1980; 66: 1432-1436Google Scholar), although the mechanism for this is unclear. There is some debate regarding the cause of the deposition. Several investigators have proposed that the elevation of the serum IgA1 concentration is the cause of IgAN (24Nomoto Y. Sakai H. Arimori S. Am. J. Clin. Pathol. 1979; 71: 158-160Google Scholar, 25.Deleted in proof.Google Scholar, 26Casanueva B. Rodriguez-Valverde V. Arias M. Vallo A. Garcia-Fuentes M. Rodriquez-Soriano J. Nephron. 1986; 43: 33-37Google Scholar). To explain why IgA1, but neither IgA2 nor IgG is deposited, it was proposed thatO-glycan structures in the hinge region of IgA1 are profoundly involved in the deposition. IgA1 is characterized by a long extended polyproline structure and distinctive O-glycan side chains in its hinge region (27Torano A. Tsuzukida Y. Liu Y.S. Putnam F.W. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2301-2305Google Scholar, 28Baenziger J. Kornfeld S. J. Biol. Chem. 1974; 249: 7270-7281Google Scholar). The other immunoglobulins, except for IgA1 and IgD, do not have O-glycans in their hinge regions. Five O-glycans have been identified to attach to the hinge region of IgA1 derived from healthy individuals (28Baenziger J. Kornfeld S. J. Biol. Chem. 1974; 249: 7270-7281Google Scholar, 29Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Google Scholar). The amino acid positions of the O-glycans attached were determined, and the O-glycan structure was defined to be SAα2–3Galβ1–3GalNAcα1-Ser/Thr (29Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Google Scholar). There are many reports describing incomplete structures of O-glycans in the IgA1 hinge region of IgAN patients compared with those of normal individuals (20Mestecky J. Tomana M. Crowley-Nowick P.A. Moldoveanu Z. Julian B.A. Jackson S. Contrib. Nephrol. 1993; 104: 172-182Google Scholar, 30Iwase H. Ishii-Karakasa I. Fujii E. Hotta K. Hiki Y. Kobayashi Y. Anal. Biochem. 1992; 206: 202-205Google Scholar, 31Allen A.C. Harper S.J. Feehally J. Clin. Exp. Immunol. 1995; 100: 470-474Google Scholar, 32Allen A.C. Topham P.S. Harper S.J. Feehally J. Nephrol. Dial. Transplant. 1997; 12: 701-706Google Scholar, 33Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Kidney Int. 1997; 52: 509-516Google Scholar, 34Kokubo T. Hiki Y. Iwase H. Tanaka A. Nishikido J. Hotta K. Kobayashi Y. Nephrol. Dial. Transplant. 1999; 14: 81-85Google Scholar, 35Allen A.C. Bailey E.M. Brenchley P.E. Buck K.S. Barratt J. Feehally J. Kidney Int. 2001; 60: 969-973Google Scholar). According to such papers, the number ofO-glycans is decreased or some O-glycans are shortened in the IgA1 hinge region of IgAN patients. Iwase et al. (36Iwase H. Tanaka A. Hiki Y. Kokubo T. Sano T. Ishii-Karakasa I. Toma K. Kobayashi Y. Hotta K. J. Chromatogr. B. 1999; 724: 1-7Google Scholar, 37Iwase H. Ohkawa S. Ishii-Karakasa I. Hiki Y. Kokubo T. Sano T. Tanaka A. Toma K. Kobayashi Y. Hotta K. Biochem. Biophys. Res. Commun. 1999; 261: 472-477Google Scholar) showed by an in vitro experiment that removal of sialic acid enzymatically resulted in the self-aggregation of IgA1, which may be the cause of the deposition of IgA1.Based on the hypothesis that the disruption of O-glycan synthesis in the IgA1 hinge region is the cause of IgAN, we started an investigation of the molecular basis of O-glycan synthesis in the IgA1 hinge region. In this study, we examined, as a first step, which pp-GalNAc-T(s) determine the initiation of O-glycan synthesis among the many members cloned to date.DISCUSSIONIgAN is defined as predominant IgA1 deposits in the renal mesangium (45Berger J. Hinglais N. J. Urol. Nephrol. 1968; 74: 694-695Google Scholar). Although there is some argument regarding the pathogenesis of IgAN, incompleteness of O-glycosylation in the IgA1 hinge region is considered a possible cause of the deposition (20Mestecky J. Tomana M. Crowley-Nowick P.A. Moldoveanu Z. Julian B.A. Jackson S. Contrib. Nephrol. 1993; 104: 172-182Google Scholar, 31Allen A.C. Harper S.J. Feehally J. Clin. Exp. Immunol. 1995; 100: 470-474Google Scholar, 33Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Kidney Int. 1997; 52: 509-516Google Scholar, 46Hiki Y. Horii A. Iwase H. Tanaka A. Toda Y. Hotta K. Kobayashi Y. Contrib. Nephrol. 