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.