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

利用基因重组技术对甘露糖和N-乙酰氨基葡萄糖特异的肝脏凝集素--肝甘露聚糖结合蛋白(MBP)的功能和结构进行了研究。本研究取得的主要研究成果如下：1.根据大鼠肝脏MBP的cDNA序列，推测其一级结构。通过亲和纯化的抗大鼠肝MBP抗体筛选，从GT-11携带的大鼠肝cDNA文库中分离到大鼠肝MBP的基因克隆。双脱氧法测定的核苷酸序列显示了MBP的完整氨基酸序列(226个残基)。在MBP的NH_2-末端残基谷氨酸之前，有一个由18个氨基酸组成的疏水的信号肽。在NH_2末端附近有一个胶原样结构域，由19个重复序列组成，序列为GIY-X-Y。胶原酶消化得到的分子的COOH末端半部分保留了糖结合活性，表明糖类识别结构域位于其COOH末端150个氨基酸残基上。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有关。肝髓鞘蛋白内源性配体的鉴定。用固定化MBP柱亲和层析，从大鼠肝粗微体和原代培养的肝细胞中分离出肝凝集素的内源性配体。用特异性抗血清进行免疫印迹分析表明，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和溶酶体酶--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，肝脏MBP与糖蛋白在其生物合成过程中在细胞内的运输有关。

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.