title: Enzymatic synthesis of Gb3 and iGb3 ceramides.
abstract: Gb3 and iGb3 are physiologically important trihexosylceramides with a terminal alpha-d-Galp-(1-->4)-beta-d-Galp- and alpha-d-Galp-(1-->3)-beta-d-Galp sequence, respectively. In particular iGb3 is attracting considerable attention as it is believed to serve as a ligand for natural killer T cells. Whether or not iGb3 is present in humans and which enzyme might be responsible for its synthesis is at present a matter of lively debate. In the current investigation we evaluated human blood group B galactosyltransferase (GTB) for its ability to catalyze the formation of iGb3 from lactosylceramide and UDP-Galp. GTB is a retaining glycosyltransferase that in vivo catalyzes the transfer of galactose from UDP-Galp donors to OH-3 of Galp on the H-antigen (alpha-l-Fucp-(1-->2)-beta-d-Galp) acceptor forming the blood group B antigen. GTB tolerates modifications in donor and acceptor substrates and its ability to accept lactosides as acceptors makes it a possible candidate for iGb3 production in humans. For comparison iGb3 and Gb3 were also synthesized from the same acceptor using an alpha-(1-->3)- and alpha-(1-->4)-specific galactosyltransferase, respectively. All the enzymes tested catalyzed the desired reactions. Product characterization by NMR analysis clearly differentiated between the alpha-Galp-(1-->3)-Galp and alpha-Galp-(1-->4)-Galp product, with the GTB product being identical to that of the alpha-(1-->3)-GalT-catalyzed reaction. The rate of transfer by GTB however was very low, only 0.001% of the rate obtained with a good substrate, H antigen disaccharide (octyl alpha-l-Fucp-(1-->2)-beta-d-Galp). This is too low to account for the possible formation of the iGb3 structure in humans in vivo.
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark.
authors.forename: Dietlind
authors.initials: D
authors.lastname: Adlercreutz
authors.forename: Joel T
authors.initials: JT
authors.lastname: Weadge
authors.forename: Bent O
authors.initials: BO
authors.lastname: Petersen
authors.forename: Jens Ø
authors.initials: JØ
authors.lastname: Duus
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
authors.forename: Monica M
authors.initials: MM
authors.lastname: Palcic
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D005700:Galactosyltransferases / Q000378:metabolism*; D005915:Globosides / Q000138:chemical synthesis* / Q000737:chemistry; D006801:Humans; D007790:Lactosylceramides / Q000378:metabolism; D009345:Neisseria meningitidis / Q000201:enzymology; D014281:Trihexosylceramides / Q000138:chemical synthesis* / Q000737:chemistry; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
chemical_list: D005915:Globosides; D007790:Lactosylceramides; D014281:Trihexosylceramides; C514780:globotrihexosylceramide; C514781:isoglobotrihexosylceramide; D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases