Functional domains of the human epididymal protease inhibitor, eppin

Abstract

Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram‐negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin’s domains are involved in the protein’s antibacterial activity, only the Kunitz domain is required for selective protease inhibition.

The N-terminal glycine-rich and cysteine-rich regions are essential for antimicrobial activity of crustinPm1 from the black tiger shrimp Penaeus monodon

“…The recombinant protein with only the WAP domain also has antiproteinase activity but low antimicrobial activity [14]. In human EPPIN, the WAP domain itself has some antibacterial activity [17].…”

“…The human EPPIN is composed of a WAP domain and a Kunitz domain. The WAP domain has no antiproteinase activity against elastase like the Kunitz inhibitory domain but both domains have some antibacterial activity [17]. The mouse SWAM1 and 2 have antibacterial activity but not the antiproteinase activity [11].…”

Role of Posttranslational Protein Modifications in Epididymal Sperm Maturation and Extracellular Quality Control

“…While most of the protease inhibitors are predominantly expressed in the epididymis, their functional roles have yet to be established. However, roles in innate immunity have been proposed since the Kunitz and WFDC domains have been shown to inhibit proinfl ammatory proteases such as elastin [ 84 ]. Human epididymal protein 4 (HE4), is one of the WFDC proteins that comprise the protease inhibitor locus and though its function in the epididymis is unknown, it has become a marker for the diagnosis of ovarian cancer [ 85 ].…”

The contribution of key hydrophobic residues in ecotin to enzyme-inhibitor complex stability

“…Wild-type ecotin and the mutants were assayed for their ability to inhibit elastase (prepared as described previously 11 ), trypsin (Sigma), and chymotrypsin (Sigma) using the chromogenic substrates MeOSuc-Ala-Ala-Pro-Val-pNA, Bz-PheVal-Arg-pNa, and Suc-Ala-Ala-Pro-Phe-pNA (Bachem) respectively. In each case, the Michaelis constant (K m ) for the substrate was determined under the conditions of the experiment, i.e.…”