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Molecular Cloning and Expression of a Novel Human β-Gal-3-O-sulfotransferase That Acts Preferentially onN-Acetyllactosamine in N- andO-Glycans

2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês

10.1074/jbc.m103135200

ISSN

1083-351X

Autores

Atsushi Suzuki, Nobuyoshi Hiraoka, Masami Suzuki, Kiyohiko Angata, Anup Kumar Misra, Joseph C. McAuliffe, Ole Hindsgaul, Minoru Fukuda,

Tópico(s)

Proteoglycans and glycosaminoglycans research

Resumo

A novel cDNA-encoding galactose 3-O-sulfotransferase was cloned by screening the expressed sequence tag data base using the previously cloned cDNA encoding a galactosyl ceramide 3-O-sulfotransferase, which we term Gal3ST-1. The newly isolated cDNA encodes a novel 3-O-sulfotransferase, termed Gal3ST-3, that acts exclusively on N-acetyllactosamine present inN-glycans and core2-branched O-glycans. These conclusions were confirmed by analyzing CD43 chimeric proteins in Chinese hamster ovary cells expressing core2 β1,6-N-acetylglucosaminyltransferase. The acceptor specificity of Gal3ST-3 contrasts with that of the recently cloned galactose 3-O-sulfotransferase (Honke, K., Tsuda, M., Koyota, S., Wada, Y., Iida-Tanaka, N., Ishizuka, I., Nakayama, J., and Taniguchi, N. (2001) J. Biol. Chem. 276, 267–274), which we term Gal3ST-2 in the present study because the latter enzyme can also act on core1 O-glycan and type 1 oligosaccharides, Galβ1→3GlcNAc. Moreover, Gal3ST-3 but not Gal3ST-2 can act on Galβ1→4(sulfo→6)GlcNAc, indicating that disulfated sulfo→3Galβ1→4(sulfo→6) GlcNAc→R may be formed by Gal3ST-3 in combination with GlcNAc 6-O-sulfotransferase. Although both Gal3ST-2 and Gal3ST-3 do not act on galactosyl ceramide, Gal3ST-3 is only moderately more homologous to Gal3ST-2 (40.1%) than to Gal3ST-1 (38.0%) at the amino acid level. Northern blot analysis demonstrated that transcripts for Gal3ST-3 are predominantly expressed in the brain, kidney, and thyroid where the presence of 3′-sulfation ofN-acetyllactosamine has been reported. These results indicate that the newly cloned Gal3ST-3 plays a critical role in 3′-sulfation of N-acetyllactosamine in both O- and N-glycans. A novel cDNA-encoding galactose 3-O-sulfotransferase was cloned by screening the expressed sequence tag data base using the previously cloned cDNA encoding a galactosyl ceramide 3-O-sulfotransferase, which we term Gal3ST-1. The newly isolated cDNA encodes a novel 3-O-sulfotransferase, termed Gal3ST-3, that acts exclusively on N-acetyllactosamine present inN-glycans and core2-branched O-glycans. These conclusions were confirmed by analyzing CD43 chimeric proteins in Chinese hamster ovary cells expressing core2 β1,6-N-acetylglucosaminyltransferase. The acceptor specificity of Gal3ST-3 contrasts with that of the recently cloned galactose 3-O-sulfotransferase (Honke, K., Tsuda, M., Koyota, S., Wada, Y., Iida-Tanaka, N., Ishizuka, I., Nakayama, J., and Taniguchi, N. (2001) J. Biol. Chem. 276, 267–274), which we term Gal3ST-2 in the present study because the latter enzyme can also act on core1 O-glycan and type 1 oligosaccharides, Galβ1→3GlcNAc. Moreover, Gal3ST-3 but not Gal3ST-2 can act on Galβ1→4(sulfo→6)GlcNAc, indicating that disulfated sulfo→3Galβ1→4(sulfo→6) GlcNAc→R may be formed by Gal3ST-3 in combination with GlcNAc 6-O-sulfotransferase. Although both Gal3ST-2 and Gal3ST-3 do not act on galactosyl ceramide, Gal3ST-3 is only moderately more homologous to Gal3ST-2 (40.1%) than to Gal3ST-1 (38.0%) at the amino acid level. Northern blot analysis demonstrated that transcripts for Gal3ST-3 are predominantly expressed in the brain, kidney, and thyroid where the presence of 3′-sulfation ofN-acetyllactosamine has been reported. These results indicate that the newly cloned Gal3ST-3 plays a critical role in 3′-sulfation of N-acetyllactosamine in both O- and N-glycans. Chinese hamster ovary 3′-phosphoadenosine 5′-phosphate galactose 3-O-sulfotransferase expressed sequence tag polymerase chain reaction 2-(N-morpholino)ethanesulfonic acid core2 β1,6-N-acetylglucosaminyltransferase-1 high performance liquid chromatography p-nitrophenol the neural adhesion molecule Sulfate groups in carbohydrates play important roles in conferring highly specific functions on glycoproteins, glycolipids, and proteoglycans (1Fiete D. Srivastave V. Hindsgaul O. Baenziger J.U. Cell. 1991; 67: 1103-1110Abstract Full Text PDF PubMed Scopus (262) Google Scholar, 2Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 3Akama T.O. Nishida K. Nakayama J. Watanabe H. Ozaki K. Nakamura T. Dota A. Kawasaki S. Inoue Y. Maeda N. Yamamoto S. Fujiwara T. Thonar E.J. Shimomura Y. Kinoshita S. Tanigami A. Fukuda M.N. Nat. Genet. 2000; 26: 237-241Crossref PubMed Scopus (221) Google Scholar). One of these sulfated glycans is 3′-sulfo galactose attached to a type 2 oligosaccharide (N-acetyllactosamine), sulfo→3Galβ1→4GlcNAc→R, or attached to a type 1 oligosaccharide, sulfo→3Galβ1→3GlcNAc→R. Feizi and colleagues (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar) demonstrated that 3′-sulfo galactose, in a type 1 or type 2, fucosylated oligosaccharide, functions as an E-selectin ligand (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar). When oligosaccharides were released from ovarian cystadenoma glycoprotein and conjugated to lipids, sulfo→3Galβ1→3/4(Fucα1→4/3)GlcNAcβ1→3Gal was found to bind to Chinese hamster ovary (CHO)1 cells expressing E-selectin (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar). On the other hand, sulfo→3Galβ1→4(Fucα1→3)GlcNAc acted as a P-selectin ligand when a synthetic oligosaccharide with this structure was transferred to cell surface glycoproteins through a fucose residue by α1,3-fucosyltransferase III (5Tsuboi S. Srivastava O.P. Palcic M. Hindsgaul O. Fukuda M. Arch. Biochem. Biophys. 