Mammalian cysteine‐rich secretory proteins interact with plasma membrane Ca2+ exporter PMCA4b

Abstract

AbstractBackgroundMammalian cysteine‐rich secretory proteins (CRISPs) are predominantly expressed in the male reproductive tract. Knockout mice lacking two or more CRISPs show defects in sperm transport, sperm–egg interaction and Ca2+ homeostasis. CRISPs play redundant and specific roles via their binding partners. To understand this, a comprehensive analysis of CRISP interactome needs to be undertaken.ObjectivesThis study aimed to analyse CRISP4 binding partners on the plasma membrane of rat caudal spermatozoa.Materials and methodsTotal proteins from rat caudal spermatozoa were subjected to immunoprecipitation using anti‐CRISP4 antibody followed by liquid chromatography–mass spectrophotometry analysis. Plasma membrane localised proteins were shortlisted, and a key target was validated by co‐immunoprecipitation and co‐localisation. Co‐transfection followed by co‐immunoprecipitation was carried out for studying the interaction of full‐length as well as deletion mutants of CRISPs with human plasma membrane calcium ATPase, isoform b (hPMCA4b). Calcium assays were performed using Fura‐2‐AM. The cholesterol binding ability of different CRISPs was evaluated in silico.ResultsThe membrane‐specific interactome of rat CRISP4 (rCRISP4) from caudal spermatozoa revealed PMCA4b as a novel binding partner, and their interaction was validated in rat spermatozoa. Human CRISP1 (hCRISP1) and hCRISP3 also interacted with PMCA4b via the N‐terminal domain. Interestingly, hCRISP1 and rCRISP4 delayed PMCA4b‐mediated calcium extrusion but hCRISP3 did not. In silico analysis demonstrated that hCRISP1 and rCRISP4 have higher binding affinity towards cholesterol than hCRISP3. The secretion profile of different CRISPs also showed that the ratio of secreted to cell‐associated proteins was highest for hCRISP3.ConclusionOur study identifies PMCA4b as a target of multiple mammalian CRISPs and unravels a new role of CRISPs in regulating calcium homeostasis. Differences in the interaction of different CRISPs with cholesterol may regulate their enrichment in the lipid rafts and redistribution in the membrane post‐capacitation, thereby affecting their interaction with PMCA4b.