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W2030180824 abstract "Mucin-type O-glycans are classified according to their core structures. Among them, cores 2 and 4 are important for having N-acetyllactosamine side chains, which can be further modified to express various functional oligosaccharides. Previously, we discovered by cloning cDNAs that the core 2 branching enzyme, termed core 2 β-1,6-N-acetylglucosaminyltransferase-leukocyte type (C2GnT-L), is highly homologous to the I branching β-1,6-N-acetylglucosaminyltransferase (IGnT) (Bierhuizen, M. F. A., Mattei, M.-G., and Fukuda, M. (1993) Genes Dev.7, 468–478). Using these homologous sequences as probes, we identified an expressed sequence tag in dbEST, which has significant homology to C2GnT-L and IGnT. This approach, together with 5′and 3′ rapid amplification of cDNA ends, yielded a human cDNA that encompasses a whole coding region of an enzyme, termed C2GnT-mucin type (C2GnT-M). C2GnT-M has 48.2 and 33.8% identity with C2GnT-L and IGnT at the amino acid levels. The expression of C2GnT-M cDNA directed the expression of core 2 branched oligosaccharides and I antigen on the cell surface. Moreover, a soluble chimeric C2GnT-M had core 4 branching activity in addition to core 2 and I branching activities. A soluble chimeric C2GnT-L, in contrast, almost exclusively contains core 2 branching activity. Northern blot analysis demonstrated that the C2GnT-M transcripts are heavily expressed in colon, small intestine, trachea, and stomach, where mucin is produced. In contrast, the transcripts of C2GnT-L were more widely detected, including the lymph node and bone marrow. These results indicate that the newly cloned C2GnT-M plays a critical role inO-glycan synthesis in mucins and might have distinctly different roles in oligosaccharide ligand formation compared with C2GnT-L. Mucin-type O-glycans are classified according to their core structures. Among them, cores 2 and 4 are important for having N-acetyllactosamine side chains, which can be further modified to express various functional oligosaccharides. Previously, we discovered by cloning cDNAs that the core 2 branching enzyme, termed core 2 β-1,6-N-acetylglucosaminyltransferase-leukocyte type (C2GnT-L), is highly homologous to the I branching β-1,6-N-acetylglucosaminyltransferase (IGnT) (Bierhuizen, M. F. A., Mattei, M.-G., and Fukuda, M. (1993) Genes Dev.7, 468–478). Using these homologous sequences as probes, we identified an expressed sequence tag in dbEST, which has significant homology to C2GnT-L and IGnT. This approach, together with 5′and 3′ rapid amplification of cDNA ends, yielded a human cDNA that encompasses a whole coding region of an enzyme, termed C2GnT-mucin type (C2GnT-M). C2GnT-M has 48.2 and 33.8% identity with C2GnT-L and IGnT at the amino acid levels. The expression of C2GnT-M cDNA directed the expression of core 2 branched oligosaccharides and I antigen on the cell surface. Moreover, a soluble chimeric C2GnT-M had core 4 branching activity in addition to core 2 and I branching activities. A soluble chimeric C2GnT-L, in contrast, almost exclusively contains core 2 branching activity. Northern blot analysis demonstrated that the C2GnT-M transcripts are heavily expressed in colon, small intestine, trachea, and stomach, where mucin is produced. In contrast, the transcripts of C2GnT-L were more widely detected, including the lymph node and bone marrow. These results indicate that the newly cloned C2GnT-M plays a critical role inO-glycan synthesis in mucins and might have distinctly different roles in oligosaccharide ligand formation compared with C2GnT-L. Mucin-type glycoproteins are unique in having clusters of large numbers of O-glycans. These O-glycans containN-acetylgalactosamine residues at reducing ends, which are linked to serine or threonine in a polypeptide (1Fukuda M. Fukuda M. Molecular Glycobiology. Oxford University Press, Oxford1994: 1-52Google Scholar). These attached O-glycans can be classified into several different groups according to the core structures (2Schachter H. Brockhausen I. Allen H.J. Kisailus E.C. Glycoconjugates: Composition, Structure, and Function. Marcel Dekker, New York1992: 263-332Google Scholar). In many cells, core 1, Galβ1→3GalNAc, is the major constituent ofO-glycans. Core 1 oligosaccharides are converted to core 2 oligosaccharides Galβ1→3(GlcNAcβ1→6)GalNAc when core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) 1The abbreviations used are: C2GnT-L and -M, core 2 β-1,6-N-acetylglucosaminyltransferase-leukocyte type and -mucin type, respectively; dIGnT and cIGnT, distally acting and centrally acting I branching β-1,6-N-acetylglucosaminyltransferase, respectively; C4GnT, core 4 β-1,6-N-acetylglucosaminyltransferase; EST, expressed sequence tag; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; CHO, Chinese hamster ovary; pNP, p-nitrophenol; RH, radiation hybrid. 1The abbreviations used are: C2GnT-L and -M, core 2 β-1,6-N-acetylglucosaminyltransferase-leukocyte type and -mucin type, respectively; dIGnT and cIGnT, distally acting and centrally acting I branching β-1,6-N-acetylglucosaminyltransferase, respectively; C4GnT, core 4 β-1,6-N-acetylglucosaminyltransferase; EST, expressed sequence tag; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; CHO, Chinese hamster ovary; pNP, p-nitrophenol; RH, radiation hybrid.is present (3Fukuda M. Carlsson S.R. Klock J.C. Dell A. J. Biol. Chem. 1986; 261: 12796-12806Abstract Full Text PDF PubMed Google Scholar, 4Piller F. Piller V. Fox R.I. Fukuda M. J. Biol. Chem. 1988; 263: 15146-15150Abstract Full Text PDF PubMed Google Scholar). Various ligand carbohydrates can be formed in core 2 branched oligosaccharides. For example, sialyl Lex in mucin-type glycoproteins of blood cells can be formed only in core 2 branched