title: Enzymatic synthesis of lacto-N-difucohexaose I which binds to Helicobacter pylori.
abstract: Helicobacter pylori is known to bind with sugar chains possessing Lewis b structure. We are trying to combine oligosaccharides containing Lewis b sugar chain to water insoluble polysaccharide through some linker. Lacto-N-difucohexaose I (LNDFH I; Fucalpha1-->2Galbeta1-->3[Fucalpha1-->4]GlcNAcbeta1-->3Galbeta1-->4Glc) fits for that purpose, since it consists of Lewis b tetrasaccharide and lactose whose d-glucose residue can be utilized as a linker. We thus developed a method to synthesize this hexaose enzymatically. First, beta-1,3-N-acetylglucosaminyltransferase (beta-1,3-GnT) was partially purified from bovine blood by an established method. Using this enzyme preparation, d-GlcNAc was attached to the d-galactose residue of lactose with a beta-1,3-linkage to produce lacto-N-triose II at 44% yield. The low yield was thought to be due to contaminating N-acetylglucosaminidase that would have hydrolyzed the product, lacto-N-triose II. Next, d-galactose was attached by transglycosylation using ortho-nitrophenyl beta-d-galactopyranoside as a donor with the aid of recombinant beta-1,3-galactosidase from Bacillus circulans to generate lacto-N-tetraose (LNT) at 22% yield. l-Fucose was then linked to the d-galactose residue of LNT via an alpha-1,2-linkage using recombinant human fucosyltransferase I (FUT1) expressed in a baculovirus system (71% yield). The obtained pentasaccharide was subsequently incubated with GDP-beta-l-fucose and commercial fucosyltransferase III (FUT3) to attach l-fucose to the d-GlcNAc residue of LNT with an alpha-1,4-linkage. After purification with an activated carbon column chromatography, 1.7 mg of LNDFH I was obtained (85% yield). We thus produced LNDFH I over four enzymatic steps with a yield of 6%.
authors.affiliation: Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Akiha-ku, Niigata, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Miyazaki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sato
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Furukawa
authors.forename: Katsumi
authors.initials: K
authors.lastname: Ajisaka
mesh_terms: D000818:Animals; D001287:Attachment Sites, Microbiological; D001665:Binding Sites; D002240:Carbohydrate Sequence; D005696:Galactosidases / Q000235:genetics / Q000378:metabolism; D016480:Helicobacter pylori / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; C018991:lacto-N-difucohexaose I; C106501:N-acetylglucosaminyltransferase IGnT; D017351:N-Acetylglucosaminyltransferases; C428133:beta-1,3-N--acetylglucosaminyltransferase T3; D005696:Galactosidases