Abstract
Human α1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and hFucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced by glutamine. Western blotting analyses demonstrated that both sites in hFucTV were glycosylated, whereas in hFucTVI only one of the sites (Asn91) was glycosylated. All the single mutants and the hFucTVI N46Q/N91Q double mutant exhibited enzyme activities that did not differ considerably from the wt activities. However, the enzyme activity of the hFucTV N60Q/N105Q double mutant was reduced to approximately 40% of the wt activity. In addition, castanospermine treatment diminished the enzyme activity and hence trimming of the N-linked glycans are required for expression of full enzyme activity of both hFucTV and -VI. The present study demonstrates that both of the N-terminal potential N-glycosylation sites in hFucTV and one of the sites in hFucTVI are glycosylated. Individually, their glycosylation does not contribute considerably to expression of enzyme activity. However, elimination of both sites in hFucTV reduces the enzyme activity.
Similar content being viewed by others
References
Goelz SE, Hession C, Goff D, Griffiths B, Tizard R, Newman B, Chi-Rosso G, Lobb R, ELFT: a gene that directs the expression of an ELAM-1 ligand, Cell 63, 1349–56 (1990).
Kukowska LJ, Larsen RD, Nair RP, Lowe JB, A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group alpha(1,3/1,4) fucosyltransferase, Genes Dev 4, 1288–303 (1990).
Kumar R, Potvin B, Muller WA, Stanley P, Cloning of a human alpha (1,3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on Chinese hamster ovary cell transfectants, J Biol Chem 266, 21777–83 (1991).
Koszdin KL, Bowen BR, The cloning and expression of a human alpha-1,3 fucosyltransferase capable of forming the E-selectin ligand, Biochem Biophys Res Commun 187, 152–7 (1992).
Weston BW, Nair RP, Larsen RD, Lowe JB, Isolation of a novel human alpha (1,3)fucosyltransferase gene and molecular comparison to the human Lewis blood group alpha (1,3/1,4)fucosyltransferase gene. Syntenic, homologous, nonallelic genes encoding enzymes with distinct acceptor substrate specificities, J Biol Chem 267, 4152–60 (1992).
Weston BW, Smith PL, Kelly RJ, Lowe JB, Molecular cloning of a fourth member of a human alpha (1,3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x, and difucosyl sialyl Lewis x epitopes [published erratum appears in J Biol Chem 1993, Aug 25;268(24):18398], J Biol Chem 267, 24575–84 (1992).
Natsuka S, Gersten KM, Zenita K, Kannagi R, Lowe JB, Molecular cloning of a cDNA encoding a novel human leukocyte alpha-1,3–fucosyltransferase capable of synthesizing the sialyl Lewis x determinant [published erratum appears in J Biol Chem 1994, Aug 12;269(32):20806], J Biol Chem 269, 16789–94 (1994).
Sasaki K, Kurata K, Funayama K, Nagata M, Watanabe E, Ohta S, Hanai N, Nishi T, Expression cloning of a novel alpha 1,3–fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes, J Biol Chem 269, 14730–7 (1994).
Kaneko M, Kudo T, Iwasaki H, Ikehara Y, Nishihara S, Nakagawa S, Sasaki K, Shiina T, Inoko H, Saitou N, Narimatsu H, Alpha1,3–fucosyltransferase IX (Fuc-TIX) is very highly conserved between human and mouse; molecular cloning, characterization and tissue distribution of human Fuc-TIX, FEBS Lett 452, 237–42 (1999).
Christensen LL, Jensen UB, Bross P, Orntoft TF, The C-terminal N-glycosylation sites of the human alpha1,3/4–fucosyltransferase III,-V, and-VI (hFucTIII,-V, and-VI) are necessary for the expression of full enzyme activity, Glycobiology 10, 931–9 (2000).
Baboval T, Koul O, Smith FI, N-Glycosylation site occupancy of rat alpha-1,3–fucosyltransferase IV and the effect of glycosylation on enzymatic activity, Biochim Biophys Acta 1475, 383–9 (2000).
Nguyen AT, Holmes EH, Whitaker JM, Ho S, Shetterly S, Macher BA, Human alpha1,3/4–Fucosyltransferases. I. identification of amino acids involved in acceptor substrate binding by site-directed mutagenesis, J Biol Chem 273, 25244–9 (1998).
Legault DJ, Kelly RJ, Natsuka Y, Lowe JB, Human alpha(1,3/1,4)-fucosyltransferases discriminate between different oligosaccharide acceptor substrates through a discrete peptide fragment, J Biol Chem 270, 20987–96 (1995).
Fast DG, Jamieson JC, McCaffrey G, The role of the carbohydrate chains of Gal beta-1,4–GlcNAc alpha 2,6–sialyltransferase for enzyme activity, Biochim Biophys Acta 1202, 325–30 (1993).
Nagai K, Ihara Y, Wada Y, Taniguchi N, N-glycosylation is requisite for the enzyme activity and Golgi retention of Nacetylglucosaminyltransferase III, Glycobiology 7, 769–76 (1997).
Toki D, Sarkar M, Yip B, Reck F, Joziasse D, Fukuda M, Schachter H, Brockhausen I, Expression of stable human Oglycan core 2 beta-1,6–N-acetylglucosaminyltransferase in Sf9 insect cells, Biochem J 325, 63–9 (1997).
Martina JA, Daniotti JL, Maccioni HJ, Influence of N-glycosylation and N-glycan trimming on the activity and intracellular traffic of GD3 synthase, J Biol Chem 273, 3725–31 (1998).
Malissard M, Borsig L, Di Marco S, Grutter MG, Kragl U, Wandrey C, Berger EG, Recombinant soluble beta-1,4–galactosyltransferases expressed in Saccharomyces cerevisiae. Purification, characterization and comparison with human enzyme, Eur J Biochem 239, 340–8 (1996).
Minowa MT, Oguri S, Yoshida A, Hara T, Iwamatsu A, Ikenaga H, Takeuchi M, cDNA cloning and expression of bovine UDP-N-acetylglucosamine: alpha1, 3–D-mannoside beta1,4–N-acetylglucosaminyltransferase IV, J Biol Chem 273, 11556–62 (1998).
Haraguchi M, Yamashiro S, Furukawa K, Takamiya K, Shiku H, The effects of the site-directed removal of N-glycosylation sites from beta-1,4–N-acetylgalactosaminyltransferase on its function, Biochem J 312, 273–80 (1995).
Deng WP, Nickoloff JA, Site-directed mutagenesis of virtually any plasmid by eliminating a unique site, Anal Biochem 200, 81–8 (1992).
Goelz SE, Kumar R, Potvin B, Sundaram S, Brickelmaier M, Stanley P, Differential expression of an E-selectin ligand (SLex) by two Chinese hamster ovary cell lines transfected with the same alpha (1,3)-fucosyltransferase gene (ELFT), J Biol Chem 269, 1033–40 (1994).
Borsig L, Katopodis AG, Bowen BR, Berger EG, Trafficking and localization studies of recombinant alpha 1,3–fucosyltransferase VI stably expressed in CHO cells, Glycobiology 8, 259–68 (1998).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lotte Christensen, L., Bross, P. & Falck Ørntoft, T. Glycosylation of the N-terminal potential N- glycosylation sites in the human α1,3- fucosyltransferase V and -VI (hFucTV and -VI). Glycoconj J 17, 859–865 (2000). https://doi.org/10.1023/A:1010917229243
Issue Date:
DOI: https://doi.org/10.1023/A:1010917229243