人源类溶菌酶蛋白6的功能研究及生理特性分析 <sup>*</sup>

doi:10.13523/j.cb.20180301

中国生物工程杂志

2018, Vol. 38

Issue (3): 1-8 DOI: 10.13523/j.cb.20180301

研究报告

人源类溶菌酶蛋白6的功能研究及生理特性分析 ^*

黄鹏¹,杜望春¹,施尉珺¹,饶玉良²,孙庆文³,张宁^2*(

)

1 上海健康医学院临床医学院上海 201318
2 上海健康医学院基础医学院上海 201318
3 复旦大学生命科学院上海 200438

The Functional Studies of Human Lysozyme-like Protein 6 and Characterization of Its Physiological Properties

Peng HUANG¹,Wang-chun DU¹,Wei-jun SHI¹,Yu-liang RAO²,Qing-wen SUN³,Ning ZHANG^2*(

)

1 School of Clinical Medicine, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China
2 School of Basic Medicine, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China
3 School of Life Sciences, Fudan University, Shanghai 200438, China

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摘要：

对人源类溶菌酶蛋白6(human lysozyme-like protein 6,LYZL6)在受精过程中的作用进行研究,并对重组LYZL6蛋白(recombinant LYZL6,rLYZL6)的生理特性进行分析,从而揭示其生理功能。细胞免疫荧光法确定LYZL6定位于成熟精子头部的顶体后区域,反转录PCR(RT-PCR)分析表明精子表面的LYZL6蛋白来源于睾丸和附睾的分泌,Western blot法分析表明精子获能前后表面LYZL6的量无明显改变。半透明带结合实验和精子穿透实验分析表明兔抗LYZL6血清未明显抑制人精子结合透明带,但可明显抑制精卵融合。利用毕赤酵母表达系统成功表达了rLYZL6,使用甲壳素亲和层析和凝胶过滤层析可从发酵上清中纯化到具有生物活性的rLYZL6。酶联免疫吸附法(ELISA)分析显示rLYZL6不具有透明质酸结合能力、透明质酸水解能力和自由基清除活性,但具有较强的肽聚糖结合能力和异肽酶活性。LYZL6由睾丸和附睾分泌后定位于成熟精子头部的顶体后区域,可以参与精卵融合,并具有肽聚糖结合能力和异肽酶活性,提示LYZL6可能通过多种机制参与精子功能。

关键词： 人源类溶菌酶蛋白6; 顶体; 受精; 毕赤酵母; 异肽酶活性

Abstract:

The aim is to identify the possible performance of human lysozyme-like protein 6 (LYZL6) in fertilization and to characterize its physiological properties. Immunofluorescent staining with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa, which was secreted by testis and epididymis as demonstrated by the reverse transcription polymerase chain reaction (RT-PCR). No significant decrease of LYZL6 after capacitation was observed by Western blot analysis. Immunoneutralization of LYZL6 showed no effect on the binding of spermatozoa to the hemizona, but significantly decreased the numbers of human spermatozoa fused to zona-free hamster eggs in a dose-dependent manner. The Pichia expression system was utilized to produce recombinant LYZL6 (rLYZL6). After induction with methanol, rLYZL6 was purified from the fermentation supernatant by chitin affinity in combination with gel-filtration chromatography. In vitro assays indicated that rLYZL62 possessed no hyaluronan-binding ability, hyaluronidase activity and free radical scavenging activity, but peptidoglycan-binding ability and isopeptidase activity. In conclusion, LYZL6, a human sperm-related protein is reported, not only plays a role in sperm-egg fusion but also has peptidoglycan-binding ability and isopeptidase activity, suggesting it might contribute to diverse sperm functions.

Key words: Human lysozyme-like protein 4 Acrosome Fertilization Pichia pastoris Isopeptidase activity

收稿日期: 2017-10-09 出版日期: 2018-04-04

ZTFLH:

Q492.4

基金资助: * 上海市卫生计生委科研课题(201740161)、上海市自然科学基金(15ZR1421800)资助项目

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引用本文:

黄鹏,杜望春,施尉珺,饶玉良,孙庆文,张宁. 人源类溶菌酶蛋白6的功能研究及生理特性分析 ^*[J]. 中国生物工程杂志, 2018, 38(3): 1-8.

Peng HUANG,Wang-chun DU,Wei-jun SHI,Yu-liang RAO,Qing-wen SUN,Ning ZHANG. The Functional Studies of Human Lysozyme-like Protein 6 and Characterization of Its Physiological Properties. China Biotechnology, 2018, 38(3): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180301 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I3/1

表1 研究所用引物序列

图1 精子细胞LYZL4免疫荧光染色

图2 LYZL6在男性生殖系统中的表达谱分析

图3 精子获能前后LYZL6 的Western blot分析

图4 抗LYZL6血清对精卵融合的影响 (n=3)

图5 抗LYZL6血清对精子透明带结合的影响 (n=8)

图6 发酵液上清SDS-PAGE电泳

图7 纯化产物SDS-PAGE

图8 肽聚糖结合能力测定

图9 透明质酸结合能力测定

图10 透明质酸水解活性测定

图11 自由基清除活性测定

图12 异肽酶活性测定

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