The role of human lysozyme-like protein 4 in fertilization and its enzymatic properties / 中华男科学杂志

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.

Immunogenicity of recombinant Lugurus zone pellucida-3 expressed in E.coli and efficacy of its antiserum to affect in vitro mouse sperm-egg binding in vitro / 中国免疫学杂志

Objective:Present study was aimed to analyze the immunogenicity of recombinant Lugurus zona pellucida-3 protein (r-LZP3) expressed in E.coli,and to evaluate the efficacy of its antiserum to affect in vitro mouse sperm-egg binding.Methods:Female mouse were immunized using LZP3 DNA vaccine for priming,and boosted with r-LZP3 protein.The antibody response level of LZP3 antisera was determined by ELISA.The immunoreactivity and specificity of the anti-LZP3 antisera were tested by immunoblot, indirect immunofluorescence and immunohistochemistry using native mouse ZP.A sperm-zone pellucida binding assay was used to evaluate the efficacy of anti-LZP3 antisera to inhibit mouse sperm-egg binding in vitro.Results: The result of ELISA showed that LZP3 DNA vaccine was able to induce higher antibody titers in mouse.Antiserum could specifically recognize or bind to r-LZP3 protein expressed in E.coli and native mouse ZP in vitro.The LZP3 antisera and r-LZP3 protein also inhibited sperm-egg binding in vitro.Condusion:These results show that r-LZP3 protein is of strong immunogenicity.