oligosaccharides such as NeuNAcα2→3Galβ1→4(Fucα1→3) GlcNAcβ1→6(NeuNAcα2→3Galβ1→3)GalNAcα→serine/ threonine (3Fukuda M. Carlsson S.R. Klock J.C. Dell A. J. Biol. Chem. 1986; 261: 12796-12806Abstract Full Text PDF PubMed Google Scholar, 5Maemura K. Fukuda M. J. Biol. Chem. 1992; 267: 24379-24386Abstract Full Text PDF PubMed Google Scholar, 6Hemmerich S. Leffler H. Rosen S.D. J. Biol. Chem. 1995; 270: 12035-12047Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 7Wilkins P.P. McEver R.P. Cummings R.D. J. Biol. Chem. 1996; 271: 18732-18742Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar).Those sialyl Lex and sulfated sialyl Lex inO-glycans have been shown to be preferential ligands for P- and L-selectin (6Hemmerich S. Leffler H. Rosen S.D. J. Biol. Chem. 1995; 270: 12035-12047Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 7Wilkins P.P. McEver R.P. Cummings R.D. J. Biol. Chem. 1996; 271: 18732-18742Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 8Lasky L.A. Singer M.S. Dowbenko D. Imai Y. Henzel W.J. Grimley C. Fennie C. Gillett N. Watson S.R. Rosen S.D. Cell. 1992; 69: 927-938Abstract Full Text PDF PubMed Scopus (581) Google Scholar, 9Tsuboi S. Isogai Y. Hada N. King J.K. Hindsgaul O. Fukuda M. J. Biol. Chem. 1996; 271: 27213-27216Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Lowe J.B. Fukuda M. Hindsgaul O. Molecular Glycobiology. Oxford University Press, Oxford1994: 163-205Google Scholar). It has been also shown that poly-N-acetyllactosamines can be extended from core 2 branches (3Fukuda M. Carlsson S.R. Klock J.C. Dell A. J. Biol. Chem. 1986; 261: 12796-12806Abstract Full Text PDF PubMed Google Scholar, 5Maemura K. Fukuda M. J. Biol. Chem. 1992; 267: 24379-24386Abstract Full Text PDF PubMed Google Scholar, 7Wilkins P.P. McEver R.P. Cummings R.D. J. Biol. Chem. 1996; 271: 18732-18742Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 11Hanisch F.G. Uhlenbruck G. Peter K.J. Egge H. Dabrowski J. Dabrowski U. J. Biol. Chem. 1989; 264: 261-267Google Scholar, 12Yousefi 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). Poly-N-acetyllactosamines provide the backbone for additional modifications such as sialyl Lex. Moreover, a linear poly-N-acetyllactosamine (Galβ1→4GlcNAcβ1→3)n can be converted to branched poly-N-acetyllactosamine, Galβ1→4GlcNAcβ1→3 (Galβ1→4GlcNAcβ1→6)Gal→R, by the I branching enzyme IGnT (13Fukuda M. Fukuda M.N. Hakomori S. J. Biol. Chem. 1979; 254: 3700-3703Abstract Full Text PDF PubMed Google Scholar, 14Piller F. Cartron J.P. Maranduba A. Veyrieres A. Leroy Y. Fournet B. J. Biol. Chem. 1984; 259: 13385-13390Abstract Full Text PDF PubMed Google Scholar). These linear and branched poly-N-acetyllactosamines represent, respectively, the i and I antigens, which are expressed in a cell type-specific manner (1Fukuda M. Fukuda M. Molecular Glycobiology. Oxford University Press, Oxford1994: 1-52Google Scholar). It has been shown that there exist two IGnTs, which use two different sets of acceptor substrates (15Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H.L. Van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar, 17Gu J. Nishikawa A. Fujii S. Gasa S. Taniguchi N. J. Biol. Chem. 1992; 267: 2994-2999Abstract Full Text PDF PubMed Google Scholar).In the gastrointestinal tract, oligosaccharides with core 3, GlcNAcβ1→3GalNAc, can be frequently found. These tissues also contain core 4, Galβ1→4GlcNAcβ1→6(GlcNAcβ1→3)GalNAc, which was originally discovered in sheep gastric mucosa (18Hounsell E.F. Fukuda M. Powell M.E. Feizi T. Hakomori S. Biochem. Biophys. Res. Commun. 1980; 92: 1143-1150Crossref PubMed Scopus (33) Google Scholar). Core 4 is formed from core 3 by core 4 β-1,6-N-acetylglucosaminyltransferase (C4GnT). It has been reported that the amount of core 4 oligosaccharides is reduced in colonic carcinoma cells, whereas the amount of core 2 oligosaccharides is maintained or increased (19Yang J.-M. Byrd J.C. Siddik 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, 20Vavasseur F. Yang J.M. Dole K. Paulsen H. Brockhausen I. Glycobiology. 1995; 5: 351-357Crossref PubMed Scopus (63) Google Scholar). More recently, the increase in the transcript of C2GnT was found to be associated with the progression of colonic carcinomas (21Shimodaira K. Nakayama J. Nakayama N. Hasebe O. Katsuyama T. Fukuda M. Cancer Res. 1997; : 5201-5206PubMed Google Scholar).To understand the roles of these oligosaccharides in normal and pathological conditions, it is essential to understand the regulation of their biosynthesis. To this end, we previously cloned cDNAs encoding C2GnT (termed in the present study C2GnT-leukocyte type (C2GnT-L)) from human HL-60 promyelocytic cells (22Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar) and IGnT from human PA-1 embryonic carcinoma cells (23Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). C2GnT-L and IGnT share homologous sequences in three regions of their catalytic domains (23Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). Neither C2GnT-L nor IGnT, however, forms core 4 structure. On the other hand, a C2GnT purified from mucin-producing cells, termed C2GnT-mucin (C2GnT-M) type, was reported to contain C2GnT and C4GnT activities in the same enzyme (2Schachter H. Brockhausen I. Allen H.J. Kisailus E.C. Glycoconjugates: Composition, Structure, and Function. Marcel Dekker, New York1992: 263-332Google Scholar, 15Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H.L. Van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar). In the present study we describe the isolation of cDNA encoding such an enzyme using EST sequence, which has strong homology with C2GnT-L and IGnT. We found that this newly cloned enzyme is unique in having C2GnT activity as well as core 4 and I branching activities. We termed this new enzyme C2GnT-M, because it is mainly expressed