2000; 374: 100-106Crossref PubMed Scopus (11) Google Scholar). These studies suggest that 3′-sulfo galactose on the cell surface plays a role in carbohydrate-protein interactions, including those involved with selectin.Recently, a comparison of the amino acid sequences of cloned sulfotransferases demonstrated that there is a weak but discernible homologous sequence motif among Golgi-associated sulfotransferases (6Ong E. Yeh J.C. Ding Y. Hindsgaul O. Fukuda M. J. Biol. Chem. 1998; 273: 5190-5195Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 7Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (232) Google Scholar, 8Kakuta Y. Pedersen L.G. Pedersen L.C. Negishi M. Trends Biochem. Sci. 1998; 23: 129-130Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 9Ong E. Yeh J.C. Ding Y. Hindsgaul O. Pedersen L.C. Negishi M. Fukuda M. J. Biol. Chem. 1999; 274: 25608-25612Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In particular, the amino acid sequences that are responsible for binding 5′-phosphosulfate and 3′-phosphate groups of the donor substrate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), are well conserved and are often highly homologous to each other among those that share the same acceptor specificity (10Uchimura K. Muramatsu H. Kadomatsu K. Fan Q.W. Kurosawa N. Mitsuoka C. Kannagi R. Habuchi O. Muramatsu T. J. Biol. Chem. 1998; 273: 22577-22583Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 11Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar, 13Xia G. Evers M.R. Kang H-G. Schachner M. Baenziger J.U. J. Biol. Chem. 2000; 276: 38402-38409Abstract Full Text Full Text PDF Scopus (57) Google Scholar, 14Okuda T. Mita S. Yamauchi S. Fukuta M. Nakano H. Sawada T. Habuchi O. J. Biol. Chem. 2000; 275: 40605-40613Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 15Hiraoka, N., Misra, A., Belot, F., Hindsgaul, O., and Fukuda, M. (2001)Glycobiology, in press.Google Scholar). Previously, galactosyl ceramide 3′-sulfotransferase, which forms a sulfatide, sulfo→3Gal→ceramide, has been cloned based on the amino acid sequence of purified protein (16Honke K. Tsuda M. Hirahara Y. Ishii A. Makita A. Wada Y. J. Biol. Chem. 1997; 272: 4864-4868Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Because this enzyme, which we now term Gal3ST-1, is thought not to add a sulfate to glycoproteins (17Lo-Guidice J. Perini J. Lafitte J. Ducourouble M. Roussel P. Lamblin G. J. Biol. Chem. 1995; 270: 27544-27550Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), galactose 3-O-sulfotransferase was molecularly cloned by searching for an enzyme homologous to Gal3ST-1. This reported enzyme, which we now term Gal3ST-2, has the unique property of adding a sulfate on both type 1 and type 2 oligosaccharides and core1O-glycans, Galβ1→3GalNAcα1→R (18Honke K. Tsuda M. Koyota S. Wada Y. Iida-Tanaka N. Ishizuka I. Nakayama J. Taniguchi N. J. Biol. Chem. 2001; 276: 267-274Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). On the other hand, the structures of O-linked oligosaccharides containing 3′-sulfo galactose reported to date show that 3′-sulfo galactose is present in N-acetyllactosamine in core2O-glycans, sulfo→3Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAcα1→R (19Lamblin G. Rahmoune H. Wieruszeski J.-M. Lhermitte M. Strecker G. Roussel P. Biochem. J. 1991; 275: 199-206Crossref PubMed Scopus (43) Google Scholar); core3 O-glycans, sulfo→3Galβ1→4GlcNAcβ1→ 3GalNAc (20Capon C. Leroy Y. Wieruszeski J.-M. Ricart G. Strecker G. Montreuil J. Fournet B. Eur. J. Biochem. 1989; 182: 139-152Crossref PubMed Scopus (74) Google Scholar); and core1 extended structures, sulfo→ 3Galβ1→4GlcNAcβ1→3Gaβ1→3GalNAc (21Capon C. Wieruszeski J.-M. Lemoine J. Byrd J.C. Leffler H. Kim Y.S. J. Biol. Chem. 1997; 272: 31957-31968Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Moreover, no 3′-sulfo galactose in core1 O-glycans such as sulfo→3Galβ1→ 3GalNAcα1→R has been previously reported. Similarly, galactose 3-O-sulfotransferase in human respiratory mucosa was found to act exclusively onN-acetyllactosamine in core2-branched O-glycans and not on core1 O-glycans (17Lo-Guidice J. Perini J. Lafitte J. Ducourouble M. Roussel P. Lamblin G. J. Biol. Chem. 1995; 270: 27544-27550Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The presence of 3′-sulfo galactose in N-glycans has been extensively studied in human, bovine, and porcine thyroglobulins, and these studies showed that 3′-sulfo galactose is present in N-acetyllactosamine in complex N-glycans (22Edge A. Spiro R. J. Biol. Chem. 1984; 259: 4710-4713Abstract Full Text PDF PubMed Google Scholar, 23Spiro R.G. Bhoyroo V.D. J. Biol. Chem. 1988; 263: 14351-14358Abstract Full Text PDF PubMed Google Scholar, 24Waard P.D. Koorevaar A. Kamerling J.P. Vliegenthart J.F.G. J. Biol. Chem. 1991; 266: 4237-4243Abstract Full Text PDF PubMed Google Scholar). Notably, galactose 3-O-sulfotransferase present in the thyroid was found to act only on N-acetyllactosamine but not on type 1 oligosaccharides, which differs from the properties of Gal3ST-2 (25Kato Y. Spiro R.G. J. Biol. Chem. 1989; 264: 3364-3371Abstract Full Text PDF PubMed Google Scholar). Although no glycoprotein acceptor was tested for Gal3ST-2, these results suggested that there is another galactose 3-O-sulfotransferase (Gal3ST) yet to be identified.In the present study, we first identified a novel cDNA by screening the EST data base for cDNAs related to human Gal3ST-1 (16Honke K. Tsuda M. Hirahara Y. Ishii A. Makita A. Wada Y. J. Biol. Chem. 1997; 272: 4864-4868Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The expression of a full-length cDNA revealed that this cDNA encodes a novel galactose 3-O-sulfotransferase, termed Gal3ST-3, that adds a