Identification of a Caltrin receptor protein in rat spermatozoa

MIRANDA SABRINA D. ; MONTI MARIELA R.; NOVELLA M.L.; ARGARAÑA CARLOS E.; CORONEL CARLOS E.

Congreso; XLV Reunión Anual de SAIB; 2010

Caltrin (calcium transport inhibitor) is a small and basic protein secreted by the seminal vesicles. During ejaculation, rat caltrin (6.2 kDa) specifically binds to the acrosomal region of the sperm head and inhibits sperm Ca2+ uptake. Thus, it prevents the spontaneous acrosomal exocytosis and protects the integrity required for sperm-egg interaction. The aim of this work was to identify the molecules responsible of the specific binding of caltrin to the sperm surface. The presence of receptor molecules was first examined by chemical cross-linking using NHS and EDC reagents to covalently bind caltrin to the receptors. Then, sperm extracts were subjected to SDS-PAGE and Western blotting with anti-caltrin antibodies. An immunoreactive band of 55 kDa was revealed suggesting the presence of a 49 kDa caltrin-receptor protein. This protein was isolated by affinity chromatography using recombinant caltrin bound to the fusion protein intein. Caltrin-intein complex expressed in E. coli was bound to chitin and then pour into a column. Sperm proteins extracted with 1 % Triton X-100 were loaded in the column and those retained were eluted with 1M NaCl and then analyzed by SDS-PAGE and MALDI TOF/MS. An enriched band of 48 kDa was identified as HongrES 1, an epididymal protein secreted by epithelial cells of the cauda. Like caltrin, HongrES 1 is synthesized under strict androgenic control.