Expression Cloning of a Human Sulfotransferase That Directs the Synthesis of the HNK-1 Glycan on the Neural Cell Adhesion Molecule and Glycolipids

The HNK-1 carbohydrate is expressed on various adhesion molecules in the nervous system and is suggested to play a role in cell-cell and cell-substratum interactions. Here we describe the isolation and functional expression of a cDNA encoding a human sulfotransferase that synthesizes the HNK-1 carbohydrate epitope. A mutant Chinese hamster ovary cell line, Lec2, which stably expresses human neural cell adhesion molecule (N-CAM) (Lec2-NCAM), was first established. Lec2-NCAM was co-transfected with a human fetal brain cDNA library, a cDNA encoding the rat glucuronyltransferase that forms a precursor of the HNK-1 carbohydrate, and a vector encoding the polyoma large T antigen. The transfected Lec2-NCAM cells expressing the HNK-1 glycan were enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA encoding a sulfotransferase, HNK-1ST, that directs the expression of the HNK-1 carbohydrate epitope on the cell surface. The deduced amino acid sequence indicates that the enzyme is a type II membrane protein. Sequence analysis revealed that there is a short amino acid sequence in the presumed catalytic domain, which is highly homologous to the corresponding sequence in other Golgi-associated sulfotransferases so far cloned. The amount of HNK-1ST transcript is high in fetal brain compared with fetal lung, kidney, and liver. Expression of HNK-1ST resulted in the formation of the HNK-1 epitope on N-CAM and a soluble chimeric form of HNK-1ST was shown to add a sulfate group to a precursor, GlcAβ1→3Galβ1→4GlcNAcβ1→R, forming sulfo→3GlcAβ1→3Galβ1→4GlcNAcβ1→R. The results combined together indicate that the cloned HNK-1ST directs the synthesis of the HNK-1 carbohydrate epitope on both glycoproteins and glycolipids in the nervous tissues. The HNK-1 carbohydrate is expressed on various adhesion molecules in the nervous system and is suggested to play a role in cell-cell and cell-substratum interactions. Here we describe the isolation and functional expression of a cDNA encoding a human sulfotransferase that synthesizes the HNK-1 carbohydrate epitope. A mutant Chinese hamster ovary cell line, Lec2, which stably expresses human neural cell adhesion molecule (N-CAM) (Lec2-NCAM), was first established. Lec2-NCAM was co-transfected with a human fetal brain cDNA library, a cDNA encoding the rat glucuronyltransferase that forms a precursor of the HNK-1 carbohydrate, and a vector encoding the polyoma large T antigen. The transfected Lec2-NCAM cells expressing the HNK-1 glycan were enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA encoding a sulfotransferase, HNK-1ST, that directs the expression of the HNK-1 carbohydrate epitope on the cell surface. The deduced amino acid sequence indicates that the enzyme is a type II membrane protein. Sequence analysis revealed that there is a short amino acid sequence in the presumed catalytic domain, which is highly homologous to the corresponding sequence in other Golgi-associated sulfotransferases so far cloned. The amount of HNK-1ST transcript is high in fetal brain compared with fetal lung, kidney, and liver. Expression of HNK-1ST resulted in the formation of the HNK-1 epitope on N-CAM and a soluble chimeric form of HNK-1ST was shown to add a sulfate group to a precursor, GlcAβ1→3Galβ1→4GlcNAcβ1→R, forming sulfo→3GlcAβ1→3Galβ1→4GlcNAcβ1→R. The results combined together indicate that the cloned HNK-1ST directs the synthesis of the HNK-1 carbohydrate epitope on both glycoproteins and glycolipids in the nervous tissues. Neural cells express unique carbohydrates that are often shared by immune cells (1Jessell T.M. Hynes M.A. Dodd J. Annu. Rev. Neurosci. 1985; 13: 227-255Google Scholar, 2Schachner M. Martini R. Trends Neurosci. 