Nakagawa H, Hama Y, Sumi T, Li S C, Li Y T
Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Saga 840-8502, Japan.
Adv Exp Med Biol. 2001;491:171-84. doi: 10.1007/978-1-4615-1267-7_13.
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 b --> 6GalNAc-ol, KDNalpha2 --> 3(GalNAcbeta1 --> 14)GalNAc-ol, KDNalpha2 --> 6(GalNAcalpha1 --> 3)GalNAc-ol, KDNalpha2 --> 6(Gal3alpha1--> 3)GalNAc-ol, and NeuAcalpha2 --> 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.
人们已经广泛认识到，鱼类的黏液层具有多种重要的物理、化学和生理功能。黏液层的主要成分之一是黏液糖蛋白。我们发现，泥鳅（Misgurnus anguillicaudatus）皮肤黏液中的唾液酸主要由KDN组成。随后，我们从泥鳅皮肤黏液中分离出含KDN的糖蛋白，并对其化学性质和结构进行了表征。泥鳅黏液糖蛋白通过DEAE - 纤维素色谱、核酸酶P1处理和Sepharose CL - 6B凝胶过滤从泥鳅皮肤黏液的Tris - HCl缓冲液提取物中纯化得到。纯化后的黏液糖蛋白含有38.5%的KDN、0.5%的NeuAc、25.0%的GalNAc、3.5%的Gal、0.5%的GlcNAc和28%的氨基酸。用肌动蛋白酶对该糖蛋白进行彻底消化后，得到一种糖含量更高、苏氨酸（Thr）和丝氨酸（Ser）含量更高的糖肽。这种糖肽的分子大小约为完整糖蛋白的1/12。这些结果表明，大约11个高度糖基化的多肽单元通过非糖基化肽串联连接形成糖蛋白分子。通过碱性硼氢化钠处理从泥鳅黏液糖蛋白中释放出的寡糖糖醇，经Sephadex G - 25凝胶过滤和HPLC分离。纯化后的糖链经分析为b --> 6GalNAc - ol、KDNα2 --> 3(GalNAcβ1 --> 14)GalNAc - ol、KDNα2 --> 6(GalNAcα1 --> 3)GalNAc - ol、KDNα2 --> 6(Gal3α1--> 3)GalNAc - ol和NeuAcα2 --> 6Gal NAc - ol。据估计，一个泥鳅黏液糖蛋白分子含有超过500条含KDN的糖链，这些糖链通过GalNAc与蛋白质核心的苏氨酸和丝氨酸残基相连。