in mucin-producing tissues.DISCUSSIONIn the present study, we have isolated a cDNA encoding a novel C2GnT, C2GnT-M. C2GnT-M is unique in catalyzing more than one enzymatic reaction, forming core 4 branches and I branches in addition to core 2 branches. Previously, fucosyltransferase III and ST3Gal III, an α-2,3-sialyltransferase, were shown to add, respectively, fucose and sialic acid residues to both Galβ1→4GlcNAc and Galβ1→3 GlcNAc acceptors (35Prieels J.P. Monnom D. Dolmans M. Beyer T.A. Hill R.L. J. Biol. Chem. 1981; 256: 10456-10463Abstract Full Text PDF PubMed Google Scholar, 36Kukowska-Latallo J.F. Larsen R.D. Nair R.P. Lowe J.B. Genes Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (469) Google Scholar, 37Wen D.X. Livingstone B.D. Medzihradszky K.F. Kelm S. Burlingame A.L. Paulson J.C. J. Biol. Chem. 1992; 267: 21011-21019Abstract Full Text PDF PubMed Google Scholar). As far as we know, we are the first to demonstrate in a definitive manner that a single enzyme catalyzes three different but related reactions.C2GnT-M is almost exclusively expressed in small and large intestines, trachea, stomach, and thyroid among tissues examined (Fig. 5), thus likely involved in the synthesis of mucin-type oligosaccharides. Accordingly, we termed this new enzyme C2GnT-M (mucin-type). It is not apparent why the C2GnT-M is also expressed in thyroid. However, it was recently demonstrated that a major glycoprotein in calf thyroid contains core 2 branched O-glycans and I-branchedN-glycans (38Edge A.S.B. Spiro R.G. Arch. Biochem. Biophys. 1997; 343: 73-80Crossref PubMed Scopus (11) Google Scholar), indicating that thyroid synthesizes mucin-type O-glycans.It is noteworthy that C2GnT-M exhibits a significant activity of C4GnT. This finding is consistent with previous reports that the presence of C4GnT activity is always associated with the presence of C2GnT activity when various mucin-producing tissues were examined (15Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H.L. Van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar, 19Yang J.-M. Byrd J.C. Siddik 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, 20Vavasseur F. Yang J.M. Dole K. Paulsen H. Brockhausen I. Glycobiology. 1995; 5: 351-357Crossref PubMed Scopus (63) Google Scholar). It is very likely that C2GnT-M is responsible for C4GnT activity in these tissues. C2GnT-L, on the other hand, lacks C4GnT activity (Fig. 4). In small intestine, colon, and stomach, significant amounts of the transcripts for C2GnT-L and C2GnT-M were detected (Fig. 5), suggesting that both enzymes contribute to the C2GnT activity in these tissues. In fact, recent studies on C2GnT-L knock-out mice demonstrated that a residual C2GnT activity exists in small and large intestines among tissues examined after C2GnT-L is inactivated (39Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; (in press)PubMed Google Scholar). It is most likely that C2GnT-M cloned in the present study is responsible for this activity.C2GnT-M is also unique in transferring a GlcNAc residue preferentially to predistal galactose residues of GlcNAcβ1→3 Galβ1→4 GlcNAcβ1→R, forming GlcNAcβ1→3(GlcNAcβ1→6)Galβ1→4GlcNAcβ1→R. Although the presence of this enzyme activity, dIGnT, was reported previously (14Piller F. Cartron J.P. Maranduba A. Veyrieres A. Leroy Y. Fournet B. J. Biol. Chem. 1984; 259: 13385-13390Abstract Full Text PDF PubMed Google Scholar, 15Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H.L. Van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar, 17Gu J. Nishikawa A. Fujii S. Gasa S. Taniguchi N. J. Biol. Chem. 1992; 267: 2994-2999Abstract Full Text PDF PubMed Google Scholar), a cDNA encoding an enzyme acting more as a dIGnT than as a cIGnT was not reported before. The activities of dIGnT reported in hog gastric mucosa and rat liver (14Piller F. Cartron J.P. Maranduba A. Veyrieres A. Leroy Y. Fournet B. J. Biol. Chem. 1984; 259: 13385-13390Abstract Full Text PDF PubMed Google Scholar, 17Gu J. Nishikawa A. Fujii S. Gasa S. Taniguchi N. J. Biol. Chem. 1992; 267: 2994-2999Abstract Full Text PDF PubMed Google Scholar) may be attributable to the newly cloned C2GnT-M. However, those enzymatic activities reported previously had much less C2GnT or C4GnT activity compared with dIGnT activity, whereas the opposite is true for C2GnT-M. It is thus likely that both IGnT and C2GnT-M contribute to dIGnT activity in these tissues. Our cloned enzyme most closely resembles the C2GnT preparation isolated from bovine trachea (16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar). However, dIGnT activity of the bovine trachea enzyme preparation was almost 40% of its C2GnT activity, whereas it is only 7% of C2GnT activity in the cloned C2GnT-M, although similar acceptors were used in both studies. On the other hand, it is noteworthy that the ratio of C2GnT and dIGnT activities in LS180, HT29, and NCI498 colonic carcinomas (20Vavasseur F. Yang J.M. Dole K. Paulsen H. Brockhausen I. Glycobiology. 1995; 5: 351-357Crossref PubMed Scopus (63) Google Scholar) is similar to that of C2GnT-M. Further studies are necessary to clarify this discrepancy.The results obtained in the present study reveal that the genes of C2GnT-M, C2GnT-L, and IGnT are located in chromosome 15, q22, chromosome 9, q13, and chromosome 6, p24, respectively. The chromosomal mapping of C2GnT-L and IGnT genes was corroborated by a recent report using the Genebridge 4 RH panel, which was developed independently from the Stanford G3 RH panel (40Deloukas P. Schuler G.D. Gyapay G. Beasley E.M. Soderlund C. Rodriguez-Tomé P. Hui L. Matise T.C. McKusick K.B. Beckmann J.S. Bentolila S. Bilhoreau M.-T. Birren B.B. Browne J. Butler A. Castle A.B. Chiannilkulchai N. Clee C. Day P.J.R. Dehejia A. Dibling T. Drouot N. Duprat S. Fizames C. Fox S. Gelling S. Green L. Harrison P. Hocking R.E. Holloway E. Hunt S. Keil S. Lijnzaad P. Louis-Dit-Sully C. Ma J. Mendis A. Miller J. Morissette J. Muselet D. Nusbaum H.C. Peck A. Rozen S. Simon D. Slonim D.K. Staples R. Stein L.D. Stewart E.A. Suchard M.A. Thangarajah T. Vega-Xzarny N. Webber C. Wu X. Hudson J. Auffray C. Nomura N. Sikela J.M. Polymeropoulos M.H. James M.R. Lander E.S. Hudson T.J. Myers R.M. Cox D.R. Weissenbach J. Boguski M.S. Bentley D.R. Science. 1998; 282: 744-746Crossref PubMed Scopus (586) Google Scholar). The gene locus for IGnT was originally reported to be in chromosome 9, q21 (23Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). The reason for this discrepancy in the IGnT chromosomal mapping is not clear but may be related to a more sensitive method used in the present study. Similar to the results obtained in the present study, the genes for two highly related polysialyltransferases, PST and STX, were located at different chromosomes by fluorescence in situ hybridization (41Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). These results, as a whole, suggest that Golgi-associated glycosyltransferases diverged relatively early in evolution.In the present study, we could detect dIGnT activity in the IGnT cloned (23Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar), as shown in Fig. 4, although the previous studies detected only cIGnT activity (34Mattila P. Salminen H. Hirvas L. Niittymäki J. Salo H. Niemelä R. Fukuda M. Renkonen O. Renkonen R. J. Biol. Chem. 1998; 273: 27633-27639Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This discrepancy is probably attributable to the fact that the chimeric soluble IGnT was assayed after concentration of the culture supernatant in the present study, whereas a culture supernatant was directly used in the previous study (34Mattila P. Salminen H. Hirvas L. Niittymäki J. Salo H. Niemelä R. Fukuda M. Renkonen O. Renkonen R. J. Biol. Chem. 1998; 273: 27633-27639Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). These results, as a whole, indicate that in vitro assay of glycosyltransferase is a very powerful tool for detecting activity. On the other hand, the expression of I antigen by C2GnT-M, assessed by immunofluorescent staining was apparently as much as the I antigen expressed by IGnT when the full-length cDNAs were expressed in CHO cells (Fig. 3). This is strikingly in contrast to the results that C2GnT-M had only 7% activity of dIGnT compared with C2GnT activity when measured by in vitro assay (Fig. 4). These results suggest that the enzymatic activities measured by in vitroassay may not quantitatively reflect its actual activities in cells. It is possible that the protein A portion of the chimeric protein may bring a constraint on its catalytic domain. Future studies will be important to determine how C2GnT-M synthesizes these different oligosaccharides in a given cell. Mucin-type glycoproteins are unique in having clusters of large numbers of O-glycans. These O-glycans containN-acetylgalactosamine residues at reducing ends, which are linked to serine or threonine in a polypeptide (1Fukuda M. Fukuda M. Molecular Glycobiology. Oxford University Press, Oxford1994: 1-52Google Scholar). These attached O-glycans can be classified into several different groups according to the core structures (2Schachter H. Brockhausen I. Allen H.J. Kisailus E.C. Glycoconjugates: Composition, Structure, and Function. Marcel Dekker, New York1992: 263-332Google Scholar). In many cells, core 1, Galβ1→3GalNAc, is the major constituent ofO-glycans. Core 1 oligosaccharides are converted to core 2 oligosaccharides Galβ1→3(GlcNAcβ1→6)GalNAc when core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) 1The abbreviations used are: C2GnT-L and -M, core 2 β-1,6-N-acetylglucosaminyltransferase-leukocyte type and -mucin type, respectively; dIGnT and cIGnT, distally acting and centrally acting I branching β-1,6-N-acetylglucosaminyltransferase, respectively; C4GnT, core 4 β-1,6-N-acetylglucosaminyltransferase; EST, expressed sequence tag; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; CHO, Chinese hamster ovary; pNP, p-nitrophenol; RH, radiation hybrid. 1The abbreviations used are: C2GnT-L and -M, core 2 β-1,6-N-acetylglucosaminyltransferase-leukocyte type and -mucin type, respectively; dIGnT and cIGnT, distally acting and centrally acting I branching β-1,6-N-acetylglucosaminyltransferase, respectively; C4GnT, core 4 β-1,6-N-acetylglucosaminyltransferase; EST, expressed sequence tag; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; CHO, Chinese hamster ovary; pNP, p-nitrophenol; RH, radiation hybrid.is present (3Fukuda M. Carlsson S.R. Klock J.C. Dell A. J. Biol. Chem. 1986; 261: 12796-12806Abstract Full Text PDF PubMed Google Scholar, 4Piller F. Piller V. Fox R.I. Fukuda M. J. Biol. Chem. 1988; 263: 15146-15150Abstract Full Text PDF PubMed Google Scholar). Various ligand carbohydrates can be formed in core 2 branched oligosaccharides. For example, sialyl Lex in mucin-type glycoproteins of blood cells can be formed only in core 2 branched oligosaccharides such as NeuNAcα2→3Galβ1→4(Fucα1→3) GlcNAcβ1→6(NeuNAcα2→3Galβ1→3)GalNAcα→serine/ threonine (3Fukuda M. Carlsson S.R. Klock J.C. Dell A. J. Biol. Chem. 1986; 261: 12796-12806Abstract Full Text PDF PubMed Google Scholar, 5Maemura K. Fukuda M. J. Biol. Chem. 1992; 267: 24379-24386Abstract Full Text PDF PubMed Google Scholar, 6Hemmerich S. Leffler H. Rosen S.D. J. Biol. Chem. 1995; 270: 12035-12047Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 7Wilkins P.P. McEver R.P. Cummings R.D. J. Biol. Chem. 1996; 271: 18732-18742Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Those sialyl Lex and sulfated sialyl Lex inO-glycans have been shown to be preferential ligands for P- and L-selectin (6Hemmerich S. Leffler H. Rosen S.D. J. Biol. Chem. 