Expression Cloning of a cDNA Encoding a Sulfotransferase Involved in the Biosynthesis of the HNK-1 Carbohydrate Epitope

Abstract

The HNK-1 carbohydrate epitope is expressed on several neural adhesion glycoproteins and as a glycolipid, and is involved in cell interactions. The structural element of the epitope common to glycoproteins and glycolipids has been determined to be sulfate-3-GlcAβ1→ 3Galβ1→4GlcNAc. The glucuronyltransferase and sulfotransferase are considered to be the key enzymes in the biosynthesis of this epitope because the rest of the structure occurs often in glycoconjugates. Here we describe the isolation of the rat sulfotransferase cDNA via an expression cloning strategy. The clone finally isolated predicts a protein of 356 amino acids, with characteristics of a type II transmembrane protein and with no sequence similarity to other known sulfotransferases. Both the enzyme expressed as a soluble fusion protein and homogenates of cells transfected with the full-length cDNA could transfer sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid. The HNK-1 carbohydrate epitope is expressed on several neural adhesion glycoproteins and as a glycolipid, and is involved in cell interactions. The structural element of the epitope common to glycoproteins and glycolipids has been determined to be sulfate-3-GlcAβ1→ 3Galβ1→4GlcNAc. The glucuronyltransferase and sulfotransferase are considered to be the key enzymes in the biosynthesis of this epitope because the rest of the structure occurs often in glycoconjugates. Here we describe the isolation of the rat sulfotransferase cDNA via an expression cloning strategy. The clone finally isolated predicts a protein of 356 amino acids, with characteristics of a type II transmembrane protein and with no sequence similarity to other known sulfotransferases. Both the enzyme expressed as a soluble fusion protein and homogenates of cells transfected with the full-length cDNA could transfer sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid. The carbohydrate antigen recognized by the monoclonal antibody HNK-1 was originally described as a marker for human natural killer cells (1Abo T. Balch C.M. J. Immunology. 1981; 127: 1024-1029PubMed Google Scholar). Later it was shown to be expressed predominantly on glycolipids and glycoproteins from nervous tissue (2McGarry R.C. Helfand S.L. Quarles R.H. Roder J.C. Nature. 1983; 306: 376-378Crossref PubMed Scopus (370) Google Scholar, 3Ilyas A.A. Quarles R.H. Brady R.O. Biochem. Biophys. Res. Commun. 1984; 122: 1206-1211Crossref PubMed Scopus (71) Google Scholar, 4Kruse J. Mailhammer R. Wernecke H. Faissner A. Sommer I. Goridis C. Schachner M. Nature. 1984; 311: 153-155Crossref PubMed Scopus (585) Google Scholar, 5Yuen 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 (112) Google Scholar). The expression pattern of the HNK-1 carbohydrate in both the central and peripheral nervous system is spatially and developmentally regulated (6Wernecke H. Lindner J. Schachner M. J. Neuroimmunol. 1985; 9: 115-130Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 7Holley J.A. Yu R.K. Dev Neurosci. 1987; 9: 105-119Crossref PubMed Scopus (42) Google Scholar, 8Prasadarao N. Koul O. Tobet S.A. Chou D.K. Jungalwala F.B. J. Neurochem. 1990; 55: 2024-2030Crossref PubMed Scopus (40) Google Scholar, 9Chou D.K. Prasadarao N. Koul O. Jungalwala F.B. J. Neurochem. 1991; 57: 852-859Crossref PubMed Scopus (53) Google Scholar, 10Löw K. Orberger G. Schmitz B. Martini R. Schachner M. Eur. J. Neurosci. 1994; 6: 1773-1781Crossref PubMed Scopus (41) Google Scholar, 11Jungalwala F.B. Neurochem. Res. 1994; 19: 945-957Crossref PubMed Scopus (111) Google Scholar). The HNK-1 carbohydrate epitope is carried by many, but not all, neural recognition glycoproteins and is involved in homo- and heterophilic binding of these proteins (for a review, see Ref. 12Schachner M. Martini R. Trends Neurosci. 1995; 18: 183-191Abstract Full Text PDF PubMed Scopus (181) Google Scholar). Of special interest is the association of the epitope with Schwann cells myelinating motor but not sensory axons (10Löw K. Orberger G. Schmitz B. Martini R. Schachner M. Eur. J. Neurosci. 1994; 6: 1773-1781Crossref PubMed Scopus (41) Google Scholar), where it may be involved in the preferential reinervation of muscle nerves by motor axons after lesion (13Martini R. Xin Y. Schmitz B. Schachner M. Eur. J. Neurosci. 