Molecular cloning of a sperm fucosidase cDNA of Halocynthia roretzi

精子与卵黄外壳的特异性结合是许多动物受精的必要步骤。在两个海鞘物种，Halocynthia roretzi和Ciona ninteinalis中，糖蛋白中酸性糖链的L-聚焦基残基似乎对卵磷脂外壳的精子受体活性至关重要。聚焦酶特异性抑制剂阻断精子结合，聚焦胺亲和柱在正常海水中对精子提取物有较好的吸附作用。由此可见，精子聚焦酶在精子与卵磷脂被膜结合过程中起重要作用。本研究利用α - l -聚焦酶的保守区，采用RT-PCR的方法，分离到了编码褐藻精子α - l -聚焦酶的cDNA （HrFuc）。并与人、大鼠和盘状盘状盘的聚焦酶序列进行比较。制备了针对大肠杆菌lacZ-HrFuc融合蛋白的多克隆抗体。Western分析显示该抗体与33 kDa精子蛋白杂交。该抗体抑制褐藻的受精。因此，克隆的cDNA似乎与精子-卵磷脂外壳结合密切相关。

Specific binding of spermatozoa to the vitelline coat is a necessary step for fertilization in various animals. In two ascidian species, Halocynthia roretzi and Ciona intestinalis, L-fucosyl residues of acidic saccharide chains in glycoproteins appear to beessential for the sperm-receptor activity of the vitelline coat. Sperm binding was blocked by specific inhibitors against fucosidase, and the fucosylamine affinity column adsorbed the sperm extract appreciably in normal sea water. It is thus suggested that sperm fucosidase play an important role in sperm binding to the vitelline coat.In the present study, we isolated the cDNA (HrFuc) encoding Halocynthia roretzi sperm alpha-L-fucosidase by RT-PCR method using the conservative region of alpha-L-fucosidase. The nucleotide and fucosidase were compared with the human, rat and Discoidium fucosidase sequences. The polyclonal antibody was prepared against a lacZ-HrFuc fusion protein made in Escherichia coli. Western analysis showed that this antibody crossed to 33 kDa sperm proteins. And this antibody inhibited ferterization in Halocynthia roretzi. Then, the cloned cDNA seems to be closedly corelated with the sperm-vitelline coat binding.