Purification and Characterization of a Glucuronyltransferase Involved in the Biosynthesis of the HNK-1 Epitope on Glycoproteins from Rat Brain
1998; Elsevier BV; Volume: 273; Issue: 46 Linguagem: Inglês
10.1074/jbc.273.46.30295
ISSN1083-351X
AutoresKoji Terayama, Takashi Seiki, Akemi Nakamura, Kanae Matsumori, Satoru Ohta, Shogo Oka, Mutsumi Sugita, Toshisuke Kawasaki,
Tópico(s)Cellular transport and secretion
ResumoThe glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins was purified to an apparent homogeneity from the Nonidet P-40 extract of 2-week postnatal rat forebrain by sequential chromatographies on CM-Sepharose CL-6B, UDP-GlcA-Sepharose 4B, asialo-orosomucoid-Sepharose 4B, Matrex gel Blue A, Mono Q, HiTrap chelating, and HiTrap heparin columns. The purified enzyme migrated as a 45-kDa protein upon SDS-polyacrylamide gel electrophoresis under reducing conditions, but eluted as a 90-kDa protein upon Superose gel filtration in the presence of Nonidet P-40, suggesting that the enzyme forms homodimers under non-denatured conditions. The enzyme transferred glucuronic acid to various glycoprotein acceptors bearing terminal N-acetyllactosamine structure such as asialo-orosomucoid, asialo-fetuin, and asialo-neural cell adhesion molecule, whereas little activity was detected to paragloboside, a precursor glycolipid of the HNK-1 epitope on glycolipids. These results suggested that the enzyme is specifically associated with the biosynthesis of the HNK-1 epitope on glycoproteins. Sphingomyelin was specifically required for expression of the enzyme activity. Stearoyl-sphingomyelin (18:0) was the most effective, followed by palmitoyl-sphingomyelin (16:0) and lignoceroyl-sphingomyelin (24:0). Interestingly, activity was demonstrated only for sphingomyelin with a saturated fatty acid,i.e. not for that with an unsaturated fatty acid, regardless of the length of the acyl group . The glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins was purified to an apparent homogeneity from the Nonidet P-40 extract of 2-week postnatal rat forebrain by sequential chromatographies on CM-Sepharose CL-6B, UDP-GlcA-Sepharose 4B, asialo-orosomucoid-Sepharose 4B, Matrex gel Blue A, Mono Q, HiTrap chelating, and HiTrap heparin columns. The purified enzyme migrated as a 45-kDa protein upon SDS-polyacrylamide gel electrophoresis under reducing conditions, but eluted as a 90-kDa protein upon Superose gel filtration in the presence of Nonidet P-40, suggesting that the enzyme forms homodimers under non-denatured conditions. The enzyme transferred glucuronic acid to various glycoprotein acceptors bearing terminal N-acetyllactosamine structure such as asialo-orosomucoid, asialo-fetuin, and asialo-neural cell adhesion molecule, whereas little activity was detected to paragloboside, a precursor glycolipid of the HNK-1 epitope on glycolipids. These results suggested that the enzyme is specifically associated with the biosynthesis of the HNK-1 epitope on glycoproteins. Sphingomyelin was specifically required for expression of the enzyme activity. Stearoyl-sphingomyelin (18:0) was the most effective, followed by palmitoyl-sphingomyelin (16:0) and lignoceroyl-sphingomyelin (24:0). Interestingly, activity was demonstrated only for sphingomyelin with a saturated fatty acid,i.e. not for that with an unsaturated fatty acid, regardless of the length of the acyl group . neural cell adhesion molecule asialo-orosomucoid glucuronyltransferase glycoprotein-specific GlcAT 2-(N-morpholino)ethanesulfonic acid phosphatidylcholine phosphatidylinositol phosphatidylserine phosphatidylethanolamine sphingomyelin phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis. Various cell surface carbohydrate moieties are thought to be involved in cell-to-cell interactions (1Rutishauser U. Acheson A. Hall A.K. Mann D.M. Sunshine J. Science. 1988; 240: 53-57Crossref PubMed Scopus (670) Google Scholar, 2Jessell T.M. Hynes M.A. Dodd J. Annu. Rev. Neurosci. 1990; 13: 227-255Crossref PubMed Scopus (180) Google Scholar). The HNK-1 carbohydrate epitope, which is recognized by HNK-1 monoclonal antibody, is found on many neural cell adhesion molecules, such as neural cell adhesion molecule (NCAM)1 (3Kruse J. Mailhammer R. Wernecke H. Faissner A. Sommer I. Goridis C. Schachner M. Nature. 1984; 311: 153-155Crossref PubMed Scopus (580) Google Scholar), myelin-associated glycoproteins (4McGarry R.C. Helfand S.L. Quarles R.H. Roder J.C. Nature. 1983; 306: 376-378Crossref PubMed Scopus (366) Google Scholar), L1 (3Kruse J. Mailhammer R. Wernecke H. Faissner A. Sommer I. Goridis C. Schachner M. Nature. 1984; 311: 153-155Crossref PubMed Scopus (580) Google Scholar), transiently expressed axonal glycoprotein-1 (5Dodd J. Morton S.B. Karagogeos D. Yamamoto M. Jessell T.M. Neuron. 1988; 1: 105-116Abstract Full Text PDF PubMed Scopus (639) Google Scholar), and P0 (6Bollensen E. Schachner M. Neurosci. Lett. 1987; 82: 77-82Crossref PubMed Scopus (113) Google Scholar), and some proteoglycans (7Krueger Jr., R.C. Hennig A.K. Schwartz N.B. J. Biol. Chem. 1992; 267: 12149-12161Abstract Full Text PDF PubMed Google Scholar). Expression of the HNK-1 carbohydrate epitope is spatially and temporally regulated during development, and its highest expression is seen at the stages where the neural networks are constructed in the central and peripheral nervous systems (8Bronner-Fraser M. Dev. Biol. 1986; 115: 44-55Crossref PubMed Scopus (367) Google Scholar, 9Yoshihara Y. Oka S. Watanabe Y. Mori K. J. Cell Biol. 1991; 115: 731-744Crossref PubMed Scopus (57) Google Scholar, 10Schwarting G.A. Jungalwala F.B. Chou D.K. Boyer A.M. Yamamoto M. Dev. Biol. 1987; 120: 65-76Crossref PubMed Scopus (141) Google Scholar). The HNK-1 epitope is presumed to be involved in cell-to-cell interactions such as cell adhesion (11Keilhauer G. Faissner A. Schachner M. Nature. 1985; 316: 728-730Crossref PubMed Scopus (408) Google Scholar), migration (12Bronner-Fraser M. Dev. Biol. 1987; 123: 321-331Crossref PubMed Scopus (145) Google Scholar), and neurite extension (13Martini R. Xin Y. Schmitz B. Schachner M. Eur. J. Neurosci. 1992; 4: 628-639Crossref PubMed Scopus (173) Google Scholar). The epitope is expressed not only on glycoproteins but also on glycolipids. The structures of the HNK-1 reactive glycolipids are: SGGL-1 (HSO3-GlcAβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glcβ1-Cer) and SGGL-2 (HSO3-GlcAβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glcβ1-Cer) (14Chou D.K.H. 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, 15Ariga 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 inner unit of their glycolipids, Galβ1–4GlcNAcβ1, is commonly found in mammalian glycoproteins and glycolipids, and the unique feature of this epitope is the terminal sulfo-3-glucuronyl group. This structure was shown to be essential not only for immunoreactivity with HNK-1 monoclonal antibodies (16Ilyas A.A. Chou D.K.H. Jungalwala F.B. Costello C. Quarles R.H. Cancer Res. 1988; 48: 1512-1516PubMed Google Scholar) but also for their functions (17Schmitz B. Schachner M. Ito Y. Nakano T. Ogawa T. Glycoconjugate J. 1994; 11: 345-352Crossref PubMed Scopus (43) Google Scholar). Therefore, in order to study the functions of the HNK-1 carbohydrate epitope during construction of the central and peripheral nervous systems, it is important to characterize the glucuronyltransferase that transfers glucuronic acid from uridine 5′-diphosphoglucuronic acid to the terminalN-acetyllactosamine structure of glycoproteins and glycolipids. In our previous study, it was demonstrated that there are two types of glucuronyltransferases associated with the biosynthesis of the HNK-1 carbohydrate epitope in rat brain, one for glycolipid acceptors (18Kawashima C. Terayama K. Ii M. Oka S. Kawasaki T. Glycoconjugate J. 1992; 9: 307-314Crossref PubMed Scopus (21) Google Scholar) and the other for glycoprotein acceptors (19Oka S. Terayama K. Kawashima C. Kawasaki T. J. Biol. Chem. 1992; 267: 22711-22714Abstract Full Text PDF PubMed Google Scholar). Similar glucuronyltransferase activities were found in chick (20Das K.K. Basu M. Basu S. Chou D.K.H. Jungalwala F.B. J. Biol. Chem. 1991; 266: 5238-5243Abstract Full Text PDF PubMed Google Scholar) and rat (21Chou D.K.H. Flores S. Jungalwala F.B. J. Biol. Chem. 1991; 266: 17941-17947Abstract Full Text PDF PubMed Google Scholar) brains with paragloboside as an acceptor. In this study, a glucuronyltransferase specific for glycoprotein acceptors (GlcAT-P) was purified to apparent homogeneity from postnatal 2-week rat forebrains by means of various column chromatographies. The enzyme is a 45-kDa protein, which requires sphingomyelin (SM) for the expression of its transferase activity. UDP-[14C]GlcA (10.2 GBq/mmol) was purchased from ICN Radiochemicals. UDP-GlcA, UDP-GlcNAc, ATP, GlcA, Nonidet P-40, and benzamidine were from Nakalai Tesque Inc. (Kyoto, Japan). MES was from Dojindo (Kumamoto, Japan). UDP, aprotinin, and pepstatin A were purchased from Sigma. Heparin and phenylmethylsulfonyl fluoride (PMSF) were from Wako Chemicals (Osaka, Japan). Heparin sulfate, hyaluronic acid, chondroitin, and neolactotetraosyl-ceramide (nLc-Cer) were from Seikagaku Corp. (Tokyo, Japan).N-Acetyllactosamine was provided by Yaizu Suisan Kagaku Kogyo Inc. (Yaizu, Japan). Orosomucoid was provided by Dr. M. Wickerhauser of the American Red Cross Research Center (Bethesda, MD). ASOR (asialo-orosomucoid) was prepared by hydrolysis of orosomucoid with 0.05 m H2SO4 for 1 h at 80 °C (22Kawasaki T. Ashwell G. J. Biol. Chem. 1976; 251: 1296-1302Abstract Full Text PDF PubMed Google Scholar). Dye Matrex Blue A-agarose was obtained from Amicon (Davers, MA). CM-Sepharose CL-6B, EAH-Sepharose 4B, and CNBr-activated Sepharose 4B resins, and Mono Q, HiTrap chelating, and HiTrap heparin columns were from Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden). An anti-rat NCAM monoclonal antibody (AF11) (23Ono K. Asou H. Yamada M. Tokunaga A. Neurosci. Res. 1992; 15: 221-223Crossref PubMed Scopus (18) Google Scholar) was kindly provided by Dr. Katsuhiko Ono (Shimane Medical College, Japan). Postnatal 2-week Wistar rats were purchased from Oriental Bio-service (Kyoto, Japan). The Wistar rats were anesthetized with diethylether and then sacrificed. Their brains were removed and immediately frozen on dry ice. The frozen rat brains were stored at −80 °C. ASOR-Sepharose 4B and anti-NCAM antibody-conjugated Sepharose 4B were performed by coupling ASOR (300 mg) to CNBr-activated Sepharose 4B (30 ml) and AF11 antibody (5 mg) to CNBr-Sepharose 4B (5 ml), respectively, according to the procedure described previously (19Oka S. Terayama K. Kawashima C. Kawasaki T. J. Biol. Chem. 1992; 267: 22711-22714Abstract Full Text PDF PubMed Google Scholar). UDP-GlcA-Sepharose 4B was prepared by coupling UDP-GlcA (1 g) to EAH-Sepharose 4B (100 ml) according to the procedure described by Anttinen and Kivurikko (24Anttinen H. Kivirikko K.I. Biochim. Biophys. Acta. 1976; 429: 750-758Crossref PubMed Scopus (26) Google Scholar). Glucuronyltransferase activity toward glycoprotein acceptors was measured essentially as described previously (19Oka S. Terayama K. Kawashima C. Kawasaki T. J. Biol. Chem. 1992; 267: 22711-22714Abstract Full Text PDF PubMed Google Scholar) with slight modification. Incubation was carried out 37 °C for 3 h in an assay mixture comprising 20 μg of ASOR, 100 μm UDP-[14C]GlcA (2×105dpm), 200 mm MES buffer, pH 6.5, 20 mmMnCl2, 0.5 mm ATP, 0.2% (v/v) Nonidet P-40, and 2 μl of a 2% Nonidet P-40 extract of rat forebrain, which had been treated at 100 °C for 3 min, in a final volume of 50 μl. After incubation, the assay mixture was spotted onto a 2.5-cm Whatman No. 1 disc and the radioactivity of [14C]GlcA-ASOR on the discs was counted with a liquid scintillation counter (Beckman LS-6000). Protein was quantitated with a Micro-BCA protein assay kit (Pierce) unless otherwise stated. Bovine serum albumin was used as a standard. Homogenizing buffer consisted of 20 mm MES buffer, pH 6.5, 0.32 m sucrose, 1 mm EDTA, 0.1% (v/v) β-mercaptoethanol, 1 μg/ml aprotinin, 0.7 μg/ml pepstatin A, 10 μg/ml benzamidine, 0.1 mg/ml PMSF. Extracting buffer consisted of the homogenizing buffer containing 0.5% (v/v) Nonidet P-40 instead of sucrose. Buffer A consisted of 20 mm MES buffer, pH 6.5, 0.5% Nonidet P-40. Buffer B consisted of 20 mm MES buffer, pH 6.5, 0.5% Nonidet P-40, 1 mmEDTA. Buffer C consisted of 10 mm MES buffer, pH 6.5, 0.5m NaCl, 0.1% Nonidet P-40, 1 mm EDTA. Buffer D consisted of 10 mm MES buffer, pH 6.5, 0.1% Nonidet P-40, 20 mm MnCl2, 0.5 m NaCl, 0.1 mm UDP, 20% glycerol. Buffer E consisted of 10 mm MES buffer, pH 6.5, 0.1% Nonidet P-40, 10 mm EDTA, 0.5 m NaCl, 20% glycerol. Buffer F consisted of 10 mm MES buffer, pH 6.5, 0.1% Nonidet P-40, 0.25 m NaCl. Buffer G consisted of 10 mm MES buffer, pH 6.5, 0.1% Nonidet P-40,1 m NaCl. Buffer H consisted of 20 mm Tris/HCl buffer, pH 8.0, 0.1% Nonidet P-40, 10 mm NaCl. Buffer I consisted of 20 mmTris/HCl buffer, pH 8.0, 0.1% Nonidet P-40, 0.5 m NaCl. Buffer J consisted of 10 mm MES buffer, pH 6.5, 0.1% Nonidet P-40, 0.1 m NaCl. Buffer K consisted of 10 mm MES butter, pH6.5, 0.1% Nonidet P-40, 1 mNaCl. Preparation of the enzyme source and the following purification procedure were carried out at 4 °C. Sixty frozen rat forebrains (60 g) were thawed and homogenized with five volumes of the homogenizing buffer. Each homogenate was centrifuged at 10,000 × g for 10 min, and the resulting supernatant was centrifuged at 105,000 ×g for 1 h. The pellet was suspended in five volumes of the extracting buffer for 1 h, and then the suspension was centrifuged at 105,000 × g for 1 h. The pellet was reextracted once more with three volumes of the extracting buffer. To the combined extracts glycerol was added to give a final concentration of 20%. The resulting solution (Nonidet P-40 extract) could be stored at −20 °C for at least 2 months without loss of activity. CM-Sepharose CL-6B (50 ml) equilibrated with buffer A was added to the Nonidet P-40 extract prepared above. After shaking for 1 h, the suspension was filtered through a glass filter (CM-unbound fraction). The CM-unbound fraction was applied to a UDP-GlcA-Sepharose 4B column (100 ml; 5 × 5.5 cm), which had been equilibrated with 500 ml of buffer B. After washing the column, the enzyme was eluted with buffer C. The flow-through fraction was applied to a UDP-GlcA-Sepharose 4B column once more, and the enzyme was eluted with buffer C. To the combined eluate, glycerol, MnCl2, and UDP were added to final concentrations of 20%, 20 mm, and 0.1 mm, respectively. The eluate from the UDP-GlcA-Sepharose 4B column was applied to an ASOR-Sepharose 4B column (30 ml; 5 × 1.8 cm), which had been equilibrated with buffer D. After washing the column with buffer D and then buffer E, glucuronyltransferase activity was eluted with 200 ml of buffer E containing 10 mm N-acetyllactosamine. The eluate from the ASOR-Sepharose 4B column was dialyzed against buffer F. The dialysate was applied to a Matrex gel Blue A column (2 ml; 1.5 × 1.2 cm), which had been equilibrated with buffer F. The enzyme was recovered by elution with buffer G. The eluate from the Matrex gel Blue A column was dialyzed against buffer H, and then the dialysate was applied to a Mono Q column (1 ml; 0.6 × 5.2 cm), which had been equilibrated with buffer H. Elution of the enzyme was carried out with buffer H containing a linear gradient of NaCl, from 10 mm to 1 m. The eluate from the Mono Q column was applied to a HiTrap chelating column (1 ml; 1.0 × 2.4 cm), which had been chelated with copper ions and equilibrated with buffer I. The enzyme was eluted with buffer I containing a linear gradient of glycine, from 0 to 30 mm. The eluate from the HiTrap chelating column was dialyzed against buffer J, and then applied to a HiTrap heparin column, which had been equilibrated with the same buffer. The enzyme was eluted with buffer J containing a gradient of NaCl, from 0.1 to 1.5 m. Enzyme fractions were treated with dithiothreitol at a final concentration of 100 mm prior to SDS-PAGE. SDS-PAGE was performed with a 10% polyacrylamide gel and the buffer system of Laemmli (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). The protein bands were stained with a silver stain kit (Wako, Osaka, Japan). Approximately 100 ng of the purified enzyme was applied to a Superose 12 column (25 ml; 1 × 30 cm), which had been equilibrated with buffer K. The flow rate was 0.4 ml/min, and fractions of 0.4 ml were collected. Total lipids were extracted from lyophilized rat brains (8.4 g) twice with 200 ml of chloroform-methanol, 2:1 (v/v), and then once with 250 ml of chloroform-methanol, 1:1 (v/v). The chloroform-methanol extract was subjected to Folch's partitioning (26Folch J. Ascoli I. Less M. Meath J.A. LeBaron F.N. J. Biol. Chem. 1951; 191: 833-841Abstract Full Text PDF PubMed Google Scholar). The lower phase (1.87 g) was charged onto a DEAE-Sephadex A-25 column (2 × 40 cm, acetate form; Pharmacia LKB Biotechnology) equilibrated with chloroform-methanol-water, 30:60:8 (v/v), and then the column was eluted successively with the same solvent (five bed volumes) and with 0.45 m ammonium acetate in methanol (five bed volumes). One-eighth of the unbound lipid fraction was applied to an Alumina column (1 × 20 cm, ICN Alumina N-Super I; ICN Biomedicals GmbH, Eschwege, Germany). The column was successively eluted with 200 ml each of chloroform, chloroform-methanol, 95:5 (v/v), and chloroform-methanol 80:20 (v/v), and then chloroform-methanol-water, 60:40:10 (v/v). Phosphatidylcholine (PC, 23 mg) was recovered from the chloroform-methanol (95:5) eluate in an almost homogeneous state, a mixture (40 mg) of PC, SM, and lyso-PC from the chloroform-methanol (80:20) eluate, and a mixture of phosphatidylethanolamine (PE) and lyso-PE from the chloroform-methanol-water eluate. Separation of PE and lyso-PE was carried out by Iatrobeads column (1 × 60 cm, 6RS-8060; Iatron Laboratory, Tokyo, Japan) chromatography. The column was eluted with a solvent mixture of chloroform-methanol-water, 65:25:4 (v/v). The yields of PE and lyso-PE fractions were 14.9 and 7.0 mg, respectively. The phosphatidylserine (PS) and phosphatidylinositol (PI) fractions, bound to the DEAE-Sephadex A-25 column, and then recovered by elution with 0.45 m ammonium acetate in methanol, were subjected to Iatrobeads column (2 × 60 cm, 6RS-8060) chromatography. The column was eluted with a solvent mixture of chloroform-methanol-water, 65:25:4 (v/v). The yields of the PS and PI fractions were 42 and 20 mg, respectively. One-fourth of the unbound lipid fraction recovered on DEAE-Sephadex A-25 chromatography was applied to a QAE-Sephadex A-25 (OH− form; Pharmacia LKB Biotechnology) column (2.5 × 20 cm) equilibrated with chloroform-methanol-water, 30:60:8 (v/v). The unbound lipid fraction from the column was evaporated, acetylated, and then fractionated on a Florisil column (1 × 20 cm, 60–100 mesh; Floriden Co., New York, NY) by the method of Saito and Hakomori (27Saito T. Hakomori S. J. Lipid Res. 1971; 12: 257-259Abstract Full Text PDF PubMed Google Scholar) with slight modification. The acetylated neutral glycolipid fraction and the acetylated SM fraction were deacetylated with 0.5 m KOH in methanol at 37 °C for 6 h. The yield of the deacetylated neutral glycolipid fraction containing ceramide mono-, di-, and trisaccharides was 2.7 mg. The deacetylated SM fraction was applied to an Iatrobeads column (4.6 mm × 25 cm, 6RS-8010), and the column was eluted with a solvent mixture of chloroform-methanol-water, 65:25:4 (v/v), at 40 °C. SMs were prepared by N-acylation of lyso-SM (sphingosylphosphorylcholine) with fatty acylchlorides (28Kopaczyk K.C. Radin N.S. J. Lipid Res. 1965; 6: 140-145Abstract Full Text PDF PubMed Google Scholar). SM isolated from bovine brain was partially hydrolyzed with 6m HCl-butanol, 1:1 (v/v), at 100 °C for 1 h for preparation of lyso-SM (29Kaller H. Biochem. Z. 1961; 334: 451-456PubMed Google Scholar, 30Hara A. Taketomi T. J. Biochem. 1983; 94: 1715-1718Crossref PubMed Scopus (4) Google Scholar). A mixture of 5 mg of lyso-SM, 0.4 ml of tetrahydrofuran, and 0.5 ml of 50% aqueous sodium acetate was added to about 5 mg of fatty acylchloride (stearoylchloride, oleoylchloride, lignoceroylchloride, or nervonoylchloride; Funacoshi Co., Tokyo, Japan), and the reaction mixture was vigorously stirred for 2 h at 20 °C. The synthetic product was treated with 0.1 mmethanolic NaOH and then purified by high performance liquid chromatography as described above. The purified SM was identified by TLC and infrared spectroscopy. The yields were 2.1 mg forN-stearoyl-, 2.5 mg for N-oleoyl-, 3.0 mg forN-lignoceroyl-, and 3.1 mg for N-nervonoyl-SM, respectively. Frozen and thawed postnatal 2-week rat brains were homogenized with a Positron homogenizer in five volumes of 20 mm Tris buffer, pH 7.5, containing 0.15 m NaCl, 1 mm EDTA, 0.1 mg/ml PMSF, 10 μm leupeptin, and 10 μg/ml trypsin inhibitor. The homogenate was centrifuged at 105,000 × g for 1 h, and the resulting pellet was suspended in five volumes of the same buffer containing 0.5% Nonidet P-40 to extract NCAM. After stirring for 1 h, the suspension was centrifuged at 105,000 × g for 30 min. From the supernatant, NCAM was purified on an AF11-Sepharose 4B column (5 ml). NCAM bound to the column was eluted with 0.1 m diethylamine containing 0.1 m NaCl, 0.1% deoxycholic acid, and 1 mm EDTA. For desialization of the purified NCAM, 10 μg of NCAM was treated with 50 milliunits of neuraminidase (Seikagaku Co., Tokyo, Japan) at 37 °C for 4 h. The results of purification of a glucuronyltransferase from rat brain are summarized in Table I. A preliminary study indicated that more than half of the glucuronyltransferase activity in the homogenate was recovered in the 105,000 ×g pellet, suggesting that the enzyme is mostly associated with the microsomal fraction. Treatment of the pellet with either 1m NaCl or phosphatidylinositol-phospholipase C did not release the enzyme activity, suggesting that the enzyme is an intrinsic membrane protein. Extraction of the pellet with 0.5% Nonidet P-40 released the glucuronyltransferase activity into the soluble fraction.Table IPurification of the glucuronyltransferase specific for glycoprotein acceptorsTotal proteinTotal activitySpecific activityYieldPurificationmgnmol/minnmol/mg/min%-fold0.5% Nonidet P-40 extract68,1002320.003411001CM-Sepharose CL-6B63,9002530.003961091.2UDP-GlcA-Sepharose 4B12,7002150.0078792.62.3ASOR-Sepharose 4B2.7821.37.65(9.16)1-aThe assay was carried out in the presence of N-acetyllactosamine and glycerol, both of which inhibited the glucuronyltransferase activity significantly.(2,250)1-aThe assay was carried out in the presence of N-acetyllactosamine and glycerol, both of which inhibited the glucuronyltransferase activity significantly.DyeMatrex Gel Blue A1.1588.376.838.022,500Mono Q0.092532.935614.2104,000HiTrap chelating0.03221.86819.4200,000HiTrap heparin (1st)0.004411.52,6005.0768,000HiTrap heparin (2nd)0.00229.44,3004.01,220,0001-a The assay was carried out in the presence of N-acetyllactosamine and glycerol, both of which inhibited the glucuronyltransferase activity significantly. Open table in a new tab As the first step of purification, the Nonidet P-40 extract was subjected to CM-Sepharose CL-6B cation exchange chromatography. Essentially all the glucuronyltransferase activity was recovered in the unbound fraction, with a specific activity increase of 1.2-fold. The next step of the purification involved UDP-GlcA affinity chromatography. A preliminary experiment indicated that essentially all (97%) the glucuronyltransferase activity toward glycolipid acceptors, with neolactotetraose-phenyl-C14H29(nLc-PA14) as a substrate (19Oka S. Terayama K. Kawashima C. Kawasaki T. J. Biol. Chem. 1992; 267: 22711-22714Abstract Full Text PDF PubMed Google Scholar), was recovered in the pass-through fraction, while a larger portion of the activity toward glycoprotein acceptors, with ASOR as a substrate, was recovered in the eluate fraction. Consistent with this preliminary result, approximately 93% of the glucuronyltransferase activity in the CM-Sepharose CL-6B-unbound fraction was recovered in the eluate fraction with a purification of about 2-fold. The third step of the purification involved ASOR-conjugated Sepharose affinity chromatography. Among several buffers tested, a buffer containing 10 mm N-acetyllactosamine was effective for elution of the activity from the column. BecauseN-acetyllactosamine and glycerol inhibited the glucuronyltransferase activity, the apparent yield of the enzymatic activity in the eluate fraction was very low (9% that of the Nonidet P-40 extract; see Table I). However, after dialysis, the activity recovered to more than 30% that of the Nonidet P-40 extract. The purification achieved with this ASOR-Sepharose 4B affinity chromatography was over 1,000-fold. The fourth step of the purification involved dye ligand affinity chromatography. A Matrex gel Blue A column was found to retain the glucuronyltransferase activity. 