1995; 111: 73-84Google Scholar). Unlike IgA2 or IgG, IgA1 has a structurally exceptional hinge portion that comprises a proline-rich sequence and possesses multiple O-glycans. In the past few years, with the progress of MS analysis, capillary electrophoresis and so on, it has been well elucidated that the number of carbohydrate residues in the IgA1 hinge region is reduced in IgAN patients compared with normal controls (47Odani H. Hiki Y. Takahashi M. Nishimoto A. Yasuda Y. Iwase H. Shinzato T. Maeda K. Biochem. Biophys. Res. Commun. 2000; 271: 268-274Google Scholar, 48Hiki Y. Odani H. Takahashi M. Yasuda Y. Nishimoto A. Iwase H. Shinzato T. Kobayashi Y. Maeda K. Kidney Int. 2001; 59: 1077-1085Google Scholar, 49Novak J. Tomana M. Kilian M. Coward L. Kulhavy R. Barnes S. Mestecky J. Mol. Immunol. 2000; 37: 1047-1056Google Scholar). Furthermore, it has been reported that incompletely glycosylated IgA1 tends to aggregate in vitro (36Iwase H. Tanaka A. Hiki Y. Kokubo T. Sano T. Ishii-Karakasa I. Toma K. Kobayashi Y. Hotta K. J. Chromatogr. B. 1999; 724: 1-7Google Scholar, 50Iwase H. Tanaka A. Hiki Y. Kokubo T. Sano T. Ishii-Karakasa I. Hisatani K. Kobayashi Y. Hotta K. Anal. Biochem. 2001; 288: 22-27Google Scholar).pp-GalNAc-Ts, UDP-galactose:N-acetylgalactosamine-α-Ser/Thr β3-galactosyltransferase (core 1 β3-Gal-T; C1Gal-T), and α2,3- and α2,6-sialyltransferases (STs) are essential for the complete synthesis of O-glycans in the IgA1 hinge region. To date, at least 11 human pp-GalNAc-Ts (6White T. Bennett E.P. Takio K. Sorensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Google Scholar, 7Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Google Scholar, 8Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Google Scholar, 9Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadimitriou J. Hollingsworth M.A. Merkx G. van Kessel A.G. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Google Scholar, 10Bennett 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: 25362-25370Google Scholar, 11Bennett E.P. Hassan H. Hollingsworth M.A. Clausen H. FEBS Lett. 1999; 460: 226-230Google Scholar, 12White K.E. Lorenz B. Evans W.E. Meitinger T. Strom T.M. Econs M.J. Gene (Amst.). 2000; 246: 347-356Google Scholar, 13Toba S. Tenno M. Konishi M. Mikami T. Itoh N. Kurosaka A. Biochim. Biophys. Acta. 2000; 1493: 264-268Google Scholar, 14Guo J.M. Zhang Y. Cheng L. Iwasaki H. Wang H. Kubota T. Tachibana K. Narimatsu H. FEBS Lett. 2002; 524: 211-218Google Scholar, 15Schwientek 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: 22623-22638Google Scholar, 16Cheng L.M. Tachibana K. Zhang Y. Guo J.-M. Tachibana K. Kameyama A. Wang H. Hiruma T. Iwasaki H. Togayachi A. Kudo T. Narimatsu H. FEBS Lett. 2002; 531: 115-121Google Scholar), 2 human C1Gal-Ts that generate core 1 structure, Galβ1–3GalNAcα1-Ser/Thr (51Ju T. Brewer K. D'Souza A. Cummings R.D. Canfield W.M. J. Biol. Chem. 2002; 277: 178-186Google Scholar, 52Kudo T. Iwai T. Kubota T. Iwasaki H. Hiruma T. Inaba N. Zhang Y. Gotoh M. Togayachi A. Narimatsu H. J. Biol. Chem. 2002; 277: 47724-47731Google Scholar), 2 human α2,3STs (53Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 17872-17878Google Scholar, 54Kim Y.J. Kim K.S. Kim S.H. Kim C.H. Ko J.H. Choe I.S. Tsuji S. Lee Y.C. Biochem. Biophys. Res. Commun. 1996; 228: 324-327Google Scholar) and 2 α2,6STs (55Ikehara Y. Kojima N. Kurosawa N. Kudo T. Kono M. Nishihara S. Issiki S. Morozumi K. Itzkowitz S. Tsuda T. Nishimura S.I. Tsuji S. Narimatsu H. Glycobiology. 