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Antimicrobial Activity of Human EPPIN, an Androgen-Regulated, Sperm-Bound Protein with a Whey Acidic Protein Motif¹

Laboratories for Reproductive Biology,³ Department of Pediatrics,⁴ Department of Cell and Developmental Biology,⁵ University of North Carolina, Chapel Hill, North Carolina 27599-7500

The role of epididymal sperm-binding proteins in reproductive tract immunity is now well recognized in addition to their role in sperm maturation. Spermatozoa acquire forward motility and fertilizing ability during their passage through the epididymis, where they acquire a wide variety of proteins belonging to different classes. Previously, we demonstrated that EPPIN (epididymal protease inhibitor), an androgen-regulated, sperm-binding protein containing protease-inhibitory motifs, is expressed specifically in the testis and epididymis. In the present study, we investigated the antibacterial activity of EPPIN against Escherichia coli and the mechanism of antimicrobial action. EPPIN exhibited dose- and time-dependent antibacterial activity that was relatively insensitive to salt. However, EPPIN lost its antibacterial activity completely on reduction and alkylation of its cysteines, indicating the importance of disulfide bonds for its activity. EPPIN permeabilized the outer and inner membranes of E. coli, which is consistent with its ability to induce striking morphological alterations of E. coli membranes as shown by scanning electron microscopy. EPPIN did not cause disruption of eukaryotic membranes in the rat erythrocyte hemolytic assay. The present results indicate that EPPIN has a role in the innate immune system of human epididymis.

¹ Supported by the Consortium for Industrial Collaboration in Contraceptive Research Program of the Contraceptive Research and Development Program, Eastern Virginia Medical School. The views expressed by the authors do not necessarily reflect the views of the Contraceptive Research and Development Program or the Consortium for Industrial Collaboration in Contraceptive Research. This work is also supported by NIH Grants R37-HD04466, by National Institute of Child Health and Human Development/NIH through cooperative agreement U54-HD35041 as part of the Specialized Cooperative Centers Program in Reproduction Research, and by the Fogarty International Center Training and Research in Population and Health Grant D43TW=/HD00627.

² Correspondence: Susan H. Hall, Laboratories for Reproductive Biology, CB 7500, Department of Pediatrics, University of North Carolina, Chapel Hill, NC 27599-7500. FAX: 919 966 2203; shh{at}med.unc.edu

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