Analysis of sperm proteins involved in mammalian fertilization

多种碳水化合物识别机制在受精过程中起作用。我们已经证明了几个识别卵细胞透明带糖蛋白的分子在猪精子头上表达。本课题对猪精子的碳水化合物识别分子进行了研究。用n -乙酰乳胺-寡糖探针包裹塑料孔，将猪精子的洗剂溶解质膜涂在塑料孔上，两个分子（70 kDa和40 kDa）与孔结合，寡糖的加入抑制了它们的结合。这两种分子也被证明与含有n -乙酰乳胺寡糖的asialo- α -1-酸糖蛋白有亲和力。测定了两种蛋白色氨酸片段的部分氨基酸序列。同源性分析表明，70和40 kDa分子为ADAM（崩解素和金属蛋白酶蛋白）家族蛋白的成员。克隆编码该蛋白的cDNA并测定其核苷酸序列。然后，用酵母制备了猪ADAM4重组蛋白，纯化后的蛋白与整合素具有亲和力。在哺乳动物细胞表面表达的猪ADAM5重组蛋白也被证实与亚洲乳母蛋白结合，而不与无乳乳母蛋白结合，这表明ADAM5是一种识别n -乙酰乳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.