1995; 270: 12035-12047Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 7Wilkins P.P. McEver R.P. Cummings R.D. J. Biol. Chem. 1996; 271: 18732-18742Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 8Lasky L.A. Singer M.S. Dowbenko D. Imai Y. Henzel W.J. Grimley C. Fennie C. Gillett N. Watson S.R. Rosen S.D. Cell. 1992; 69: 927-938Abstract Full Text PDF PubMed Scopus (581) Google Scholar, 9Tsuboi S. Isogai Y. Hada N. King J.K. Hindsgaul O. Fukuda M. J. Biol. Chem. 1996; 271: 27213-27216Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Lowe J.B. Fukuda M. Hindsgaul O. Molecular Glycobiology. Oxford University Press, Oxford1994: 163-205Google Scholar). It has been also shown that poly-N-acetyllactosamines can be extended from core 2 branches (3Fukuda M. Carlsson S.R. Klock J.C. Dell A. J. Biol. Chem. 1986; 261: 12796-12806Abstract Full Text PDF PubMed Google Scholar, 5Maemura K. Fukuda M. J. Biol. Chem. 1992; 267: 24379-24386Abstract Full Text PDF PubMed Google Scholar, 7Wilkins P.P. McEver R.P. Cummings R.D. J. Biol. Chem. 1996; 271: 18732-18742Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 11Hanisch F.G. Uhlenbruck G. Peter K.J. Egge H. Dabrowski J. Dabrowski U. J. Biol. Chem. 1989; 264: 261-267Google Scholar, 12Yousefi 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). Poly-N-acetyllactosamines provide the backbone for additional modifications such as sialyl Lex. Moreover, a linear poly-N-acetyllactosamine (Galβ1→4GlcNAcβ1→3)n can be converted to branched poly-N-acetyllactosamine, Galβ1→4GlcNAcβ1→3 (Galβ1→4GlcNAcβ1→6)Gal→R, by the I branching enzyme IGnT (13Fukuda M. Fukuda M.N. Hakomori S. J. Biol. Chem. 1979; 254: 3700-3703Abstract Full Text PDF PubMed Google Scholar, 14Piller F. Cartron J.P. Maranduba A. Veyrieres A. Leroy Y. Fournet B. J. Biol. Chem. 1984; 259: 13385-13390Abstract Full Text PDF PubMed Google Scholar). These linear and branched poly-N-acetyllactosamines represent, respectively, the i and I antigens, which are expressed in a cell type-specific manner (1Fukuda M. Fukuda M. Molecular Glycobiology. Oxford University Press, Oxford1994: 1-52Google Scholar). It has been shown that there exist two IGnTs, which use two different sets of acceptor substrates (15Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H.L. Van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar, 17Gu J. Nishikawa A. Fujii S. Gasa S. Taniguchi N. J. Biol. Chem. 1992; 267: 2994-2999Abstract Full Text PDF PubMed Google Scholar). In the gastrointestinal tract, oligosaccharides with core 3, GlcNAcβ1→3GalNAc, can be frequently found. These tissues also contain core 4, Galβ1→4GlcNAcβ1→6(GlcNAcβ1→3)GalNAc, which was originally discovered in sheep gastric mucosa (18Hounsell E.F. Fukuda M. Powell M.E. Feizi T. Hakomori S. Biochem. Biophys. Res. Commun. 1980; 92: 1143-1150Crossref PubMed Scopus (33) Google Scholar). Core 4 is formed from core 3 by core 4 β-1,6-N-acetylglucosaminyltransferase (C4GnT). It has been reported that the amount of core 4 oligosaccharides is reduced in colonic carcinoma cells, whereas the amount of core 2 oligosaccharides is maintained or increased (19Yang J.-M. Byrd J.C. Siddik 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, 20Vavasseur F. Yang J.M. Dole K. Paulsen H. Brockhausen I. Glycobiology. 1995; 5: 351-357Crossref PubMed Scopus (63) Google Scholar). More recently, the increase in the transcript of C2GnT was found to be associated with the progression of colonic carcinomas (21Shimodaira K. Nakayama J. Nakayama N. Hasebe O. Katsuyama T. Fukuda M. Cancer Res. 1997; : 5201-5206PubMed Google Scholar). To understand the roles of these oligosaccharides in normal and pathological conditions, it is essential to understand the regulation of their biosynthesis. To this end, we previously cloned cDNAs encoding C2GnT (termed in the present study C2GnT-leukocyte type (C2GnT-L)) from human HL-60 promyelocytic cells (22Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar) and IGnT from human PA-1 embryonic carcinoma cells (23Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). C2GnT-L and IGnT share homologous sequences in three regions of their catalytic domains (23Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). Neither C2GnT-L nor IGnT, however, forms core 4 structure. On the other hand, a C2GnT purified from mucin-producing cells, termed C2GnT-mucin (C2GnT-M) type, was reported to contain C2GnT and C4GnT activities in the same enzyme (2Schachter H. Brockhausen I. Allen H.J. Kisailus E.C. Glycoconjugates: Composition, Structure, and Function. Marcel Dekker, New York1992: 263-332Google Scholar, 15Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H.L. Van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar). In the present study we describe the isolation of cDNA encoding such an enzyme using EST sequence, which has strong homology with C2GnT-L and IGnT. We found that this newly cloned enzyme is unique in having C2GnT activity as well as core 4 and I branching activities. We termed this new enzyme C2GnT-M, because it is mainly expressed in mucin-producing tissues. DISCUSSIONIn the present study, we have isolated a cDNA encoding a novel C2GnT, C2GnT-M. C2GnT-M is unique in catalyzing more than one enzymatic reaction, forming core 4 branches and I branches in addition to core 2 branches. Previously, fucosyltransferase III and ST3Gal III, an α-2,3-sialyltransferase, were shown to add, respectively, fucose and sialic acid residues to both Galβ1→4GlcNAc and Galβ1→3 GlcNAc acceptors (35Prieels J.P. Monnom D. Dolmans M. Beyer T.A. Hill R.L. J. Biol. Chem. 1981; 256: 10456-10463Abstract Full Text PDF PubMed Google Scholar, 36Kukowska-Latallo J.F. Larsen R.D. Nair R.P. Lowe J.B. Genes Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (469) Google Scholar, 37Wen D.X. Livingstone B.D. Medzihradszky K.F. Kelm S. Burlingame A.L. Paulson J.C. J. Biol. Chem. 1992; 267: 21011-21019Abstract Full Text PDF PubMed Google Scholar). As far as we know, we are the first to demonstrate in a definitive manner that a single enzyme catalyzes three different but related reactions.C2GnT-M is almost exclusively expressed in small and large intestines, trachea, stomach, and thyroid among tissues examined (Fig. 5), thus likely involved in the synthesis of mucin-type oligosaccharides. Accordingly, we termed this new enzyme C2GnT-M (mucin-type). It is not apparent why the C2GnT-M is also expressed in thyroid. However, it was recently demonstrated that a major glycoprotein in calf thyroid contains core 2 branched O-glycans and I-branchedN-glycans (38Edge A.S.B. Spiro R.G. Arch. Biochem. Biophys. 