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Extended Core 1 and Core 2 Branched O-Glycans Differentially Modulate Sialyl Lewis x-type L-selectin Ligand Activity

2003; Elsevier BV; Volume: 278; Issue: 11 Linguagem: Inglês

10.1074/jbc.m212756200

ISSN

1083-351X

Autores

Junya Mitoma, Bronislawa Petryniak, Nobuyoshi Hiraoka, Jiunn‐Chern Yeh, John B. Lowe, Minoru Fukuda,

Tópico(s)

Proteoglycans and glycosaminoglycans research

Resumo

It has been established that sialyl Lewis x in core 2 branched O-glycans serves as an E- and P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis x in extended core 1 O-glycans, NeuNAcα2→3Galβ1→4(Fucα1→3(sulfo→6))GlcNAcβ1→ 3Galβ1→3GalNAcα1→Ser/Thr, functions as an L-selectin ligand in high endothelial venules. Extended core 1O-glycans can be synthesized when a core 1 extension enzyme is present. In this study, we first show that β1,3-N-acetylglucosaminyltransferase-3 (β3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different β3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding β3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 β1,6-N-acetylglucosaminyltransferase I (Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either β3GlcNAcT-3 or Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of sialyl Lewis x in extended core 1 and core 2 branchedO-glycans. In a rolling assay, CHO cells expressing sialyl Lewis x in extended core 1 O-glycans supported a significant degree of shear-dependent tethering and rolling of neutrophils and lymphocytes, although less than CHO cells expressing sialyl Lewis x in core 2 branched O-glycans. These results indicate that sialyl Lewis x in extended core 1 O-glycans can function as an L-selectin ligand and is potentially involved in neutrophil adhesion on neutrophils bound to activated endothelial cells. It has been established that sialyl Lewis x in core 2 branched O-glycans serves as an E- and P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis x in extended core 1 O-glycans, NeuNAcα2→3Galβ1→4(Fucα1→3(sulfo→6))GlcNAcβ1→ 3Galβ1→3GalNAcα1→Ser/Thr, functions as an L-selectin ligand in high endothelial venules. Extended core 1O-glycans can be synthesized when a core 1 extension enzyme is present. In this study, we first show that β1,3-N-acetylglucosaminyltransferase-3 (β3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different β3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding β3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 β1,6-N-acetylglucosaminyltransferase I (Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either β3GlcNAcT-3 or Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of sialyl Lewis x in extended core 1 and core 2 branchedO-glycans. In a rolling assay, CHO cells expressing sialyl Lewis x in extended core 1 O-glycans supported a significant degree of shear-dependent tethering and rolling of neutrophils and lymphocytes, although less than CHO cells expressing sialyl Lewis x in core 2 branched O-glycans. These results indicate that sialyl Lewis x in extended core 1 O-glycans can function as an L-selectin ligand and is potentially involved in neutrophil adhesion on neutrophils bound to activated endothelial cells. Mucin-type O-glycans are unique in having clusters of large numbers of O-glycans. These O-glycans contain N-acetylgalactosamine residues at reducing ends, which are linked to serine or threonine residues in a polypeptide (1Fukuda M. Fukuda M. Hindsgaul O. Molecular