Purification and characterization of protein D/E, a putative sperm-binding protein involved in fertilization

We describe a method for the efficient purification of a 32 Kd glycoprotein from rat epididymal tissue. The glycoprotein was purified by gel filtration, ion-exchange, affinity, and reverse phase high pressure liquid chromatography. The highly purified glycoprotein was radiolabeled with an iodinatable, cleavable, photoreactive cross-linking agent, 1-[N-(2-hydrox-5-azidobenzoyl)-2-aminoethyl]- 4-( -hydroxysuccinimidyl)-succinate (HAHS). The soluble radiolabled glycoprotein was bound to washed epididymal spermatozoa in a time-dependent, saturable, and reversible manner. Scatchard analysis demonstrates that there are approximately 3,403 binding sites/spermatozoon. The binding efficiency (K-d) for spermatozoa was similar to 2.0 x 10(-10) M. The function of this glycoprotein was verified by using an in vivo artificial insemination fertilization assay. The fertility rate for control spermatozoa was similar to 53%, but the rate for spermatozoa exposed to polyclonal anti-glycoprotein antibodies was only 5%. These data suggest that the binding of the glycoprotein to the surface of rat spermatozoa is mediated by a receptor-type mechanism and is involved in the fertilization process.