| Summary: | Lympho-Epithelial Kazal-Type-related Inhibitor (LEKTI) is a serine protease inhibitor that contains a total of fifteen potential inhibitory domains and exists as single- and multi-domain fragments in the uppermost layers of the epidermis. In this study, a novel ProteinChip Array method was developed to investigate the interactions between LEKTI and target proteases, with the aim of identifying novel targets of LEKTI in the skin. Using this method, multiple proteins, including several kallikreins, were shown to bind to different forms of recombinant LEKTI on the ProteinChip array surface. By using conventional inhibitory assays, it was confirmed that the interactions corresponded to actual levels of inhibition and kallikrein 8 was identified as a potential target of LEKTI. Numerous binding proteins were also detected from whole epidermal extracts. Overall, ProteinChip Array technology can provide a much more rapid and simple process for studying protein: protein interactions compared to more conventional techniques such as yeast 2 hybrids. In addition, a label-free quantitative proteomic method was used to create a map of the skin proteome, by allowing the identification and quantitation of all major proteins in the skin barrier. The cysteine protease, caspase 14 was revealed as a potential target of LEKTI in the skin. By using standard inhibitory assays, LEKTI was found to demonstrate potent inhibition against caspase 14. This is the first study to report LEKTI as a cysteine protease inhibitor in addition to its recognised role as a serine protease inhibitor and implicated a possible function for LEKTI in epidermal hydration and differentiation. Furthermore, the label-free quantitative proteomic method that was developed has great potential for applications in diagnostics and in studies investigating the aetiology of disease.
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