1995; 18: 183-191Google Scholar). One of them is the HNK-1 carbohydrate epitope, originally discovered by a monoclonal antibody raised againsthuman natural killer cells (3Abo T. Balch C.M. J. Immunol. 1981; 127: 1024-1029Google Scholar), although its role in immune cells is not known. The functional significance of the HNK-1 carbohydrate was first recognized as an auto-antigen involved in peripheral demyelinative neuropathy. The structural analysis of glycolipids reacting with these auto-antibodies led to the discovery that the HNK-1 epitope is sulfo→3GlcAβ1→3Galβ1→4GlcNAcβ1→R (4Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Google Scholar, 5Ariga T. Kohriyama T. Freddo L. Latov N. Saito M. Kon K. Ando S. Suzuki M. Hemling M.E. Rinehart Jr., K.L. Kusunoki S. Yu R.K. J. Biol. Chem. 1987; 262: 848-853Google Scholar). By using HNK-1-specific antibodies and carbohydrate structural studies, the HNK-1 glycan has been found in a number of neural cell adhesion molecules including N-CAM, 1The abbreviations used are: N-CAM, the neural cell adhesion molecule; GlcAT-P, glycoprotein-specific glucuronyltransferase; HNK-1ST, HNK-1 sulfotransferase; PCR, polymerase chain reaction; PAPS, 3′-phosphoadenosine 5′-phosphosulfate.1The abbreviations used are: N-CAM, the neural cell adhesion molecule; GlcAT-P, glycoprotein-specific glucuronyltransferase; HNK-1ST, HNK-1 sulfotransferase; PCR, polymerase chain reaction; PAPS, 3′-phosphoadenosine 5′-phosphosulfate.myelin-associated glycoprotein, L1, contactin, and P0 (2Schachner M. Martini R. Trends Neurosci. 1995; 18: 183-191Google Scholar, 6McGarry R.C. Helfand S.L. Quarles R.H. Roder J.C. Nature. 1983; 306: 376-378Google Scholar, 7Kruse J. Mailhammer R. Wernecke H. Faissner A. Sommer I. Goridis C. Schachner M. Nature. 1984; 311: 153-155Google Scholar, 8Gennarini G. Rougon G. Vitiello F. Corsi P. Di Benedetta C. Goridis C. J. Neurosci. Res. 1989; 22: 1-12Google Scholar, 9Voshol H. van Zuylen C.W.E.M. Orberger G. Vliegenthart J.F.G. Schachner M. J. Biol. Chem. 1996; 271: 22957-22960Google Scholar). The studies, using either monoclonal antibodies or isolated carbohydrates, demonstrated that the HNK-1 glycan is involved in cell-cell and cell-substratum interactions (10Keilhauer G. Faissner A. Schachner M. Nature. 1985; 316: 728-730Google Scholar, 11Mohan P.S. Chou D.K.H. Jungalwala F.B. J. Neurochem. 1990; 54: 2024-2031Google Scholar). In one study, the inhibition by HNK-1 oligosaccharide was abolished by desulfation of the HNK-1 glycan indicating the critical role of the sulfate group (11Mohan P.S. Chou D.K.H. Jungalwala F.B. J. Neurochem. 1990; 54: 2024-2031Google Scholar). The expression of the HNK-1 epitope is spatially and developmentally regulated, and is found on migrating neural crest cells, cerebellum, and myelinating Schwann cells in motor neurons but not on those in the sensory neurons (12Bronner-Fraser M. Dev. Biol. 1986; 115: 44-55Google Scholar, 13Eisenman L.M. Hawkes R. J. Comp. Neurol. 1993; 335: 586-605Google Scholar, 14Martini R. Xin Y. Schmitz B. Schachner M. Eur. J. Neurosci. 1992; 4: 628-639Google Scholar). In addition, the HNK-1 carbohydrate was shown to bind to P- and L-selectins (15Needham L.K. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1359-1363Google Scholar), suggesting that the interactions between immune cells and the nervous system may be mediated through the binding of the HNK-1 carbohydrate in neural cells. The HNK-1 carbohydrate is synthesized in a stepwise manner by the addition of a β-1,3-linked glucuronic acid to a precursorN-acetyllactosamine followed by the addition of a sulfate group to GlcAβ1→3Galβ1→4GlcNAc→R (16Jungalwala F.B. Neurochem. Res. 1994; 19: 945-957Google Scholar, 17Chou D.K.H. Jungalwala F.B. J. Biol. Chem. 1993; 268: 330-336Google Scholar). Recently, Terayamaet al. (18Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Google Scholar) reported the cloning of a β-1,3-glucuronyltransferase, GlcAT-P, that forms an HNK-1 precursor carbohydrate, GlcAβ1→3Galβ1→4GlcNAcβ1→R, in