sulfate exclusively in the 3′-position of galactose in N-acetyllactosamine in both N- andO-glycans but not on a type 1 Galβ1→3GlcNAc or core1 Galβ1→3GalNAc structure. When CD43 (leukosialin) was tested as an acceptor, Gal3ST-3 preferentially acted onN-acetyllactosamine present in core2-branchedO-glycans, whereas Gal3ST-2 acted on both core1- and core2-branched O-glycans. Moreover, we show that this novel enzyme is expressed almost exclusively in the thyroid, kidney, and brain, in contrast to the previously reported ubiquitous expression of Gal3ST-2 (18Honke K. Tsuda M. Koyota S. Wada Y. Iida-Tanaka N. Ishizuka I. Nakayama J. Taniguchi N. J. Biol. Chem. 2001; 276: 267-274Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar).DISCUSSIONThe present study describes the isolation of a novel cDNA encoding galactose 3-O-sulfotransferase by searching the EST data base for cDNAs homologous to the human galactosyl ceramide 3-O-sulfotransferase, Gal3ST-1 (16Honke K. Tsuda M. Hirahara Y. Ishii A. Makita A. Wada Y. J. Biol. Chem. 1997; 272: 4864-4868Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Gal3ST-1 adds a sulfate to a β-galactose residue linked to ceramide, whereas Gal3ST-3 adds a sulfate to a β-galactose linked to N-acetylglucosamine through a 1,4-linkage. Previously, Gal3ST-2 was also cloned based on its similarity to Gal3ST-1, but the acceptor specificities of Gal3ST-2 and Gal3ST-3 differ substantially. Although Gal3ST-3 acts exclusively on N-acetyllactosamine, Gal3ST-2 can act also on type 1 oligosaccharide, Galβ1→ 3GlcNAc, and core1 oligosaccharide, Galβ1→3(GlcNAcβ1→ 6)GalNAc. On the other hand, Gal3ST-3 can act on Galβ1→ 4(sulfo→6)GlcNAcβ1→R, but Gal3ST-2 cannot act on the same acceptor (Fig. 3). Interestingly, the best acceptor for Gal3ST-3 is poly-N-acetyllactosamine attached to a side chain derived from C-6 of α-mannose, whereas the best acceptor for Gal3ST-2 is N-acetyllactosamine attached to a side chain derived from C-2 of α-mannose (Fig. 3). Moreover, Gal3ST-3 preferentially acts on N-acetyllactosamine attached to anN-glycan mannose core, such as Galβ1→4GlcNAcβ1→6Manα1→6Manβ1→octyl, than onN-acetyllactosamine itself, whereas Gal3ST-2 does not possess this preference (Fig. 3). These results indicate that Gal3ST-2 and Gal3ST-3 act differentially on various acceptor glycoproteins.In the present study, the transcripts of Gal3ST-3 were found to be expressed selectively in the brain, kidney, and thyroid. Previously, it was reported that human thyroglobulin contains a sulfo→3Galβ1→4GlcNAcβ1→R structure in the majority ofN-glycans (23Spiro R.G. Bhoyroo V.D. J. Biol. Chem. 1988; 263: 14351-14358Abstract Full Text PDF PubMed Google Scholar). This 3′-O-sulfatedN-acetyllactosamine side chain was shown to exist in both bi-antennary and highly branched tri- and tetra-antennaryN-glycans. In porcine thyroglobulin, the majority of sulfo→3Galβ1→4GlcNAcβ1→R are present in side chains derived from the 6-position of α-mannose. Moreover, the same glycoprotein contains a Galβ1→4(sulfo→6)GlcNAc side chain in a portion of the N-glycans (24Waard P.D. Koorevaar A. Kamerling J.P. Vliegenthart J.F.G. J. Biol. Chem. 1991; 266: 4237-4243Abstract Full Text PDF PubMed Google Scholar). As shown previously, sulfo→6GlcNAc→R is first formed from a GlcNAc→R structure and then converted to Galβ1→4(sulfo→6)GlcNAc→R (11Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar). Galβ1→4(sulfo→6)GlcNAc→R can then be converted by Gal3ST-3 to sulfo→3Galβ1→4(sulfo→6)GlcNAc→R, considering that Gal3ST-3 can act on Galβ1→4(sulfo→6)GlcNAc→R. In the structural studies described above, no multisulfated N-glycans were analyzed (24Waard P.D. Koorevaar A. Kamerling J.P. Vliegenthart J.F.G. J. Biol. Chem. 1991; 266: 4237-4243Abstract Full Text PDF PubMed Google Scholar). It is possible that sulfo→3Galβ1→4(sulfo→6)GlcNAc→R may be found with further analysis of highly sulfated N-glycans in thyroglobulin.It has been reported that a major glycoprotein in calf thyroid contains core2-branched O-glycans (44Edge A.S.B. Spiro R.G. Arch. Biochem. Biophys. 1997; 343: 73-80Crossref PubMed Scopus (11) Google Scholar) and that core2 branches in the thyroid are likely synthesized by C2GnT-1 and C2GnT-2 (45Yeh J.-C. Ong E. Fukuda M. J. Biol. Chem. 1999; 274: 3215-3221Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 46Schwientek T. Nomoto M. Levery S.B. Merkx G. van Kessel A.G. Bennett E.P. Hollingsworth M.A. Clausen H. J. Biol. Chem. 1999; 274: 4504-4512Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Core2-branched oligosaccharides from calf thyroid apparently lack 3′-sulfate galactose. In contrast to human thyroglobulin, the presence of 3′-sulfate galactose in calf thyroglobulin is minimal, presumably due to the competition with the strong α1,3-galactosyltransferase activity in calf thyroid (44Edge A.S.B. Spiro R.G. Arch. Biochem. Biophys. 1997; 343: 73-80Crossref PubMed Scopus (11) Google Scholar). It has also been reported that galactose 3-O-sulfotransferase in the thyroid acts only onN-acetyllactosamine as does Gal3ST-3 (25Kato Y. Spiro R.G. J. Biol. Chem. 1989; 264: 3364-3371Abstract Full Text PDF PubMed Google Scholar). These results indicate that Gal3ST-3 is most likely responsible for the formation of sulfo→ 3Galβ1→4GlcNAc attached to bothN-glycans and core2- branched O-glycans synthesized in the thyroid.Although it was reported that the transcripts of Gal3ST-2 are expressed in various tissues, the amount of transcripts was relatively low in brain and kidney when estimated by reverse transcription PCR (18Honke K. Tsuda M. Koyota S. Wada Y. Iida-Tanaka N. Ishizuka I. Nakayama J. Taniguchi N. J. Biol. Chem. 2001; 276: 267-274Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Because the transcripts of Gal3ST-3, on the other hand, are highly expressed in brain and kidney, it is likely that GalST-3 plays a major role in the brain and kidney in addition to the thyroid. Previously, the existence of two different galactosyl 3-O-sulfotransferases has been reported: one (A) acts on core1 O-glycans, whereas the other (B) acts onN-acetyllactosamine (47Chandrasekaran E.V. Jain R.K. Vig R. Matta K.L. Glycobiology. 