Molecular Cloning and Characterization of UDP-GlcNAc:Lactosylceramide β1,3-N-Acetylglucosaminyltransferase (β3Gn-T5), an Essential Enzyme for the Expression of HNK-1 and Lewis X Epitopes on Glycolipids

• DOI: 10.1074/jbc.m011369200
• Publication Date: 2002-07-26T15:09:24Z
• Authors: Akira Togayachi; Tomohiro Akashima; Reiko Ookubo; Takashi Kudo; Shoko Nishihara; Hiroko Iwasaki; Ayumi Natsume; Hiroyuki Mio; Jin-ichi Inokuchi; Tatsuro Irimura; Katsutoshi Sasaki; Hisashi Narimatsu
• Keywords: Lactosylceramide; Cloning (programming)

A new member of the UDP-N-acetylglucosamine:β-galactose β1,3-N-acetylglucosaminyltransferase (β3Gn-T) family having β3Gn-T motifs was cloned from rat and human cDNA libraries named β3Gn-T5 based on its position in a phylogenetic tree. We concluded that is most feasible candidate for lactotriaosylceramide (Lc3Cer) synthase, an important enzyme which plays key role synthesis lacto- or neolacto-series carbohydrate chains glycolipids. exhibited strong activity to transfer GlcNAc glycolipid substrates, such as lactosylceramide (LacCer) neolactotetraosylceramide (nLc4Cer; paragloboside), resulting Lc3Cer neolactopentaosylceramide (nLc5Cer), respectively. marked decrease LacCer increase nLc4Cer detected Namalwa cells stably expressing β3Gn-T5. This indicated exerted synthesize LacCer, followed by conversion via endogenous galactosylation. The following four findings further supported synthase. 1) transcript levels various were consistent with synthase those cells. 2) presented tissues cultured 3) expression up-regulated stimulation retinoic acid down-regulated 12-O-tetradecanoylphorbol-13-acetate HL-60 4) changes during brain development determined. Points 2, 3, 4 reported previously. UDP-GlcNAc:β-galactose UDP-galactose:β-N-acetylglucosamine β1,3-galactosyltransferase uridine diphosphate-N-acetylglucosamine monoclonal antibody base pair(s) polymerase chain reaction open reading frame high performance thin layer chromatography sulfoglucuronylglycolipid glycosphingolipid Lewis b x stage-specific embryonal antigen-1 (Galβ1–4Glcβ1–1Cer) (GlcNAcβ1–3Galβ1–4Glcβ1–1Cer) (paragloboside: Galβ1–4GlcNAcβ1–3Galβ1–4Glcβ1–1Cer) GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glcβ1–1Cer Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glcβ1–1Cer pyridylaminated 2-aminobenzamide lacto-N-neotetraose (Galβ1–4GlcNAcβ1–3Galβ1–4Glc) lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc) lacto-N-fucopentaose-II (Galβ1–3(Fucα1–4)GlcNAcβ1–3Galβ1–4Glc) lacto-N-fucopentaose-III (Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4Glc) lacto-N-fucopentaose-V (Galβ1–3GlcNAcβ1–3Galβ1–4(Fucα1–3)Glc) lacto-N-difucohexaose-II (Galβ1–3(Fucα1–4)GlcNAcβ1–3Galβ1–4(Fucα1–3)Glc) N-acetyllactosamine (Galβ1–4GlcNAc) Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc (Galβ1–4GlcNAcβ1–3)2Galβ1–4GlcNAc (Galβ1–4GlcNAcβ1–3)3Galβ1–4GlcNAc (Galβ1–4GlcNAcβ1–3)4Galβ1–4GlcNAc reverse transcriptase-polymerase lactosylceramide:α2,3-sialyltransferase To date, three members (β3Gn-T)1 (β3Gn-T2, -T3, -T4) (1Shiraishi N. Natsume A. Togayachi Endo T. Akashima Yamada Y. Imai Nakagawa S. Koizumi Sekine Narimatsu H. Sasaki K. J. Biol. Chem. 2001; 276: 3498-3507Abstract Full Text PDF PubMed Scopus (118) Google Scholar, 2Zhou D. Dinter Gutierrez Gallego R. Kamerling