1999; 9: 1213-1224Google Scholar, 56Samyn-Petit B. Krzewinski-Recchi M.A. Steelant W.F. Delannoy P. Harduin-Lepers A. Biochim. Biophys. Acta. 2000; 1474: 201-211Google Scholar) have been cloned. However, it is not known which glycosyltransferases are involved in the biosynthesis of O-glycans in the IgA1 hinge region. As an initial attempt to answer this question, we identified which pp-GalNAc-T(s) are responsible for the biosynthesis ofO-glycans in the IgA1 hinge region. pp-GalNAc-T2 showed the strongest pp-GalNAc-T activity toward the IgA1 hinge region among all pp-GalNAc-Ts we examined, and transferred GalNAcs to selective Ser/Thr residues that had been identified to be glycosylated in vivo(29Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Google Scholar). Thus, pp-GalNAc-T2 was determined to be merely responsible for the initiation of O-glycan synthesis in the IgA1 hinge region in this study.Six pp-GalNAc-Ts, pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9, were found to be expressed in IgA-positive cells. This expression profile was almost similar to that of the B cells and an IgA myeloma cell line. We examined the specificity and the relative activity of the six enzymes toward a hinge peptide (HRP) that mimicked the human IgA1 hinge region.The retention time of HRP glycosylated by pp-GalNAc-Ts on reversed phase-HPLC became shorter dependent on the number of GalNAcs because of the hydrophilicity. As the number of incorporated GalNAc residues increased, the retention time decreased. In addition, we could separate the reaction products having the same number of GalNAcs dependent on the positions where GalNAc was added, such as P1 versus P2, P4 versus P5, and P7 versus P8.As summarized in Fig. 6, two initial products, P1 and P2, were identified to have a GalNAc at Ser-11 or Thr-7, respectively, followed by the addition at Thr-15 as a secondary reaction. Thr-7 and Thr-15 are typical threonine residues encoded in the mucin box,XTPXP. Thus, the specificity of pp-GalNAc-T2 is somehow in agreement with the mucin box rule (40Yoshida A. Suzuki M. Ikenaga H. Takeuchi M. J. Biol. Chem. 1997; 272: 16884-16888Google Scholar), although Ser-11 is not consistent with the rule. The existence of these two initial products suggested that there are at least two synthetic pathways. The next product from P2 was P3; however, the product derived from P1 was not found, and similarly some products lacked intermediates. These transient products were thought to exist as small peaks that we could not fractionate, and such small transient peaks may be immediately glycosylated to the following product. P5 was considered to be generated from both of the initial products. P4, which has a GalNAc at Thr-12, was produced from P3; however, the products from P4 were not detected until the end of the reaction. P4 may be a dead-end product of the reaction.It has also been reported that the initialO-glycosylation of a peptide substrate influences the subsequent glycosylation, probably due to conformational change of the peptide and the accessibility of pp-GalNAc-Ts for particular acceptor sites (57Kirnarsky L. Nomoto M. Ikematsu Y. Hassan H. Bennett E.P. Cerny R.L. Clausen H. Hollingsworth M.A. Sherman S. Biochemistry. 1998; 37: 12811-12817Google Scholar). There are two consecutive Ser and Thr residues, such as Ser-3/Thr-4 and Ser-11/Thr-12, in HRP. Although one of the consecutive residues was glycosylated, no products in which both consecutive residues were glycosylated were found except for P11. This result indicated that pp-GalNAc-T2 cannot glycosylate the sites next to GalNAc-attached sites within HRP. This phenomenon is consistent with the previous result using a peptide substrate that mimicked the tandem repeat portion of MUC2 (41Iida S. Takeuchi H. Hassan H. Clausen H. Irimura T. FEBS Lett. 1999; 449: 230-234Google Scholar, 58Kato K. Takeuchi H. Miyahara N. Kanoh A. Hassan H. Clausen H. Irimura T. Biochem. Biophys. Res. Commun. 2001; 287: 110-115Google Scholar). This is contrast to the activity of pp-GalNAc-T13 which preferentially transfers GalNAc to all three consecutive Ser/Thr residues in a synthetic peptide derived from syndecan-3 (59Zhang Y. Iwasaki H. Wang H. Kudo T. Kalka T.B. Hennet T. Kubota T. Cheng L. Inaba N. Gotoh M. Togayachi A. Guo J. Hisatomi H. Nakajima K. Nishihara S. Nakamura M. Marth J.D. Narimatsu H. J. Biol. Chem. 2002; 277 (in press)Google Scholar).Mattu et al. (29Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Google Scholar) analyzed O-glycans in the IgA1 hinge region that was prepared from serum IgA1 of healthy individuals. They indicated that O-glycans were attached at five positions, which