CHARACTERIZATION OF A DEAMINATED NEURAMINIC ACID-CONTAINING GLYCOPROTEIN FROM THE SKIN MUCUS OF THE LOACH MISGURNUS-ANGUILLICAUDATUS

Using NMR spectroscopy and mass spectrometry, the major sialic acid of the skin mucus of the leach, Misgurnus anguillicaudatus was found to be 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN). We have subse quently devised a method to isolate a KDN-containing glycoprotein prep aration from leach skin mucus. The method involves the sonication of t he skin mucus with 0.05 M Tris-HCl, pH 8.0, to solubilize the glycopro tein, followed by DE52-cellulose chromatography of the extract, Nuclea se P-1 treatment, and Sepharose CL-4B gel filtration. The purified gly coprotein 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 compos ition of this glycoprotein preparation revealed that it is unusually r ich in Thr, and 6 amino acids, Thr, Ser, Glu (or Gin), Pro, Gly and Al a, account for 83% of the total amino acids. This glycoprotein is extr emely poor in Cys, Met, Tyr, Phe, Arg, and Trp. Treatment of this glyc oprotein with alkali resulted in the destruction of 83% of Thr suggest ing that most of the sugar chains in this glycoprotein are linked thro ugh Thr. Alkaline borohydride treatment of 100 mg of the glycoprotein preparation, followed by Sephadex G-25 (superfine) gel filtration, yie lded 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-ol. KDN alpha 2-->GalNAc is structurally s imilar to NeuAc alpha 2-->6GalNAc found in ovine submaxillary glycopro tein while II represents a novel structure which contains two sialic a cids linked to a GalNAc through both alpha 2-->3 and alpha 2-->6 linka ges. This structure has never been found in mucin type glycoproteins i ncluding mammalian epithelial mucin glycoproteins. This is the first r eport of the presence of a mucin type glycoprotein which contains ISDN instead of NeuAc or NeuGc in fish skin mucus.