Huang Peng, Li Wen-Shu, Yang Zhi-Fang, Xu Yi-Xin, Bao Jian-Ying, Cao Xiao-E, Zhou Pei-Hua, Li Kun, Yu Long
School of Basic Medicine, Shanghai College of Medicine and Health Sciences, Shanghai 201318, China.
College of Arts and Sciences, Shanghai New York University, Shanghai 200122, China.
Zhonghua Nan Ke Xue. 2016 Jul;22(7):584-590.
To study the expression of human lysozyme-like protein 6 (LYZL6) in the male reproductive system and its physiological role.
The recombinant P. pastoris strain was cultured and induced with methanol to express LYZL6, followed by purification using chitin affinity chromatography. The bactericidal activity of the recombinant LYZL6 was observed by bilayer agar plate diffusion assay, and then the recombinant protein was used as an immunogen to generate polyclonal antibodies, whose specificity was examined by ELISA. The distribution of LYZL6 in the human tissue and semen was identified by Western blotting and the subcellular localization in the testis was investigated by immunohistochemistry.
At pH 5.6, recombinant LYZL6 exhibited a high bacteriolytic activity against M. lysodeikticus. ELISA analysis showed that the anti-LYZL6 polyclonal antibodies could bind the recombinant protein with a high specificity. Western blot manifested the expression of LYZL6 in the testis and epididymis, higher in the former than in the latter. LYZL6 was also detected in the sperm protein extract, while protein bands were not observed in the seminal plasma. Immunodetection with a specific antiserum localized the LYZL6 protein in the late spermatocytes and round spermatids.
LYZL6 has a higher bacteriolytic activity under low pH condition and is bound to spermatozoa after secreted in the testicular epithelia, suggesting that LYZL6 could act as a potential hydrolase for carbohydrates in zona pellucida penetration.
研究人溶菌酶样蛋白6（LYZL6）在男性生殖系统中的表达及其生理作用。
培养重组巴斯德毕赤酵母菌株，用甲醇诱导表达LYZL6，然后通过几丁质亲和层析进行纯化。采用双层琼脂平板扩散法观察重组LYZL6的杀菌活性，然后将重组蛋白用作免疫原制备多克隆抗体，通过酶联免疫吸附测定法检测其特异性。通过蛋白质免疫印迹法鉴定LYZL6在人体组织和精液中的分布，通过免疫组织化学法研究其在睾丸中的亚细胞定位。
在pH 5.6时，重组LYZL6对溶壁微球菌表现出高溶菌活性。酶联免疫吸附测定分析表明，抗LYZL6多克隆抗体能高度特异性地结合重组蛋白。蛋白质免疫印迹显示LYZL6在睾丸和附睾中表达，前者表达水平高于后者。在精子蛋白提取物中也检测到LYZL6，而在精浆中未观察到蛋白条带。用特异性抗血清进行免疫检测将LYZL6蛋白定位在晚期精母细胞和圆形精子细胞中。
LYZL6在低pH条件下具有较高的溶菌活性，在睾丸上皮细胞分泌后与精子结合，提示LYZL6可能作为一种潜在的水解酶参与透明带穿透过程中的碳水化合物水解。
