Functional domains of the human epididymal protease inhibitor, eppin

Authors

  • Maelíosa T. C. McCrudden,

    1.  School of Biological Sciences, Medical Biology Centre, Queen’s University Belfast, UK
    2.  School of Chemistry and Chemical Engineering, Queen’s University Belfast, UK
    3.  School of Medicine and Dentistry, Queen’s University Belfast, UK
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  • Tim R. Dafforn,

    1.  School of Biosciences, The University of Birmingham, Edgbaston, UK
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  • David F. Houston,

    1.  School of Biological Sciences, Medical Biology Centre, Queen’s University Belfast, UK
    2.  School of Chemistry and Chemical Engineering, Queen’s University Belfast, UK
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  • Philip T. Turkington,

    1.  School of Chemistry and Chemical Engineering, Queen’s University Belfast, UK
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  • David J. Timson

    1.  School of Biological Sciences, Medical Biology Centre, Queen’s University Belfast, UK
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D. J. Timson, School of Biological Sciences, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK
Fax: +44 28 9097 5877
Tel: +44 28 9097 5875
E-mail: d.timson@qub.ac.uk

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.

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