correspond to Thr-4, Thr-7, Ser-9, Ser-11, and Thr-15 in our peptide, HRP. Our results indicated that pp-GalNAc-T2 is able to transfer GalNAc residues to almost all possible glycosylation sites in HRP. However, a tremendous amount of the enzyme and a very long incubation were required to glycosylate all such positions. Because a FAM-labeled short peptide was used in this study, the three-dimensional structure might be different from the physiological structure of the IgA1 molecule, and this may influence the enzyme specificities in an artificial manner. The enzyme reaction was performed in vitro; therefore, the acceptor substrate specificity of pp-GalNAc-T2 under in vitro conditions may not necessarily reflect those of pp-GalNAc-T2 in the cells. However, the glycosylated sites of P1 to P7 except for P4 and P6 were not inconsistent with the results obtained by analyzing natural IgA1 derived from human serum (29Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Google Scholar). Supposing that the pathways up to P7 production reflect thein vivo O-glycosylation, this is consistent with the finding of Mattu et al. (29Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Google Scholar). P6 is not consistent with the results Mattu et al. (29Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Google Scholar) because it contained GalNAc at Ser-19; however, Ser-19 may be easily glycosylated because it exists at the end of an artificial peptide and may be difficult to be glycosylated in the hinge region of natural IgA1 molecule.On the other hand, all five pp-GalNAc-Ts other than pp-GalNAc-T2 showed very weak and almost negligible activity toward HRP, and their specificities were totally different from those of pp-GalNAc-T2. In addition, they produced PX as an initial product that was mono-glycosylated at Thr-12. The Thr-12 glycosylation is not consistent with the previous report (29Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Google Scholar). These pp-GalNAc-Ts probably do not function in physiological O-glycosylation of the IgA1 in the cells.From the above, we concluded that pp-GalNAc-T2 is merely responsible for the initiation of O-glycosylation in the IgA1 hinge region among the 10 pp-GalNAc-Ts that have been reported to date. A single enzyme, pp-GalNAc-T2, can transfer GalNAc to all five positions in the hinge region. This is quite an interesting exception from the general rule of O-glycosylation, because it has been believed that O-glycosylation on a protein is determined by a combination of multiple pp-GalNAc-Ts expressed in the cell. In the case of O-glycosylation for mucins that possess a large number of O-glycans, it is probably true that multiple pp-GalNAc-Ts are involved in the initiation of O-glycan synthesis on a single mucin.The present study is the first report on the elucidation ofO-glycosylation in the IgA1 hinge region. Up-regulation or down-regulation of the pp-GalNAc-T2 activity in the cells may lead to the increase or decrease in the number of O-glycans in IgA1, respectively. Based on the pathways in Fig. 6, we can predict that the decrease of pp-GalNAc-T2 activity will result in the loss ofO-glycan in the order of Thr-4, Ser-9, Thr-15, and Thr-7 or Ser-11. To elucidate the molecular mechanisms of incompleteO-glycosylation of IgA1 of IgAN patients, more investigation will be required not only for pp-GalNA-Ts but also for core 1 Gal-T(s) and ST(s). O-Glycan is a general term for one of the carbohydrate chains, of which synthesis is initiated by a transfer of GalNAc, xylose, mannose, or fucose to a serine or threonine residue in proteins. In mammalian cells, O-glycans are mostly produced by a transfer of GalNAc through the activity of UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase (pp-GalNAc-T). 