1997; 343: 73-80Crossref PubMed Scopus (11) Google Scholar), indicating that thyroid synthesizes mucin-type O-glycans.It is noteworthy that C2GnT-M exhibits a significant activity of C4GnT. This finding is consistent with previous reports that the presence of C4GnT activity is always associated with the presence of C2GnT activity when various mucin-producing tissues were examined (15Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H.L. Van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar, 19Yang J.-M. Byrd J.C. Siddik 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, 20Vavasseur F. Yang J.M. Dole K. Paulsen H. Brockhausen I. Glycobiology. 1995; 5: 351-357Crossref PubMed Scopus (63) Google Scholar). It is very likely that C2GnT-M is responsible for C4GnT activity in these tissues. C2GnT-L, on the other hand, lacks C4GnT activity (Fig. 4). In small intestine, colon, and stomach, significant amounts of the transcripts for C2GnT-L and C2GnT-M were detected (Fig. 5), suggesting that both enzymes contribute to the C2GnT activity in these tissues. In fact, recent studies on C2GnT-L knock-out mice demonstrated that a residual C2GnT activity exists in small and large intestines among tissues examined after C2GnT-L is inactivated (39Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; (in press)PubMed Google Scholar). It is most likely that C2GnT-M cloned in the present study is responsible for this activity.C2GnT-M is also unique in transferring a GlcNAc residue preferentially to predistal galactose residues of GlcNAcβ1→3 Galβ1→4 GlcNAcβ1→R, forming GlcNAcβ1→3(GlcNAcβ1→6)Galβ1→4GlcNAcβ1→R. Although the presence of this enzyme activity, dIGnT, was reported previously (14Piller F. Cartron J.P. Maranduba A. Veyrieres A. Leroy Y. Fournet B. J. Biol. Chem. 1984; 259: 13385-13390Abstract Full Text PDF PubMed Google Scholar, 15Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H.L. Van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar, 17Gu J. Nishikawa A. Fujii S. Gasa S. Taniguchi N. J. Biol. Chem. 1992; 267: 2994-2999Abstract Full Text PDF PubMed Google Scholar), a cDNA encoding an enzyme acting more as a dIGnT than as a cIGnT was not reported before. The activities of dIGnT reported in hog gastric mucosa and rat liver (14Piller F. Cartron J.P. Maranduba A. Veyrieres A. Leroy Y. Fournet B. J. Biol. Chem. 1984; 259: 13385-13390Abstract Full Text PDF PubMed Google Scholar, 17Gu J. Nishikawa A. Fujii S. Gasa S. Taniguchi N. J. Biol. Chem. 1992; 267: 2994-2999Abstract Full Text PDF PubMed Google Scholar) may be attributable to the newly cloned C2GnT-M. However, those enzymatic activities reported previously had much less C2GnT or C4GnT activity compared with dIGnT activity, whereas the opposite is true for C2GnT-M. It is thus likely that both IGnT and C2GnT-M contribute to dIGnT activity in these tissues. Our cloned enzyme most closely resembles the C2GnT preparation isolated from bovine trachea (16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar). However, dIGnT activity of the bovine trachea enzyme preparation was almost 40% of its C2GnT activity, whereas it is only 7% of C2GnT activity in the cloned C2GnT-M, although similar acceptors were used in both studies. On the other hand, it is noteworthy that the ratio of C2GnT and dIGnT activities in LS180, HT29, and NCI498 colonic carcinomas (20Vavasseur F. Yang J.M. Dole K. Paulsen H. Brockhausen I. Glycobiology. 1995; 5: 351-357Crossref PubMed Scopus (63) Google Scholar) is similar to that of C2GnT-M. Further studies are necessary to clarify this discrepancy.The results obtained in the present study reveal that the genes of C2GnT-M, C2GnT-L, and IGnT are located in chromosome 15, q22, chromosome 9, q13, and chromosome 6, p24, respectively. The chromosomal mapping of C2GnT-L and IGnT genes was corroborated by a recent report using the Genebridge 4 RH panel, which was developed independently from the Stanford G3 RH panel (40Deloukas P. Schuler G.D. Gyapay G. Beasley E.M. Soderlund C. Rodriguez-Tomé P. Hui L. Matise T.C. McKusick K.B. Beckmann J.S. Bentolila S. Bilhoreau M.-T. Birren B.B. Browne J. Butler A. Castle A.B. Chiannilkulchai N. Clee C. Day P.J.R. Dehejia A. Dibling T. Drouot N. Duprat S. Fizames C. Fox S. Gelling S. Green L. Harrison P. Hocking R.E. Holloway E. Hunt S. Keil S. Lijnzaad P. Louis-Dit-Sully C. Ma J. Mendis A. Miller J. Morissette J. Muselet D. Nusbaum H.C. Peck A. Rozen S. Simon D. Slonim D.K. Staples R. Stein L.D. Stewart E.A. Suchard M.A. Thangarajah T. Vega-Xzarny N. Webber C. Wu X. Hudson J. Auffray C. Nomura N. Sikela J.M. Polymeropoulos M.H. James M.R. Lander E.S. Hudson T.J. Myers R.M. Cox D.R. Weissenbach J. Boguski M.S. Bentley D.R. Science. 