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

糖结构的解析以及与乳脂肪球膜蛋白抗体反应的未知糖蛋白的生化和免疫学表征

基本信息

批准号：
05660129
负责人：
KANNO Choemon
金额：
$ 1.41万
依托单位：
Utsunomiya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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.

1.制备了抗牛乳脂球膜主要组成蛋白PAS-4糖蛋白的单克隆抗体，命名为KAS-4。另外，在乳清蛋白中发现了一些与KAS-4反应的蛋白质，也被命名为GP-88。结论：1. GP-88为糖蛋白，SDS-PAGE测得其分子量为88，000。用Protein G亲和层析和KAS-4亲和层析从乳清蛋白中纯化出GP-88，纯化后的GP-88在SDS-PAGE上为单一条带，经N-聚糖酶处理后的GP-88分子量为57，000。比较了GP-88和PAS-4的生化特性.另外，在用三氟甲磺酸和N-聚糖酶处理后，通过免疫印迹检测KAS-4对GP-88和PAS-4的表位。用C8和C18反相柱对胰蛋白酶消化后的片段进行HPLC分析，并通过ELISA检测到GP-88和PAS-4的一个抗KAS-4活性肽.测定了不同泌乳期乳清中GP-88和乳脂球膜中PAS-4的含量。结果表明，GP-88的含量促进了乳凝的进行，而PAS-4的含量降低.通过肼水解从GP-88中除去N-连接的糖链，并通过HPLC上的大小估计、用外切糖苷酶的连续水解、乙酰解和GC-MS上的甲基化分析来分析糖链的结构。