title: Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme.
abstract: The sialyltranferase ST3Gal-V transfers a sialic acid to lactosylceramide. We investigated the role of each of the N-glycans modifying mouse ST3Gal-V (mST3Gal-V) by measuring the in vitro enzyme activity of Chinese hamster ovary (CHO) cells transfected with ST3Gal-V cDNA or its mutants. By examining mutants of mST3Gal-V, in which each asparagine was replaced with glutamine (N180Q, N224Q, N334Q), we determined that all three sites are N-glycosylated and that each N-glycan is required for enzyme activity. Despite their importance, N-glycosylation sites in ST3Gal-V are not conserved among species. Therefore, we considered whether the function in the activity that is performed in mST3Gal-V by the N-glycan could be substituted for by specific amino acid residues selected from the ST3Gal-V of other species or from related sialyltransferases (ST3Gal-I, -II, -III, and -IV), placed at or near the glycosylation sites. To this end, we constructed a series of interspecies mutants for mST3Gal-V, specifically, mST3Gal-V-H177D-N180S (medaka or tetraodon type), mST3Gal-V-N224K (human type), and mST3Gal-V-T336Q (zebrafish type). The ST3Gal-V activity of these mutants was quite similar to that of the wild-type enzyme. Thus, we have demonstrated here that the N-glycans on mST3Gal-V are required for activity but can be substituted for specific amino acid residues placed at or near the glycosylation sites. We named this method SUNGA (substitution of N-glycan functions in glycosyltransferases by specific amino acids). Furthermore, we verified that the ST3Gal-V mutant created using the SUNGA method maintains its high activity when expressed in Escherichia coli thereby establishing the usefulness of the SUNGA method in exploring the function of N-glycans in vivo.
authors.affiliation: Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Uemura
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kurose
authors.forename: Tomoko
authors.initials: T
authors.lastname: Suzuki
authors.forename: Sayaka
authors.initials: S
authors.lastname: Yoshida
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
authors.forename: Masaki
authors.initials: M
authors.lastname: Saito
authors.forename: Masataka
authors.initials: M
authors.lastname: Horiuchi
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Igarashi
authors.forename: Jin-ichi
authors.initials: J
authors.lastname: Inokuchi
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008358:Mannose / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D011134:Polysaccharides; D012799:Sialyltransferases; C021343:haematoside synthetase; D008358:Mannose