Identification of CRISP2 from human sperm as PSP94‐binding protein and generation of CRISP2‐specific anti‐peptide antibodies

Abstract

Cysteine-rich secretory proteins (CRISPs) are mainly found in the mammalian male reproductive tract and reported to be involved at different stages of fertilization. CRISPs have been shown to interact with prostate secretory protein of 94 amino acids (PSP94) from diverse sources, and the binding of these evolutionarily conserved proteins across species is proposed to be of functional significance. Of the three mammalian CRISPs, PSP94-CRISP3 interaction is well characterized, and specific binding sites have been identified; whereas, CRISP2 has been shown to interact with PSP94 in vitro. Interestingly, human CRISP3 and CRISP2 proteins are closely related showing 71.4% identity. In this study, we identified CRISP2 as a potential binding protein of PSP94 from human sperm. Further, we generated antisera capable of specifically detecting CRISP2 and not CRISP3. In this direction, specific peptides corresponding to the least conserved ion channel regulatory region were synthesized, and polyclonal antibodies were generated against the peptide in rabbits. The binding characteristics of the anti-CRISP2 peptide antibody were evaluated using competitive ELISA. Immunoblotting experiments also confirmed that the peptide was able to generate antibodies capable of detecting the mature CRISP2 protein present in human sperm lysate. Furthermore, this anti-CRISP2 peptide antibody also detected the presence of native CRISP2 on sperm.Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Structural and molecular biology of PSP94 Its significance in prostate pathophysiology

“…Interestingly, human CRISP-3 and CRISP-2 share 71.4. percent sequence identity. In this direction, using affinity pull down, we identified CRISP-2 as one of the probable binding proteins from human sperm to bind to PSP94 (135). Further, we pursued the development of indigenous anti-peptide antibodies against the least conserved ICR region of the CRISP domain in an attempt to distinguish human CRISP-2 from CRISP-3.…”

Prostate secretory protein 94 inhibits sterol binding and export by the mammalian CAP protein CRISP2 in a calcium-sensitive manner

“…In contrast to CRISP3, PSP94 expression is downregulated in prostate cancer, suggesting a protective role in prostate carcinogenesis (21)(22)(23). PSP94 also interacts with another CRISP family member, CRISP2, also referred to as Tpx-1 (testis-specific protein 1), a non-glycosylated CRISP family member produced during spermatogenesis, which shares 71% sequence identity with CRISP3 (24,25). CRISP2 regulates calcium influx through ryanodine receptors to modulate sperm flagellar motility (11,26,27).…”

The Ubiquitin-Proteasome System Does Not Regulate the Degradation of Porcine β-Microseminoprotein during Sperm Capacitation

“…Sperm MSMB is probably involved in the interactions between spermatozoa and zona pellucida at fertilization as well as in the regulation of sperm hyperactivation at the time of sperm capacitation [11]. In a more recent study, sperm MSMB has been found to associate with CRISPs (cysteine-rich secretory proteins) [17] implicated in gamete binding and fusion [18].…”