1992; 4: 628-639Crossref PubMed Scopus (176) Google Scholar, 14Martini R. Schachner M. Brushart T.M. J. Neurosci. 1994; 14: 7180-7191Crossref PubMed Google Scholar). Determination of the structure of the glycolipid (15Chou 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) and glycoprotein (16Voshol H. van Zuylen C.W.E.M. Orberger G. Vliegenthart J.F.G. Schachner M. J. Biol. Chem. 1996; 271: 22957-22960Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) forms has shown that both carry sulfate-3-GlcAβ1→3Galβ1→4GlcNAc at the nonreducing end. The minimal requirement for recognition by HNK-1 is unknown, but the antibody only binds to the sulfated form(17Ilyas A.A. Chou D.K. Jungalwala F.B. Costello C. Quarles R.H. J. Neurochem. 1990; 55: 594-601Crossref PubMed Scopus (63) Google Scholar). Several other monoclonal antibodies have been isolated that recognize identical or similar structures(4Kruse J. Mailhammer R. Wernecke H. Faissner A. Sommer I. Goridis C. Schachner M. Nature. 1984; 311: 153-155Crossref PubMed Scopus (585) Google Scholar, 18Noronha A.B. Ilyas A. Antonicek H. Schachner M. Quarles R.H. Brain Res. 1986; 385: 237-244Crossref PubMed Scopus (75) Google Scholar); of these, L2–412 is important for this study, because it also recognizes the non-sulfated form of the carbohydrate(19Schmitz B. Schachner M. Ito Y. Nakano T. Ogawa T. Glycoconjugate J. 1994; 11: 345-352Crossref PubMed Scopus (43) Google Scholar). The key enzymes in the biosynthesis of HNK-1 carbohydrates are a glucuronyltransferase (20Chou D.K.H. Flores S. Jungalwala F.B. J. Biol. Chem. 1991; 266: 17941-17947Abstract Full Text PDF PubMed Google Scholar, 21Oka S. Terayama K. Kawashima C. Kawasaki T. J. Biol. Chem. 1992; 267: 22711-22714Abstract Full Text PDF PubMed Google Scholar), transferring GlcA in β1→3 linkage to a terminal galactose, and a sulfotransferase (22Chou D.K.H. Jungalwala F.B. J. Biol. Chem. 1993; 268: 330-336Abstract Full Text PDF PubMed Google Scholar), responsible for coupling sulfate to the C-3 position of this GlcA residue. A cDNA encoding the glucuronyltransferase involved in the biosynthesis of at least the HNK-1 glycoprotein epitope has recently been cloned (23Terayama 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). We describe here the cloning of a cDNA coding for a sulfotransferase active on terminal glucuronic acid residues and whose expression can render cells immunoreactive with HNK-1 antibody when cotransfected with a glucuronyltransferase cDNA. CHOP2 cells (24Cummings L. Warren C.E. Granovsky M. Dennis J.W. Biochem. Biophys. Res. Commun. 1993; 195: 814-822Crossref PubMed Scopus (8) Google Scholar) were grown in minimal essential medium α supplemented with 10% fetal calf serum, penicillin/streptomycin, and 200 μg/ml G418 (all from Life Technologies, Basel, Switzerland). For transfections, G418 was omitted. Hybridoma supernatants containing antibodies HNK-1 (mouse, Ref. 1Abo T. Balch C.M. J. Immunology. 1981; 127: 1024-1029PubMed Google Scholar) and L2–412 (rat, Ref. 4Kruse J. Mailhammer R. Wernecke H. Faissner A. Sommer I. Goridis C. Schachner M. Nature. 1984; 311: 153-155Crossref PubMed Scopus (585) Google Scholar) were produced as described(18) and used without further purification. The glucuronyltransferase cDNA was in the mammalian expression vector pEF-BOS (23Terayama 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). Poly(A)+ RNA from cerebral cortex of newborn rats was converted to double-stranded cDNA using a kit from Stratagene, and a 1–3 kb fraction prepared by gel electrophoresis was cloned into the vector pXMD1 (25Kluxen F.-W. Bruns C. Lübbert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4618-4622Crossref PubMed Scopus (194) Google Scholar). Plates with transformed colonies were replicated to nitrocellulose filters, which were then further processed for storage as filter “sandwiches” at −80 °C (26Hanahan D. Meselson M. Gene. 1980; 10: 63-67Crossref PubMed Scopus (503) Google Scholar). Bacteria remaining on the plates were regrown overnight and collected for preparation of plasmid DNA. Subpools were prepared by cutting replica sandwiches into 10–12 pieces and regrowing the bacteria from one side of the sandwich. For production of a fusion protein, the sulfotransferase cDNA downstream from the MscI site at nucleotide 319 (see below) was subcloned into the filled EcoRI site of plasmid pPROTA (27Sanchez-Lopez R. Nicholson R. Gesnel M.C. Matrisian L.M. Breathnach R. J. Biol. Chem. 1988; 263: 11892-11899Abstract Full Text PDF PubMed Google Scholar). In cotransfection experiments, 2 parts of test plasmids were supplemented with 1 part plasmid coding for glucuronyltransferase (23Terayama 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). CHOP2 cells were transfected using DEAE-dextran (28Kluxen F.