Analysis of sperm proteins involved in mammalian fertilization

一种多糖识别机制在受精过程中起作用。我们已经发现，猪精子头部表达了几种识别卵透明质酸糖蛋白的分子，本项目对猪精子上的碳水化合物识别分子进行了研究。当将洗涤剂溶解的猪精子质膜应用于涂有N-乙酰乳胺-寡糖探针的塑料威尔斯孔时，两种分子（70 kDa和40 kDa）与威尔斯孔结合，并且通过添加寡糖抑制结合。两种分子也显示对含有N-乙酰乳糖胺-寡糖的脱唾液酸-α-1-酸性糖蛋白具有亲和力。测定了这两种蛋白的胰蛋白酶肽段的部分氨基酸序列。肽序列的同源性搜索表明，70和40 kDa的分子是ADAM（一种去整合素和金属蛋白酶蛋白）家族蛋白的成员。克隆编码蛋白质的cDNA，并测定其核苷酸序列。利用酵母菌表达猪ADAM 4重组蛋白，纯化后的蛋白对整合素具有亲和性。在哺乳动物细胞表面表达的猪ADAM 5的重组蛋白也被证明与去唾液酸胎球蛋白结合，但不与半乳糖胎球蛋白结合，这表明ADAM 5是一种识别N-乙酰乳糖胺结构的碳水化合物识别分子。

A multiple carbohydrate recognition mechanism is working in fertilization. We have shown that several moleculesrecognizing egg zona pellucida glycoproteins are expressed on boar sperm heads.In this project, carbohydrate recognition molecules on boar sperm were investigated. When detergent-solubilized plasma membranes of boar sperm were applied to plastic wells coated with the N-acetyllactoamine-oligosaccharide probe, two molecules(70 kDa and 40 kDa) bound to the wells and binding was inhibited by addition of the oligosaccharides. Both molecules were also shown to have affinity to asialo-alpha-1-acid glycoprotein which contains N-acetyllactosamine-oligosacharides. Partial amino acid sequences of tryptic peptide fragments of the two proteins were determined. Homology search of the peptide sequences suggested that the 70 and 40 kDa molecules are members of ADAM(a disintegrin and metalloprotease protein)family protein. cDNA encoding the proteins were cloned and their nucleotide sequences were determined. Then, the recombinant proteins of porcine ADAM4 were produced by yeast and the purified proteins were shown to have affinity to integrins. The recombinant proteins of porcine ADAM5 expressed on the surface of mammalian cells were also shown to bind to asialo-fetun but not to agalacto-fetuin, suggesting that ADAM5 is a carbohydrate recognition molecule recognizing the N-acetyllactosamine structure.