Molecular Cloning and Characterization of a Novel UDP-GlcNAc:GalNAc-peptide β1,3-N-Acetylglucosaminyltransferase (β3Gn-T6), an Enzyme Synthesizing the Core 3 Structure of O-Glycans

Abstract

The core 3 structure of the O-glycan, GlcNAcβ1–3GalNAcα1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide β1,3-N- acetylglucosaminyltransferase (β3Gn-T; core 3 synthase). The core 3 structure is restricted in its occurrence to mucins from specific tissues such as the stomach, small intestine, and colon. A partial sequence encoding a novel member of the human β3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it β3Gn-T6. The putative amino acid sequence of β3Gn-T6 retains the β3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of β3Gn-T6 expressed in insect cells showed β3Gn-T activity toward GalNAcα-p-nitrophenyl and GalNAcα1-serine/threonine. The β1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. β3Gn-T6 effectively transferred a GlcNAc to the GalNAc residue on MUC1 mucin, resulting in the synthesis of a core 3 structure. Real time PCR analysis revealed that the β3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that β3Gn-T6 is the most logical candidate for the core 3 synthase, which plays an important role in the synthesis of mucin-type O-glycans in digestive organs. The core 3 structure of the O-glycan, GlcNAcβ1–3GalNAcα1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide β1,3-N- acetylglucosaminyltransferase (β3Gn-T; core 3 synthase). The core 3 structure is restricted in its occurrence to mucins from specific tissues such as the stomach, small intestine, and colon. A partial sequence encoding a novel member of the human β3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it β3Gn-T6. The putative amino acid sequence of β3Gn-T6 retains the β3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of β3Gn-T6 expressed in insect cells showed β3Gn-T activity toward GalNAcα-p-nitrophenyl and GalNAcα1-serine/threonine. The β1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. β3Gn-T6 effectively transferred a GlcNAc to the GalNAc residue on MUC1 mucin, resulting in the synthesis of a core 3 structure. Real time PCR analysis revealed that the β3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that β3Gn-T6 is the most logical candidate for the core 3 synthase, which plays an important role in the synthesis of mucin-type O-glycans in digestive organs. GalNAc transferase(s) β1,3-N-acetylgalactosaminyltransferase β1,3-galactosyltransferase β1,3-N-acetylglucosaminyltransferase β1,3-glycosyltransferase Galβ1–3GalNAcα1-serine/threonine Galβ1–3(GlcNAcβ1–6)GalNAcα1-serine/threonine GlcNAcβ1–3GalNAcα1-serine/threonine GlcNAcβ1–3(GlcNAcβ1–6)GalNAcα1-serine/threonine GlcNAcβ1–6GalNAcα1-serine/threonine fluorescein isothiocyanate glyceraldehyde-3-phosphate dehydrogenase high performance liquid chromatography high performance thin layer chromatography Lewis x matrix-assisted laser desorption/ionization time-of-flight neuraminic acid (sialic acid) open reading frame p-nitrophenyl sialyl Lewis x (NeuAcα2–3Galβ1–4(Fucα1–3)GlcNAc) NeuAcα2–6GalNAcα1-serine/threonine GalNAcα1-serine/threonine bovine submaxillary gland mucin Mucins constitute a heterogeneous group of glycoproteins having O-linked carbohydrate chains. Some are membrane-associated glycoproteins in epithelial cells, others are secreted from these cells (1.Williams S.J. 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Gastroenterology. 1996; 110: 1354-1367Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 12.Kim Y.S. Keio J. Med. 1998; 47: 10-18Crossref PubMed Scopus (19) Google Scholar). Mucin-type glycoproteins have clusters of a multitude of O-glycans whose synthesis is initiated by various UDP-GalNAc:polypeptide GalNAc-transferases (polypeptide-GalNAc-Ts1). After the addition of a GalNAc residue to a serine and/or threonine residue by polypeptide-GalNAc-T, sugar residues are sequentially transferred to the GalNAc on the peptide to form O-glycans.O-Glycans can be classified into several different groups according to their core structure. As illustrated in Fig. 1, there are at least eight core types. Core 1, Galβ1–3GalNAcα1-serine/threonine, is the major constituent of O-glycans in many cells. Core 