Glycobiology. Oxford University Press, Oxford1994: 1-52Google Scholar). These attached O-glycans can be classified into several different groups according to the core structure (2Schachter H. Brockhausen I. Allen H.J. Kisailus E.C. Glycoconjugates: Composition, Structure, and Function. Marcel Decker, Inc., New York1992: 263-332Google Scholar). In many cells, a structure called 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 β1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT) 1The abbreviations used are: Core2GlcNAcT-I, core 2 β1,6-N-acetylglucosaminyltransferase I; HEV, high endothelial venule(s); LSST, L-selectin ligand sulfotransferase; FucT-VII, fucosyltransferase VII; β3GlcNAcT, β1,3-N-acetylglucosaminyltransferase; CHO, Chinese hamster ovary; RT, reverse transcription; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting; HPLC, high performance liquid chromatography 1The abbreviations used are: Core2GlcNAcT-I, core 2 β1,6-N-acetylglucosaminyltransferase I; HEV, high endothelial venule(s); LSST, L-selectin ligand sulfotransferase; FucT-VII, fucosyltransferase VII; β3GlcNAcT, β1,3-N-acetylglucosaminyltransferase; CHO, Chinese hamster ovary; RT, reverse transcription; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting; HPLC, high performance liquid chromatography is present (3Piller F. Piller V. Fox R.I. Fukuda M. J. Biol. Chem. 1988; 263: 15146-15150Google Scholar, 4Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Google Scholar). Various ligand carbohydrates can be formed in core 2 branched oligosaccharides. For example, sialyl Lewis x in mucin-type glycoproteins of blood cells can be found in core 2 branched oligosaccharides such as NeuNAcα2→3Galβ1→4(Fucα1→3)GlcNAcβ1→6(NeuNAcα2→ 3Galβ1→3)GalNAcα1→Ser/Thr (see Fig. 1) (5Fukuda M. Carlsson S.R. Klock J.C. Dell A. J. Biol. Chem. 1986; 261: 12796-12806Google Scholar, 6Wilkins P.P. McEver R.P. Cummings R.D. J. Biol. Chem. 1996; 271: 18732-18742Google Scholar). When leukocytes are recruited to inflammatory sites, E- and P-selectins expressed in activated endothelial cells recognize α2→3-sialylated, α1→3-fucosylated O-glycans, allowing leukocytes to eventually extravasate (7Lowe J.B. Stoolman L.M. Nair R.P. Larsen R.D. Berhend T.L. Marks R.M. Cell. 1990; 63: 475-484Google Scholar, 8Phillips M.L. Nudelman E. Gaeta F.C.A. Perez M. Singhai A.K. Hakomori S.I. Paulson J.C. Science. 1990; 250: 1130-1132Google Scholar, 9Walz G. Aruffo A. Kolanus W. Bevilacqua M. Seed B. Science. 1990; 250: 1132-1135Google Scholar). It has also been demonstrated that L-selectin in neutrophils mediates neutrophil rolling on neutrophils adherent to activated endothelial cells (10Bargatze R.F. Kurk S. Butcher E.C. Jutila M.A. J. Exp. Med. 1994; 180: 1785-1792Google Scholar, 11Alon R. Fuhlbrigge R.C. Finger E.B. Springer T.A. J. Cell Biol. 1996; 135: 849-865Google Scholar). PSGL-1 (P-selectin glycoproteinligand-1) contributes to this process, although the nature of the glycan moieties that decorate this and other potential L-selectin ligands in neutrophils has not been well characterized. On the other hand, L-selectin in lymphocytes recognizes 6-sulfosialyl Lewis x in core 2 branched O-glycans, NeuNAcα2→3Galβ1→4(Fucα1→3(sulfo→6))GlcNAcβ1→ 6(Galβ1→3)GalNAcα1→Ser/Thr, which are expressed in high endothelial venules (HEV) in lymph nodes (12Hiraoka 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: 79-89Google Scholar, 13Tangemann K. Bistrup A. Hemmerich S. Rosen S.D. J. Exp. Med. 