Proteolytic Activation Cascade of the Netherton Syndrome–Defective Protein, LEKTI, in the Epidermis: Implications for Skin Homeostasis

Abstract

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is the defective protein of the ichthyosiform condition Netherton syndrome (NS). Strongly expressed in the most differentiated epidermal layers, LEKTI is a serine protease inhibitor synthesized as three different high-molecular-weight precursors, which are rapidly processed into shorter fragments and secreted extracellularly. LEKTI polypeptides interact with several proteases to regulate skin barrier homeostasis as well as inflammatory and/or immunoallergic responses. Here, by combining antibody mapping, N-terminal sequencing, and site-specific mutagenesis, we defined the amino-acid sequence of most of the LEKTI polypeptides physiologically generated in human epidermis. We also identified three processing intermediates not described so far. Hence, a proteolytic cascade model for LEKTI activation is proposed. We then pinpointed the most effective fragments against the desquamation-related kallikreins (KLKs) and we proved that LEKTI is involved in stratum corneum shedding as some of its polypeptides inhibit the KLK-mediated proteolysis of desmoglein-1. Finally, we quantified the individual LEKTI fragments in the uppermost epidermis, showing that the ratios between LEKTI polypeptides and active KLK5 are compatible with a fine-tuned inhibition. These findings are relevant both to the understanding of skin homeostasis regulation and to the design of novel therapeutic strategies for NS.

A synonymous mutation in SPINK5 exon 11 causes Netherton syndrome by altering exonic splicing regulatory elements

“…LEKTI bioactive fragments D10D15 (65 and 68 kDa band), D10D13 (42 kDa), D6D9 (37 kDa), D7D9 (30 kDa) and D8D9 (23 kDa) are detected in the keratinocyte-conditioned medium of a normal individual (C). 13 The same pattern is observed in the patient's sample (P), although the intensity of each band is extremely reduced. Equal protein loading is assessed by Ponceau-S staining of the blotted membrane (lower panel).…”

“…To evaluate the LEKTI protein expression level, secreted proteins were concentrated from conditioned medium of differentiated keratinocytes by acetone precipitation, separated on 12% SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting, using the anti-D7D12 polyclonal antibody that recognizes the central region of LEKTI. 13 All the expected LEKTI bioactive fragments were detected in the patient's keratinocyte-conditioned medium, although their expression was strongly reduced compared with the control, confirming NS diagnosis (Figure 1b).…”

Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome

“…To investigate epidermal barrier function in Tg KLK5 mice, we first examined the ability of the skin to prevent penetration of an external dye solution in a whole mount assay. Tg KLK5 neonates exhibited more patches of inhibits several members of the kallikrein related peptidase (KLK) serine protease family (KLK5, KLK7, and KLK14; Deraison et al, 2007;Fortugno et al, 2011). Additionally, LEKTI has been recently suggested to inhibit caspase 14 (Bennett et al, 2010).…”

“…Collectively, these proteases are known to hold prominent roles in the SC. KLK5, KLK7, and KLK14 contribute to desquamation by degrading desmosomal cad herins such as desmoglein 1 and desmocollin 1 (Caubet et al, 2004;Fortugno et al, 2011) whereas caspase 14 is needed to complete the processing of filaggrin (Flg; Denecker et al, 2007;Hoste et al, 2011). KLK5 and KLK14 are also able to stimulate proinflammatory and proallergic signals via protease activated receptor 2 (PAR2; Stefansson et al, 2008;Briot et al, 2009) and KLK5 and KLK7 are linked to the maturation of cathelicidin antimicrobial peptides (Yamasaki et al, 2006).…”

“…Similarly, LEKTI has emerged as a key regulator of epidermal prote olysis. Synthesized as a large (145 kD) polypro tein containing 15 Kazal domains, LEKTI is cleaved by furin to liberate a series of smaller fragments comprising one or more inhibitory units that target distinct protease subsets (Bitoun et al, 2003;Fortugno et al, 2011). LEKTI of rapid dehydration (Yang et al, 2004;Descargues et al, 2005;Hewett et al, 2005).…”