KDN-containing glycoprotein from loach skin mucus

Abstract

It has been widely recognized that the mucus coat of fish plays a variety of important physical, chemical, and physiological functions. One of the major constituents of the mucus coat is mucus glycoprotein. We found that sialic acids in the skin mucus of the loach, Misgurnus anguillicaudatus, consisted predominantly of KDN. Subsequently, we isolated KDN-containing glycoprotein from loach skin mucus and characterized its chemical nature and structure. Loach mucus glycoprotein was purified from the Tris-HCl buffer extract of loach skin mucus by DEAE-cellulose chromatography, Nuclease P1 treatment, and Sepharose CL-6B gel filtration. The purified mucus glycoprotein was found to contain 38.5 % KDN, 0.5 % NeuAc, 25.0 % GalNAc, 3.5 % Gal, 0.5% GlcNAc and 28 % amino acids. Exhaustive Actinase digestion of the glycoprotein yielded a glycopeptide with a higher sugar content and higher Thr and Ser contents. The molecular size of this glycopeptide was approximately 1/12 of the intact glycoprotein. These results suggest that approximately 11 highly glycosylated polypeptide units are linked in tandem through nonglycosylated peptides to form the glycoporotein molecule. The oligosaccharide alditols liberated from the loach mucus glycoprotein by alkaline borohydride treatment were separated by Sephadex G-25 gel filtration and HPLC. The purified sugar chains were analyzed by permethylation analysis, 1H-NMR spectroscopy, and glycosidase digestion. Their structures were established to be: KDNα2→6(KDNα2 →3)GalNAc-ol, KDNα2→6GalNAc-ol, KDNα2→3(GalNAcβ1→4)GalNAc-ol, KDNα2 →6(GalNAcα1→3)GalNAc-ol, KDNα2→6(Galβ1→3)GalNAc-ol, and NeuAcα2→6Gal NAc-ol. It is estimated that one loach mucus glycoprotein molecule contains more than 500 KDN-containing sugar chains that are linked to Thr and Ser residues of the protein core through GalNAc.