Biosynthesis of HNK-1 Glycans on O-Linked Oligosaccharides Attached to the Neural Cell Adhesion Molecule (NCAM)
2002; Elsevier BV; Volume: 277; Issue: 20 Linguagem: Inglês
10.1074/jbc.m201312200
ISSN1083-351X
AutoresEdgar Ong, Misa Suzuki, Frédéric Belot, Jiunn‐Chern Yeh, Isabelle Franceschini, Kiyohiko Angata, Ole Hindsgaul, Minoru Fukuda,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoThe HNK-1 glycan, sulfo→3GlcAβ1→3Galβ1→4GlcNAcβ1→R, is highly expressed in neuronal cells and apparently plays critical roles in neuronal cell migration and axonal extension. The HNK-1 glycan synthesis is initiated by the addition of β1,3-linked GlcA to N-acetyllactosamine followed by sulfation of the C-3 position of GlcA. The cDNAs encoding β1,3-glucuronyltransferase (GlcAT-P) and HNK-1 sulfotransferase (HNK-1ST) have been recently cloned. Among various adhesion molecules, the neural cell adhesion molecule (NCAM) was shown to contain HNK-1 glycan on N-glycans. In the present study, we first demonstrated that NCAM also bears HNK-1 glycan attached to O-glycans when NCAM contains the O-glycan attachment scaffold, muscle-specific domain, and is synthesized in the presence of core 2 β1,6-N-acetylglucosaminyltransferase, GlcAT-P, and HNK-1ST. Structural analysis of the HNK-1 glycan revealed that the HNK-1 glycan is attached on core 2 branched O-glycans, sulfo→3GlcAβ1→3Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAc. Using synthetic oligosaccharides as acceptors, we found that GlcAT-P and HNK-1ST almost equally act on oligosaccharides, mimicking N- and O-glycans. By contrast, HNK-1 glycan was much more efficiently added to N-glycans than O-glycans when NCAM was used as an acceptor. These results are consistent with our results showing that HNK-1 glycan is minimally attached to O-glycans of NCAM in fetal brain, heart, and the myoblast cell line, C2C12. These results combined together indicate that HNK-1 glycan can be synthesized on core 2 branched O-glycans but that the HNK-1 glycan is preferentially added on N-glycans over O-glycans of NCAM, probably because N-glycans are extended further than O-glycans attached to NCAM containing the muscle-specific domain. The HNK-1 glycan, sulfo→3GlcAβ1→3Galβ1→4GlcNAcβ1→R, is highly expressed in neuronal cells and apparently plays critical roles in neuronal cell migration and axonal extension. The HNK-1 glycan synthesis is initiated by the addition of β1,3-linked GlcA to N-acetyllactosamine followed by sulfation of the C-3 position of GlcA. The cDNAs encoding β1,3-glucuronyltransferase (GlcAT-P) and HNK-1 sulfotransferase (HNK-1ST) have been recently cloned. Among various adhesion molecules, the neural cell adhesion molecule (NCAM) was shown to contain HNK-1 glycan on N-glycans. In the present study, we first demonstrated that NCAM also bears HNK-1 glycan attached to O-glycans when NCAM contains the O-glycan attachment scaffold, muscle-specific domain, and is synthesized in the presence of core 2 β1,6-N-acetylglucosaminyltransferase, GlcAT-P, and HNK-1ST. Structural analysis of the HNK-1 glycan revealed that the HNK-1 glycan is attached on core 2 branched O-glycans, sulfo→3GlcAβ1→3Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAc. Using synthetic oligosaccharides as acceptors, we found that GlcAT-P and HNK-1ST almost equally act on oligosaccharides, mimicking N- and O-glycans. By contrast, HNK-1 glycan was much more efficiently added to N-glycans than O-glycans when NCAM was used as an acceptor. These results are consistent with our results showing that HNK-1 glycan is minimally attached to O-glycans of NCAM in fetal brain, heart, and the myoblast cell line, C2C12. These results combined together indicate that HNK-1 glycan can be synthesized on core 2 branched O-glycans but that the HNK-1 glycan is preferentially added on N-glycans over O-glycans of NCAM, probably because N-glycans are extended further than O-glycans attached to NCAM containing the muscle-specific domain. Neural cell adhesion molecules undergo critical post-translational modifications that modulate their affinity and in some cases alter their specificity for their cognate ligands (1Edelman G.M. Crossin K.L. Annu. Rev. Biochem. 