title: Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures.
abstract: Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.
authors.affiliation: The Noguchi Institute, 1-8-1, Kaga, Itabashi, Tokyo 173-0003, Japan.
authors.forename: Katsuji
authors.initials: K
authors.lastname: Haneda
authors.forename: Midori
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Mizuka
authors.initials: M
authors.lastname: Tagashira
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Inazu
authors.forename: Kazunori
authors.initials: K
authors.lastname: Toma
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Isogai
authors.forename: Masayuki
authors.initials: M
authors.lastname: Hori
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Makoto
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Kaoru
authors.initials: K
authors.lastname: Takegawa
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002116:Calcitonin / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D004524:Eels; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D051381:Rats
chemical_list: D009844:Oligosaccharides; D002116:Calcitonin; D016695:Glycosyltransferases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase