Human Acrosin and Fertilization: Identification and localization of potencial functional sites by molecular secuence analisis.

VAZQUEZ-LEVIN, MH; GHIRINGHELLI, DP; ZAHN, A; RIVAROLA V; CHARREAU, EH

Boston - Estados Unidos

Congreso; 52nd Annual Meeting of the American Society for Reproductive Medicine.; 1996

American Society for Reproductive Medicine

Objectives: Acrosin, a testis-specific serine proteinase localized on the sperm acrosome of all mammalian species studied, is involved in sperm penetration of the zona pellucida (ZP) during fertilization, by binding to and digesting the egg´s extracellular matrix. Acrosin is synthesized as a proenzyme and apparently activated during the acrosome reaction. The purpose of the present study was to perform a molecular analysis on the aminoacidic sequence deduced from the nucleotide sequence of the human acrosin gene, in an attempt to identify and localize specific sequences associ-ated with acrosin activation and function(s) during fertilization. In addition, a múltiple sequence alignment was done to identify conservation of functional sites in other acrosin species previously characterized at the molecular level (mouse, rat, boar, guinea pig, rabbit). Design/Materials and Methods: The aminoacidic sequence of human acrosin was deduced from a nucleotide sequence previously reported (Vázquez et al, Eur. J. Bio-chem., 1992), and a computational analysis using the PC/ GENE package for sequence analysis, and múltiple sequence alignment was done. Results: The analysis confirmed the localization of aminoacids (aa) participating in the proteinase active site, located on His-69, Asp:123, and Ser-221, and highly conserved within all species evaluated. In addition, three consensus sequences for binding to sulfated glycoconjugates were localized in on 158-169, 190-201, and 217-231, Moreover, a consensué sequence for binding to the ZP re-cently described in boar acrosin (Dev. Biol. 1995, 168:575) was localized in aa 362-369, and conserved in all other species. The study revealed the prescence of three potential phosphorylation sites for Protein Kinase C in aa 48 (Ser/His/ Arg), 287 (Thr/Thr/Arg), and 356 (Thr/Thr/Lys). Moreover, it was found a potential phosphorylation site for a tyrosine kinase in aa 235 (Ala/Tyr/Val/Val). These sites were partially conserved on the other species analyzed. It may be interesting to point out that the human acrosome reaction has been shown to involve signal transduction pathways with protein phosphorylation by sperm protein kinases. Finally, a Tyrosine sulfation site was identified in aa 388 (His/Tyr/Asp/Met). Previous reports have shown that protein,tyrosine sulfation serve numerous functions, and could be involved in acrosin transport, and/or regulating its activation. Conclusión: The study has confirmed the localization of acrosin protease active site, and has localized a group of domains for binding to glycoconjugates. Moreover, the analysis has identified the presence of potential phosphorylation and sulfation sites that could play a key role in acrosin transport and localization in the acrosome, as well as in acrosin activation and its participation in the process of sperm-egg interaction during fertilization.