1997; 7: 753-768Crossref PubMed Scopus (27) Google Scholar, 48Kuhns W. Jain R.K. Matta K.L. Paulsen H. Baker M. Geyer R. Brockhausen I. Glycobiology. 1995; 5: 689-697Crossref PubMed Scopus (40) Google Scholar). Although it is not straightforward to correlate these findings with the acceptor specificities of the cloned enzymes, it appears that A and B correspond to Gal3ST-2 and Gal3ST-3, respectively. On the other hand, the acceptor specificity of Gal3ST-3 appears to be identical to the enzyme described in human airways (17Lo-Guidice J. Perini J. Lafitte J. Ducourouble M. Roussel P. Lamblin G. J. Biol. Chem. 1995; 270: 27544-27550Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), whereas Gal3ST-2, which acts on type 1 oligosaccharides, is most likely responsible for the formation of sulfo→3Galβ1→3(Fucα1→4)GlcNAcβ1→R (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar, 49Tsuji H. Hong J. Kim Y. Ikehara Y. Narimatsu H. Irimura T. Biochem. Biophys. Res. Commun. 1998; 253: 374-381Crossref PubMed Scopus (24) Google Scholar).It has been reported that the sulfo→3Galβ1→4(Fucα1→ 3)GlcNAc→R structure serves as an E-selectin ligand (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar, 50Green P.J. Tamatani T. Watanabe T. Miyasaka M. Hasegawa A. Kiso M. Yuen C.-T. Stoll M.S. Feizi T. Biochem. Biophys. Res. Commun. 1992; 188: 244-251Crossref PubMed Scopus (174) Google Scholar). The same oligosaccharide was, on the other hand, shown to be a ligand for P-selectin but not for E-selectin when a synthetic oligosaccharide attached to GDP-fucose was transferred to the cell surface (5Tsuboi S. Srivastava O.P. Palcic M. Hindsgaul O. Fukuda M. Arch. Biochem. Biophys. 2000; 374: 100-106Crossref PubMed Scopus (11) Google Scholar). Recently, it has been shown that 6-sulfo sialyl Lewis x, NeuNAcα2→3Galβ1→4[Fucα1→3(sulfo→6)]GlcNAcβ1→R, is a potent physiological ligand for L-selectin (11Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar). It will be of significance to determine if a 3′-sulfate analogue, sulfo→3Galβ1→4[Fucα1→3(sulfo→6)]GlcNAcβ1→R, also serves as an L-selectin ligand. Considering the specificity of Gal3ST-3, sulfation should occur first onN-acetylglucosamine and then on galactose, forming sulfo→3Galβ1→4(sulfo→6)GlcNAcβ1→R. This product would then be converted to sulfo→3Galβ1→4[Fucα1→3(sulfo→6)]GlcNAc by α1,3-fucosyltransferase. It is anticipated that all of these different oligosaccharides containing 3′-sulfo galactose can be synthesized in cells with the necessary cDNAs, including those encoding Gal3ST-3.In this context, it is noteworthy that Gal3ST-3 has a relatively narrow acceptor specificity compared with Gal3ST-2. Due to this distinct acceptor specificity, it is likely that the expression of Gal3ST-3 results in the formation of sulfo→3Galβ1→4GlcNAc→R and related structures in a well-defined set of carbohydrates attached to glycoproteins. It is thus expected that the cDNA encoding Gal3ST-3, cloned in the present study, will be a powerful tool to determine the structure/function of sulfo→3Galβ1→4GlcNAc→R and related structures. Sulfate groups in carbohydrates play important roles in conferring highly specific functions on glycoproteins, glycolipids, and proteoglycans (1Fiete D. Srivastave V. Hindsgaul O. Baenziger J.U. Cell. 1991; 67: 1103-1110Abstract Full Text PDF PubMed Scopus (262) Google Scholar, 2Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 3Akama T.O. Nishida K. Nakayama J. Watanabe H. Ozaki K. Nakamura T. Dota A. Kawasaki S. Inoue Y. Maeda N. Yamamoto S. Fujiwara T. Thonar E.J. Shimomura Y. Kinoshita S. Tanigami A. Fukuda M.N. Nat. Genet. 2000; 26: 237-241Crossref PubMed Scopus (221) Google Scholar). One of these sulfated glycans is 3′-sulfo galactose attached to a type 2 oligosaccharide (N-acetyllactosamine), sulfo→3Galβ1→4GlcNAc→R, or attached to a type 1 oligosaccharide, sulfo→3Galβ1→3GlcNAc→R. Feizi and colleagues (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar) demonstrated that 3′-sulfo galactose, in a type 1 or type 2, fucosylated oligosaccharide, functions as an E-selectin ligand (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar). When oligosaccharides were released from ovarian cystadenoma glycoprotein and conjugated to lipids, sulfo→3Galβ1→3/4(Fucα1→4/3)GlcNAcβ1→3Gal was found to bind to Chinese hamster ovary (CHO)1 cells expressing E-selectin (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar). On the other hand, sulfo→3Galβ1→4(Fucα1→3)GlcNAc acted as a P-selectin ligand when a synthetic oligosaccharide with this structure was transferred to cell surface glycoproteins through a fucose residue by α1,3-fucosyltransferase III (5Tsuboi S. Srivastava O.P. Palcic M. Hindsgaul O. Fukuda M. Arch. Biochem. Biophys. 