J.P. Vliegenthart J.F. Berger E.G. Hennet Proc. Natl. Acad. Sci. U. 1999; 96 (; Correction (2000) 97, 11673–11975): 406-411Crossref (87) Scholar) five (β3Gal-T) (β3Gal-T1, -T2, -T4, -T5) have been identified (3Sasaki, K., Sasaki, E., Kawashima, Hanai, N., Nishi, T., Hasegawa, M. (1994), Japan patent 0618759 940705.Google 4Amado Almeida Carneiro F. Leverly S.B. Holmes E.H. Nomoto Hollingsworth M.A. Hassan Schwientek Nielsen P.A. Bennett E.P. Clausen 1998; 278: 12770-12778Abstract (160) 5Kolbinger Streiff M.B. Katopodis A.G. 273: 433-440Abstract (58) 6Isshiki Kudo Nishihara Watanabe Kubota Kitajima Shiraishi Andoh 274: 12499-12507Abstract (129) Scholar). All them share amino (β3Gn-T β3Gal-T motifs) regions catalytic domain. first, β3Gn-T, cloning method using anti-i (7Sasaki Kurata-Miura Ujita Angata Nishi Fukuda 1997; 94: 14294-14299Crossref (134) However, this unique it does not although transfers Gal β1,3-linkage, polylactosamine chains. It iGn-T Thereafter, β3Gn-T1 isolated structural similarity (2Zhou previously additional β3Gn-Ts, β3Gn-T2, are also structurally related sequence recently corrected Zhou et al. (see Ref. identical β3Gn-T2 us So, total i.e. iGn-T, β3Gn-T2(-T1), described date. avoid confusion, we note fact, but do change names used study. By transfection experiments vitro enzymatic analysis, these β3Gn-Ts found exhibit catalyzes chains, activity. HNK-1 reacts sulfoglucuronyl epitope, SO43-GlcAβ1–3Galβ1–4GlcNAc-R, expressed several glycoproteins involved neural cell recognition, two neolactoglycolipids, (SGGL)-1 -2 (SGGL-1, SO43-GlcAβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glcβ1–1ceramide; SGGL-2, SO43-GlcAβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glcβ1–1ceramide) (8Chou K.H. Ilyas A.A. Evans J.E. Quarles R.H. Jungalwala F.B. Biochem. Biophys. Res. Commun. 1985; 128: 383-388Crossref (170) 9Chou D.K. Costello C. 1986; 261: 11717-11725Abstract Neurobiologists suggested series studies epitope functions cell-cell interaction molecule complete nervous system (10Nair S.M. Zhao Z. Chou Tobet S.A. Neuroscience. 85: 759-771Crossref (23) 11Nair Prasadarao Comp. Neurol. 1993; 332: 282-292Crossref (24) 12Nair Neurochem. 68: 1286-1297Crossref (35) 13Oka Yakugaku Zasshi. 118: 431-446Crossref (4) 14Chou 8508-8515Abstract developmentally spatially regulated system. In cerebellum, SGGLs biphasic manner, initial peak at around postnatal days (PD) 1–3, second PD20 (15Jungalwala 1994; 19: 945-957Crossref (111) adult kept constant (16Prasadaro Koul O. 1990; 55: 2024-2030Crossref (40) 17Chou Flores 54: 1589-1597Crossref (13) Immunohistochemical localized specific some biological neuron guidance (14Chou 15Jungalwala 18Chou 1988; 50: 1655-1658Crossref (25) cerebral cortex, peaked embryonic day (ED) 19 then decreased until PD 5, had almost completely disappeared 20. Carbohydrate extended stepwise reactions catalyzed glycosyltransferases. (19Chou 1991; 266: 17941-17947Abstract 20Chou 268: 21727-21733Abstract 21Chou 330-336Abstract 22Chou 62: 307-314Crossref (14) developmental each correlation SGGL brain. demonstrated residue (LacCer; Galβ1–4Glcβ1–1Cer) β1–3-linkage (GlcNAcβ1–3Galβ1–4Glcβ1–1Cer), developing brain, because only well correlated expression. (Lex; CD15) structure, α1,3-fucosyl-N-acetyllactosamine, Galβ1–4(Fucα1–3)GlcNAcβ1-R, region specifically (23Allendoerfer K.L. Durairaj Matthews G.A. Patterson P.H. Dev. 211: 208-219Crossref (41) 24Ashwell K.W. Mai J.K. Int. Neurosci. 