2 is formed from core 1 by the addition of GlcNAc to GalNAc with a β1,6-linkage. Core 3, GlcNAcβ1–3GalNAcα-serine/threonine, is restricted in its occurrence to mucins from specialized tissues such as the colon. Core 4 is formed from core 3 by the addition of GlcNAc to GalNAc with a β1,6-linkage. Each core structure is expressed differentially in conjunction with the differentiation and malignant transformation of various cells and tissues (13.Piller F. Piller V. Fox R.I. Fukuda M. J. Biol. Chem. 1988; 263: 15146-15150Abstract Full Text PDF PubMed Google Scholar, 14.Yang 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: 873-884Crossref PubMed Scopus (144) Google Scholar, 15.Yousefi S. Higgins E. Daoling Z. Pollex-Kruger A. Hindsgaul O. Dennis J.W. J. Biol. Chem. 1991; 266: 1772-1782Abstract Full Text PDF PubMed Google Scholar, 16.Fukuda M. Cancer Res. 1996; 56: 2237-2244PubMed Google Scholar, 17.Brockhausen I. Yang J.M. Burchell J. Whitehouse C. Taylor- Papadimitriou J. Eur. J. 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The loss of the core 3 structure in cancerous tissues and cancer cell lines may be caused by down-regulation of the expression of the core 3 synthase. Core 3 activity was detected in normal colonic tissue; however, the enzyme responsible for synthesizing the core 3 structure has not been purified. We have been studying the molecular cloning of each member of the β1,3-galactosyltransferase (β3Gal-T) and β1,3-N-acetylglucosaminyltransferase (β3Gn-T) families (21.Sasaki K. Watanabe E. Kawashima K. Sekine S. Dohi T. Oshima M. Hanai N. Nishi T. Hasegawa M. J. Biol. Chem. 1993; 268: 22782-22787Abstract Full Text PDF PubMed Google Scholar, 22.Kolbinger F. Streiff M.B. Katopodis A.G. J. Biol. Chem. 1998; 273: 433-440Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 23.Amado M. Almeida R. Carneiro F. Levery S.B. Holmes E.H. Nomoto M. Hollingsworth M.A. Hassan H. Schwientek T. Nielsen P.A. Bennett E.P. Clausen H. J. Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar). We found that of the and families amino acid motifs in of the S. Togayachi A. Kudo T. Nishihara S. Watanabe M. Kubota T. Kitajima M. Shiraishi N. Sasaki K. Andoh T. Narimatsu H. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). each member in its for the and which is with a They be in a group named the β1,3-glycosyltransferase We the core 3 to be a member of the family it the synthesis of of the which showed activity to core the data to novel having the and cloned and a member of family We β3Gn-T6 as the most logical candidate for the core 3 synthase. We a of the expressed sequence data and and in amino acid sequence to the open reading frame of N. A. Togayachi A. T. T. N. S. S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the human DNA data found that the in the of each other in a having a the of this novel which be to that of was not in the this the of was a for the and for the A DNA by the of was an The DNA a of the sequence of the a putative and a a encoding the of a novel family member was by PCR the of human as a and into The putative of β3Gn-T6 was expressed as a secreted with a peptide in insect cells according to the of DNA encoding the putative of β3Gn-T6 was by PCR the of human as a The PCR was with a according to the The and with and to the The was and the The was between the and of the the the was transferred between the and of by the is an expression from and a encoding a peptide of human the peptide and a for the cells with the the β3Gn-T6 and DNA was from the cells. insect cells with the was according to the of of the other also as N. A. Togayachi A. T. T. N. S. S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. R. Kudo T. Nishihara S. H. A. H. J. T. Sasaki K. Narimatsu H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). insect cells with each and at the was to secreted β3Gn-T with the The with The with 1 the was with 1 and and to for analysis a membrane The membrane was with and with the Each β3Gn-T was to the according to the of The reaction for activity an of a of and the After at for the product was by various The activity was by in of a reaction and substrate. The in from and from the a as The was by with of and with of The enzyme