glycoproteins (18Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Google Scholar). As a part of our systematic studies on neural cell glycoconjugates (19Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Google Scholar, 20Nakayama J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Google Scholar, 21Nakayama J. Fukuda M.N. Hirabayashi Y. Kanamori A. Sasaki K. Nishi T. Fukuda M. J. Biol. Chem. 1996; 271: 3684-3691Google Scholar, 22Angata K. Nakayama J. Fredette B. Chong K. Ranscht B. Fukuda M. J. Biol. Chem. 1997; 272: 7182-7190Google Scholar, 45Ong E. Nakayama J. Angata K. Reyes L. Katsuyama T. Arai Y. Fukuda M. Glycobiology. 1998; 8 (in press)Google Scholar), we describe herein the expression cloning of HNK-1 sulfotransferase, HNK-1ST. Using the cDNA isolated, the expression profile of the HNK-1ST transcripts was compared with that of GlcAT-P transcripts for various fetal and adult tissues. We also demonstrate the HNK-1ST activity in vivo and in vitro using N-CAM, synthetic oligosaccharides and glycolipids as acceptors. A mutant cell line of Chinese hamster ovary cells, Lec2, was used as recipient cells. β-Glucuronylation of N-acetyllactosamines is extremely efficient in Lec2 cells (18Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Google Scholar) because sialylation is absent in this cell line (23Deutscher S.L. Nuwayhid N. Stanley P. Briles E.I. Hirschberg C.B. Cell. 1984; 39: 295-299Google Scholar). Lec2 cells were first transfected with pHβAPr-1-neo-NCAM 140 (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: 1119-1130Google Scholar) and a stable cell line expressing human N-CAM, Lec2-NCAM, was selected as described before (19Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Google Scholar). For cloning of GlcAT-P, the cDNA was synthesized from poly(A)+ RNA of rat brain (CLONTECH) using a reverse transcription-PCR kit (Stratagene). Using the cDNAs synthesized as templates, PCR was performed to amplify the GlcAT-P sequence under the conditions described previously (21Nakayama J. Fukuda M.N. Hirabayashi Y. Kanamori A. Sasaki K. Nishi T. Fukuda M. J. Biol. Chem. 1996; 271: 3684-3691Google Scholar). The 5′- and 3′-primers correspond to nucleotides −32 to −10 and nucleotides 1047–1027, respectively, of the reported rat GlcAT-P sequence (18Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Google Scholar). The 5′- and 3′-primers also contain HindIII and XhoI sites, respectively. The PCR product was digested withHindIII and XhoI, then cloned into pcDNA3, resulting in pcDNA3-GlcAT-P. Lec2-NCAM cells were found to be negative for the HNK-1 antigen after pcDNA3GlcAT-P was transiently expressed. Lec2-NCAM cells were thus co-transfected with 18 μg of a human fetal brain cDNA library in pcDNAI (19Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Google Scholar), 6 μg of pcDNA3-GlcAT-P, and 6 μg of pPSVE1-PyE harboring the polyoma large T cDNA (25Bierhuizen M.F.A. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Google Scholar), using LipofectAMINETM (Life Technologies, Inc.) as described previously (19Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Google Scholar). After 62 h, the transfected cells were dissociated into monodispersed cells using the enzyme-free cell dissociation solution (Hanks' based, purchased from Cell and Molecular Technologies, Lavellette, NJ), followed by fluorescence-activated cell sorting of the HNK-1-positive cells using anti-HNK-1 monoclonal antibody (Becton Dickinson). Plasmid DNA from the sorted cells was isolated by the Hirt (26Hirt B. J. Mol. Biol. 1967; 26: 365-369Google Scholar) procedure and amplified in the host bacteriaEscherichia coli MC1061/P3 in the presence of ampicilin and tetracycline. The pcDNAI vector contains the supF suppressor tRNA, so that MC1061/P3 cells containing pcDNAI are resistant to both ampicillin and tetracycline. In contrast, MC1061/P3 cells harboring pcDNA3GlcAT-P or pPSVE1-PyE