15: 883-889Crossref (11) 25Ashwell Cell Tissue 289: 17-23Crossref 26Chou Suzuki Glycoconj. 1996; 13: 295-305Crossref (18) 27Mai Winking Ashwell 404: 197-211Crossref 28Mai Krajewski Reifenberger G. Genderski B. Lensing-Hohn 88: 847-858Crossref (21) 29Streit Yuen C.T. Loveless R.W. Lawson A.M. Finne Schmitz Feizi Stern C.D. 66: 834-844Crossref (78) 30Sajdel-Sulkowska E.M. Acta Biochim. Pol. 45: 781-790Crossref (17) 31Gocht Struckhoff Lhler Histol. Histopathol. 11: 1007-1028PubMed Lex recognition highly organized structures central (30Sajdel-Sulkowska 32Gotz Wizenmann Reinhardt Lumsden Price Neuron. 16: 551-564Abstract (81) 33Yoshida-Noro Heasman Goldstone Vickers L. Wylie Glycobiology. 9: 1323-1330Crossref (26) GSLs synthesizes root structure carrying (34Dasgupta Hogan E.L. Spicer S.S. 367-375Crossref Thus, interest terms their biosynthetic regulation. respect hematopoietic differentiation (35Nakamura Tsunoda Sakoe Gu Nishikawa Taniguchi Saito 1992; 267: 23507-23514Abstract cells, promyelocytic leukemic line, capable bidirectional into monocytoid granulocytoid lineage GM3 (lactosylceramide:α2,3-sialyltransferase), acceptor substrate (LacCer); Galβ1–4Glcβ1–1Cer, enzymes determining flow different directions. During monocytic induced (TPA) treatment, ganglioside markedly increased up-regulation down-regulation contrast, granulocytic all-trans-retinoic (RA), Stults Macher (36Stults C.L. B.A. Arch 303: 125-133Crossref (19) mature myeloid toward both leading types neolacto-neutral while lymphoid nLc4Cer, LacCer. lack synthesizing explains absence GSLs. controlling so roles many present study, characterized fifth (β3Gn-T5), likely tumor lines RPMI 1640 medium (Life Technologies, Inc., Rockville, MD) supplemented 10% fetal bovine serum. donated purchased American Type Culture Collection (ATCC, Manassas, VA), Riken Bank (RCB, Tsukuba, Japan), Research (JCRB, Tokyo, Immunobiological laboratory (IBL, Fujioka, Dai-Nippon Pharmaceutical Co., Ltd. (DNP, Osaka, Japan). MAb 1B2 (anti-N-acetyllactosamine) nLc6Cer (neolactohexaosylceramide) (IgM) 37Young Jr., W.W. Portoukalian Hakomori 1981; 256: 10967-10972Abstract Scholar), cytometry immuno-TLC assays, hybridoma 15% supernatant culture experiments. kindly provided Seikagaku Kogyo Co. Ltd (Tokyo, constructed shank library random sequencing project. sequencing, encoding partial novel (738 bp) did encode full ORF. But shared known β3Gal-Ts β3Gn-Ts. Colo205 previous study (6Isshiki screened probe isolate full-length possessed 3.7-kilobase pair insert DNA. clone principle behind competitive RT-PCR quantification transcripts detail our 38Kudo Ikehara Morozumi Nakamura Lab. Inv. 78: 797-811PubMed Total cellular RNAs fromCLONTECH. Those extracted purified laboratory. As measurement genes,β3Gn-T2, primers PCR conditions Regarding gene, standard DNA plasmid subcloning ORF pBluescript SK(−) vector. competitor small deletion (245 pairs) within follows. double digested appropriate restriction endonucleases, MscI andBglII, blunt-ended T4 polymerase. self-ligation deleted plasmid. RT-PCR, primer set: forward primer, 5′-TCTTATGACTGCTGATGATGACAT-3′, 5′-CTTTAGGATCTGTAGCATTCTTCC-3′. Expression glycosyltransferase genes subcloned pAMo papers 7Sasaki 39Sasaki E. Kawashima Dohi Oshima Hanai Hasegawa 22782-22787Abstract 40Sasaki Kurata Kojima Kurosawa Ohta Tsuji 269: 15950-15956Abstract 41Sasaki Funayama Nagata 14730-14737Abstract 42Kudo Kaneko Hiraga 26729-26738Abstract (99) construction vector inserted theβ3Gn-T5 fragment excised SK(−), flanked SfiI linker theSfiI site Each fourβ3Gn-T transfected electroporation These selected presence geneticin (G418) Inc.) concentration 0.8 mg/ml. Stable transfectant obtained after 25 exposure geneticin. measured means RT-PCR. For 1.0 × 108 β3Gn-Tgene collected, washed twice phosphate-buffered saline, lyophilized. glycolipids lyophilized Crude first chloroform/methanol (2:1, v/v), chloroform/methanol/water (30:60:8, v/v/v). Samples dried N2 evaporator dissolved methanol, subjected mild alkaline treatment 0.1 n KOH/methanol 40 °C 2 h, neutralized 1 acetic acid. After free fatty acids removed n-hexane, remaining fractions Folch's partition. lower neutral analysis. Neutral equivalent 107 applied lane TLC. separated TLC (HPTLC Kieselgel 60, 5641; MERK, Germany) mixtures (60:35:8, v/v/v) analysis performed putative domain -T4 secreted protein fused FLAG peptide insect 1.1-kilobase COOH-terminal portion (amino 39 378) amplified PCR. Platinum Pfx Polymerase Inc.), according supplier's manual. 5′ 3′ sequences withBamHI XbaI sequences, respectively, create sites. follows: 5′-CGGGATCCATTGTGAGCCATATGAAGTCATAT-3′, 5′-GCTCTAGATGACAGTGAAAACATACAACATTC-3′. between BamHI sites Subsequently, NotI, NotI pVL1393-F2 yield pVL1393-F2G5. derived pVL1393 (Pharmingen) contains signal immunoglobulin (MHFQVQIFSFLLISASVIMSRG) (DYKDDDDK). prepared recombinant viruses Sf21 infected individual virus multiplicity infection 10 incubated 27 72 h conditioned media containing proteins peptide. Bacu3GnT readily anti-FLAG M1 resin (Sigma) eluted 50 mm TBS (50 mmTris-HCl, pH 7.4, 150 NaCl), mmCaCl2 buffer (pH 7.4). Bacu3GnT2, Bacu3GnT3, Bacu3GnT4, Bacu3GnT5. SDS-polyacrylamide gel electrophoresis transferred Immobilon PVDF membrane (Millipore, Bedford, MA). probed antibody, stained ECL Western blotting detection reagents (Amersham Pharmacia Biotech). intensity positive bands densitometer (43Togayachi Iwasaki Higashiyama Kodama Nakamori Cancer. 83: 70-79Crossref determine relative amounts protein. Two soluble produced baculoexpression membrane-bound form homogenates other lines, activities substrates. Bacu3Gn-T adjusted same amount above assay. solubilized 20 HEPES 7.2) 2% Triton X-100, μg reaction. Various oligosaccharides fluorescently labeled (2AB) (PA) 44Nishihara Tawada Ito FEBS Lett. 462: 289-294Crossref (77) acceptors. assayed 20-μl mixture sodium cacodylate 7.2), UDP-GlcNAc, MnCl2, 0.4% CF-54, μm 2AB-acceptor PA-acceptor substrate, source. incubation 37 16 terminated boiling 3 min, diluted 80 μl water. centrifugation 15,000 rpm 5 filtered Ultrafree-MC column (Millipore). 10-μl aliquot liquid TSK-gel ODS-80TS QA (4.6 300 mm; Tosoh, products mmammonium acetate 4.0) 7% methanol rate ml/min monitored fluorescence spectrophotometer, JASCO FP-920 (JASCO, Japan) conducted assay incorporation radioactive sugar 25-μl mmsodium 480 175 nCi UDP-[14C]GlcNAc Biotech), nmol 200 water m KCl added. centrifuged min. Radioactive s