Functional domains of the human epididymal protease inhibitor, eppin.
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.
McCrudden
Maelíosa T C
MT
School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, 97 Lisburn Road, Belfast, UK.
Dafforn
Tim R
TR
Houston
David F
DF
Turkington
Philip T
PT
Timson
David J
DJ
D020816:Amino Acid Motifs; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D004579:Electron Transport; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D053491:Proteinase Inhibitory Proteins, Secretory / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D000900:Anti-Bacterial Agents; C431553:Eppin protein, human; D053491:Proteinase Inhibitory Proteins, Secretory