2000; 374: 100-106Crossref PubMed Scopus (11) Google Scholar). These studies suggest that 3′-sulfo galactose on the cell surface plays a role in carbohydrate-protein interactions, including those involved with selectin. Recently, a comparison of the amino acid sequences of cloned sulfotransferases demonstrated that there is a weak but discernible homologous sequence motif among Golgi-associated sulfotransferases (6Ong E. Yeh J.C. Ding Y. Hindsgaul O. Fukuda M. J. Biol. Chem. 1998; 273: 5190-5195Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 7Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (232) Google Scholar, 8Kakuta Y. Pedersen L.G. Pedersen L.C. Negishi M. Trends Biochem. Sci. 1998; 23: 129-130Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 9Ong E. Yeh J.C. Ding Y. Hindsgaul O. Pedersen L.C. Negishi M. Fukuda M. J. Biol. Chem. 1999; 274: 25608-25612Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In particular, the amino acid sequences that are responsible for binding 5′-phosphosulfate and 3′-phosphate groups of the donor substrate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), are well conserved and are often highly homologous to each other among those that share the same acceptor specificity (10Uchimura K. Muramatsu H. Kadomatsu K. Fan Q.W. Kurosawa N. Mitsuoka C. Kannagi R. Habuchi O. Muramatsu T. J. Biol. Chem. 1998; 273: 22577-22583Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 11Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar, 13Xia G. Evers M.R. Kang H-G. Schachner M. Baenziger J.U. J. Biol. Chem. 2000; 276: 38402-38409Abstract Full Text Full Text PDF Scopus (57) Google Scholar, 14Okuda T. Mita S. Yamauchi S. Fukuta M. Nakano H. Sawada T. Habuchi O. J. Biol. Chem. 2000; 275: 40605-40613Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 15Hiraoka, N., Misra, A., Belot, F., Hindsgaul, O., and Fukuda, M. (2001)Glycobiology, in press.Google Scholar). Previously, galactosyl ceramide 3′-sulfotransferase, which forms a sulfatide, sulfo→3Gal→ceramide, has been cloned based on the amino acid sequence of purified protein (16Honke K. Tsuda M. Hirahara Y. Ishii A. Makita A. Wada Y. J. Biol. Chem. 1997; 272: 4864-4868Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Because this enzyme, which we now term Gal3ST-1, is thought not to add a sulfate to glycoproteins (17Lo-Guidice J. Perini J. Lafitte J. Ducourouble M. Roussel P. Lamblin G. J. Biol. Chem. 1995; 270: 27544-27550Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), galactose 3-O-sulfotransferase was molecularly cloned by searching for an enzyme homologous to Gal3ST-1. This reported enzyme, which we now term Gal3ST-2, has the unique property of adding a sulfate on both type 1 and type 2 oligosaccharides and core1O-glycans, Galβ1→3GalNAcα1→R (18Honke K. Tsuda M. Koyota S. Wada Y. Iida-Tanaka N. Ishizuka I. Nakayama J. Taniguchi N. J. Biol. Chem. 2001; 276: 267-274Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). On the other hand, the structures of O-linked oligosaccharides containing 3′-sulfo galactose reported to date show that 3′-sulfo galactose is present in N-acetyllactosamine in core2O-glycans, sulfo→3Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAcα1→R (19Lamblin G. Rahmoune H. Wieruszeski J.-M. Lhermitte M. Strecker G. Roussel P. Biochem. J. 1991; 275: 199-206Crossref PubMed Scopus (43) Google Scholar); core3 O-glycans, sulfo→3Galβ1→4GlcNAcβ1→ 3GalNAc (20Capon C. Leroy Y. Wieruszeski J.-M. Ricart G. Strecker G. Montreuil J. Fournet B. Eur. J. Biochem. 1989; 182: 139-152Crossref PubMed Scopus (74) Google Scholar); and core1 extended structures, sulfo→ 3Galβ1→4GlcNAcβ1→3Gaβ1→3GalNAc (21Capon C. Wieruszeski J.-M. Lemoine J. Byrd J.C. Leffler H. Kim Y.S. J. Biol. Chem. 1997; 272: 31957-31968Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Moreover, no 3′-sulfo galactose in core1 O-glycans such as sulfo→3Galβ1→ 3GalNAcα1→R has been previously reported. Similarly, galactose 3-O-sulfotransferase in human respiratory mucosa was found to act exclusively onN-acetyllactosamine in core2-branched O-glycans and not on core1 O-glycans (17Lo-Guidice J. Perini J. Lafitte J. Ducourouble M. Roussel P. Lamblin G. J. Biol. Chem. 1995; 270: 27544-27550Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The presence of 3′-sulfo galactose in N-glycans has been extensively studied in human, bovine, and porcine thyroglobulins, and these studies showed that 3′-sulfo galactose is present in N-acetyllactosamine in complex N-glycans (22Edge A. Spiro R. J. Biol. Chem. 1984; 259: 4710-4713Abstract Full Text PDF PubMed Google Scholar, 23Spiro R.G. Bhoyroo V.D. J. Biol. Chem. 1988; 263: 14351-14358Abstract Full Text PDF PubMed Google Scholar, 24Waard P.D. Koorevaar A. Kamerling J.P. Vliegenthart J.F.G. J. Biol. Chem. 1991; 266: 4237-4243Abstract Full Text PDF PubMed Google Scholar). Notably, galactose 3-O-sulfotransferase present in the thyroid was found to act only on N-acetyllactosamine but not on type 1 oligosaccharides, which differs from the properties of Gal3ST-2 (25Kato Y. Spiro R.G. J. Biol. Chem. 1989; 264: 3364-3371Abstract Full Text PDF PubMed Google Scholar). Although no glycoprotein acceptor was tested for Gal3ST-2, these results suggested that there is another galactose 3-O-sulfotransferase (Gal3ST) yet to be identified. In the present study, we first identified a novel cDNA by screening the EST data base for cDNAs related to human Gal3ST-1 (16Honke K. Tsuda M. Hirahara Y. Ishii A. Makita A. Wada Y. J. Biol. Chem. 1997; 272: 4864-4868Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The expression of a full-length cDNA revealed that this cDNA encodes a novel galactose 3-O-sulfotransferase, termed Gal3ST-3, that adds a sulfate exclusively in the 3′-position of galactose in N-acetyllactosamine in both N- andO-glycans but not on a type 1 Galβ1→3GlcNAc or core1 Galβ1→3GalNAc structure. When CD43 (leukosialin) was tested as an acceptor, Gal3ST-3 preferentially acted onN-acetyllactosamine present in core2-branchedO-glycans, whereas Gal3ST-2 acted on both core1- and core2-branched O-glycans. Moreover, we show that this novel enzyme is expressed almost exclusively in the thyroid, kidney, and brain, in contrast to the previously reported ubiquitous expression of Gal3ST-2 (18Honke K. Tsuda M. Koyota S. Wada Y. Iida-Tanaka N. Ishizuka I. Nakayama J. Taniguchi N. J. Biol. Chem. 2001; 276: 