1991; 60: 155-190Crossref PubMed Scopus (653) Google Scholar, 2Schachner M. Martini R. Trends Neurosci. 1995; 18: 183-191Abstract Full Text PDF PubMed Scopus (181) Google Scholar). In particular, many neural cell adhesion molecules are heavily glycosylated, and the extent and composition of attached carbohydrates modify their adhesive properties. Among those, polysialic acid and HNK-1 are particularly notable. Polysialic acid is mostly attached to NCAM, 1The abbreviations used are: NCAMneural cell adhesion moleculeGlcAglucuronic acidMSDmuscle-specific domainHNK-1STHNK-1 sulfotransferaseGlcAT-Pβ1,3-glucuronyltransferaseVASEvariable alternatively spliced exonPAPS3′-phosphoadenosine 5′-phosphosulfateGnT-Iβ1,2-N-acetylglucosaminyltransferase ICore2GlcNAcTcore 2 β1,6-N-acetylglucosaminyltransferaseConAconcanavalin A and polysialylated NCAM is abundantly present in embryonic brain (1Edelman G.M. Crossin K.L. Annu. Rev. Biochem. 1991; 60: 155-190Crossref PubMed Scopus (653) Google Scholar, 2Schachner M. Martini R. Trends Neurosci. 1995; 18: 183-191Abstract Full Text PDF PubMed Scopus (181) Google Scholar). In the adult brain, polysialylated NCAM is restricted to certain tissues, such as the hippocampus and olfactory bulb, where neuronal regeneration persists. Polysialic acid is a linear homopolymer of α-2,8-linked sialic acid residues formed on α-2,3- or α-2,6-linked sialic acid in N-acetyllactosamine of N-glycans (3Finne J. J. Biol. Chem. 1982; 257: 11966-11970Abstract Full Text PDF PubMed Google Scholar, 4Kudo M. Kitajima K. Inoue S. Shiokawa K. Morris R.R. Dell A. Inoue Y. J. Biol. Chem. 1996; 271: 32667-32677Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 5Angata K. Suzuki M. McAuliffe J. Ding Y. Hindsgaul O. Fukuda M. J. Biol. Chem. 2000; 275: 18594-18601Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 6Nakayama J. Angata K. Ong E. Katsuyama T. Fukuda M. Pathol. Int. 1998; 48: 665-677Crossref PubMed Scopus (64) Google Scholar). Polysialic acid is attached to two N-glycosylation sites in the fifth immunoglobulin-like domain in NCAM and thought to attenuate the adhesive property of NCAM (7Nelson R.W. Bates P.A. Rutishauser U. J. Biol. Chem. 1995; 270: 17171-17179Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 8Angata K. Suzuki M. Fukuda M. J. Biol. Chem. 1998; 273: 28524-28532Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 9von der Ohe M. Wheeler S.F. Wuhrer M. Harvey D.J. Liedtke S. Mühlenhoff M. Gerardy-Schahn R. Geyer H. Dwek R.A. Geyer R. Wing D.R. Schachner M. Glycobiology. 2002; 12: 47-63Crossref PubMed Scopus (63) Google Scholar). neural cell adhesion molecule glucuronic acid muscle-specific domain HNK-1 sulfotransferase β1,3-glucuronyltransferase variable alternatively spliced exon 3′-phosphoadenosine 5′-phosphosulfate β1,2-N-acetylglucosaminyltransferase I core 2 β1,6-N-acetylglucosaminyltransferase concanavalin A The HNK-1 carbohydrate epitope was originally defined by a monoclonal antibody raised against human natural killer (HNK) cells (10Abo T. Balch C.M. J. Immunol. 1981; 127: 1024-1029PubMed Google Scholar). In nervous tissues, the HNK-1 carbohydrate was first recognized as an autoantigen involved in peripheral demyelinative neuropathy (11Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar, 12Ariga T. Kohriyama T. Freddo L. Latov N. Saito M. Kon K. Ando S. Suzuki M. Hemling M.E. Rinehart K.L. Kusunoki S. Yu R.K. J. Biol. Chem. 1987; 262: 848-853Abstract Full Text PDF PubMed Google Scholar). The structural analysis of glycolipids reacting with these autoantibodies demonstrated that the HNK-1 epitope is sulfo→3GlcAβ1→3Galβ1→4GlcNAcβ1→R (11Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar, 12Ariga T. Kohriyama T. Freddo L. Latov N. Saito M. Kon K. Ando S. Suzuki M. Hemling M.E. Rinehart K.L. Kusunoki S. Yu R.K. J. Biol. Chem. 1987; 262: 848-853Abstract Full Text PDF PubMed Google Scholar). HNK-1 glycan is widely distributed in glycoproteins, glycolipids, and proteoglycans (2Schachner M. Martini R. Trends Neurosci. 