1998; 282: 744-746Crossref PubMed Scopus (586) Google Scholar). The gene locus for IGnT was originally reported to be in chromosome 9, q21 (23Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). The reason for this discrepancy in the IGnT chromosomal mapping is not clear but may be related to a more sensitive method used in the present study. Similar to the results obtained in the present study, the genes for two highly related polysialyltransferases, PST and STX, were located at different chromosomes by fluorescence in situ hybridization (41Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). These results, as a whole, suggest that Golgi-associated glycosyltransferases diverged relatively early in evolution.In the present study, we could detect dIGnT activity in the IGnT cloned (23Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar), as shown in Fig. 4, although the previous studies detected only cIGnT activity (34Mattila P. Salminen H. Hirvas L. Niittymäki J. Salo H. Niemelä R. Fukuda M. Renkonen O. Renkonen R. J. Biol. Chem. 1998; 273: 27633-27639Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This discrepancy is probably attributable to the fact that the chimeric soluble IGnT was assayed after concentration of the culture supernatant in the present study, whereas a culture supernatant was directly used in the previous study (34Mattila P. Salminen H. Hirvas L. Niittymäki J. Salo H. Niemelä R. Fukuda M. Renkonen O. Renkonen R. J. Biol. Chem. 1998; 273: 27633-27639Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). These results, as a whole, indicate that in vitro assay of glycosyltransferase is a very powerful tool for detecting activity. On the other hand, the expression of I antigen by C2GnT-M, assessed by immunofluorescent staining was apparently as much as the I antigen expressed by IGnT when the full-length cDNAs were expressed in CHO cells (Fig. 3). This is strikingly in contrast to the results that C2GnT-M had only 7% activity of dIGnT compared with C2GnT activity when measured by in vitro assay (Fig. 4). These results suggest that the enzymatic activities measured by in vitroassay may not quantitatively reflect its actual activities in cells. It is possible that the protein A portion of the chimeric protein may bring a constraint on its catalytic domain. Future studies will be important to determine how C2GnT-M synthesizes these different oligosaccharides in a given cell. In the present study, we have isolated a cDNA encoding a novel C2GnT, C2GnT-M. C2GnT-M is unique in catalyzing more than one enzymatic reaction, forming core 4 branches and I branches in addition to core 2 branches. Previously, fucosyltransferase III and ST3Gal III, an α-2,3-sialyltransferase, were shown to add, respectively, fucose and sialic acid residues to both Galβ1→4GlcNAc and Galβ1→3 GlcNAc acceptors (35Prieels J.P. Monnom D. Dolmans M. Beyer T.A. Hill R.L. J. Biol. Chem. 1981; 256: 10456-10463Abstract Full Text PDF PubMed Google Scholar, 36Kukowska-Latallo J.F. Larsen R.D. Nair R.P. Lowe J.B. Genes Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (469) Google Scholar, 37Wen D.X. Livingstone B.D. Medzihradszky K.F. Kelm S. Burlingame A.L. Paulson J.C. J. Biol. Chem. 1992; 267: 21011-21019Abstract Full Text PDF PubMed Google Scholar). As far as we know, we are the first to demonstrate in a definitive manner that a single enzyme catalyzes three different but related reactions. C2GnT-M is almost exclusively expressed in small and large intestines, trachea, stomach, and thyroid among tissues examined (Fig. 5), thus likely involved in the synthesis of mucin-type oligosaccharides. Accordingly, we termed this new enzyme C2GnT-M (mucin-type). It is not apparent why the C2GnT-M is also expressed in thyroid. However, it was recently demonstrated that a major glycoprotein in calf thyroid contains core 2 branched O-glycans and I-branchedN-glycans (38Edge A.S.B. Spiro R.G. Arch. Biochem. Biophys. 1997; 343: 73-80Crossref PubMed Scopus (11) Google Scholar), indicating that thyroid synthesizes mucin-type O-glycans. It is noteworthy that C2GnT-M exhibits a significant activity of C4GnT. This finding is consistent with previous reports that the presence of C4GnT activity is always associated with the presence of C2GnT activity when various mucin-producing tissues were examined (15Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H.L. Van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar, 19Yang J.-M. Byrd J.C. Siddik 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, 20Vavasseur F. Yang J.M. Dole K. Paulsen H. Brockhausen I. Glycobiology. 1995; 5: 351-357Crossref PubMed Scopus (63) Google Scholar). It is very likely that C2GnT-M is responsible for C4GnT activity in these tissues. C2GnT-L, on the other hand, lacks C4GnT activity (Fig. 4). In small intestine, colon, and stomach, significant amounts of the transcripts for C2GnT-L and C2GnT-M were detected (Fig. 5), suggesting that both enzymes contribute to the C2GnT activity in these tissues. In fact, recent studies on C2GnT-L knock-out mice demonstrated that a residual C2GnT activity exists in small and large intestines among tissues examined after C2GnT-L is inactivated (39Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; (in press)PubMed Google Scholar). It is most likely that C2GnT-M cloned in the present study is responsible for this activity. C2GnT-M is also unique in transferring a GlcNAc residue preferentially to predistal galactose residues of GlcNAcβ1→3 Galβ1→4 GlcNAcβ1→R, forming GlcNAcβ1→3(GlcNAcβ1→6)Galβ1→4GlcNAcβ1→R. Although the presence of this enzyme activity, dIGnT, was reported previously (14Piller F. Cartron J.P. Maranduba A. Veyrieres A. Leroy Y. Fournet B. J. Biol. Chem. 1984; 259: 13385-13390Abstract Full Text PDF PubMed Google Scholar, 15Brockhausen I. Matta K.L. Orr J. Schachter H. Koenderman A.H.L. Van den Eijnden D.H. Eur. J. Biochem. 