Epididymal C4b-binding protein is processed and degraded during transit through the duct and is not essential for fertility

Abstract

C4b-binding protein (C4BP) is known as one of the circulating complement regulators that prevents excessive activation of the host-defense complement system. We have reported previously that C4BP is expressed abundantly in the rodent epididymis, one of the male reproductive organs connecting the testis and vas deferens, where immature spermatozoa acquire their motility and fertilizing ability during their transit through the duct. Epididymal C4BP (EpC4BP) is synthesized androgen-dependently by the epithelial cells, secreted into the lumen, and bound to the outer membrane of the passing spermatozoa. In this study, we found that EpC4BP is secreted as a large oligomer, similar to the serum C4BP, but is digested during the epididymal transit and is almost lost from both the luminal fluid and the sperm surface in the vas deferens. Such a processing pattern is not known in serum C4BP, suggesting that EpC4BP and serum C4BP might have different functional mechanisms, and that there is a novel function of EpC4BP in reproduction. In addition, the disappearance of EpC4BP from the sperm surface prior to ejaculation suggests that EpC4BP works only in the epididymis and would not work in the female reproductive tract to protect spermatozoa from complement attack. Next, we generated C4BP-deficient (C4BP−/−) mice to examine the possible role of EpC4BP in reproduction. However, the C4BP−/− mice were fertile and no significant differences were observed between the C4BP−/− and wild-type mouse spermatozoa in terms of morphology, motility, and rate of the spontaneous acrosome reaction. These results suggest that EpC4BP is involved in male reproduction, but not essential for sperm maturation.

C4b-binding protein: The good, the bad and the deadly. Novel functions of an old friend

“…Complement complexes are involved and seem to be important in healthy pregnancies, while the role is yet unclear [79]. Unexpectedly, transgenic mice lacking C4BP appear to have an overall normal phenotype [80]. However, we observed that homozygous C4BP knockout mice often die during second or third pregnancy (unpublished observation).…”

C4b‐binding protein negatively regulates TLR4/MD‐2 response but not TLR3 response

“…C57BL/6 C4BP-deficient mice were provided by MI Nonaka [15]. HEK293 cells stably expressing mouse TLR4, mouse MD-2, and mouse CD14 (HEK293-TLR4/MD-2/CD14), and those stably expressing mouse TLR3 (HEK293-TLR3) were purchased from Invi-voGen.…”

“…These RAW264.7 cells were cultured in complete DMEM supplemented with 1 lgÁmL À1 puromycin. C57BL/6 C4BP-deficient mice were provided by MI Nonaka [15].…”

C4b binding protein negatively regulates TLR1/2 response

“…C57BL/6 C4BP-deficient mice were provided by Dr MI Nonaka. 18 Detection and microsequencing of 25-kDa molecule Cell surface biotinylation of A20 cells were performed as described previously. 19 Briefly, cells ($5 Â 10 7 cells/ ml) were suspended in saline containing 100 mM HEPES (pH 8.0), and then biotinylated with sulfosuccinimidobiotin (Pierce, Rockford, IL, USA) at a final concentration of 0.5 mg/ml.…”