267-274Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). DISCUSSIONThe present study describes the isolation of a novel cDNA encoding galactose 3-O-sulfotransferase by searching the EST data base for cDNAs homologous to the human galactosyl ceramide 3-O-sulfotransferase, Gal3ST-1 (16Honke K. Tsuda M. Hirahara Y. Ishii A. Makita A. Wada Y. J. Biol. Chem. 1997; 272: 4864-4868Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Gal3ST-1 adds a sulfate to a β-galactose residue linked to ceramide, whereas Gal3ST-3 adds a sulfate to a β-galactose linked to N-acetylglucosamine through a 1,4-linkage. Previously, Gal3ST-2 was also cloned based on its similarity to Gal3ST-1, but the acceptor specificities of Gal3ST-2 and Gal3ST-3 differ substantially. Although Gal3ST-3 acts exclusively on N-acetyllactosamine, Gal3ST-2 can act also on type 1 oligosaccharide, Galβ1→ 3GlcNAc, and core1 oligosaccharide, Galβ1→3(GlcNAcβ1→ 6)GalNAc. On the other hand, Gal3ST-3 can act on Galβ1→ 4(sulfo→6)GlcNAcβ1→R, but Gal3ST-2 cannot act on the same acceptor (Fig. 3). Interestingly, the best acceptor for Gal3ST-3 is poly-N-acetyllactosamine attached to a side chain derived from C-6 of α-mannose, whereas the best acceptor for Gal3ST-2 is N-acetyllactosamine attached to a side chain derived from C-2 of α-mannose (Fig. 3). Moreover, Gal3ST-3 preferentially acts on N-acetyllactosamine attached to anN-glycan mannose core, such as Galβ1→4GlcNAcβ1→6Manα1→6Manβ1→octyl, than onN-acetyllactosamine itself, whereas Gal3ST-2 does not possess this preference (Fig. 3). These results indicate that Gal3ST-2 and Gal3ST-3 act differentially on various acceptor glycoproteins.In the present study, the transcripts of Gal3ST-3 were found to be expressed selectively in the brain, kidney, and thyroid. Previously, it was reported that human thyroglobulin contains a sulfo→3Galβ1→4GlcNAcβ1→R structure in the majority ofN-glycans (23Spiro R.G. Bhoyroo V.D. J. Biol. Chem. 1988; 263: 14351-14358Abstract Full Text PDF PubMed Google Scholar). This 3′-O-sulfatedN-acetyllactosamine side chain was shown to exist in both bi-antennary and highly branched tri- and tetra-antennaryN-glycans. In porcine thyroglobulin, the majority of sulfo→3Galβ1→4GlcNAcβ1→R are present in side chains derived from the 6-position of α-mannose. Moreover, the same glycoprotein contains a Galβ1→4(sulfo→6)GlcNAc side chain in a portion of the N-glycans (24Waard P.D. Koorevaar A. Kamerling J.P. Vliegenthart J.F.G. J. Biol. Chem. 1991; 266: 4237-4243Abstract Full Text PDF PubMed Google Scholar). As shown previously, sulfo→6GlcNAc→R is first formed from a GlcNAc→R structure and then converted to Galβ1→4(sulfo→6)GlcNAc→R (11Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar). Galβ1→4(sulfo→6)GlcNAc→R can then be converted by Gal3ST-3 to sulfo→3Galβ1→4(sulfo→6)GlcNAc→R, considering that Gal3ST-3 can act on Galβ1→4(sulfo→6)GlcNAc→R. In the structural studies described above, no multisulfated N-glycans were analyzed (24Waard P.D. Koorevaar A. Kamerling J.P. Vliegenthart J.F.G. J. Biol. Chem. 1991; 266: 4237-4243Abstract Full Text PDF PubMed Google Scholar). It is possible that sulfo→3Galβ1→4(sulfo→6)GlcNAc→R may be found with further analysis of highly sulfated N-glycans in thyroglobulin.It has been reported that a major glycoprotein in calf thyroid contains core2-branched O-glycans (44Edge A.S.B. Spiro R.G. Arch. Biochem. Biophys. 1997; 343: 73-80Crossref PubMed Scopus (11) Google Scholar) and that core2 branches in the thyroid are likely synthesized by C2GnT-1 and C2GnT-2 (45Yeh J.-C. Ong E. Fukuda M. J. Biol. Chem. 1999; 274: 3215-3221Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 46Schwientek T. Nomoto M. Levery S.B. Merkx G. van Kessel A.G. Bennett E.P. Hollingsworth M.A. Clausen H. J. Biol. Chem. 1999; 274: 4504-4512Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Core2-branched oligosaccharides from calf thyroid apparently lack 3′-sulfate galactose. In contrast to human thyroglobulin, the presence of 3′-sulfate galactose in calf thyroglobulin is minimal, presumably due to the competition with the strong α1,3-galactosyltransferase activity in calf thyroid (44Edge A.S.B. Spiro R.G. Arch. Biochem. Biophys. 1997; 343: 73-80Crossref PubMed Scopus (11) Google Scholar). It has also been reported that galactose 3-O-sulfotransferase in the thyroid acts only onN-acetyllactosamine as does Gal3ST-3 (25Kato Y. Spiro R.G. J. Biol. Chem. 1989; 264: 3364-3371Abstract Full Text PDF PubMed Google Scholar). These results indicate that Gal3ST-3 is most likely responsible for the formation of sulfo→ 3Galβ1→4GlcNAc attached to bothN-glycans and core2- branched O-glycans synthesized in the thyroid.Although it was reported that the transcripts of Gal3ST-2 are expressed in various tissues, the amount of transcripts was relatively low in brain and kidney when estimated by reverse transcription PCR (18Honke K. Tsuda M. Koyota S. Wada Y. Iida-Tanaka N. Ishizuka I. Nakayama J. Taniguchi N. J. Biol. Chem. 2001; 276: 267-274Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Because the transcripts of Gal3ST-3, on the other hand, are highly expressed in brain and kidney, it is likely that GalST-3 plays a major role in the brain and kidney in addition to the thyroid. Previously, the existence of two different galactosyl 3-O-sulfotransferases has been reported: one (A) acts on core1 O-glycans, whereas the other (B) acts onN-acetyllactosamine (47Chandrasekaran E.V. Jain R.K. Vig R. Matta K.L. Glycobiology. 1997; 7: 753-768Crossref PubMed Scopus (27) Google Scholar, 48Kuhns W. Jain R.K. Matta K.L. Paulsen H. Baker M. Geyer R. Brockhausen I. Glycobiology. 