title: Characterization of a deaminated neuraminic acid-containing glycoprotein from the skin mucus of the loach, Misgurnus anguillicaudatus.
abstract: Using NMR spectroscopy and mass spectrometry, the major sialic acid of the skin mucus of the loach, Misgurnus anguillicaudatus was found to be 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN). We have subsequently devised a method to isolate a KDN-containing glycoprotein preparation from loach skin mucus. The method involves the sonication of the skin mucus with 0.05 M Tris-HCl, pH 8.0, to solubilize the glycoprotein, followed by DE52-cellulose chromatography of the extract, Nuclease P1 treatment, and Sepharose CL-4B gel filtration. The purified glycoprotein preparation was found to contain 38.5% KDN, 0.4% NeuAc, 24.6% GalNAc, 3.3% Gal, and 28.2% amino acids (w/w). The amino acid composition of this glycoprotein preparation revealed that it is unusually rich in Thr, and 6 amino acids, Thr, Ser, Glu (or Gln), Pro, Gly and Ala, account for 83% of the total amino acids. This glycoprotein is extremely poor in Cys, Met, Tyr, Phe, Arg, and Trp. Treatment of this glycoprotein with alkali resulted in the destruction of 83% of Thr suggesting that most of the sugar chains in this glycoprotein are linked through Thr. Alkaline borohydride treatment of 100 mg of the glycoprotein preparation, followed by Sephadex G-25 (superfine) gel filtration, yielded two major oligosaccharide alditols, I (15.4 mg) and II (15.6 mg). Using liquid secondary ion mass spectrometry and methylation analysis, I was identified to be KDN alpha 2-->6GalNAc-ol and II, KDN alpha 2-->6(KDN alpha 2-->3)-GalNAc-o1. KDN alpha 2-->6GalNAc is structurally similar to NeuAc alpha 2-->GalNAc found in ovine submaxillary glycoprotein while II represents a novel structure which contains two sialic acids linked to a GalNAc through both alpha 2-->3 and alpha 2-->6 linkages. This structure has never been found in mucin type glycoproteins including mammalian epithelial mucin glycoproteins. This is the first report of the presence of a mucin type glycoprotein which contains KDN instead of NeuAc or NeuGc in fish skin mucus.
authors.affiliation: Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kimura
authors.forename: Y
authors.initials: Y
authors.lastname: Hama
authors.forename: T
authors.initials: T
authors.lastname: Sumi
authors.forename: M
authors.initials: M
authors.lastname: Asakawa
authors.forename: B N
authors.initials: BN
authors.lastname: Rao
authors.forename: A P
authors.initials: AP
authors.lastname: Horne
authors.forename: S C
authors.initials: SC
authors.lastname: Li
authors.forename: Y T
authors.initials: YT
authors.lastname: Li
authors.forename: H
authors.initials: H
authors.lastname: Nakagawa
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002240:Carbohydrate Sequence; D003531:Cypriniformes; D003641:Deamination; D006023:Glycoproteins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009093:Mucus / Q000737:chemistry*; D009438:Neuraminic Acids / Q000032:analysis*; D009844:Oligosaccharides / Q000032:analysis; D012867:Skin / Q000737:chemistry*; D013402:Sugar Alcohols / Q000032:analysis
chemical_list: D000596:Amino Acids; D006023:Glycoproteins; D009438:Neuraminic Acids; D009844:Oligosaccharides; D013402:Sugar Alcohols