1999; 190: 935-942Google Scholar, 14Mitsuoka C. Sawada-Kasugai M. Ando-Furui K. Izawa M. Nakanishi H. Nakamura S. Ishida H. Kiso M. Kannagi R. J. Biol. Chem. 1998; 273: 11225-11233Google Scholar). Such recognition leads to lymphocyte adhesion to HEV, allowing lymphocyte movement from the vascular to the lymphatic system. The formation of 6-sulfosialyl Lewis x depends on 6-sulfotransferase, designated L-selectin ligand sulfotransferase (LSST) or high endothelial cellN-acetylglucosamine 6-O-sulfotransferase (12Hiraoka 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: 79-89Google Scholar, 15Bistrup 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: 899-910Google Scholar, 16Hemmerich S. Bistrup A. Singer M. van Zante A. Lee J.K. Tsay D. Peters M. Carminati J.L. Brennan T.J. Carver-Moore K. Leviten M. Fuentes M. Ruddle N.H. Rosen S.D. Immunity. 2001; 15: 237-247Google Scholar). The roles of sialyl Lewis x in core 2 branched O-glycans have been demonstrated by analyzing mutant mice with deficient Core2GlcNAcT-I, obtained through homologous recombination (17Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; 9: 881-890Google Scholar). Leukocytes derived from null mutant mice display significantly reduced adhesion to L-, P-, and E-selectins, demonstrating that ligands for these selectins are mainly carried by core 2 branchedO-glycans. By contrast, in these mice, lymphocyte adhesion to HEV in lymph nodes is only marginally impaired, and MECA-79 antibody staining that decorates HEV is not reduced (17Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; 9: 881-890Google Scholar). Recent studies reveal that L-selectin ligand activity remaining after abrogation of Core2GlcNAcT-I is due to the activity of 6-sulfosialyl Lewis x in extended core 1 O-glycans, NeuNAcα2→3Galβ1→4[Fucα1→3(sulfo→6)]GlcNAcβ1→ 3Galβ1→3GalNAcα1→Ser/Thr (18Yeh J.-C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Cell. 2001; 105: 957-969Google Scholar). Moreover, a minimum MECA-79 epitope was found to be a 6-sulfo structure in the extended core 1O-glycan, and MECA-79 antibody binds efficiently to 6-sulfosialyl Lewis x-containing extended core 1 O-glycans (18Yeh J.-C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Cell. 2001; 105: 957-969Google Scholar). These findings are consistent with previous findings that MECA-79 antibody inhibits lymphocyte adhesion to HEV without removal of sialic acid (19Streeter P.R. Rouse B.T. Butcher E.C. J. Cell Biol. 1988; 107: 1853-1862Google Scholar) or fucose and that MECA-79 staining remains after expression of fucose is abrogated by inactivation of fucosyltransferase VII (FucT-VII) (20Maly P. Thall A. Petryniak B. Rogers C.E. Sith P.L. Marks R.M. Kelly R.J. Gersten K.M. Cheng G. Saunders T.L. Camper S.A. Camphausen R.T. Sullivan F.X. Isogai Y. Hindsgaul O. von Andrian U.H. Lowe J.B. Cell. 1996; 86: 643-653Google Scholar). Extended core 1 structure is synthesized by core 1 β1,3-N-acetylglucosaminyltransferase (β3GlcNAcT), which adds β1,3-linked N-acetylglucosamine to Galβ1→3GalNAcα1→R (Fig. 1). A cDNA encoding β3GlcNAcT was first cloned by expression cloning, and the encoded protein was designated both i-antigen forming β1,3-N-acetylglucosaminyltransferase (21Sasaki K. Kurata-Miura K. Ujita M. Angata K. Nakagawa S. Sekine S. Nishi T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14294-14299Google Scholar) and β3GlcNAcT-1 (22Shiraishi N. Natsume A. Togayachi A. Endo T. Akashima T. Yamada Y. Imai N. Nakagawa S. Koizumi S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; 276: 3498-3507Google Scholar, 23Fukuda M. Taniguchi N. Honke K. Fukuda M. Handbook of Glycosyltransferases and Related Genes. Springer-Verlag, Berlin2002: 114-124Google Scholar). Expression of β3GlcNAcT-1 does not, however, result in the synthesis of extended core 1 structure (18Yeh J.