Expression and Structure-Function Analysis of DE, a Sperm Cysteine-Rich Secretory Protein That Mediates Gamete Fusion1

Abstract

Rat sperm epididymal glycoprotein DE belongs to the cysteine-rich secretory protein (CRISP) family and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. To investigate the molecular mechanisms underlying the role of DE in gamete fusion, in the present work we expressed DE in a prokaryotic system, and examined the relevance of carbohydrates and disulfide bonds for the biological activity of the protein. Immunofluorescence and sperm-egg fusion assays carried out in the presence of recombinant DE (recDE) revealed that this protein exhibits the ability to bind to the DE-egg binding sites and to inhibit gamete fusion, as does native DE (nDE). Comparison of the proteins indicated, however, that the inhibitory ability of recDE was significantly lower than that of nDE. This difference would not be due to the lack of carbohydrates in the bacterially expressed protein because enzymatically deglycosylated nDE was as able as the untreated protein to inhibit gamete fusion. To examine whether disulfide bridges are involved in DE activity, the presence of sulfhydryls in nDE and recDE was evaluated by the biotin-maleimide technique. Results indicated that, unlike nDE, in which all cysteines are involved in disulfide bonds, recDE contains free thiol groups. Subsequent experiments showed that reduction of nDE with dithiothreitol significantly decreased the ability of the protein to inhibit gamete fusion. Together, these results indicate that whereas carbohydrates do not have a role in DE-mediated gamete fusion, disulfide bridges are required for full biological activity of the protein. To our knowledge, this is the first study reporting the relevance of structural components for the function of a CRISP member.

Participation of cysteine-rich secretory proteins (CRISP) in mammalian sperm-egg interaction

“…However, the molecular mechanisms involved in these interactions remained unknown. Structure-function studies using a recombinant CRISP1 (recCRISP1) protein expressed in a prokaryotic system showed that the activity of the protein does not involve carbohydrates, and resides in the polypeptide region of the molecule (Ellerman et al, 2002). However, rCRISP1 does not present any known functional domain that could explain its involvement in gamete fusion.…”

Immunocontraceptive properties of recombinant sperm protein DE: implications for the development of novel contraceptives

“…Recombinant maltose-binding protein (MBP) and recombinant DE fused to MBP (recDE) were expressed in Escherichia coli and purified as previously described (20). Native DE (nDE) was purified following the procedures described elsewhere (20).…”

“…Native DE (nDE) was purified following the procedures described elsewhere (20). Reduction and alkylation of DE was carried out according to Ellerman et al (20).…”

“…Our laboratory succeeded in expressing a functional DE molecule (recDE) in a prokaryotic system. Like the native protein (nDE), recDE is able to bind to the egg surface and inhibit gamete fusion (20). However, because it is expressed in a prokaryotic system, recDE is not glycosylated and presents a lower content of disulfide bridges when compared with nDE (20), opening the possibility that the recombinant molecule exhibits immunogenic properties different from those of the native folded protein.…”

TheCaenorhabditis elegans spe-38gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization

“…After sperm have penetrated the egg coat, direct gamete cell-cell interactions can occur (Foltz and Lennarz, 1993;Kaji and Kudo, 2004). In mammals, sperm proteins that mediate egg binding and fusion are thought to include the surface-associated protein DE (Ellerman et al, 2002), the immunoglobulin-like protein Izumo (Inoue et al, 2005), and the sperm ADAM proteins (fertilin α, fertilin β and cyritestin) (Evans, 2001). On the egg side of the equation, integrins (Evans, 2001), GPI-anchored proteins (Alfieri et al, 2003) and the tetraspanin CD9 (Kaji et al, 2000) are thought to mediate sperm binding and/or fusion.…”