1 m NaCl was effective for dissociating the enzyme from the column, with a 10-fold increase in the specific activity. The fifth step of the purification involved Mono Q anion exchange chromatography. The glucuronyltransferase activity was mainly eluted in fractions 43–47 (Fig. 1), in which the concentration of NaCl was around 0.4 m. Because of the low amount of proteins in each fraction, protein quantification and SDS-PAGE were carried out with several (five to seven) fractions combined. Five-fold purification was achieved through this step. In the following purification steps (HiTrap chelating and HiTrap heparin columns), the protein concentrations were determined in the same way. The sixth step of the purification involved HiTrap chelating metal chelate affinity chromatography. Mono Q eluate fractions 43–47 were pooled and applied to a HiTrap chelating column, which had been chelated with Cu2+. The glucuronyltransferase activity was eluted at a glycine concentration of 15 mm (fractions 22–28 in Fig. 2). Two-fold purification was achieved at this step. The last step of the purification involved HiTrap heparin affinity chromatography, utilizing the inhibitory activity of heparin, as shown in Table II. The glucuronyltransferase bound to the column and was eluted in fractions 43–47 at a NaCl concentration of around 0.7 m (Fig. 3 A). Upon SDS-PAGE, a major band of 45 kDa was observed, with a few minor bands. Since these minor bands were predominant components in the previous fractions,i.e. fractions 38–42, the HiTrap heparin affinity chromatography was repeated. The glucuronyltransferase thus obtained gave a single band corresponding to 45 kDa upon SDS-PAGE (Fig. 3 B).Table IISubstrate specificity of the purified enzyme, as measured by inhibition assayingInhibitorConcentrationInhibition%N-Acetyllactosamine5 mm95Lacto-N-biose5 mm0.0Lactose5 mm23nLc-Cer5 mm13Heparin1.0 mg/ml90Heparan sulfate1.0 mg/ml36Hyaluronic acid1.0 mg/ml9.5Chondroitin1.0 mg/ml0.0GlcA10 mm0.0UDP10 mm98UDP-GlcA10 mm99UDP-GlcNAc10 mm67CMP-NeuAc10 mm1.5The glucuronyltransferase activity was measured as described under “Experimental Procedures” in the presence of various inhibitors. The concentration of ASOR in the assay mixture was changed from 20 μg to 5 μg. The data are the mean values for three determinations in a representative experiment. Open table in a new tab The glucuronyltransferase activity was measured as described under “Experimental Procedures” in the presence of various inhibitors. The concentration of ASOR in the assay mixture was changed from 20 μg to 5 μg. The data are the mean values for three determinations in a representative experiment. The 45-kDa protein was shown to have a SH-group specifically protected by UDP-GlcA when the eluate fraction on Matrex gel Blue A column chromatography was treated withN-maleimidopropionyl-biocytin (MPB) according to the procedure of Pukazhenthi et al. (31Pukazhenthi B.S. Muniappa N. Vijay I.K. J. Biol. Chem. 1993; 268: 6445-6452Abstract Full Text PDF PubMed Google Scholar). Based on these results, the 45-kDa protein was tentatively concluded to be a glucuronyltransferase. This conclusion was finally confirmed by our recent successful cDNA cloning of a HNK-1-associated glucuronyltransferase on the basis of the partial amino acid sequence of the purified protein (32Terayama 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). Thus, the glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoprotein acceptors (GlcAT-P) was purified to apparent homogeneity from postnatal 2-week rat forebrains, with 1,200,000-fold purification and a 4.0% overall recovery (Table I). Superose 12 gel filtration chromatography of the purified glucuronyltransferase indicated that the molecular mass of the enzyme was approximately 90 kDa, as shown in Fig. 4. This value is almost 2 times larger than that determined by SDS-PAGE under reducing conditions (45 kDa, Fig. 3 B), suggesting that the enzyme occurs as a homodimer of 45-kDa polypeptides under non-denaturing conditions. In order to study the substrate specificity of purified enzyme, we analyzed the effects of various compounds on the glucuronyltransferase activity toward ASOR (Table II).N-Acetyllactosamine at the concentration of 5 mmhad a very potent (95%) inhibitory effect. In contrast, lacto-N-biose (Galβ1–3GlcNAc) and lactose (Galβ1–4Glc) had no or little effect on the enzymatic activity (0% and 23%, respectively), indicating that the enzyme recognizes not only the terminal sugars on the acceptor molecules but also the penultimate sugars and their linkage positions. Hyaluronic acid and chondroitin had little or no inhibitory effect (9.5% and 0%, respectively), whereas heparin and heparan sulfate decreased the enzymatic activity (90% and 36%, respectively). These results may indicate that heparin and heparan sulfate act as acceptors for the enzyme but that hyaluronic acid and chondroitin