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

doi:10.13523/j.cb.20180301

China Biotechnology

2018, Vol. 38

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

Orginal Article

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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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

Received: 09 October 2017 Published: 04 April 2018

ZTFLH:

Q492.4

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	Peng HUANG
	Wang-chun DU
	Wei-jun SHI
	Yu-liang RAO
	Qing-wen SUN
	Ning ZHANG

Cite this article:

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.

URL:

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

Table 1 Primers used in this study

Fig. 1 Immunofluorescence staining of LYZL4 in spermatozoa

Fig. 2 Expression profile of LYZL6 in male reproductive system
M: DL2000 DNA marker; 1,2: Sperm cDNA; 3: Testicular cDNA; 4:Epididymal cDNA

Fig.3 Western blot analysis of LYZL6 in spermatozoa before and after capacitation

Fig. 4 Effect of LYZL6 antiserum on sperm-egg fusion (n=3)
* P< 0.05 vs the control group

Fig.5 Effect of LYZL6 antiserum on sperm-hemizona binding (n=8)

Fig.6 SDS-PAGE of the fermentation supernatant
M: Molecular weight marker; 1: Supernatant before induction; 2-6: Supernatant after induction

Fig.7 SDS-PAGE of purified product
M:Molecular weight marker; 1-2:Purified rLYZL6

Fig.8 Assays of peptidoglycan binding ability
* P<0.05 vs the LYZ group

Fig.9 Assays of hyaluronan binding ability
* P<0.05 vs the HAase group and the LYZ group

Fig.10 Assays of hyaluronan hydrolyzing activity
* P<0.05 vs the HAase group

Fig.11 Assays of free radical scavenging activity
* P<0.05 vs the LYZ group

Fig.12 Assays of isopeptidase activity
* P<0.05 vs the LYZ group


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