1995; 18: 183-191Abstract Full Text PDF PubMed Scopus (181) Google Scholar, 11Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar, 12Ariga T. Kohriyama T. Freddo L. Latov N. Saito M. Kon K. Ando S. Suzuki M. Hemling M.E. Rinehart K.L. Kusunoki S. Yu R.K. J. Biol. Chem. 1987; 262: 848-853Abstract Full Text PDF PubMed Google Scholar, 13Margolis R.K. Ripellino J.A. Goossen B. Steinbrich R. Margolis R.U. Biochem. Biophys. Res. Commun. 1987; 145: 1142-1148Crossref PubMed Scopus (57) Google Scholar). The expression of HNK-1 glycan is spatially and developmentally regulated and found on migrating neuronal crest cells, cerebellum, and myelinating Schwann cells in motor neurons but not on those in the sensory neurons (14Bronner-Fraser M. Dev. Biol. 1986; 115: 44-55Crossref PubMed Scopus (367) Google Scholar, 15Schwarting G.A. Jungalwala F.B. Chou D.K. Boyer A.M. Yamamoto M. Dev. Biol. 1987; 120: 65-76Crossref PubMed Scopus (142) Google Scholar, 16Martini R. Schachner M. Brushart T.M. J. Neurosci. 1994; 14: 7180-7191Crossref PubMed Google Scholar). Although it has been reported that HNK-1 glycan is attached to the N-glycosylation site in the third immunoglobulin-like domain of NCAM, the recent studies showed that HNK-1 glycan is attached to the second, third, fifth, and sixth N-glycosylation sites (17Liedtke S. Geyer H. Wuhrer M. Geyer R. Frank G. Gerardy-Schahn R. Zähringer U. Schachner M. Glycobiology. 2001; 11: 373-384Crossref PubMed Scopus (86) Google Scholar). HNK-1 apparently plays critical roles in neural cell migration and axonal extension. HNK-1 glycolipids coated on plates facilitated neurite outgrowth, whereas no facilitation of neurite outgrowth was observed on sulfatide, 3′-sulfogalactosyl ceramide. The effect of HNK-1 glycan was abolished once the sulfate group was removed (18Martini R. Xin Y. Schmitz B. Schachner M. Eur. J. Neurosci. 1992; 4: 628-639Crossref PubMed Scopus (176) Google Scholar, 19Kunemund V. Jungalwala F.B. Fischer G. Chou D.K.H. Keilhauer G. Schachner M. J. Cell Biol. 1998; 106: 213-223Crossref Scopus (323) Google Scholar). The HNK-1 glycan was identified in NCAM and N- glycans isolated from P0 glycoprotein (20Voshol H. van Zuylen C.W.E.M. Orberger G. Vliegenthart J.F.G. Schachner M. J. Biol. Chem. 1996; 271: 22957-22969Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 21Kruse J. Mailhammer R. Wernecke H. Faissner A. Sommer I. Goridis C. Schachner M. Nature. 1984; 311: 153-155Crossref PubMed Scopus (585) Google Scholar). It was also found as rather unusual structures of O-linked oligosaccharides, sulfo→3GlcAβ1→3Galβ1→4GlcNAcβ1→2Man, which then are attached to serine or threonine (22Yuen C-T. Chai W. Loveless R.W. Lawson A.M. Margolis R.U. Feizi T. J. Biol. Chem. 1997; 272: 8924-8931Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). This glycan can be attached to dystroglycan, and its defect may cause muscular dystrophy (23Yoshida A. Kobayashi K. Manya H. Taniguchi K. Kano H. Mizuno M. Inazu T. Mitsuhashi H. Takahashi S. Takeuchi M. Herrmann R. Straub V. Talim B. Voit T. Topaloglu H. Toda T. Endo T. Dev. Cell. 2001; 1: 717-724Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). NCAM has numerous isoforms due to alternative splicing of precursor mRNAs. Among them, NCAM(MSD) contains an additional domain, the so-called muscle-specific domain (MSD), consisting of 37 amino acids between two fibronectin type III-like domains (24Dickson G. Gower H.J. Barton C.H. Prentice H.M. Elsom V.L. Moore S.E. Cox R.D. Quinn C. Putt W. Walsh F.S. Cell. 1987; 50: 1119-1130Abstract Full Text PDF PubMed Scopus (168) Google Scholar). This NCAM(MSD) is mostly present in skeletal myotubes and often anchored to the plasma membrane through glycophosphatidylinositol (24Dickson G. Gower H.J. Barton C.H. Prentice H.M. Elsom V.L. Moore S.E. Cox R.D. Quinn C. Putt W. Walsh F.S. Cell. 1987; 50: 1119-1130Abstract Full Text PDF PubMed Scopus (168) Google Scholar). The MSD is highly enriched with serine, threonine and proline and O-linked oligosaccharides were shown to attach to this domain (25Walsh F.S. Parekh R.B. Moore S.E. Dickson G. Barton C.H. Gower H.J. Dwek R.A. Rademacher T.W. Development. 