-W. Lübbert H. Anal. Biochem. 1993; 208: 352-356Crossref PubMed Scopus (45) Google Scholar) but without chloroquine treatment. Glutaraldehyde-fixed monolayers of transfected cells were stained with HNK-1 or L2–412 antibody, followed by HRP-conjugated secondary antibody (goat anti-mouse for HNK-1 and goat anti-rat for L2–412, both from Jackson ImmunoResearch) and color development with 3-amino-9-ethylcarbazole. CHOP2 cells were harvested by scraping in phosphate-buffered saline 2 days after transfection with the sulfotransferase cDNA plasmid. After centrifugation, the cells from an 80-cm2 flask were taken up in 250 μl of 100 mm Bis-TRIS, pH 6.6, containing mixed protease inhibitors (chymostatin/pepstatin A/leupeptin/antipain/aprotinin, all at 10 μg/ml). Aliquots were stored at −20 °C and only thawed once. Cerebral cortex of 7-day-old rats was homogenized in the same buffer (25% w/v) and similarly aliquoted. The sulfotransferase assays were done in 100 mm Bis-TRIS (pH 6.6), 10 mm MnCl2, 2.5 mm ATP, and 0.1% Triton X-100, in a final volume of 20 μl including 10 μl of the transfected cell homogenate (50 μg protein) or 5 μl of 25% brain homogenate (30 μg protein), 100 pmol of [35S]PAPS 1The abbreviations used are: PAPS, 3′-phosphoadenosine-5′-phosphosulfate; Galβ-pNP, 4-nitrophenyl-β-d-galactose; GlcAβ1→3Galβ-R, 2-heptanoylamidoethyl-(3-O-β-d-glucuronyl)-β-d-galactose; GlcAβ-pNP, 4-nitrophenyl-β-d-glucuronic acid; HPTLC, high performance-thin layer chromatography; EST, expressed sequence tag. (900 Bq; from New England Nuclear, diluted with unlabeled PAPS from Sigma), and 10 nmol of acceptor substrate. The acceptors used were 4-nitrophenyl-β-d-galactose (Galβ-pNP), 4-nitrophenyl-β-d-glucuronic acid (GlcAβ-pNP) (both Fluka) or 2-heptanoylamidoethyl-(3-O-β-d-glucuronyl)-β-d-galactose (GlcAβ1→3Galβ-R). 2A. V. Kornilov, L. O. Kononov, A. A. Sherman, and N. E. Nifant'ev, unpublished data. The mixture was incubated for 2 h at 37 °C with mild shaking. Then 100 μl of 4:6, water:methanol was added to the samples and centrifuged, and the residue was re-extracted with 100 μl of 1:1 water:methanol. Combined supernatants were dried, redissolved in 10 μl of 1:1, water:methanol, and run on aluminum-supported HPTLC plates (Silica Gel 60, Merck) in 5:4:1, chloroform:methanol:0.25% KCl/water. Activity was assessed using a Phosphorimager (Molecular Dynamics) or by autoradiography. For the protein A fusion protein, the medium of transfected cells was replaced 1 day after transfection by medium with 5% low immunoglobulin calf serum (Life Technologies, Basel, Switzerland) and incubated for 2 more days. The medium (20 ml) was filtered (5 μm) and incubated overnight with 100 μl of human IgG-agarose beads (Sigma) after addition of 0.05% sodium azide and mixed protease inhibitors (see above). The beads were washed 3 times with 1 ml of 100 mm Bis-TRIS, pH 6.6, containing 0.05% sodium azide, 1 mm MnCl2, and 1 mg/ml bovine serum albumin and then stored in this buffer at 4 °C in the same volume as the cell pellets. Assays were carried out directly with the beads as with cell homogenates, except that Triton X-100 was omitted. For expression screening, we used the cell line CHOP2 (24Cummings L. Warren C.E. Granovsky M. Dennis J.W. Biochem. Biophys. Res. Commun. 