title: Strategy for the fine characterization of glycosyltransferase specificity using isotopomer assembly.
abstract: Glycosylation, which represents the most complex posttranslational modification (PTM) event during protein maturation, has a vital role in biological processes. Glycan biosynthesis is orchestrated by numerous glycosyltransferases, each displaying different selectivities for multiple reaction sites. The precise specificities of these enzymes have been difficult to study because of the lack of available substrates of defined structure and problems associated with the analyses. Moreover, the analysis of glycans is extremely difficult owing to the structural complexity of the glycan chain. Here we describe a new strategy for the fine characterization of enzyme specificity using substrate isotopomer assemblies. Because isotopomer assemblies contain a sugar residue that is position-specifically labeled with a stable isotope, we can use tandem mass spectrometry (MS/MS) to assign the structure of positional isomers generated by glycosylation. We demonstrated the analysis of substrate specificities of five beta4-galactosyltransferases (beta4GalT-I, -II, -III, -IV and -V) using our strategy.
authors.affiliation: Glycogene Function Team of Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ito
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kameyama
authors.forename: Takashi
authors.initials: T
authors.lastname: Sato
authors.forename: Masako
authors.initials: M
authors.lastname: Sukegawa
authors.forename: Hide-Ki
authors.initials: HK
authors.lastname: Ishida
authors.forename: Hisashi
authors.initials: H
authors.lastname: Narimatsu
mesh_terms: D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005700:Galactosyltransferases / Q000737:chemistry; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011134:Polysaccharides; D016695:Glycosyltransferases; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I