Molecular cloning of a sperm fucosidase cDNA of Halocynthia roretzi

精子与卵黄膜的特异性结合是各种动物受精的必要步骤。在两种海鞘（Halocynthiaroretzi和Cionaerodiata）中，糖蛋白中酸性糖链的L-岩藻糖基残基似乎对卵黄被膜的精子受体活性至关重要。精子结合被岩藻糖苷酶的特异性抑制剂阻断，岩藻糖胺亲和柱在正常海水中吸附精子提取物。本研究利用罗氏盐爪鱼精子α-L-岩藻糖苷酶基因保守区，通过RT-PCR方法克隆了罗氏盐爪鱼精子α-L-岩藻糖苷酶的cDNA（HrFuc）。将核苷酸和岩藻糖苷酶与人、大鼠和盘状藻岩藻糖苷酶序列进行比较。制备针对在大肠杆菌中制备的lacZ-HrFuc融合蛋白的多克隆抗体。Western分析表明，该抗体与33 kDa精子蛋白杂交。该抗体对罗氏盐萼的受精有抑制作用。因此，克隆的cDNA可能与精子-卵黄膜结合密切相关。

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.