1995; 5: 689-697Crossref PubMed Scopus (40) Google Scholar). Although it is not straightforward to correlate these findings with the acceptor specificities of the cloned enzymes, it appears that A and B correspond to Gal3ST-2 and Gal3ST-3, respectively. On the other hand, the acceptor specificity of Gal3ST-3 appears to be identical to the enzyme described in human airways (17Lo-Guidice J. Perini J. Lafitte J. Ducourouble M. Roussel P. Lamblin G. J. Biol. Chem. 1995; 270: 27544-27550Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), whereas Gal3ST-2, which acts on type 1 oligosaccharides, is most likely responsible for the formation of sulfo→3Galβ1→3(Fucα1→4)GlcNAcβ1→R (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar, 49Tsuji H. Hong J. Kim Y. Ikehara Y. Narimatsu H. Irimura T. Biochem. Biophys. Res. Commun. 1998; 253: 374-381Crossref PubMed Scopus (24) Google Scholar).It has been reported that the sulfo→3Galβ1→4(Fucα1→ 3)GlcNAc→R structure serves as an E-selectin ligand (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar, 50Green P.J. Tamatani T. Watanabe T. Miyasaka M. Hasegawa A. Kiso M. Yuen C.-T. Stoll M.S. Feizi T. Biochem. Biophys. Res. Commun. 1992; 188: 244-251Crossref PubMed Scopus (174) Google Scholar). The same oligosaccharide was, on the other hand, shown to be a ligand for P-selectin but not for E-selectin when a synthetic oligosaccharide attached to GDP-fucose was transferred to the cell surface (5Tsuboi S. Srivastava O.P. Palcic M. Hindsgaul O. Fukuda M. Arch. Biochem. Biophys. 2000; 374: 100-106Crossref PubMed Scopus (11) Google Scholar). Recently, it has been shown that 6-sulfo sialyl Lewis x, NeuNAcα2→3Galβ1→4[Fucα1→3(sulfo→6)]GlcNAcβ1→R, is a potent physiological ligand for L-selectin (11Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar). It will be of significance to determine if a 3′-sulfate analogue, sulfo→3Galβ1→4[Fucα1→3(sulfo→6)]GlcNAcβ1→R, also serves as an L-selectin ligand. Considering the specificity of Gal3ST-3, sulfation should occur first onN-acetylglucosamine and then on galactose, forming sulfo→3Galβ1→4(sulfo→6)GlcNAcβ1→R. This product would then be converted to sulfo→3Galβ1→4[Fucα1→3(sulfo→6)]GlcNAc by α1,3-fucosyltransferase. It is anticipated that all of these different oligosaccharides containing 3′-sulfo galactose can be synthesized in cells with the necessary cDNAs, including those encoding Gal3ST-3.In this context, it is noteworthy that Gal3ST-3 has a relatively narrow acceptor specificity compared with Gal3ST-2. Due to this distinct acceptor specificity, it is likely that the expression of Gal3ST-3 results in the formation of sulfo→3Galβ1→4GlcNAc→R and related structures in a well-defined set of carbohydrates attached to glycoproteins. It is thus expected that the cDNA encoding Gal3ST-3, cloned in the present study, will be a powerful tool to determine the structure/function of sulfo→3Galβ1→4GlcNAc→R and related structures. The present study describes the isolation of a novel cDNA encoding galactose 3-O-sulfotransferase by searching the EST data base for cDNAs homologous to the human galactosyl ceramide 3-O-sulfotransferase, Gal3ST-1 (16Honke K. Tsuda M. Hirahara Y. Ishii A. Makita A. Wada Y. J. Biol. Chem. 1997; 272: 4864-4868Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Gal3ST-1 adds a sulfate to a β-galactose residue linked to ceramide, whereas Gal3ST-3 adds a sulfate to a β-galactose linked to N-acetylglucosamine through a 1,4-linkage. Previously, Gal3ST-2 was also cloned based on its similarity to Gal3ST-1, but the acceptor specificities of Gal3ST-2 and Gal3ST-3 differ substantially. Although Gal3ST-3 acts exclusively on N-acetyllactosamine, Gal3ST-2 can act also on type 1 oligosaccharide, Galβ1→ 3GlcNAc, and core1 oligosaccharide, Galβ1→3(GlcNAcβ1→ 6)GalNAc. On the other hand, Gal3ST-3 can act on Galβ1→ 4(sulfo→6)GlcNAcβ1→R, but Gal3ST-2 cannot act on the same acceptor (Fig. 3). Interestingly, the best acceptor for Gal3ST-3 is poly-N-acetyllactosamine attached to a side chain derived from C-6 of α-mannose, whereas the best acceptor for Gal3ST-2 is N-acetyllactosamine attached to a side chain derived from C-2 of α-mannose (Fig. 3). Moreover, Gal3ST-3 preferentially acts on N-acetyllactosamine attached to anN-glycan mannose core, such as Galβ1→4GlcNAcβ1→6Manα1→6Manβ1→octyl, than onN-acetyllactosamine itself, whereas Gal3ST-2 does not possess this preference (Fig. 3). These results indicate that Gal3ST-2 and Gal3ST-3 act differentially on various acceptor glycoproteins. In the present study, the transcripts of Gal3ST-3 were found to be expressed selectively in the brain, kidney, and thyroid. Previously, it was reported that human thyroglobulin contains a sulfo→3Galβ1→4GlcNAcβ1→R structure in the majority ofN-glycans (23Spiro R.G. Bhoyroo V.D. J. Biol. Chem. 1988; 263: 14351-14358Abstract Full Text PDF PubMed Google Scholar). This 3′-O-sulfatedN-acetyllactosamine side chain was shown to exist in both bi-antennary and highly branched tri- and tetra-antennaryN-glycans. In porcine thyroglobulin, the majority of sulfo→3Galβ1→4GlcNAcβ1→R are present in side chains derived from the 6-position of α-mannose. Moreover, the same glycoprotein contains a Galβ1→4(sulfo→6)GlcNAc side chain in a portion of the N-glycans (24Waard P.D. Koorevaar A. Kamerling J.P. Vliegenthart J.F.G. J. Biol. Chem. 1991; 266: 4237-4243Abstract Full Text PDF PubMed Google Scholar). As shown previously, sulfo→6GlcNAc→R is first formed from a GlcNAc→R structure and then converted to Galβ1→4(sulfo→6)GlcNAc→R (11Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar). Galβ1→4(sulfo→6)GlcNAc→R can then be converted by Gal3ST-3 to sulfo→3Galβ1→4(sulfo→6)GlcNAc→R, considering that Gal3ST-3 can act on Galβ1→4(sulfo→6)GlcNAc→R. In the structural studies described above, no multisulfated N-glycans were analyzed (24Waard P.D. Koorevaar A. Kamerling J.P. Vliegenthart J.F.G. J. Biol. Chem. 1991; 266: 4237-4243Abstract Full Text PDF PubMed Google Scholar). It is possible that sulfo→3Galβ1→4(sulfo→6)GlcNAc→R may be found with further analysis of highly sulfated N-glycans in thyroglobulin. It has been reported that a major glycoprotein in calf thyroid contains core2-branched O-glycans (44Edge A.S.B. Spiro R.G. Arch. Biochem. Biophys. 