do not. However, the purified enzyme did not show any transferase activity toward any of these glycosaminoglycans (data not shown). With regard to donor specificity, UDP and UDP-GlcA exhibited strong inhibitory effects (99% and 98%, respectively), followed by UDP-GlcNAc (67%). In contrast, GlcA and CMP-NeuAc had no or little effect on the activity (0.0% and 1.5%, respectively). These results suggest that the enzyme principally recognizes the terminal non-reducing N-acetyllactosamine structure in the sugar chains on glycoproteins and the nucleotide portion of UDP-GlcA. In order to determine the effect of the polypeptide portion of the acceptor glycoconjugates, various asialo-glycoproteins and glycolipids were tested as acceptors, as described under “Experimental Procedures.” The purified enzyme sufficiently transferred the glucuronic acid to asialo-NCAM and asialo-fetuin (72% and 87% of that in the case of ASOR, respectively). In contrast, asialo-thyroglobulin, which contains high mannose-type sugar chains, as well as complex-type sugar chains, was a poor acceptor of the enzyme. Interestingly, GlcAT-P did not show any activity toward paragloboside, a precursor glycolipid of the HNK-1 epitope. The dependence of the rate of the glucuronyltransferase reaction on the concentrations of ASOR and UDP-GlcA was examined, and their kinetic parameters were analyzed by Lineweaver-Burk plotting (data not shown). The K m values for ASOR and UDP-GlcA were 1.9 and 22 μm, respectively. TheV max value of the enzyme (4.5 units/mg) is comparable to those reported for the purified glycosyltransferases from the Golgi apparatus, such as GM2/GD2 N-acetylgalactosaminyltransferase (3.6 units/mg) (33Hashimoto Y. Sekine M. Iwasaki K. Suzuki A. J. Biol. Chem. 1993; 268: 25857-25864Abstract Full Text PDF PubMed Google Scholar), α2,6-sialyltransferase (8.2 units/mg) (34Weinstein J. de Souza-e-Silva U. Paulson J.C. J. Biol. Chem. 1982; 257: 13835-13844Abstract Full Text PDF PubMed Google Scholar), α1,3-galactosyltransferase (4.3 units/mg) (35Blanken W.M. Van den Eijnden D.H. J. Biol. Chem. 1985; 260: 12927-12934Abstract Full Text PDF PubMed Google Scholar), and β1,2-N-acetylgalactosaminyltransferase (28 units/mg) (36Nishikawa Y. Pegg W. Paulson H. Schachter H. J. Biol. Chem. 1988; 263: 8270-8281Abstract Full Text PDF PubMed Google Scholar). The effects of various divalent cations on the glucuronyltransferase activity were determined. Among those tested, Mn2+activated the enzyme most effectively. Co2+ and Mg2+ showed 20% and 14% of the activity of Mn2+, respectively. Ca2+, Ba2+, Ni2+, Cu2+, and Zn2+ had no effect on the enzyme at all (data not shown). To our surprise, during the process of purification, the enzyme activity disappeared almost completely at the step of Matrex gel Blue A affinity chromatography (with approximately 22,500-fold purification). However, the activity was recovered when an aliquot of an Nonidet P-40 extract was added to the assay mixture, suggesting that GlcAT-P requires some kinds of activator(s) for its catalytic activity. This presumed activator was stable on heating at 100 °C for 3 min. Therefore, we carried out the enzyme assay in the presence of a saturating amount of the heat-treated Nonidet P-40 extract in the following purification steps, as described under “Experimental Procedures.” We tried to identify the activator present in postnatal 14-day rat brains. First, we found that the chloroform-methanol extract of rat brains can substitute for the heat-inactivated Nonidet P-40 extract. Upon Folch partitioning (26Folch J. Ascoli I. Less M. Meath J.A. LeBaron F.N. J. Biol. Chem. 1951; 191: 833-841Abstract Full Text PDF PubMed Google Scholar), the organic solvent layer (Folch lower phase) activated the enzyme effectively in a saturable manner, but the upper phase did not (Fig. 5, Aand B). These lines of evidence indicated that the activator is a kind of lipid. Then, the respective lipid components were prepared from the Folch's lower phase according to the procedure described under “Experimental Procedures,” and their stimulatory activity was measured. Table III shows the amount of each lipid that gives 50% of the full activity (in the presence of heat-treated Nonidet P-40 extract). It is clear that SM caused recovery of the enzymatic activity most effectively (5.2 μg for 50% recovery), followed by PC. In contrast, PE, PS, PI, and neutral glycolipids did not have a positive effect. It should be noted that almost all the stimulatory activity in the Folch lower phase was accounted for by SM.Table IIIEffects of various lipids on the catalytic activity of the partially purified glucuronyltransferaseLipidAmount of lipid giving 50% activationμgSphingomyelin5.2Phosphatidylcholine91.4Phosphatidylethanolamine>600Phosphatidylserine>1200Phosphatidylinositol>1200Neutral glycolipids>1200The glucuronyltransferase activity was measured as described under “Experimental Procedures” in the presence of various amounts of lipids purified from the Folch lower phase. The data indicate the amounts of lipids required to give 50% of the full activity observed in the presence of a saturating amount of the heat-treated Nonidet P-40 extract. Open table in a new tab The glucuronyltransferase activity was measured as described under “Experimental Procedures” in the presence of various amounts of lipids purified from the Folch lower phase. The data indicate the amounts of lipids required to give 50% of the full activity observed in the presence of a saturating amount of the heat-treated Nonidet P-40 extract. In order to determine the effect of the fatty acid composition on the stimulatory activity, several SM molecules with a single species of acyl group were synthesized, as described under “Experimental Procedures,” and their activities were compared. Interestingly, the stimulatory activity of SM was extremely affected by the fatty acid composition (Table IV). As for the length of the acyl group, stearoyl-SM (18:0) was the most effective activator, followed by palmitoyl-SM (16:0) and lignoceroyl-SM (24:0). More interestingly, SM with a saturated acyl group activated the enzyme remarkably, while that with an unsaturated acyl group did not show any stimulatory activity regardless of the length of the acyl group. The most abundant acyl group in rat brain SM is the stearoyl group (37Abe T. Norton W.T. J. Neurochem. 1974; 23: 1025-1036Crossref PubMed Scopus (92) Google Scholar), which is in fact the most effective acyl group as an activator. Phosphatidylcholine, which has phosphocholine as a hydrophilic group like SM, stimulated the enzyme activity, although the activity was 20 times less than that of SM. It should be noted, in this context, that more than 90% of the phosphatidylcholine in the rat brain has an unsaturated acyl group. Ceramide, on the other hand, had no stimulatory effect, even if it had a stearoyl acyl group.Table IVEffects of various acyl groups of SM on the activity of the partially purified glucuronyltransferaseLipidAmount of lipid giving 50% activationμgSphingomyelin (16:0)4.2Sphingomyelin (18:0)3.8Sphingomyelin (18:1)>100Sphingomyelin (24:0)10.5Sphingomyelin (24:1)>100Ceramide>100The glucuronyltransferase activity in the eluate obtained on Matrex gel Blue A column chromatography was measured as described under “Experimental Procedures” in the presence of various amounts of lipids. The data indicate the amounts of lipids required to give 50% of the control value (in the presence of the heat-treated Nonidet P-40 extract). Open table in a new tab The glucuronyltransferase activity in the eluate obtained on Matrex gel Blue A column chromatography was measured as described under “Experimental Procedures” in the presence of various amounts of lipids. The data indicate the amounts of lipids required to give 50% of the control value (in the presence of the heat-treated Nonidet P-40 extract). Recently, some phospholipids were reported to stimulate the activities of glycosyltransferases, such as hepatic glucuronyltransferase (38Zakim D. Cantor M. Eibl H. J. Biol. Chem. 1988; 263: 5164-5169Abstract Full Text PDF PubMed Google Scholar), β1–4-galactosyltransferase (39Mitranic M.M. Boggs J.M. Moscarello M.A. J. Biol. Chem. 1983; 258: 8630-8636Abstract Full Text PDF PubMed Google Scholar), and α2–3-sialyltransferase (40Westcott K.R. Wolf C.C. Hill R.L. J. Biol. Chem. 1985; 260: 13109-13115Abstract Full Text PDF PubMed Google Scholar). GlcAT-P was activated dramatically in the presence of SM. Many glycosyltransferases that have so far been cloned are type II transmembrane proteins located in the Golgi apparatus (41Gillespie W. Kelm S. Paulson J. J. Biol. Chem. 1992; 267: 21004-21010Abstract Full Text PDF PubMed Google Scholar). GlcAT-P is also believed to be a type II transmembrane protein in the Golgi or ER membrane, on the basis of its characteristic hydropathy profile (32Terayama 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 also the requirement of detergents for its solubilization. Two phospholipids, which contain phosphocholine as a hydrophilic group, stimulated the enzymatic activity in common, suggesting that the phosphocholine group of these phospholipids interacts with the luminal portion of the enzyme through an electrostatic interaction. Interestingly, in the cellular membrane system, SM is localized predominantly in the outer leaflet of the plasma membrane and the Golgi lumen (42Nilsson O.S. Dallner G. Biochim. Biophys. Acta. 1977; 464: 453-458Crossref PubMed Scopus (86) Google Scholar). This is in good agreement with the putative location of the catalytic domain of this glucuronyltransferase, the Golgi lumen. These lines of evidence suggest that expression of the HNK-1 epitope on glycoproteins can be regulated not only by the expression of the enzyme protein but also by the micro-circumstances around the enzyme, especially by SM. We thank Hiroko Yamaguchi and Yasuko Nagao for their excellent secretarial assistance.
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