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Studies on the structure and function of the liver mannan-binding protein by utilizing gene recombinant technology.

利用基因重组技术研究肝甘露聚糖结合蛋白的结构和功能。

基本信息

批准号：
61480429
负责人：
KAWASAKI Toshisuke
金额：
$ 3.65万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1988
项目状态：
已结题

项目摘要

The function and structure of the liver mannan-binding protein (MBP), a hepatic lectin specific for mannose and N-acetylglucosamine, were investigated by utilizing gene recombinant technology. The major findings obtained in this sutdy are as follows.1. The primary structure of rat liver MBP was deduced from its cDNA sequence. cDNA clones encoding rat liver MBP were isolated from a rat liver cDNA library carried in gt 11 by screening with an affinity purified anti-rat liver MBP antibody. The nucleotide sequence of the cDNA determined by the dideoxymethod revealed the complete amino acid sequence of the MBP (226 residues). The NH_2-terminal residue of the MBP, glutamic acid, was preceded by a signal peptide consisting of hydrophobic strecth of 18 amino acids. Near the NH_2-terminal, there was a collagen-like domain, which consisted of 19 repeats of the sequence, Giy-X-Y. The COOH-terminal half of the molecule obtained by collagenase digestion retained the sugar binding activity, revealing that the carbohydrate recognizing domain is located in its COOH terminal 150 amino acid residues. Treatment with reducing agents inactivated the lectin but this effect was reversed by air oxidation, indicating that disulfide bonds are associated with the expression of the binding activity.2. Identification of endogenous liginds of the liver MBP. Endogenous ligands for the hepatic lectin were isolated from rat liver rough microsomes and primary cultured hepatocytes by affinity chromatography on an immobilized MBP column. Western blotting using specific antisera revealed that biosynthetic intermediates of serum glycoproteins, alpha_1-acid glycoprotein, alpha_1-macroglobulin, and alpha_1-antitrypsin, and a lysosomal enzyme,beta-glucuronidase were the major constituents of the endogenous ligands. These results suggest that the liver MBP is associated with the transport of glycoproteins within the cells during the process of their biosynthesis.

利用基因重组技术研究了肝甘露聚糖结合蛋白（MBP）的结构和功能。本研究的主要结果如下：1.根据大鼠肝MBP的cDNA序列推测其一级结构。通过用亲和纯化的抗大鼠肝MBP抗体筛选，从gt 11中携带的大鼠肝cDNA文库中分离编码大鼠肝MBP的cDNA克隆。通过双脱氧法测定的cDNA核苷酸序列揭示了MBP的完整氨基酸序列（226个残基）。MBP的NH_2端残基谷氨酸前有一个由18个氨基酸组成的疏水信号肽。在NH_2端附近有一个胶原样结构域，由19个重复序列Giy-X-Y组成。通过胶原酶消化获得的分子的COOH末端的一半保留了糖结合活性，揭示了碳水化合物识别结构域位于其COOH末端150个氨基酸残基。用还原剂处理使凝集素失活，但这种作用被空气氧化逆转，表明二硫键与结合活性的表达相关.肝MBP的内源性配体的鉴定。用MBP亲和层析法从大鼠肝粗微粒体和原代培养的肝细胞中分离肝凝集素的内源性配体。使用特异性抗血清的Western印迹显示，血清糖蛋白、α_1-酸性糖蛋白、α_1-巨球蛋白和α_1-抗胰蛋白酶的生物合成中间体以及溶酶体酶β-葡萄糖醛酸苷酶是内源性配体的主要成分。这些结果表明，肝MBP与糖蛋白在细胞内的转运过程中，其生物合成过程中。