title: Chemo-enzymatic synthesis and structure-activity study of artificially N-glycosylated eel calcitonin derivatives with a complex type oligosaccharide.
abstract: Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-beta-N-acetylglucosaminidase from Mucor hiemalis . The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation.
authors.affiliation: The Noguchi Institute, 1-8-1, Kaga, Itabashi, Tokyo 173-0003, Japan. toma@noguchi.or.jp
authors.forename: Katsuji
authors.initials: K
authors.lastname: Haneda
authors.forename: Mizuka
authors.initials: M
authors.lastname: Tagashira
authors.forename: Eiichi
authors.initials: E
authors.lastname: Yoshino
authors.forename: Midori
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Inazu
authors.forename: Kazunori
authors.initials: K
authors.lastname: Toma
authors.forename: Hideki
authors.initials: H
authors.lastname: Iijima
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Isogai
authors.forename: Masayuki
authors.initials: M
authors.lastname: Hori
authors.forename: Shinji
authors.initials: S
authors.lastname: Takamatsu
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Fujibayashi
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Makoto
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D002116:Calcitonin / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002942:Circular Dichroism; D006031:Glycosylation; D008297:Male; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship
chemical_list: D009844:Oligosaccharides; D002116:Calcitonin