1989; 105: 803-811PubMed Google Scholar). It has been shown that NCAM(MSD) from C2C12 myoblast cell line contains mucin-type O-linked oligosaccharides ±NeuNAcα2→3Galβ1→3(±NeuNAcα2→6)GalNAcα→Thr/Ser (25Walsh F.S. Parekh R.B. Moore S.E. Dickson G. Barton C.H. Gower H.J. Dwek R.A. Rademacher T.W. Development. 1989; 105: 803-811PubMed Google Scholar). However, no studies have addressed whether O-glycans in NCAM contain an HNK-1-capping structure. The HNK-1 glycan is synthesized in a stepwise manner by the addition of a β1,3-linked glucuronic acid to precursor N- acetyllactosamine by β1,3-glucosaminyltransferase (GlcAT-P) followed by the addition of a sulfate group by HNK-1 sulfotransferase (HNK-1ST) to GlcAβ1→3Galβ1→4GlcNAc→R, forming sulfo→3GlcAβ1→3Galβ1→4GlcNAc→R (26Chou D.K.H. Jungalwala F.B. J. Biol. Chem. 1993; 268: 330-336Abstract Full Text PDF PubMed Google Scholar). Recently, the cDNAs encoding GlcAT-P and HNK-1ST have been cloned (27Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar, 28Bakker H. Friendmann I. Oka S. Kawasaki T. Nifant'ev N. Schachner M. Mantei N. J. Biol. Chem. 1997; 272: 29942-29946Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 29Ong 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). In mucin-type O-glycans, N-acetyllactosamine can be formed when core 2 branch, GlcNAcβ1→6(Galβ1→3) GalNAcα1→R, is synthesized by core 2 β1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT, Fig. 1, Ref. 30Bierhuizen M.F.A. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar). These results prompted us to examine how the HNK-1 glycan is synthesized in mucin-type O-linked oligosaccharides attached to NCAM. In this report, we first present evidence that HNK-1 glycan can be formed in O-glycans attached to the MSD in NCAM. We found that HNK-1 glycan is attached to core 2 branched O-glycans but not to core 1 O-glycans present in NCAM. We determined the HNK-1 glycan structure in mucin-type O-linked oligosaccharides as sulfo→3GlcAβ1→3Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAc. Finally, we found that HNK-1 glycans can be formed on N-glycans more efficiently than O-glycans on NCAM, although GlcAT-P and HNK-1ST utilize O-glycan and N-glycan oligosaccharides almost equally as an acceptor. pIG-NCAM·IgG and pIG-NCAM(MSD)·IgG encoding NCAM·IgG and NCAM(MSD)·IgG chimeric proteins, respectively, were constructed from pIG-NCAM(VASE, MSD)·IgG, originally provided by Dr. David Simmons at Oxford University (31Simmons D.L. Hartly D. Cellular Interactions in Development. Oxford University Press, Oxford1993: 93-127Google Scholar), as described previously (32Franceschini I. Angata K. Ong E. Hong A. Doherty P. Fukuda M. Glycobiology. 2001; 11: 231-239Crossref PubMed Scopus (35) Google Scholar). All products derived from these different vectors were found to react with anti-NCAM antibody (Eric-1) by Western blotting, confirming the sequences and sizes of the products (32Franceschini I. Angata K. Ong E. Hong A. Doherty P. Fukuda M. Glycobiology. 2001; 11: 231-239Crossref PubMed Scopus (35) Google Scholar). The cDNA encoding rat β1,3-glucuronyltransferase, GlcAT-P (27Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar), was cloned as described previously using reverse transcription-PCR and subcloned into pcDNA3, resulting in pcDNA3(neo)-GlcAT-P (29Ong 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). The cDNA encoding a catalytic domain of GlcAT-P was obtained by PCR using pcDNA3(neo)-GlcAT-P as a template. 5′- and 3′-primers for the PCR are 5′-CGGATCCCAGCCTCGCACCTCTGCTTGCT-3′ (the BamHI site is underlined, and the rest is nucleotides 120–141 of GlcAT-P) and 5′-ACTCGAGTCAGATCTCCACCGAGGGGTC-3′ (the XhoI site is underlined, and the rest of the sequence is nucleotides 1044–1024 of GlcAT-P). After BamHI and XhoI digestion, the cDNA fragment was ligated into the same sites of pcDNAI-A, which harbors a signal peptide and IgG-binding domain of protein A, resulting in pcDNAI-A·GlcAT-P. The cDNA encoding a human sulfotransferase that transfers a sulfate group from PAPS to glucuronylated N-acetyllactosamine precursors was cloned into pcDNA3 as described previously, resulting in pcDNA3-HNK-1ST (29Ong 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). Similarly, pcDNAI-A·HNK-1ST was prepared as described before (33Ong 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 (41) Google Scholar). pcDNAI encoding core 2 β1,6-N-acetylglucosaminyltransferase, Core2GlcNAcT-I (30Bierhuizen M.F.A. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar), was cloned into the pcDNA3.1(Zeo), resulting pcDNA3.1(Zeo)-Core2GlcNAcT-I. A cDNA encoding β1,2-N-acetylglucosaminyltransferase I (GnT-I) cloned in pcDNA3.1 (34Kumar R. Yang J. Larsen R.D. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9948-9952Crossref PubMed Scopus (142) Google Scholar, 35Sarkar M. Hull E. Nishikawa Y. Simpson R.J. Moritz R.L. Dunn R. Schachter H. Proc. Natil. Acad. Sci. U. S. A. 1991; 88: 234-238Crossref PubMed Scopus (103) Google Scholar) was a kind gift of Dr. Pamela Stanley. To avoid the formation of HNK-1 in N-glycans, the majority of the experiments were carried out using a mutant Chinese hamster ovary cell line, Lec1. Since Lec1 cells are deficient in GnT-I, all of the N-glycans synthesized in this cell line remain as high mannose oligosaccharides (36Stanley P. Narashimhan S. Siminovitch L. Schachter H. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3323Crossref PubMed Scopus (160) Google Scholar). Lec1 cells were found to lack core 2 β1,6-N-acetylglucosaminyltransferase as in other Chinese hamster ovary cells (30Bierhuizen M.F.A. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar). Lec1 cells were stably transfected with pcDNA3.1(Zeo)-Core2GlcNAcT-I as described before (29Ong 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). Lec1-core 2 cells were first selected in the presence of zeocin (Invitrogen) and then by immunofluorescent staining using tomato lectin, which reacts with N-acetyllactosamine and poly-N-acetyllactosamines (37Merkle R.K. Cummings R.D. J. Biol. Chem. 1987; 262: 8179-8189Abstract Full Text PDF PubMed Google Scholar). As shown previously, the formation of core 2 branches results in the formation of small amounts of poly-N-acetyllactosamine (38Bierhuizen M.F. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar). To confirm the integration of human Core2GlcNAcT-I cDNA into Lec1 cell chromosome, reverse transcription-PCR was carried out on poly(A)+ RNA derived from Lec1-core 2 cells. The primers for this reverse transcription-PCR were designed in exon 1 and exon 2, avoiding the amplification of hamster genomic DNA harboring Core2GlcNAcT. Lec1-core 2 cells were transfected with pcDNA3(neo)-GlcAT-P and selected in the presence of G418 and zeocin, and Lec1-core2·GlcA were chosen after immunostaining using M6749 antibody (39Obata K. Tanaka H. Neurosci. Res. 1988; 6: 131-142Crossref PubMed Scopus (25) Google Scholar), which reacts with both nonsulfated and sulfated forms of HNK-1 carbohydrates (27Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar,29Ong 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). Those cells were further transfected with pcDNA3.1(hyg)-HNK-1ST and selected in the presence of G418, zeocin, and hygromycin. Those cells expressing a significant amount of HNK-1 glycan, assessed by immunostaining with anti-HNK-1 antibody (Becton Dickinson), were chosen and designated as Lec1-core 2·HNK-1. As the second antibody, fluorescein isothiocyanate-conjugated goat (Fab′)2 fragment specific to mouse IgG (for anti-NCAM antibody) or IgM (for HNK-1 antibody and M6749 antibody) was used. In parallel, Lec1 cells stably expressing GlcAT-P or HNK-1ST alone or GlcAT-P and HNK-1ST together were established. Lec1 cells expressing the HNK-1 glycan were transiently transfected with pIG-NCAM·IgG or pIG-NCAM(MSD)·IgG with or without pcDNAI-Core2GlcNAcT-I, using LipofectAMINE Plus™ as described previously (29Ong 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). The medium was changed to serum-free medium 24 h after the transfection and cultured for an additional 48 h. NCAM·IgG in the cultured medium was adsorbed to protein A-agarose as described before (8Angata K. Suzuki M. Fukuda M. J. Biol. Chem. 1998; 273: 28524-28532Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). NCAM·IgG molecules eluted from the medium were subjected to SDS-polyacrylamide (5%) gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The blot was then incubated with anti-NCAM antibody, anti-HNK-1 antibody, or M6749 antibody, with a secondary antibody as described before (29Ong 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). Lec2 cells expressing GlcAT-P and HNK-1ST, Lec2-HNK-1, were established as described previously (29Ong 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). Lec2-HNK-1 cells were selected using anti-HNK-1 monoclonal antibody. Lec2 cells expressing GlcAT-P were similarly established and selected by immunofluorescent staining using M6749 monoclonal antibody. Lec2 cells do not synthesize sialylated oligosaccharides due to the defect in CMP-sialic acid transporter in the Golgi (40Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I. Hirschberg C.B. Cell. 1984; 39: 295-299Abstract Full Text PDF PubMed Scopus (198) Google Scholar). Lec1-core 2·HNK-1 cells were transiently transfected with pIG-NCAM(MSD)·IgG or pIG-NCAM·IgG. Twenty-four hours after the transfection, the medium was replaced with sulfate-free or glucose-free RPMI 1640 medium containing dialyzed 10% fetal calf serum in the presence of Na2[35S]O4 (100 μCi/ml), [6-3H]GlcNH2 (10 μCi/ml), and [6-3H]Gal (20 μCi/ml) or [6-3H]GlcNH2 (10 μCi/ml) or Na2[35S]O4 (100 μCi/ml) alone (41Hiraoka 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 (212) Google Scholar). After a 48-h incubation, the spent medium was harvested, and NCAM·IgG was isolated as described above. After confirming the purity of the isolated samples by SDS-polyacrylamide gel electrophoresis and fluorography, the samples were subjected to oligosaccharide characterization. Metabolically labeled NCAM(MSD)·IgG was digested with Pronase, and labeled glycoproteins were isolated by Sephadex G-25 gel filtration as described previously (41Hiraoka 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 (212) Google Scholar). O-Glycan-containing glycopeptides were separated from high mannose glycopeptides and other glycans using ConA-Sepharose affinity chromatography. The purified glycopeptides were subjected to alkaline reductive treatment to release O-linked oligosaccharides as described previously (41Hiraoka 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 (212) Google Scholar). Oligosaccharides were applied to a Poly-Prep column (Bio-Rad) containing 2 ml of anion-exchange QAE-Sephadex A-25. The resin was equilibrated with 2 mm pyridine-acetate buffer, pH 5.5, and eluted in a stepwise manner with 6 ml each of 70 mm, 250 mm, and 1 m NaCl in 2 mmpyridine-acetate buffer, pH 5.5. Fractions (1 ml) were collected, and aliquots were taken for determination of radioactivity. The oligosaccharides eluted with 1 m NaCl were digested with Arthrobacter sialidase (Sigma) to remove sialic acid and subjected to QAE-Sephadex A-25 column chromatography. Those eluted with 1 m NaCl after sialidase treatment were subjected to structural characterization. The O-linked glycans were subjected to solvolysis to remove a sulfate group as described previously (41Hiraoka 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 (212) Google Scholar, 42Nagasawa K. Inoue Y. Tokuyasu T. J. Biochem. (Tokyo). 