-C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Cell. 2001; 105: 957-969Google Scholar). By screening expressed sequence tag data bases using the cDNA encoding the now designated β1,3-galactosyltransferase-6 as a probe, cDNA encoding core 1-β3GlcNAcT was identified (18Yeh J.-C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Cell. 2001; 105: 957-969Google Scholar). This cloning was possible because it was reported that β1,3-galactosyltransferase and β3GlcNAcT are highly homologous proteins (24Zhou D. Dinter A. Gutierrez Gallego R. Kamerling J.P. Vliegenthart J.F. Berger E.G. Hennet T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 406-411Google Scholar). In parallel, at least five additional β3GlcNAcTs have been molecularly identified based on similarity to the previously cloned β1,3-galactosyltransferase or to β3GlcNAcT-2 (22Shiraishi N. Natsume A. Togayachi A. Endo T. Akashima T. Yamada Y. Imai N. Nakagawa S. Koizumi S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; 276: 3498-3507Google Scholar, 25Togayachi A. Akashima T. Ookubo R. Kudo T. Nishihara S. Iwasaki H. Natsume A. Mio H. Inokuchi J. Irimura T. Sasaki K. Narimatsu H. J. Biol. Chem. 2001; 276: 22032-22040Google Scholar, 26Henion T.R. Zhou D. Wolfer D.P. Jungalwala G.B. Hennet T. J. Biol. Chem. 2001; 276: 30261-30269Google Scholar, 27Iwai T. Inaba N. Naundorf A. Zhang Y. Gotoh M. Iwasaki H. Kudo T. Togayachi A. Ishizuka Y. Nakanishi H. Narimatsu H. J. Biol. Chem. 2002; 277: 12802-12809Google Scholar, 28Kataoka K. Huh N. Biochem. Biophys. Res. Commun. 2002; 294: 843-848Google Scholar). These results indicate that core 1-β3GlcNAcT belongs to the β3GlcNAcT gene family, and it is thus designated β3GlcNAcT-3. β3GlcNAcT-3 transcripts are highly expressed in the small intestine, colon, and placenta and are moderately expressed in various tissues, including the liver, kidney, pancreas, and prostate (18Yeh J.-C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Cell. 2001; 105: 957-969Google Scholar). It is not known whether blood cells express β3GlcNAcT-3. In this study, we address the function and expression pattern of β3GlcNAcT-3. First, we show that β3GlcNAcT-3 was the only enzyme that significantly formed extended core 1 among highly related β3GlcNAcTs. We then show that β3GlcNAcT-3 transcripts were present in both human neutrophils and lymphocytes, but that these cells lacked LSST. Transfection studies using FucT-VII and β3GlcNAcT-3 or Core2GlcNAcT-I showed that extended core 1 could be synthesized in Chinese hamster ovary (CHO) cells and that extended core 1 structure was fucosylated by FucT-VII more efficiently than core 2 branches. Finally, we show that sialyl Lewis x synthesized in extended core 1 served as an L-selectin ligand, although it is apparently less potent than sialyl Lewis x in core 2 branches. cDNA cloning of β3GlcNAcT-3 was described previously (18Yeh J.-C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Cell. 2001; 105: 957-969Google Scholar). cDNAs encoding human β3GlcNAcT-1 (21Sasaki K. Kurata-Miura K. Ujita M. Angata K. Nakagawa S. Sekine S. Nishi T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14294-14299Google Scholar), β3GlcNAcT-2 (22Shiraishi N. Natsume A. Togayachi A. Endo T. Akashima T. Yamada Y. Imai N. Nakagawa S. Koizumi S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; 276: 3498-3507Google Scholar), β3GlcNAcT-4 (22Shiraishi N. Natsume A. Togayachi A. Endo T. Akashima T. Yamada Y. Imai N. Nakagawa S. Koizumi S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; 276: 3498-3507Google Scholar), β3GlcNAcT-5 (25Togayachi A. Akashima T. Ookubo R. Kudo T. Nishihara S. Iwasaki H. Natsume A. Mio H. Inokuchi J. Irimura T. Sasaki K. Narimatsu H. J. Biol. Chem. 2001; 276: 22032-22040Google Scholar, 26Henion T.R. Zhou D. Wolfer D.P. Jungalwala G.B. Hennet T. J. Biol. Chem. 2001; 276: 30261-30269Google Scholar), β3GlcNAcT-6 (27Iwai T. Inaba N. Naundorf A. Zhang Y. Gotoh M. Iwasaki H. Kudo T. Togayachi A. Ishizuka Y. Nakanishi H. Narimatsu H. J. Biol. Chem. 2002; 277: 12802-12809Google Scholar), and β3GlcNAcT-7 (28Kataoka K. Huh N. Biochem. Biophys. Res. Commun. 2002; 294: 843-848Google Scholar) were cloned by reverse transcription (RT)-PCR using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) (29Barnes W.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2216-2220Google Scholar). RT-PCR primers were as follows: β3GlcNAcT-1, 5′-CGAGAGCCATGCAGATGTCCTAC-3′ (5′-primer) and 5′-AAGGGCTCAGCAGCGTCGGGGAG-3′ (3′-primer); β3GlcNAcT-2, 5′-GACAAGATATGAGAAATGAGTGTTGG-3′ (5′-primer) and 5′-TTTTAGCATTTTAAATGAGCACTCTGC-3′ (3′-primer); β3GlcNAcT-4, 5′-AGCACGGAGACAGTCTCCAGCTG-3′ (5′-primer) and 5′-AGGCATCAATTTCGCATCACGATAG-3′ (3′-primer); β3GlcNAcT-5, 5′-AGACTTGAGTGGATATGAGAATGTTG-3′ (5′-primer) and 5′-AAGTACTATTAGATAAACGCAGCCCT-3′ (3′-primer); β3GlcNAcT-6, 5′-ACGCTCAAGCACCTGCACTTGCT-3′ (5′-primer) and 5′-ACTGGCCTCAGGAGACCCGGTG-3′ (3′-primer); and β3GlcNAcT-7, 5′-GCCGCCATGTCGCTGTGGAAGA-3′ (5′-primer) and 5′GGGTCAGAGCACCTGGAGCTTG-3′ (3′-primer). As templates, we used a human fetal brain cDNA library in pcDNA1 (30Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Google Scholar) for β3GlcNAcT-1, human esophagus cDNA in a human digestive system multiple tissue cDNA panel (BD Biosciences) for β3GlcNAcT-2, an SK-N-MC cell cDNA library (see below) for β3GlcNAcT-4, human pituitary gland Marathon-Ready cDNA (BD Biosciences) for β3GlcNAc-5, human stomach cDNA in a human digestive system multiple tissue cDNA panel (BD Biosciences) for β3GlcNAcT-6, and human colon Marathon-Ready cDNA (BD Biosciences) for β3GlcNAcT-7. A cDNA library of SK-N-MC neuroblastoma cells was prepared by isolation of total RNA with TRIzol (Invitrogen), followed by first-strand cDNA synthesis using SuperScript II RNase H− reverse transcriptase (Invitrogen). PCR products were first inserted into pCR2.1-TOPO (Invitrogen). In the case of β3GlcNAcT-1, -2, -4, -6, and -7, cDNAs in pCR2.1-TOPO were digested with EcoRI and inserted into the dephosphorylated EcoRI site of pcDNA3.1(N). pcDNA3.1(N) is a vector created by the digestion of pcDNA3.1/Zeo with SphI and BspLU11I (Roche Molecular Biochemicals), followed by filling in and self-ligation to remove the Zeocin resistance gene and f1 origin. For β3GlcNAcT-5, cDNA in pCR2.1-TOPO was digested with EcoRI andScaI and subcloned into theEcoRI-EcoRV sites of pcDNA3.1(N). Similarly, β3GlcNAcT-3 was cloned into the HindIII-XhoI sites of pcDNA1.1, resulting in pcDNA1.1-β3GlcNAcT-3. cDNA encoding LSST was cloned as described previously (12Hiraoka 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: 79-89Google Scholar, 15Bistrup 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: 899-910Google Scholar). To determine whether all of β3GlcNAcTs direct the synthesis of poly-N-acetyllactosamine synthesis, HeLa cells were transiently transfected with one of the pcDNA3.1(N)-β3GlcNAcTs or pcDNA1.1-β3GlcNAcT-3. Thirty-six hours after transfection, cells were dissociated into monodispersed cells using an enzyme-free cell dissociation solution (Hanks' balanced saline solution-based) purchased from Cell and Molecular Technologies. Monodispersed cells were incubated with human anti-i serum (Dench) (31Feizi T. Childs R.A. Watanabe K. Hakomori S.I. J. Exp. Med. 1979; 149: 975-980Google Scholar), followed by affinity-purified fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgM antibodies (Pierce). The stained cells were subjected to FACS analysis using a FACScan (BD Biosciences) as described previously (32Ohyama C. Tsuboi S. Fukuda M. EMBO J. 1999; 18: 1516-1525Google Scholar). HeLa cells were chosen as recipient cells for transfection because the molecular mass of lysosome-associated membrane protein-1, a major carrier of poly-N-acetyllactosamines (33Fukuda M. J. Biol. Chem. 1991; 266: 21327-21330Google Scholar), is the smallest among Namalwa, HL-60, CHO, HepG2, and HeLa cells. The results indicated that HeLa cells express minimum amounts of β3GlcNAcTs because poly-N-acetyllactosaminylated lysosome-associated membrane protein-1 displays a higher molecular mass than lysosome-associated membrane protein-1 containing minimum amounts of poly-N-acetyllactosamine (33Fukuda M. J. Biol. Chem. 1991; 266: 21327-21330Google Scholar). To determine which β3GlcNAcT directs expression of MECA-79 antigen, Lec2 cells were transiently transfected with pcDNA1-LSST and one of the pcDNA3.1(N)-β3GlcNAcTs or pcDNA1.1-β3GlcNAcT-3. Thirty-six hours after transfection, cells were dissociated into monodispersed cells using the cell dissociation solution as described above. Monodispersed cells were incubated with MECA-79 antibody (BD Biosciences) (19Streeter P.R. Rouse B.T. Butcher E.C. J. Cell Biol. 1988; 107: 1853-1862Google Scholar), followed by affinity-purified FITC-conjugated goat anti-rat IgM antibody (ICN Biochemicals). The stained cells were subjected to FACS analysis as described above. CHO mutant Lec2 cells lack a functional Golgi CMP-sialic acid transporter; and therefore, sialylation is absent in Lec2 cells (34Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I. Hirschberg C.B. Cell. 1984; 39: 295-299Google Scholar). The absence of sialylation facilitates core 1 extension because core 1 extension and sialylation compete with each other for the same acceptor, Galβ1→3GalNAcα1→R. To determine which β3GlcNAcT can add β1,3-N-acetylglucosamine to core 1, Galβ1→3GalNAcα1→R, Lec2 cells were transiently transfected with pZeoSV-PSGL-1 (kindly provided by Dr. Richard Cummings) and vectors encoding β3GlcNAcT using LipofectAMINE as described previously (12Hiraoka 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: 79-89Google Scholar). The ratio of pZeoSV-PSGL-1 and β3GlcNAcT cDNA was 1:5 (w/w) to achieve efficient modification of PSGL-1 by β3GlcNAcT. Forty-eight hours after transfection, cells were harvested in phosphate-buffered saline with a cell scraper. The cells were subjected to three cycles of freezing and thawing to disrupt the plasma membrane. The membrane fraction was collected by centrifugation at 12,000 × g