1986; 157: 463-474Crossref PubMed Scopus (62) Google Scholar, 16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar, 17Gu J. Nishikawa A. Fujii S. Gasa S. Taniguchi N. J. Biol. Chem. 1992; 267: 2994-2999Abstract Full Text PDF PubMed Google Scholar), a cDNA encoding an enzyme acting more as a dIGnT than as a cIGnT was not reported before. The activities of dIGnT reported in hog gastric mucosa and rat liver (14Piller F. Cartron J.P. Maranduba A. Veyrieres A. Leroy Y. Fournet B. J. Biol. Chem. 1984; 259: 13385-13390Abstract Full Text PDF PubMed Google Scholar, 17Gu J. Nishikawa A. Fujii S. Gasa S. Taniguchi N. J. Biol. Chem. 1992; 267: 2994-2999Abstract Full Text PDF PubMed Google Scholar) may be attributable to the newly cloned C2GnT-M. However, those enzymatic activities reported previously had much less C2GnT or C4GnT activity compared with dIGnT activity, whereas the opposite is true for C2GnT-M. It is thus likely that both IGnT and C2GnT-M contribute to dIGnT activity in these tissues. Our cloned enzyme most closely resembles the C2GnT preparation isolated from bovine trachea (16Ropp P.A. Little M.R. Cheng P.W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar). However, dIGnT activity of the bovine trachea enzyme preparation was almost 40% of its C2GnT activity, whereas it is only 7% of C2GnT activity in the cloned C2GnT-M, although similar acceptors were used in both studies. On the other hand, it is noteworthy that the ratio of C2GnT and dIGnT activities in LS180, HT29, and NCI498 colonic carcinomas (20Vavasseur F. Yang J.M. Dole K. Paulsen H. Brockhausen I. Glycobiology. 1995; 5: 351-357Crossref PubMed Scopus (63) Google Scholar) is similar to that of C2GnT-M. Further studies are necessary to clarify this discrepancy. The results obtained in the present study reveal that the genes of C2GnT-M, C2GnT-L, and IGnT are located in chromosome 15, q22, chromosome 9, q13, and chromosome 6, p24, respectively. The chromosomal mapping of C2GnT-L and IGnT genes was corroborated by a recent report using the Genebridge 4 RH panel, which was developed independently from the Stanford G3 RH panel (40Deloukas P. Schuler G.D. Gyapay G. Beasley E.M. Soderlund C. Rodriguez-Tomé P. Hui L. Matise T.C. McKusick K.B. Beckmann J.S. Bentolila S. Bilhoreau M.-T. Birren B.B. Browne J. Butler A. Castle A.B. Chiannilkulchai N. Clee C. Day P.J.R. Dehejia A. Dibling T. Drouot N. Duprat S. Fizames C. Fox S. Gelling S. Green L. Harrison P. Hocking R.E. Holloway E. Hunt S. Keil S. Lijnzaad P. Louis-Dit-Sully C. Ma J. Mendis A. Miller J. Morissette J. Muselet D. Nusbaum H.C. Peck A. Rozen S. Simon D. Slonim D.K. Staples R. Stein L.D. Stewart E.A. Suchard M.A. Thangarajah T. Vega-Xzarny N. Webber C. Wu X. Hudson J. Auffray C. Nomura N. Sikela J.M. Polymeropoulos M.H. James M.R. Lander E.S. Hudson T.J. Myers R.M. Cox D.R. Weissenbach J. Boguski M.S. Bentley D.R. Science. 1998; 282: 744-746Crossref PubMed Scopus (586) Google Scholar). The gene locus for IGnT was originally reported to be in chromosome 9, q21 (23Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). The reason for this discrepancy in the IGnT chromosomal mapping is not clear but may be related to a more sensitive method used in the present study. Similar to the results obtained in the present study, the genes for two highly related polysialyltransferases, PST and STX, were located at different chromosomes by fluorescence in situ hybridization (41Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). These results, as a whole, suggest that Golgi-associated glycosyltransferases diverged relatively early in evolution. In the present study, we could detect dIGnT activity in the IGnT cloned (23Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar), as shown in Fig. 4, although the previous studies detected only cIGnT activity (34Mattila P. Salminen H. Hirvas L. Niittymäki J. Salo H. Niemelä R. Fukuda M. Renkonen O. Renkonen R. J. Biol. Chem. 1998; 273: 27633-27639Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This discrepancy is probably attributable to the fact that the chimeric soluble IGnT was assayed after concentration of the culture supernatant in the present study, whereas a culture supernatant was directly used in the previous study (34Mattila P. Salminen H. Hirvas L. Niittymäki J. Salo H. Niemelä R. Fukuda M. Renkonen O. Renkonen R. J. Biol. Chem. 1998; 273: 27633-27639Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). These results, as a whole, indicate that in vitro assay of glycosyltransferase is a very powerful tool for detecting activity. On the other hand, the expression of I antigen by C2GnT-M, assessed by immunofluorescent staining was apparently as much as the I antigen expressed by IGnT when the full-length cDNAs were expressed in CHO cells (Fig. 3). This is strikingly in contrast to the results that C2GnT-M had only 7% activity of dIGnT compared with C2GnT activity when measured by in vitro assay (Fig. 4). These results suggest that the enzymatic activities measured by in vitroassay may not quantitatively reflect its actual activities in cells. It is possible that the protein A portion of the chimeric protein may bring a constraint on its catalytic domain. Future studies will be important to determine how C2GnT-M synthesizes these different oligosaccharides in a given cell. We thank Drs. Kiyohiko Angata, Minoru Ujita, and Shigeru Tsuboi for providing pcDNA3-A, pcDNAI-A·IGnT and pcDNAI-A·C2GnT, and pcDSRα-leukosialin, respectively, Drs. Joseph McAuliffe and Ole Hindsgaul for the synthetic oligosaccharides, Dr. Michiko Fukuda for useful discussion, and Susan Greaney for organizing the manuscript." @default.
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W2030180824 title "Molecular Cloning and Expression of a Novel β-1,6-N-Acetylglucosaminyltransferase That Forms Core 2, Core 4, and I Branches" @default.
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W2030180824 doi "https://doi.org/10.1074/jbc.274.5.3215" @default.
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