Abstract
LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6–8 and 9–12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (K
i) measured either directly in concentration-dependent studies or calculated from measured association (k
ass) and dissociation (k
dis) rate constants were 1.2–5.5 nM at pH 8.0 and 10–20 nM at pH 5.0. At pH 8.0, k
ass and k
dis values were 4.7×105 M−1 s−1 and 5.5×10−4 s−1, and at pH 5.0 they were 4.0×104 M−1 s−1 and 4.3×10−4 s−1, respectively. The low K
i and k
dis values (t
1/2 of 20–25 min) indicate tight and specific association. Only fragment 6–9′ was a good inhibitor of rhK7, demonstrating a K
i of 11 nM at pH 8.0 in a reaction that was rapidly reversible. These results show that LEKTI, at least in fragment form, is a potent inhibitor of rhK5 and that this protease may be a target of LEKTI in human skin.
Spatiotemporal expression of the serine protease inhibitor, SERPINE2, in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation