Objective: To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties.
Methods: The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods.
Results: Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity.
Conclusions: LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.
目的： 对人源类溶菌酶蛋白4（LYZL4）在受精过程中的作用和重组蛋白的酶学性质进行研究，从而揭示LYZL4的生理功能。 方法： 细胞免疫荧光法检测LYZL4在精子细胞上的亚细胞定位， RT-PCR分析精子表面LYZL4的来源，通过精子穿透试验分析LYZL4在受精过程中的作用。构建真核重组表达载体pPIC9K-LYZL4，转化毕赤酵母GS115菌株后，甲醇诱导表达;甲壳素亲和层析和凝胶过滤层析从发酵上清中纯化重组LYZL4蛋白（rLYZL4），通过Western印迹进行验证。ELISA测定rLYZL4的透明质酸结合能力，分光光度法测定rLYZL4的胞壁质酶活性、透明质酸酶活性和自由基清除能力。 结果： LYZL4免疫荧光定位于成熟精子头部的顶体膜上，RT-PCR检测表明精子表面的LYZL4蛋白来源于睾丸和附睾的分泌，兔抗LYZL4多克隆抗体可明显抑制精卵结合。毕赤酵母表达系统成功表达了rLYZL4，酶活性分析显示rLYZL4无胞壁质酶和透明质酸酶活性，但能够结合透明质酸，并具有较强的自由基清除能力。 结论： LYZL4由睾丸和附睾分泌后定位于成熟精子头部的顶体膜上，可在精卵结合过程中发挥作用，其还能够结合透明质酸并清除自由基，提示LYZL4可能作为一种多功能分子在精子保护及精卵结合过程中发挥作用。.
Keywords: acrosome; free radical; hyaluronan; sperm-egg binding; human lysozyme-like protein 4.