Sperm protein “DE” mediates gamete fusion through an evolutionary conserved site of the CRISP family

ELLERMAN, D, COHEN DJ, DA ROS, V, BUSSO D, MORGENFELD, M AND CUASNICU, PS.

Chicago, USA

Congreso; 31th Annual Meeting of American Society of Andrology.; 2006

American Society of Andrology

The first member of the Cysteine-Rich Secretory Protein (CRISP) family was described by our laboratory in the rat epididymis and it is known as protein DE or CRISP-1. Since then, numerous CRISPs exhibiting a high amino acid sequence similarity have been identified in animals, plants and fungus and proposed to play a variety of biological functions through as yet unknown mechanisms. Protein DE is a candidate to mediate gamete fusion through its binding to complementary sites on the egg surface. To elucidate the molecular mechanisms involved in this interaction, in the present work seven recombinant fragments of the protein (F1: aa 1-158, F2: 156-227, F3: 62-227; F4: 62-158; F5: 1-62; F6: 62-116, F7: 111-158 ) were bacterially expressed as MBP fused-proteins and examined for their ability to both bind to the egg surface and interfere with gamete fusion. Indirect immunofluorescence (IIF) experiments showed that oocytes incubated with F1, F3, F4 and F7, but not with F2, F5, F6 or MBP, showed the characteristic fluorescent labeling over the entire egg surface with the exception of a negative area corresponding to the plasma membrane overlying the meiotic spindle. In agreement with these results, when the fragments were present during gamete co-incubation, F1, F3, F4 and F7 produced a significant inhibition of the percentage of egg penetration (F1: 54% inhibition, F3: 65%, F4: 61%, F7: 52%) while F2, F5, F6 and MBP showed no effect (F2: 13 % inhibition, F5: 2%, F6: 0%, MBP: 1% ). Other parameters such as oocyte penetrability or sperm motility, were unaffected by the presence of the fragments. These results support the idea that the biological activity of DE would be contained in the 45-residues region spanning from amino acid 114 to 158. Interestingly, this region contains two motifs that are recognized by the PROSITE database as feature motifs of the CRISP family, denominated Signature 1 and 2. To investigate whether these signatures were responsible for the binding of the proteins to the egg surface, two peptides, P1 (GHYTQVVWNST) and P2 (FYVCHYCPGGNY), corresponding to Signature 1 and 2 respectively, were synthetized and evaluated for their ability to bind to rat zona-free eggs. IIF results indicated that while eggs incubated with P1 showed a complete negative labeling, those incubated with P2 presented a bright fluorescent labeling similar to that previously observed with F7. A scramble P2 peptide, used as control, was not able to bind to the egg surface. Finally, the ability of several CRISPs to bind to the egg was analyzed in correlation with the amino acid composition of their corresponding Signature 2 regions. This analysis revealed that in those cases where the proteins were able to bind to an egg (rat DE to mouse eggs and mouse TPX-1 to rat eggs) only two substitutions in the amino acid sequence of their Signature 2 region were detected while four substitutions were detected in those cases where the proteins were unable to interact with the eggs (human ARP to rat egg, helothermine to rat egg, DE to human egg). These observations support the participation of Signature 2 in gamete fusion and suggest that differences in the amino acid sequence of this region may be responsible for the specificity of the binding of each CRISP to its target egg. Altogether, these findings will not only contribute to a better understanding of the molecular mechanism involved in mammalian gamete fusion but also provide novel insights into the structure-function relationship of other members of the widely distributed and highly conserved CRISP family.