Resolution of saccharide structure and biochemical and immunological characterization of unknown glycoprotein which reacts with the antibody of milk fat globule membrane protein

1. 制备了牛乳脂球膜主要组成蛋白PAS-4糖蛋白单克隆抗体，命名为KAS-4。此外，在乳清蛋白中发现了一些与KAS-4发生反应的蛋白，也被命名为GP-88。GP-88为糖蛋白，sds - page估计分子量为88,000。先用蛋白G亲和层析，再用KAS-4亲和层析从乳清蛋白中纯化GP-88。纯化后的GP-88在SDS-PAGE上呈单条带。经n -聚糖酶处理的GP-88分子量估计为57000。比较了GP-88和PAS-4的生化特性。此外，经三氟甲磺酸和n -聚糖酶处理后，采用免疫印迹法检测KAS-4对GP-88和PAS-4的表位。在蛋白片段中发现了表位。此外，在C8和C18反柱的HPLC上分析胰蛋白酶消化后的片段，并通过elisa检测GP-88和PAS-4对KAS-4的活性肽。测定乳清中GP-88和乳脂球膜中PAS-4在泌乳期的含量。结果表明，GP-88含量增加，PAS-4含量降低。通过肼水解从GP-88中去除n链糖链，并通过HPLC测定糖链的大小、外糖苷酶顺序水解、乙酰解和GC-MS甲基化分析分析糖链的结构。建议的代表性结构如下所示。

1. Monoclonal antibody to PAS-4 glycoprotein which is a major constituent protein of bovine milk fat globule membrane was prepared and designated KAS-4. In addition, some proteins which react with KAS-4 was found in milk whey protein and also named as GP-88. GP-88 was glycoprotein and molecular weight was estimated to 88,000 on SDS-PAGE.2. GP-88 was purified from whey protein by affinity chromatography on Protein G and then KAS-4.Purified GP-88 was a single band on SDS-PAGE.Molecular weight of GP-88 treated with N-glycanase was estimated to 57,000. Biochemical properties of GP-88 and PAS-4 were compared.3. In addition, epitope of KAS-4 to GP-88 and PAS-4 was examined by immunoblotting after treatment with trifluoromethanesulfonic acid and N-glycanase. Epitope was found in protein moiety.Furthermore, fragments after digestion with trypsin were analyzed on HPLC of C8 and C18 reversed columus and an active peptide to KAS-4 from both GP-88 and PAS-4 was detected by ELISA.4. The contents of GP-88 in whey and PAS-4 in milk fat globlule membrane in laction periods were determined. It was found that GP-88 content increased the proceeding of laction, whereas PAS-4 content decreased.5. N-Linked saccharide chains were removed from GP-88 by hydrazine hydrolysis, and the structure of sugar chains was analyzed by size estimation on HPLC,sequential hydrolysis with exoglycosidases, acetolysis, and methylation analysis on GC-MS.A representative structure was proposed as shown in the following.