1The abbreviations used are: pp-GalNAc-T, UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase; IgA1, immunoglobulin A1; IgAN, IgA nephropathy; RT, reverse transcriptase; PBMC, peripheral blood mononuclear cell; 5-FAM, 5-carboxyfluorescein; HPLC, high performance liquid chromatography; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; m/z , molecular size; ACTH, adrenocorticoid hormone; PTH, phenylthiohydantoin; HRP, VPSTPPTPSPSTPPTPSPSK-FAM; C1Gal-T, UDP-gal:GalNAc-α-peptide β1,3-galactosyltransferase; ST, sialyltransferase 1The abbreviations used are: pp-GalNAc-T, UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase; IgA1, immunoglobulin A1; IgAN, IgA nephropathy; RT, reverse transcriptase; PBMC, peripheral blood mononuclear cell; 5-FAM, 5-carboxyfluorescein; HPLC, high performance liquid chromatography; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; m/z , molecular size; ACTH, adrenocorticoid hormone; PTH, phenylthiohydantoin; HRP, VPSTPPTPSPSTPPTPSPSK-FAM; C1Gal-T, UDP-gal:GalNAc-α-peptide β1,3-galactosyltransferase; ST, sialyltransferase O-Glycans were found in many glycoproteins, particularly in secretory glycoproteins such as mucins. Eight core structures, core 1–8, that are basal structures initiated by the GalNAc addition to peptides, are recognized in the mucin-type O-glycan. Each core structure is differentially expressed in conjunction with the differentiation and malignant transformation of various cells and tissues (1Brockhausen I. Yang J.M. Burchell J. Whitehouse C. Taylor-Papadimitriou J. Eur. J. Biochem. 1995; 233: 607-617Google Scholar, 2Piller F. Piller V. Fox R.I. Fukuda M. J. Biol. Chem. 1988; 263: 15146-15150Google Scholar, 3Fukuda M. Cancer Res. 1996; 56: 2237-2244Google Scholar, 4Yang J.M. Byrd J.C. Siddiki B.B. Chung Y.S. Okuno M. Sowa M. Kim Y.S. Matta K.L. Brockhausen I. Glycobiology. 1994; 4: 873-884Google Scholar, 5Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Google Scholar). pp-GalNAc-Ts are biologically important because they determine the number and position of mucin-type O-glycans in a protein. To date, at least 11 human pp-GalNAc-Ts, -T1, -T2, -T3, -T4, -T6, -T7, -T8, -T9, -T10, -T11, and -T12, have been identified (5–16). The proteins are 40–60% identical in their sequence and are therefore homologous. In particular, the predicted catalytic domains are highly conserved. Their substrate specificities have been examined using a variety of peptides, of which sequences are derived from native glycoproteins. They have shown different substrate specificities, different kinetic properties, and different tissue distributions, although some of them showed substantial overlaps in the assessed catalytic specificities and tissue distribution. The positions ofO-glycans in proteins are determined by a variety of pp-GalNAc-Ts expressed in the cells and their substrate specificities. Some pp-GalNAc-Ts exhibit strong “primary activity” toward peptides that have no GalNAc, whereas others, such as pp-GalNAc-T4 and -T7, prefer peptides having GalNAc residue(s) as acceptor substrates rather than the corresponding peptides with no GalNAc (11Bennett E.P. Hassan H. Hollingsworth M.A. Clausen H. FEBS Lett. 1999; 460: 226-230Google Scholar, 17Hassan H. Reis C.A. Bennett E.P. Mirgorodskaya E. Roepstorff P. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 2000; 275: 38197-38205Google Scholar, 18Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. Van Wuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Google Scholar). The “secondary activity,” i.e. GalNAc addition to a peptide already having GalNAc, of the latter may be directed by a lectin domain in their catalytic region. It is supposed that multiple GalNAc transfers to a single peptide are completed in the cells by a combination of multiple pp-GalNAc-Ts having primary activity and ones having secondary activity. Human serum IgA consists of two structurally and functionally distinct subclasses, IgA1 and IgA2, of which the ratio is about 85 and 15% of total IgA, respectively (19Mestecky J. Russell M.W. Monogr. Allergy. 1986; 19: 277-301Google Scholar, 20Mestecky J. Tomana M. Crowley-Nowick P.A. Moldoveanu Z. Julian B" @default.
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• W2051606980 title "Initiation of O-Glycan Synthesis in IgA1 Hinge Region Is Determined by a Single Enzyme, UDP-N-Acetyl-α-d-galactosamine:PolypeptideN-Acetylgalactosaminyltransferase 2" @default.
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