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Association of Eppin with Semenogelin on Human Spermatozoa¹

Laboratories for Reproductive Biology and Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Eppin (SPINLW1; GeneID, 57119) is a single-copy gene encoding a cysteine-rich protein found only in the testis and epididymis, which contains both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences. This study demonstrates that, in seminal plasma and on human spermatozoa following ejaculation, Eppin is bound to semenogelin I (Sg). Six different experimental approaches: 1) immunoprecipitation from spermatozoa and seminal plasma with anti-Eppin, 2) colocalization in semen and spermatozoa, 3) incubation of recombinant Eppin (rEppin) and rSg and immunoprecipitation with either anti-Eppin or anti-Sg, 4) far-Western blotting of Eppin and Sg, 5) Saturation binding of ¹²⁵I-Sg to Eppin, which is competed by unlabeled Sg, and 6) direct binding of ¹²⁵I-Sg to Eppin on a blot, all demonstrate that Eppin and Sg bind to each other. To study the specificity of binding, recombinant fragments of Eppin and Sg were made and demonstrate that the Eppin^75–133 C-terminal fragment binds the Sg^164–283 fragment containing the only cysteine in human Sg I (Cys-239). Reduction and carboxymethylation of Cys²³⁹ blocks binding of ¹²⁵I-rEppin, indicating that a disulfide bond may be necessary for Eppin binding. The physiological significance of the Eppin-semenogelin complex bound on the surface of ejaculate spermatozoa lies in its ability to provide antimicrobial activity for spermatozoa, which has been reported for both Eppin and semenogelin-derived peptides, and in its ability to provide for the survival and preparation of spermatozoa for fertility in the female reproductive tract.

male reproductive tract, seminal vesicles, sperm

² Correspondence: Dr. Michael G. O'Rand, Department of Cell and Developmental Biology, CB# 7090, 212 Taylor Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090. FAX: 919 966 1856; morand{at}unc.edu

³ Current address: Department of Urology, Duke University, Durham, NC 27710

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