Inactivation of the mouse sperm receptor, mZP3, by site-directed mutagenesis of individual serine residues located at the combining site for sperm
Inactivation of the mouse sperm receptor, mZP3, by site-directed mutagenesis of individual serine residues located at the combining site for sperm
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DOI：
10.1073/pnas.95.11.6193
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发表时间：
1998-05-26
影响因子：
11.1
通讯作者：
Wassarman, PM
中科院分区：
文献类型：
--
作者：
Chen, J;Litscher, ES;Wassarman, PM
为了启动受精，小鼠精子与位于透明质酸糖蛋白mZP 3的精子结合位点的Ser-（O-）连接的寡糖结合。显然，低聚糖存在于mZP 3多肽羧基末端区域聚集的五个Ser残基中的一个或多个上。在这里，每个丝氨酸残基，以及一个插入的天冬酰胺残基，转化为一个小的，非羟基氨基酸的定点诱变。然后用野生型和突变的mZP 3基因稳定转染小鼠胚胎癌（EC）细胞。在每种情况下，转染的细胞合成并分泌重组EC-mZP 3到培养基中。部分纯化的糖蛋白，并测定其体外抑制精子与排卵卵子结合的能力。与野生型EC-mZP 3相比，Ser-329、Ser-331或Ser-333突变对精子受体活性没有影响。Asn-330（一个潜在的N-连接糖基化位点）的突变对精子受体活性也没有影响。另一方面，Ser-332或Ser-334的突变，或Ser-332、Ser-333和Ser-334的突变，导致EC-mZP 3作为精子受体的完全失活。这些结果表明，Ser-332和Ser-334，在小鼠，仓鼠和人ZP 3中保守的残基，是精子受体活性所必需的。
To initiate fertilization, mouse sperm bind to Ser-(O-) linked oligosaccharides located at the sperm combining site of zona pellucida glycoprotein mZP3. Apparently, the oligosaccharides are present on one or more of five Ser residues clustered in the carboxyl-terminal region of the mZP3 polypeptide. Here, each of the Ser residues, as well as an intervening Asn residue, was converted to a small, nonhydroxy amino acid by site-directed mutagenesis. Mouse embryonal carcinoma (EC) cells were then stably transfected with the wild-type and mutated mZP3 genes. In each case, transfected cells synthesized and secreted recombinant EC-mZP3 into the culture medium. The glycoproteins were partially purified and assayed for their ability to inhibit binding of sperm to ovulated eggs in vitro. As compared with wild-type EC-mZP3, mutations of Ser-329, Ser-331, or Ser-333 had no effect on sperm receptor activity. Mutation of Asn-330, a potential N-linked glycosylation site, also had no effect on sperm receptor activity. On the other hand, mutation of either Ser-332 or Ser-334, or mutation of Ser-332, Ser-333, and Ser-334, resulted in complete inactivation of EC-mZP3 as a sperm receptor. These results suggest that Ser-332 and Ser-334, residues conserved in mouse, hamster, and human ZP3, are essential for sperm receptor activity.