are resistant to ampicillin but not to tetracycline. Because of this difference, only plasmids derived from pcDNAI were rescued and amplified by this procedure (19Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Google Scholar), allowing the isolation of plasmids responsible for the HNK-1 glycan expression. Bacteria harboring plasmids, which were isolated by the Hirt procedure, were divided into 20 plates. Plasmid DNA was prepared from each plate and separately transfected into Lec2-NCAM cells together with pcDNA3-GlcAT-P. The transfectants were screened by immunofluorescence microscopy using anti-HNK-1 antibody to identify a plasmid pool that directed the expression of the HNK-1 glycan. By narrowing down the plasmid pools using the same procedure, it was possible to isolate a single clone containing the plasmid DNA (pcDNAI-HNK-1ST) encoding a human sulfotransferase that directed the expression of the HNK-1 carbohydrate epitope. To shorten the long 3′-untranslated sequence of pcDNAI- HNK-1ST, HNK-1ST cDNA was digested utilizing an internal EcoRI site 15 nucleotides downstream of the stop codon and cloned into pcDNA3, resulting in pcDNA3-HNK-1ST (short). A truncated cDNA containing only 9 and 6 nucleotides of 5′- and 3′-untranslated sequences in addition to the coding sequence was prepared by PCR. The 5′- and 3′-primers for the PCR were 5′-GTCAAGCTTTGTGACAAACATGCACCACCAGTGGCT-3′ and 5′-GCGCTCGAGTATGCATTAGTTTAGCAAAAAGTC-3′.HindIII and XhoI sites are singly underlined, while HNK-1ST-coding sequences are doubly underlined. After restriction enzyme digestion, the PCR product was cloned into pcDNA3, yielding pcDNA3-HNK-1ST (ORF). Nucleotide sequences were determined in both strands by an automated sequencer (Applied Biosystems 377XL). Human multiple tissue Northern blots of poly(A)+ RNA (CLONTECH) were hybridized sequentially with gel-purified cDNA inserts of pcDNA3-HNK-1ST (ORF) and pcDNA3-GlcAT-P, after labeling with 32PdCTP by random oligonucleotide primers (Prime-IT II labeling kit, Stratagene). Lec2-NCAM cells were transiently transfected with pcDNAI- HNK-1ST and pcDNA3-GlcAT-P, pcDNAI-HNK-1ST or after cell were from the transfected cells and with a N-CAM monoclonal antibody K. Walsh F. J. Scholar), followed by After the were by and The was then with the anti-HNK-1 or antibody K. H. Neurosci. Res. Scholar) followed by and by an kit The cDNA encoding the catalytic of HNK-1ST was prepared by PCR using pcDNA3-HNK-1ST as a and with the cDNA encoding a sequence and the binding of A J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Google Scholar, K. E. K. S. T. M. Nishi T. M. J. Biol. Chem. 1993; 268: Scholar). The for this PCR is site is underlined, and the coding sequence of HNK-1ST is doubly underlined. The is The PCR product was digested by and XhoI then cloned into and XhoI sites of J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Google Scholar), yielding and were separately transfected to cells and the enzyme was to Inc.) as described previously J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Google Scholar). and were synthesized to the reported Y. J. Chem. 1996; 4: Scholar, Y. H. M. A. J. Chem. 1996; Scholar) with a The procedure for the synthesis of these oligosaccharides be and in for is and and was prepared by acid from its form (4Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Google Scholar) and by The of enzyme oligosaccharides or glycolipids in and D.K.H. Jungalwala F.B. J. Biol. Chem. 1993; 268: 330-336Google Scholar). After for the were to and to reverse After the with the same the product was with The was by The were by using After in the plate was to for oligosaccharides were then by with in Lec2-NCAM cells were co-transfected with a human fetal brain cDNA library in pcDNA3-GlcAT-P, and pPSVE1-PyE (25Bierhuizen M.F.A. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Google Scholar). The transfected cells were with anti-HNK-1 antibody followed by then to cell sorting. Plasmid recovered from anti-HNK-1 Lec2-NCAM cells, was to selection with sequentially a single clone containing the that directed the expression of the HNK-1 glycan was only HNK-1ST was to expression of a amount of was HNK-1ST cDNA was not expressed The cDNA encoding HNK-1ST contains an a of amino acid A that this has a type II membrane and the is by amino has been found in so far cloned H. Fukuda M. Molecular Glycobiology. Scholar). there is between the cloned HNK-1ST and sequences for other in the amino acid sequence of the doubly sequence in and has with the sequences found in other Golgi-associated sulfotransferases as sulfate human sulfotransferase, hamster sulfate and human sulfate M. K. K. M. K. T. J. Biol. Chem. 1995; Scholar, K. M. Y. A. A. Y. J. Biol. Chem. 1997; 272: Scholar, M. H. M. K. J. Biol. Chem. 1997; 272: Scholar, J. L. R.D. J. Biol. Chem. 1997; 272: Scholar). In the sequence is and amino are shared these amino acid sequences the amino acid sequence of the rat HNK-1ST reported H. I. Oka S. Kawasaki T. N. Schachner M. N. J. Biol. Chem. 1997; 272: Scholar) has the sequence as the human HNK-1ST in of amino acid sequences of The amino acid sequences of human HNK-1ST is compared with that of rat HNK-1ST hamster sulfate sulfate human sulfotransferase and human sulfate In the and amino and amino respectively. The number of the amino acid is shown both The compared sequences were reported in M. K. K. M. K. T. J. Biol. Chem. 1995; Scholar, K. M. Y. A. A. Y. J. Biol. Chem. 1997; 272: Scholar, M. H. M. K. J. Biol. Chem. 1997; 272: Scholar, J. L. R.D. J. Biol. Chem. 1997; 272: Scholar, H. I. Oka S. Kawasaki T. N. Schachner M. N. J. Biol. Chem. 1997; 272: To the the presumed a truncated cDNA from 9 of the codon was in pcDNA3, yielding pcDNA3-HNK-1ST (ORF). plasmid directed the expression of the HNK-1 carbohydrate that nucleotides the coding of HNK-1ST are sites in in the human HNK-1ST A sequence for is nucleotides followed by a from the of the the cDNA the Northern blots of poly(A)+ RNA derived from various human were transcript of was in fetal in lung, and but in liver. The same transcript was in adult and in and but in other tissues. various of the HNK-1ST transcript is expressed in the the other The transcript of GlcAT-P is expressed in both fetal and adult as shown previously for rat (18Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Google Scholar). the GlcAT-P transcript is expressed in various of the brain and it is in the and GlcAT-P is to form the HNK-1 glycan in expression of the HNK-1 carbohydrate in glycoproteins of nervous tissue may be determined by the of GlcAT-P expression. In other it is possible that HNK-1ST may other those synthesized by To HNK-1ST is of the HNK-1 epitope on N-CAM, Lec2-NCAM cells were transiently transfected with pcDNAI- HNK-1ST and pcDNA3-GlcAT-P, pcDNAI-HNK-1ST or analysis of N-CAM derived from those transfected cells demonstrated that the HNK-1 glycan was on N-CAM both HNK-1ST and GlcAT-P were expressed while the HNK-1 glycan was not expressed in the of either enzyme The expression of GlcAT-P resulted in the binding of the antibody which was shown to with both and forms of the HNK-1 carbohydrate (18Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Google Scholar, K. H. Neurosci. Res. Scholar). results that HNK-1ST forms the HNK-1 carbohydrate epitope on N-CAM a precursor is To that the cloned cDNA HNK-1ST, a soluble chimeric HNK-1ST was expressed in cells. The enzyme to was then with oligosaccharides or and As shown in a amount was to while was using the from cells or using as an was into the that the of the oligosaccharide is that of the HNK-1ST sulfate to the oligosaccharide as as to the The shown in were to followed by The product the same of a synthetic results that the cloned HNK-1ST a sulfate group to of forming of the after with HNK-1ST. was on the by the enzyme derived