1979; 86: 1323-1329Crossref PubMed Scopus (101) Google Scholar). The resultant oligosaccharides, purified by QAE-Sephadex chromatography, were digested with 125 milliunits of Escherichia coli β-glucuronidase (Sigma) in 50 μl of the reaction mixture under the same conditions as described previously (11Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar). The obtained oligosaccharides were analyzed on an AX-5 amino-bonded column using a Gilson 306 HPLC apparatus at a flow rate of 0.8 ml/min. The column was equilibrated with 90% solvent A (80% acetonitrile, 20% 15 mm KH2PO4) and 10% of solvent B (40% acetonitrile, 60% 15 mmKH2PO4). After 5 min, the elution was carried out by linear gradient from 10% of solvent B to 100% of solvent B in 60 min. The column was finally washed for 5 min with solvent B. Fractions were collected every minute, and aliquots were taken for determination of the radioactivity by scintillation counting. Oligosaccharides were oxidized with 50 mm NaIO4 in 50 mm sodium acetate buffer, pH 4.5, at room temperature, and oxidized samples were reduced as described previously (43Carlsson S.R. Sasaki H. Fukuda M. J. Biol. Chem. 1986; 261: 12787-12795Abstract Full Text PDF PubMed Google Scholar). The samples, after destroying NaBH4 by acetic acid, were passed over a small column of Dowex 50 × 8 and dried after the addition of methanol. The samples were then hydrolyzed in 0.01 m HCl at 80 °C for 1 h, neutralized with 0.1 m Tris-HCl buffer, pH 8.5, and applied to a column (1 × 120 cm) of Bio-Gel P-4 (200–400-mesh) equilibrated with 0.1 mpyridine-acetate buffer, pH 5.5. Oligosaccharides were also separated by QAE-Sephadex A-25 column chromatography as described above. To assay the activities of GlcAT-P and HNK-1ST, soluble protein A-GlcAT-P and soluble protein A-HNK-1ST were prepared and adsorbed to IgG-Sepharose as described previously (33Ong 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 (41) Google Scholar). To assay for GlcAT-P activity, the reaction mixture (100 μl) consisted of 500 μmacceptors, 0.1 m HEPES buffer, pH 6.5, 10 mmMnCl2, 10 mm of galactonic acid-γ-lactone, 50 μl of the soluble chimeric enzyme attached to beads, and 50 μm UDP-[14C]GlcA (294 mCi/mmol, PerkinElmer Life Sciences). The reaction mixture was incubated for 3 h at 37 °C, and the reaction was stopped by adding 50 mm(final concentration) EDTA. To assay for HNK-1ST activity, the donor, 3′-phosphate 5′-phospho-[35S]sulfate, and various amounts of an acceptor were incubated with a solution of the 50% suspension of the chimeric protein A-HNK-1ST attached to beads under the conditions described previously (29Ong 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, 33Ong 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 (41) Google Scholar). The reaction was stopped by 250 mm ammonium formate, pH 4, and labeled products were obtained by C18 reverse-phase chromatography as described previously (29Ong 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). The Km for GlcAT-P and HNK-1ST was determined using a Lineweaver-Burk plot at various concentrations of the acceptor (2.5–2000 μm). The acceptors GlcAβ1→3Galβ1→4GlcNAcβ1→6Manα1→6Manβ1→O(CH2)7CH3(octyl) (compound 1), GlcAβ1→3Galβ1→4GlcNAcβ1→2Manα1→6Manβ1→O(CH2)7CH3(octyl) (compound 2), and GlcAβ1→3Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAcα1→O(CH2)7CH3(octyl) (compound 3) were synthesized from precursors octyl 3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl(1→6)-2,3,4-tri-O-benzyl-α-d-mannopyranosyl(1→6)-2,3,4-tri-O-benzyl-β-d-mannopyranoside (compound 4), octyl 3,6-di-O-benzyl-2-deoxy-2-trichlor
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