1993; 195: 814-822Crossref PubMed Scopus (8) Google Scholar), a derivative of the Lec2 cell line (29Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-404Crossref PubMed Scopus (124) Google Scholar) that lacks the CMP-sialic acid transporter. This mutation results in an increase in glycoproteins and glycolipids terminating in β4-galactose, potentially increasing the amount of substrate available to the glucuronyl and subsequently acting sulfotransferase. Transfection of CHOP2 cells with the recently cloned glucuronyltransferase (23Terayama 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) indeed led to very clear surface staining of the cells with antibody L2–412, which recognizes the nonsulfated form of the carbohydrate (Fig.1 E). Plasmid DNA from 40 pools of 5,000–10,000 cerebral cortex cDNA clones was cotransfected with the glucuronyltransferase cDNA. Two days after transfection, the monolayers were stained with HNK-1, and the number of colored cells in each plate was scored. In one pool, about 20 positive cells were seen, indicating the sulfotransferase was expressed in these cells (Fig. 1 A). In two rounds of subdividing positive pools, the number of positive cells increased first to several hundred (Fig. 1 B) and then to several percent of all cells (Fig. 1 C). A single clone isolated from the last pool gave, upon cotransfection, HNK-1-positive cells at about the same frequency as seen when cells were transfected with glucuronyltransferase cDNA alone and stained with antibody L2–412 (Fig. 1, D and E). This isolated cDNA clone, therefore, is likely to encode the HNK-1 sulfotransferase. The HNK-1 reactivity after transfection with the sulfotransferase cDNA is dependent on the presence of the glucuronyltransferase. Only a very faint HNK-1 reactivity, but clearly higher than in mock transfected cells, is seen after transfection with the sulfotransferase alone (Fig. 1 F). Western blotting of proteins isolated from transfected cells (Fig.2) confirms these results. Mock transfected cells gave no signal with L2–412 or HNK-1, cells transfected with glucuronyltransferase cDNA alone showed only L2–412 reactive proteins, and cells transfected with both glucuronyltransferase and sulfotransferase cDNAs were positive with both antibodies. Faint staining of probably a single protein is seen with both L2–412 and HNK-1 in blots of proteins from cells transfected with only the sulfotransferase cDNA (Fig. 2, A andB, lanes 3), suggesting that Chinese hamster ovary cells already expose a low level of acceptor that can be used by the transfected sulfotransferase. This protein is presumably responsible for the faint HNK-1 immunostaining seen on whole cells transfected only with sulfotransferase cDNA. The presence of sulfotransferase activity was confirmed by enzyme assays in vitro. Sulfotransferase activity was determined with Galβ-pNP, GlcAβ-pNP, and GlcAβ1→3Galβ-R as acceptors (Fig. 3). Homogenates of cells transfected with the isolated sulfotransferase clone showed activity toward GlcAβ-pNP and GlcAβ1→3Galβ1-R, but not to Galβ-pNP. Homogenates of mock-transfected cells gave no sulfotransferase activity; cerebral cortex homogenate could use both GlcAβ-pNP and Galβ-pNP as acceptor, suggesting the presence of more than one sulfotransferase in this tissue. The sulfotransferase activity toward the disaccharide acceptor GlcAβ1→3Galβ-R in brain as measured from panel A, lane 4, is 154 pmol/mg-h, very close to the maximal activity measured by Chou and Jungalwala (22Chou D.K.H. Jungalwala F.B. J. Biol. Chem. 1993; 268: 330-336Abstract Full Text PDF PubMed Google Scholar) using a glycolipid acceptor. Transfected CHOP2 cells show an activity of 80 pmol/mg-h. There was no increase in activity with longer oligosaccharides (data not shown), and activity toward GlcAβ-pNP was about 5 times lower in both rat brain and CHOP2 cells. The enzyme encoded by the cloned cDNA is therefore able to perform the same transfer of sulfate to terminal β-linked GlcA residues as measured in rat cerebral cortex. To more conclusively demonstrate that the isolated cDNA clone encodes the sulfotransferase itself, we produced a fusion protein that could be readily separated from other cellular components. The part of the cDNA encoding the putative cytoplasmic and transmembrane domains was replaced by DNA encoding protein A preceded by a signal sequence. Fusion protein secreted into the medium was captured on human IgG-agarose beads, and sulfotransferase activity was determined (Fig.3 D). Activity was found with the bound protein A fusion protein, but not when the sulfotransferase cDNA was cloned in the reverse orientation or when the protein A moiety was absent. The measured enzyme activity produced per cell is about as high for the secreted protein A fusion as for the sulfotransferase measured in