1997; 343: 73-80Crossref PubMed Scopus (11) Google Scholar) and that core2 branches in the thyroid are likely synthesized by C2GnT-1 and C2GnT-2 (45Yeh J.-C. Ong E. Fukuda M. J. Biol. Chem. 1999; 274: 3215-3221Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 46Schwientek T. Nomoto M. Levery S.B. Merkx G. van Kessel A.G. Bennett E.P. Hollingsworth M.A. Clausen H. J. Biol. Chem. 1999; 274: 4504-4512Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Core2-branched oligosaccharides from calf thyroid apparently lack 3′-sulfate galactose. In contrast to human thyroglobulin, the presence of 3′-sulfate galactose in calf thyroglobulin is minimal, presumably due to the competition with the strong α1,3-galactosyltransferase activity in calf thyroid (44Edge A.S.B. Spiro R.G. Arch. Biochem. Biophys. 1997; 343: 73-80Crossref PubMed Scopus (11) Google Scholar). It has also been reported that galactose 3-O-sulfotransferase in the thyroid acts only onN-acetyllactosamine as does Gal3ST-3 (25Kato Y. Spiro R.G. J. Biol. Chem. 1989; 264: 3364-3371Abstract Full Text PDF PubMed Google Scholar). These results indicate that Gal3ST-3 is most likely responsible for the formation of sulfo→ 3Galβ1→4GlcNAc attached to bothN-glycans and core2- branched O-glycans synthesized in the thyroid. Although it was reported that the transcripts of Gal3ST-2 are expressed in various tissues, the amount of transcripts was relatively low in brain and kidney when estimated by reverse transcription PCR (18Honke K. Tsuda M. Koyota S. Wada Y. Iida-Tanaka N. Ishizuka I. Nakayama J. Taniguchi N. J. Biol. Chem. 2001; 276: 267-274Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Because the transcripts of Gal3ST-3, on the other hand, are highly expressed in brain and kidney, it is likely that GalST-3 plays a major role in the brain and kidney in addition to the thyroid. Previously, the existence of two different galactosyl 3-O-sulfotransferases has been reported: one (A) acts on core1 O-glycans, whereas the other (B) acts onN-acetyllactosamine (47Chandrasekaran E.V. Jain R.K. Vig R. Matta K.L. Glycobiology. 1997; 7: 753-768Crossref PubMed Scopus (27) Google Scholar, 48Kuhns W. Jain R.K. Matta K.L. Paulsen H. Baker M. Geyer R. Brockhausen I. Glycobiology. 1995; 5: 689-697Crossref PubMed Scopus (40) Google Scholar). Although it is not straightforward to correlate these findings with the acceptor specificities of the cloned enzymes, it appears that A and B correspond to Gal3ST-2 and Gal3ST-3, respectively. On the other hand, the acceptor specificity of Gal3ST-3 appears to be identical to the enzyme described in human airways (17Lo-Guidice J. Perini J. Lafitte J. Ducourouble M. Roussel P. Lamblin G. J. Biol. Chem. 1995; 270: 27544-27550Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), whereas Gal3ST-2, which acts on type 1 oligosaccharides, is most likely responsible for the formation of sulfo→3Galβ1→3(Fucα1→4)GlcNAcβ1→R (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar, 49Tsuji H. Hong J. Kim Y. Ikehara Y. Narimatsu H. Irimura T. Biochem. Biophys. Res. Commun. 1998; 253: 374-381Crossref PubMed Scopus (24) Google Scholar). It has been reported that the sulfo→3Galβ1→4(Fucα1→ 3)GlcNAc→R structure serves as an E-selectin ligand (4Yuen C.-T. Lawson A.M. Chai W. Larkin M. Stoll M.S. Stuart A.C. Sullivan F.X. Ahern T.J. Feizi T. Biochemistry. 1992; 31: 9126-9131Crossref PubMed Scopus (238) Google Scholar, 50Green P.J. Tamatani T. Watanabe T. Miyasaka M. Hasegawa A. Kiso M. Yuen C.-T. Stoll M.S. Feizi T. Biochem. Biophys. Res. Commun. 1992; 188: 244-251Crossref PubMed Scopus (174) Google Scholar). The same oligosaccharide was, on the other hand, shown to be a ligand for P-selectin but not for E-selectin when a synthetic oligosaccharide attached to GDP-fucose was transferred to the cell surface (5Tsuboi S. Srivastava O.P. Palcic M. Hindsgaul O. Fukuda M. Arch. Biochem. Biophys. 2000; 374: 100-106Crossref PubMed Scopus (11) Google Scholar). Recently, it has been shown that 6-sulfo sialyl Lewis x, NeuNAcα2→3Galβ1→4[Fucα1→3(sulfo→6)]GlcNAcβ1→R, is a potent physiological ligand for L-selectin (11Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar). It will be of significance to determine if a 3′-sulfate analogue, sulfo→3Galβ1→4[Fucα1→3(sulfo→6)]GlcNAcβ1→R, also serves as an L-selectin ligand. Considering the specificity of Gal3ST-3, sulfation should occur first onN-acetylglucosamine and then on galactose, forming sulfo→3Galβ1→4(sulfo→6)GlcNAcβ1→R. This product would then be converted to sulfo→3Galβ1→4[Fucα1→3(sulfo→6)]GlcNAc by α1,3-fucosyltransferase. It is anticipated that all of these different oligosaccharides containing 3′-sulfo galactose can be synthesized in cells with the necessary cDNAs, including those encoding Gal3ST-3. In this context, it is noteworthy that Gal3ST-3 has a relatively narrow acceptor specificity compared with Gal3ST-2. Due to this distinct acceptor specificity, it is likely that the expression of Gal3ST-3 results in the formation of sulfo→3Galβ1→4GlcNAc→R and related structures in a well-defined set of carbohydrates attached to glycoproteins. It is thus expected that the cDNA encoding Gal3ST-3, cloned in the present study, will be a powerful tool to determine the structure/function of sulfo→3Galβ1→4GlcNAc→R and related structures. We thank Dr. Yili Ding for useful discussion and Joseph P. Henig and Shizuka Mitoma for organizing the manuscript.

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