for 10 min. The resultant membrane fraction was first resuspended in 10 mm Tris-HCl and 1 mm EDTA (pH 8.0), and then 10% Triton X-100 was added to a final concentration of 1%. After gentle rocking at 4 °C for 15 min, the Triton X-100-soluble membrane fraction, containing PSGL-1, was obtained by centrifugation at 12,000 × g for 10 min. The membrane fraction was then lysed in sample buffer and subjected to SDS-PAGE. After blotting onto a polyvinylidene difluoride membrane filter, the blot was reacted with anti-PSGL-1 antibody (KPL-1, BD Biosciences), followed by secondary antibody; and immunoreactive PSGL-1 was visualized using enhanced Luminol reagent (PerkinElmer Life Sciences). Human neutrophils and lymphocytes were isolated from the peripheral blood of a volunteer as described previously (35Clark R.A. Nuaseef W.M. Coligan J.E. Current Protocols in Immunology. John Wiley & Sons, Inc., New York1994: 7.23.1-7.23.17Google Scholar). Briefly, blood was drawn in a syringe containing heparin, and erythrocytes were sedimented by dextran/saline solution, obtaining leukocyte-rich plasma in the upper layer. Ficoll-Paque Plus solution (Amersham Biosciences) was added beneath this layer and centrifuged. Mononuclear cells enriched with lymphocytes were isolated from the saline/Ficoll interface. The pellet from the above centrifugation was enriched with neutrophils, which were isolated after hypotonic lysis of erythrocytes. Total RNA was isolated from neutrophils and lymphocytes using TRIzol. RT-PCR of β3GlcNAcT-3 (18Yeh J.-C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Cell. 2001; 105: 957-969Google Scholar), Core2GlcNAcT-I (4Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Google Scholar), LSST (12Hiraoka 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: 79-89Google Scholar, 15Bistrup 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: 899-910Google Scholar), FucT-VII (20Maly P. Thall A. Petryniak B. Rogers C.E. Sith P.L. Marks R.M. Kelly R.J. Gersten K.M. Cheng G. Saunders T.L. Camper S.A. Camphausen R.T. Sullivan F.X. Isogai Y. Hindsgaul O. von Andrian U.H. Lowe J.B. Cell. 1996; 86: 643-653Google Scholar), and PSGL-1 (36Sako D. Chang X.J. Barone K.M. Vachino G. White H.M. Shaw G. Veldman G.M. Bean K.M. Ahern T.J. Furie B. Cell. 1993; 75: 1179-1186Google Scholar,37Moore K.L. Patel K. Dbruehl R.E. Fugang L. Johnson D.A. Lichenstein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Google Scholar) was carried out as follows. Using isolated RNA from respective cells as a template, first-strand cDNA was synthesized using RNase H− reverse transcriptase. PCR was carried out using AmpliTaq DNA polymerase (Applied Biosystems) and the following PCR primers: β3GlcNAcT-3, 5′-TTCTTCAACCTCACGCTCAAGCAG-3′ (5′-primer) and 5′-AGCATCTCATAAGGTAGGAAGCGG-3′ (3′-primer); Core2GlcNAcT-I, 5′-TGAAATGCTTGACAGGCTGCTGAG-3′ (5′-primer) and 5′-GGTGTTTCGAGTGGAGGAAGCATT-3′ (3′-primer); LSST, 5′-GGGAGATCTCATGATTGACAGTCG-3′ (5′-primer) and 5′-TAGTGGATTTGCTCAGGGACAGTC-3′ (3′-primer); FucT-VII, 5′-GTCAGCAACTTCCAGGAGCGGCA-3′ (5′-primer) and 5′-TCAAGGTCCTCATAGACTTGGCTG-3′ (3′-primer); PSGL-1, 5′-CCCTGTCCACAGAACCCAGTGC-3′ (5′-primer) and 5′-GAAGCTGTGCAGGGTGAGGTCAT-3′ (3′-primer); and glyceraldehyde-3-phosphate dehydrogenase, 5′-CCTGGCCAAGGTCATCCATGACA-3′ (5′-primer) and 5′-ATGAGGTCCACCACCCTGTTGCT-3′ (3′-primer) or 5′-GACCCCTTCATTGACCTCAACTACA-3′ (5′-primer) and 5′-ACATGGCCTCCAAGGAGTAAGA-3′ (3′-primer). The last primer pair was included in the human