from and and and and to and the of and respectively. oligosaccharides by the and and In this study, we describe the isolation of a cDNA clone encoding a human sulfotransferase, HNK-1ST, the enzyme responsible for the formation of a sulfate group to the of glucuronic acid in For this Lec2 cells were first stably transfected to express human Lec2 cells expressing N-CAM were then transiently co-transfected with a human fetal brain cDNA library in pcDNA3-GlcAT-P, and pcDNAI has the supF while both and pPSVE1-PyE contain only the plasmids derived from pcDNAI be thus rescued and amplified in containing as MC1061/P3 cells in the presence of ampicilin and B. A. Proc. Natl. Acad. Sci. U. S. A. 1987; Scholar). In N-CAM cDNA be also co-transfected transiently its vector contains only an ampicillin resistant procedure an expression cloning a vector encoding the polyoma large T antigen was stably expressed (25Bierhuizen M.F.A. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Google Scholar, M. M.F.A. Nakayama J. Glycobiology. 1996; Scholar). By the of stable which are for the expression of the carbohydrates and polyoma large T the for cloning has been The cloning of the HNK-1ST cDNA after the of GlcAT-P sequence (18Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Google Scholar) and to a single After the of our al. H. I. Oka S. Kawasaki T. N. Schachner M. N. J. Biol. Chem. 1997; 272: Scholar) reported the cloning of a rat HNK-1ST. cloning from because cells, which are Lec2 cells stably expressing the polyoma large T antigen L. M. Res. 1993; Scholar), were used as recipient cells, and the N-CAM cDNA was not we were in cloning the cDNA that the HNK-1 glycan on neural cell adhesion Lec2 cells stably expressing N-CAM were used as recipient cells in our In the the cDNAs cloned by and group the same of amino acid are between the human HNK-1ST and the cloned rat As shown in there to be a sequence various Golgi-associated is possible that the amino acid sequence may be involved in the binding of or in this sequence is not shared which has and is involved in sulfate synthesis Y. A. G. Hirschberg C.B. J. Biol. Chem. 1992; Scholar, I. B. U. L. J. Biol. Chem. 1994; Scholar). is to is in the other the sequence shown in is from the presumed binding sequences found soluble sulfotransferases C. J. 1997; Scholar). studies are to the sequence found Golgi-associated sulfotransferases a role in the studies revealed that the cloned human HNK-1ST the HNK-1 carbohydrate epitope on al. H. I. Oka S. Kawasaki T. N. Schachner M. N. J. Biol. Chem. 1997; 272: Scholar) on the other that the rat GlcAT-P and rat HNK-1ST add the acid and sulfate into a of glycoproteins in cells, although of them was results that GlcAT-P add glucuronic acid in a of glycoproteins and HNK-1ST those carbohydrates as studies also demonstrate that the transcript of HNK-1ST is in compared with that of GlcAT-P In the both the HNK-1ST and GlcAT-P transcripts are highly suggesting that the cloned HNK-1ST and GlcAT-P are responsible for the formation of the HNK-1 glycan in the nervous tissues. has been shown that there are for forming the HNK-1 precursor in glycoproteins and S. K. C. Kawasaki T. J. Biol. Chem. 1992; Scholar). In contrast, HNK-1ST cloned in the was shown to add a sulfate group to both glycoproteins and The HNK-1ST in other the brain may on acceptors. there is GlcAT-P on which may in tissue the one that has been cloned (18Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Google Scholar). studies are to this As described in the the HNK-1 carbohydrate is with a number of cell adhesion molecules in the nervous tissues. The addition of the HNK-1 glycan to these various adhesion molecules are to cell-cell and cell-substratum interactions. The HNK-1 cDNA in the be a to the expression of the HNK-1 glycan in cell allowing to the and cell of cell-cell interactions We and Walsh for the of and respectively, and for and for the