A mouse serine protease TESP5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein
A mouse serine protease TESP5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein
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DOI：
10.1074/jbc.m112470200
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发表时间：
2002-05-10
影响因子：
4.8
通讯作者：
Baba, T
中科院分区：
文献类型：
--
作者：
Honda, A;Yamagata, K;Baba, T
我们以前已经指出，至少在小鼠，精子丝氨酸蛋白酶（S）以外的顶体酶可能作用于卵透明膜的有限的蛋白水解，以创建一个运动精子的穿透途径，虽然顶体酶的参与不能完全排除。一个42 kDa的明胶水解丝氨酸蛋白酶存在于小鼠精子是一个候选酶参与精子穿透的透明体。在这项研究中，我们已经PCR扩增的EST克隆编码睾丸丝氨酸蛋白酶，称为TESP 5，然后筛选小鼠基因组DNA文库使用的DNA片段作为探针。分离的基因组克隆的DNA序列表明TESP 5基因与编码睾丸testisin和嗜酸性esp-1的基因相同。使用亲和纯化的抗TESP 5抗体的免疫化学分析显示，42-和41-kDa形式的TESP 5与5.0至5.5的等电点定位在头部，细胞质液滴，和中段的尾部附睾精子可能作为一种膜蛋白。此外，这两种形式的TESP 5被选择性地包含到Triton X-100不溶性微区，脂质筏，精子膜。这些结果表明TESP 5/testisin/esp-1和42-kDa精子丝氨酸蛋白酶之间的同一性。当HEK 293细胞被携带TESP 5的整个蛋白编码区的表达质粒转化时，产生的重组蛋白通过用蜡状芽孢杆菌磷脂酰肌醇特异性磷脂酶C处理而从细胞膜释放，表明TESP 5是锚定在细胞表面上的糖基磷脂酰肌醇。重组TESP 5的酶性质与大鼠顶体蛋白酶和胰蛋白酶的酶性质相似，但通过底物特异性和丝氨酸蛋白酶抑制剂的抑制作用而区别于大鼠顶体蛋白酶和胰蛋白酶。
We have previously indicated that at least in mouse, sperm serine protease(s) other than acrosin probably act on the limited proteolysis of egg zona pellucida to create a penetration pathway for motile sperm, although the participation of acrosin cannot be ruled out completely. A 42-kDa gelatin-hydrolyzing serine protease present in mouse sperm is a candidate enzyme involved in the sperm penetration of the zona pellucida. In this study, we have PCR-amplified an EST clone encoding a testicular serine protease, termed TESP5, and then screened a mouse genomic DNA library using the DNA fragment as a probe. The DNA sequence of the isolated genomic clones indicated that the TESP5 gene is identical to the genes coding for testicular testisin and eosinophilic esp-1. Immunochemical analysis using affinity-purified anti-TESP5 antibody revealed that 42- and 41-kDa forms of TESP5 with the isoelectric points of 5.0 to 5.5 are localized in the head, cytoplasmic droplet, and midpiece of cauda epididymal sperm probably as a membranous protein. Moreover, these two forms of TESP5 were selectively included into Triton X-100-insoluble microdomains, lipid rafts, of the sperm membranes These results show the identity between TESP5/testisin/esp-1 and the 42-kDa sperm serine protease. When HEK293 cells were transformed by an expression plasmid carrying the entire protein-coding region of TESP5, the recombinant protein produced was released from the cell membrane by treatment with Bacillus cereus phosphatidylinositol-specific phospholipase C, indicating that TESP5 is glycosylphosphatidylinositol-anchored on the cell surface. Enzymatic properties of recombinant TESP5 was similar to but distinguished from those of rat acrosin and pancreatic trypsin by the substrate specificity and inhibitory effects of serine protease inhibitors.