digestive system multiple tissue cDNA panel. PCR products were separated by electrophoresis on 1% agarose gels. To confirm that the PCR products were derived from respective transcripts, RT-PCR products were digested with restriction enzymes and subjected to electrophoresis on 2% agarose. CHO cells were first transfected with pZeoSV-PSGL-1 and selected in the presence of 100 μg/ml Zeocin (Invitrogen). CHO colonies stably expressing PSGL-1 were selected after staining with anti-PSGL-1 antibody KPL-1, establishing CHO-PSGL-1 cells. CHO-PSGL-1 cells were stably cotransfected with pcDNA1.1-β3GlcNAcT-3 (core 1 β3GlcNAcT) and pSV-hygromycin and selected in 100 μg/ml Zeocin and 400 μg/ml hygromycin B (Calbiochem). Expression of β3GlcNAcT-3 was shown by the expression of larger forms of PSGL-1 that contains extended core 1 O-glycans (see also “Results”). The resultant CHO-PSGL-1/C1 cells were then cotransfected with pCDM8-FucT-VII and pcDNA3 and cultured in the presence of 400 μg/ml Geneticin (Invitrogen). CHO-PSGL-1/C1 cells stably expressing FucT-VII were selected after staining with anti-sialyl Lewis x antibody (CSELX-1), resulting in CHO-PSGL-1/F7/C1 cells. In parallel, CHO-PSGL-1 cells were transfected with pcDNA1.1-Core2GlcNAcT-I together with pcDNA3 and cultured in the presence of 100 μg/ml Zeocin and 400 μg/ml Geneticin. Cells expressing Core2GlcNAcT-I were chosen for expressing larger forms of PSGL-1 that contains core 2 branched O-glycans. Expression of core 2 O-glycans was confirmed after transient transfection of CD43 (also called leukosialin) and staining with T305 antibody, resulting in CHO-PSGL-1/C2 cells. As reported previously, T305 reacts with core 2 branched O-glycans attached to CD43 (4Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Google Scholar, 38Piller F. Le Deist F. Weinberg K. Parkman R. Fukuda M. J. Exp. Med. 1991; 173: 1501-1510Google Scholar). CHO-PSGL-1/C2 cells were stably transfected with pCDM8-FucT-VII and pSV-hygromycin and cultured in the presence of Zeocin, hygromycin B, and Geneticin. Cells expressing FucT-VII were selected after staining with CSELX-1 antibody, resulting in CHO-PSGL-1/F7/C2 cells. As a control, CHO-PSGL-1 cells were stably transfected with pCDM8-FucT-VII and pcDNA3 and cultured in the presence of Zeocin and Geneticin. Cells expressing FucT-VII were selected after staining with CSELX-1 antibody, resulting in CHO-PSGL-1/F7 cells. cDNA encoding PSGL-1/IgG chimeric protein was constructed using pZeoSV-PSGL-1 and pcDNA3.1-IgG essentially as described previously (12Hiraoka 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: 79-89Google Scholar), resulting in pcDNA3.1-PSGL-1/IgG. CHO-PSGL-1/F7/C1, CHO-PSGL-1/F7/C2, and CHO-PSGL-1/F7 cells were transfected with pcDNA3.1-PSGL-1/IgG as described previously (12Hiraoka 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: 79-89Google Scholar). Twenty-four hours after transfection, cells were cultured in glucose-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal calf serum, 100 μm sodium pyruvate, 2 mm glutamine, 25 mm HEPES, and 20 μCi/ml [3H]glucosamine (PerkinElmer Life Sciences). PSGL-1/IgG chimeric protein was isolated using ImmunoPure immobilized protein A (Pierce) from the medium obtained after 48 h of additional culture as described previously (12Hiraoka 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: 79-89Google Scholar, 18Yeh J.-C. Hiraoka N. Petry

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