reaction was by the addition of of and the reaction was to the and with of of the product was 1 of with a and in of They on a with a of and The of the with a of group type not type type in a not type type 1 was with β3Gn-T6 in of a reaction 2 to the reaction A of was to on an The reaction with of acid and at a of at and with an at of was in of a reaction and threonine The enzyme reaction product was by as the by from and in of for NMR and NMR as as NMR at on a and are to and for A fluorescein isothiocyanate was from The peptide sequence was to that of as for Hassan H. E. P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. J. Clausen H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the cloned one of the which been E.P. Hassan H. Hollingsworth M.A. N. G. A.G. S. Clausen H. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google by We expressed it in the expression as in the to a was by of and the peptide was by in The to and analysis The in acid to a of 2 and by 1 of with 1 of in acid) on the The peptide was to have a GalNAc residue and the sequence of peptide was as an for β3Gn-T6. of the was a with β3Gn-T6 at in of a reaction and The of GlcNAc by β3Gn-T6 to glycoproteins was in of a reaction and of bovine submaxillary gland mucin of of After at for each of the reaction was to The of the with a In the analysis human and human not the time PCR as in P.M. Res. 1996; PubMed Scopus Google Scholar, J. P.M. Res. 1996; PubMed Scopus Google for of the β3Gn-T6 the of the was found to be small in from and colon. of various human tissues from for the β3Gn-T6 and the human glyceraldehyde-3-phosphate dehydrogenase as an by of a DNA the β3Gn-T6 gene encoding the putative and a DNA the The and for β3Gn-T6 as the the and the and to the PCR which for The PCR 1 at for 2 1 at for at for and for 1 PCR with an The of β3Gn-T6 transcript was to the of transcript in the We a of a member of the family as and named it β3Gn-T6. of the amino acid of is in Fig. The amino of β3Gn-T6 a typical type II membrane the as in other of an of a of and a and of residues β3Gn-T6 showed and to and and the sequence was to the putative The motifs by the of the family S. Togayachi A. Kudo T. Nishihara S. Watanabe M. Kubota T. Kitajima M. Shiraishi N. Sasaki K. Andoh T. Narimatsu H. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) in the predicted in the putative amino acid sequence of β3Gn-T6. The residues in the that be for the structure. A of acid which may be a as in a of other C. J. 1999; PubMed Scopus Google was also found in the sequence of β3Gn-T6. of was in The β3Gn-T6 gene was found to be to a sequence which was to on the human and its was of a The molecular of the is however, a major of was was detected on suggested that the be in insect cells. The of β3Gn-T6 by a analysis of and and analysis of activity toward was not The of β3Gn-T6 are in I. The activity toward was the the activity of β3Gn-T6 for is as and other are as in I. β3Gn-T6 activity toward activity for and activity toward the other β3Gn-T6 as an is with that of a F. Yang J.M. K. H. Brockhausen I. Glycobiology. 1995; PubMed Scopus Google Scholar). β3Gn-T6 activity toward the residue of group A and the core 3 and in It also showed activity toward the residue of and and in The of each of the for the activity to GlcNAc to The β3Gn-T6 activity is as other are as The of β3Gn-T6 and of the others transferred GlcNAc to its activity was In N. A. Togayachi A. T. T. N. S. S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google showed the activity for the of GlcNAc to the residue of In the the activity to GlcNAc to at that to not activity of toward not not in a of β3Gn-T6 and to activity for A product was by β3Gn-T6 and was the not In not β3Gn-T6 also transferred GlcNAc to to an to not between GlcNAc and GalNAc are to in in of mucin-type They are and the structure synthesized by the reaction product of β3Gn-T6 as an was to analysis and as as in Fig. The of enzyme product from the of in Fig. to the of core a and in Fig. suggested that β3Gn-T6 the core 3 structure from the structure by β3Gn-T6 NMR was The reaction product of β3Gn-T6 with and was by and the formed was by and NMR be and by their in their NMR as as NMR data with for the T. Nomoto M. Levery S.B. G. A.G. Bennett E.P. Hollingsworth M.A. Clausen H. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). showed the of GlcNAc to the substrate. the with a showed a typical for of the NMR of the of the product with the of the showed a of the from to the NMR confirmed the of a and that the putative for a of the We also a NMR as it one to between which are at not is the between different of the for that between and which to that sugar the O-glycans a the and specific between the peptide and sugar In can form a between of GalNAc and the of in a in the of MUC1 of threonine a and between the group and and of NMR of the β3Gn-T6 is a NMR of the reaction product in Fig. 4 in NMR a The peptide was as a for the β3Gn-T6 activity for core 3 As in Fig. β3Gn-T6 a of product in Fig. which to a with a time that of the in Fig. of and the reaction product and to of molecular by The molecular of the product was to be which that of the the activity of β3Gn-T6 to GlcNAc to and as As in Fig. β3Gn-T6 effectively transferred GlcNAc to and not to The of β3Gn-T6 expressed in various human tissues was by the time PCR As in Fig. the expression of the β3Gn-T6 was in the stomach, by the colon and small intestine. and expressed the β3Gn-T6 transcript at a The expression in the tissues Core 3 is an important enzyme in the synthesis of mucin-type O-glycans in digestive organs. The core 3 structure was in O-glycans from and colon, core 3 activity was also F. Yang J.M. K. H. Brockhausen I. Glycobiology. 1995; PubMed Scopus Google Scholar, I. Matta K.L. J. H. Biochemistry. PubMed Scopus Google Scholar). In the have and a novel which can the core 3 structure on As in the and are not the core 3 synthase. which is involved in the and of synthesis N. A. Togayachi A. T. T. N. S. S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. R. Kudo T. Nishihara S. H. A. H. J. T. Sasaki K. Narimatsu H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google activity for core 3 its expression is the core 3 structure and core 3 activity have been found in specific In not GlcNAc to is not responsible for the synthesis of core the activity is detected in normal colon is in colon cancer cell lines F. Yang J.M. K. H. Brockhausen I. Glycobiology. 1995; PubMed Scopus Google however, the transcript of in cells was that in normal colon N. A. Togayachi A. T. T. N. S. S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). also suggested that is not the core 3 synthase. β3Gn-T6 transferred GlcNAc not to also to and The activity in the was with that in the it may not the activity of β3Gn-T6 in In addition to and also for β3Gn-T6. The analysis suggested that the reaction product of β3Gn-T6 is the core We confirmed by NMR analysis that the reaction product from by β3Gn-T6 formed a the core 3 synthesizing activity on was confirmed and glycoproteins as β3Gn-T6 effectively transferred GlcNAc to and not to suggested that it transferred GlcNAc to GalNAc of the on not to the are by is of it that the synthesis of core 3 with that of In a cloned and a human N. N. Kudo T. M. Nishihara S. S. K. S. T. S. Narimatsu H. Glycobiology. 1999; PubMed Scopus Google Scholar). 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The expression of this gene was high in the stomach, and in the colon and small intestine. in mucin, core and core O-glycans to be the major core O-glycans to be the major form in colonic The synthesis of the core 1 structure by core 1 may compete with the synthesis of the core 3 structure by core 3 The expression of β3Gn-T6 transcript not with the of core the gene encoding the core 1 synthase, core 1 was and its expression was in many tissues T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. K. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The expression of core 1 was in the tissues however, among the in colon and small intestine. the was not that the expression of core is that of core in As in Fig. 1, core and are to be with Core 2 is also important for the expression of sialyl Lewis x a K. M. A. H. N. E. S. Cancer Res. 1984; Google Scholar, S. M. S. H. O. Sasaki T. PubMed Scopus Google Scholar, C. J.M. J. Byrd J.C. Leffler H. 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