cell The cloned cDNA an encoding a protein of 356 amino The protein probably is a type II transmembrane protein, as a transmembrane is close to the and the activity is to be in the or sequence similarity was the of the cloned cDNA and known than human were found that potentially encode the human of the cloned rat sulfotransferase. Several other showed a lower similarity with the rat cDNA clone and may encode two of the human indicating that probably is a human of at least Several cDNAs encoding enzymes involved in have been isolated by expression cloning M. J. 1996; 6: PubMed Scopus Google Scholar). The often used is and plasmid from transfected mammalian cells. the the after one or several rounds of plasmids are into pools and for expression of the epitope M. J. 1996; 6: PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, M. M. A. R. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). We found it more to directly a The cloned sulfotransferase cDNA was shown to HNK-1 reactivity in CHOP2 cells only in with a indicating that these two with common enzymes already in CHOP2 cells, are and for the biosynthesis of the HNK-1 epitope on not known these two enzymes are responsible for the of all HNK-1 carbohydrate in nervous tissue. the HNK-1 carbohydrate epitope in nervous is only on a number of proteins F.B. Neurochem. Res. 1994; 19: 945-957Crossref PubMed Scopus (111) Google Scholar, 12Schachner M. Martini R. Trends Neurosci. 1995; 18: 183-191Abstract Full Text PDF PubMed Scopus (181) Google Scholar), very proteins to carry the epitope after expression of the enzymes in CHOP2 cells. The in CHOP2 cells may be to of for acceptor by other as and In sulfotransferase assays showed that the cloned cDNA encodes an enzyme of transferring sulfate from PAPS to acceptor substrates containing terminal The disaccharide GlcAβ1→3Galβ-R is as an acceptor as the glycolipid used to the natural and the acceptor of the enzyme encoded by the cloned cDNA seen with brain The cloned enzyme therefore potentially of the known HNK-1 on glycolipids and glycoproteins (15Chou 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 H. van Zuylen C.W.E.M. Orberger G. Vliegenthart J.F.G. Schachner M. J. Biol. Chem. 1996; 271: 22957-22960Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). the cloned sulfotransferase showed no sequence similarity to other not to the recently cloned sulfotransferase K. M. A. T. A. J. Biochem. 1996; PubMed Scopus Google Scholar). The enzyme sulfate from PAPS to the C-3 of a very similar to that of the HNK-1 to transfer the sulfate to C-3 of GlcA cytoplasmic that use substrates all have sequence similarity Biophys. 1996; PubMed Scopus Google Scholar), it be that acting on carbohydrate also be This not found the enzymes whose cDNAs have been cloned two sulfate Y. A. G. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, I. D. B. U. L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) sequence with each M. K. K. M. K. T. O. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (122) Google Scholar), sulfotransferase K. M. A. T. A. J. Biochem. 1996; PubMed Scopus Google Scholar), and the HNK-1 sulfotransferase. all these enzymes show the same and are to be type II proteins with a cytoplasmic and a This structure is of J.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Several human are very similar to the cloned rat HNK-1 sulfotransferase and probably encode the same enzyme in no are in the both human and are found that may encode more of the these can of encode it is that for both the HNK-1 glucuronyltransferase (23Terayama 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) and the HNK-1 sulfotransferase a of to A similar may as for the R. A. R. Biol. 1995; PubMed Scopus Google Scholar), the enzymes only in acceptor be responsible for at least of the in and of HNK-1 immunostaining when are J.A. Yu R.K. Dev Neurosci. 1987; 9: 105-119Crossref PubMed Scopus (42) Google Scholar, 11Jungalwala F.B. Neurochem. Res. 1994; 19: 945-957Crossref PubMed Scopus (111) Google Scholar). The of clones for both a glucuronyltransferase and a sulfotransferase responsible for the biosynthesis of the HNK-1 epitope on the of the expression of these carbohydrate The enzymes can be used to of HNK-1 more than of in the nervous We and for and Dennis of the of for the CHOP2 cells.