The Androgen-Regulated Epididymal Sperm-Binding Protein, Human β-Defensin 118 (DEFB118) (Formerly ESC42), Is an Antimicrobial β-Defensin

Abstract

Spermatozoa bind a variety of proteins as they pass through the proximal regions of the epididymis, where they acquire forward motility and fertilizing ability. Recent evidence indicates that certain epididymis-specific secretory proteins that bind sperm have antibacterial activity and may function as part of the innate immune system. We reported earlier that ESC42, now designated human beta-defensin 118 (DEFB118), is a sperm-binding protein. In this study, we demonstrate that DEFB118 has potent antibacterial activity that is dose, time, and structure dependent. Incubation of Escherichia coli for 60 min with 10 microg/ml DEFB118 reduced bacterial survival to 20% of the control, and 25 microg/ml reduced survival to 5% of the control. DEFB118 concentrations of 50 and 100 microg/ml further reduced survival to less than 2 and 1%, respectively. A biphasic effect of salt concentration on the antibacterial activity of DEFB118 was observed. Reduction of disulfide bonds and alkylation of cysteines resulted in the complete loss of antibacterial activity. DEFB118 caused rapid permeabilization of both outer and inner membranes of E. coli and striking morphological alterations in the bacterial surfaces visible by scanning electron microscopy consistent with a membrane-disruptive mechanism of bacterial killing. In contrast, eukaryotic cell membranes were not permeabilized by DEFB118, as indicated by the rat erythrocyte hemolytic assay. Studies on DEFB118 inhibition of macromolecular synthesis and membrane permeability in E. coli were consistent with a primary effect at the cell membrane level. DEFB118 may contribute to epididymal innate immunity and protect the sperm against attack by microorganisms in the male and female reproductive tracts.

Identification, characterization, and evolution of a primate β-defensin gene cluster

“…4 Although the 20q defensins are structurally distinct from DEFB1 and DEFB2, they maintain the cationic property of the defensin signature six-cysteine array that is essential for antimicrobial action. 29 The antibacterial property is likely to be conserved throughout the cluster since the lead member, DEFB118, displays this activity 26 and the human 20q genes and macaque orthologs show greater sequence conservation than the members of other defensin clusters. Defensins of the 20p cluster exhibit long C-terminal domains in contrast to the 8p cluster short C-terminal domains.…”

“…20 Previously, we identified the novel epididymal spermbinding protein DEFB118 25 and recently showed that DEFB118 has potent antibacterial activity. 26 The potential importance of the male reproductive tract defensins in host defense as well as male reproductive functions prompted us to investigate the 20q b-defensin gene cluster. Herein we present the characterization and evolutionary analysis of human and macaque b-defensin orthologs, which give new insights into adaptive functional roles.…”

Comparative genomic analysis of a mammalian β-defensin gene cluster

“…Previously, we described the epididymal sperm binding protein DEFB118, a potent antibacterial agent and lead member of the ␤-defensin gene cluster on human chromosome 20q (38,54,81). Here, we report the discovery and characterization of the orthologous cluster in mouse.…”

Rat recombinant β-defensin 22 is a heparin-binding protein with antimicrobial activity

“…29 The C-terminal Histag might reduce the antibacterial potency of b-defensin 22, although the physiological significance of the long C-terminal domain remains unclear.…”