The 420K LEKTI variant alters LEKTI proteolytic activation and results in protease deregulation: implications for atopic dermatitis.

Lymphoepithelial Kazal-type related inhibitor (LEKTI) is a multidomain serine protease inhibitor which plays a central role in skin permeability barrier and allergy. Loss-of-function mutations in the LEKTI encoding gene SPINK5 cause Netherton syndrome, a rare and severe genetic skin disease with a profound skin barrier defect and atopic manifestations. Several studies also reported genetic association between the multifactorial disease atopic dermatitis (AD) and a frequent and non-conservative LEKTI variant, E420K, in different populations. Here, we provide evidence that the 420K variant impacts on LEKTI function by increasing the likelihood of furin-dependent LEKTI precursor cleavage within the linker region D6-D7. This results in the reversal of the cleavage priorities for LEKTI proteolytic activation and prevents the formation of the LEKTI fragment D6D9 known to display the strongest inhibitory activity against kallikrein (KLK) 5-mediated desmoglein-1 (DSG1) degradation. Using in situ and gel zymographies, we show that the modification of the subtle balance in LEKTI inhibitory fragments leads to enhanced KLK5, KLK7 and elastase-2 (ELA-2) activities in 420KK epidermis. By immunohistochemistry and western blot analyses, we found that increased epidermal protease activity correlates with reduced DSG1 protein expression and accelerated profilaggrin proteolysis. All changes determined by the presence of residue 420K within the LEKTI sequence likely contribute to defective skin barrier permeability. Remarkably, LEKTI 420KK epidermis displays an increased expression of the proallergic cytokine thymic stromal lymphopoietin (TSLP). This is the first functional evidence supporting association studies which identified the 420K LEKTI variant as a predisposing factor to AD, in combination with other genetic and environmental factors.

SPINK5, the defective gene in netherton syndrome, encodes multiple LEKTI isoforms derived from alternative pre-mRNA processing.

The multidomain serine protease inhibitor lymphoepithelial Kazal-type related inhibitor (LEKTI) represents a key regulator of the proteolytic events occurring during epidermal barrier formation and hair development, as attested by the severe autosomal recessive ichthyosiform skin condition Netherton syndrome (NS) caused by mutations in its encoding gene, serine protease inhibitor Kazal-type 5 (SPINK5). Synthesized as a proprotein, LEKTI is rapidly cleaved intracellularly, thus generating a number of potentially bioactive fragments that are secreted. Here, we show that SPINK5 generates three classes of transcripts encoding three different LEKTI isoforms, which differ in their C-terminal portion. In addition to the previously described 15 domain isoform, SPINK5 encodes a shorter LEKTI isoform composed of only the first 13 domains, as well as a longer isoform carrying a 30-amino-acid residue insertion between the 13th and 14th inhibitory domains. We demonstrate that variable amounts of SPINK5 alternative transcripts are detected in all SPINK5 transcriptionally active tissues. Finally, we show that in differentiated cultured human keratinocytes all SPINK5 alternative transcripts are translated into protein and that the LEKTI precursors generate distinct secreted C-terminal proteolytic fragments from a similar cleavage site. Since several data indicate a biological role for the pro-LEKTI-cleaved polypeptides, we hypothesize that the alternative processing of the SPINK5 pre-messenger RNA represents an additional mechanism to further increase the structural and functional diversity of the LEKTI bioactive fragments.

Corneodesmosomal cadherins are preferential targets of stratum corneum trypsin- and chymotrypsin-like hyperactivity in Netherton syndrome.

SPINK5 (serine protease inhibitor Kazal-type 5), encoding the protease inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor), is the defective gene in Netherton syndrome (NS), a severe inherited keratinizing disorder. We have recently demonstrated epidermal protease hyperactivity in Spink5(-/-) mice resulting in desmosomal protein degradation. Herein, we investigated the molecular mechanism underlying the epidermal defect in 15 patients with NS. We demonstrated that, in a majority of patients, desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) were dramatically reduced in the upper most living layers of the epidermis. These defects were associated with premature degradation of corneodesmosomes. Stratum corneum tryptic enzyme (SCTE)-like and stratum corneum chymotryptic enzyme (SCCE)-like activities were increased, suggesting that these proteases participate in the premature degradation of corneodesmosomal cadherins. SCTE and SCCE expression was extended to the cell layers where Dsg1 and Dsc1 immunostaining was reduced. In contrast, a subset of six patients with normal epidermal protease activity or residual LEKTI expression displayed apparently normal cadherin expression and less severe disease manifestations. This suggests a degree of correlation between cadherin degradation and clinical severity. This work further supports the implication of premature corneodesmosomal cadherin degradation in the pathogenesis of NS and provides evidence for additional factors playing a role in disease expression.

Proteolytic activation cascade of the Netherton syndrome-defective protein, LEKTI, in the epidermis: implications for skin homeostasis.

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.

Immunofluorescence analysis of villous trophoblasts: a tool for prenatal diagnosis of inherited epidermolysis bullosa with pyloric atresia.

Genetic mutations invalidating the genes for integrin alpha6beta4 and, in some cases, plectin are associated with junctional and simplex epidermolysis bullosa with pyloric atresia (PA-JEB and PA-EBS), respectively. These recessive inherited conditions are characterized by pregnancies with fetal bullae, pyloric atresia, polyhydramnios, and neonatal mucocutaneous blistering, which often results in early postnatal demise. To date, first-trimester DNA-based prenatal diagnosis is not applicable to affected kindred carrying as yet unidentified genetic mutations. Here, we show that first-trimester chorionic villi strongly express both integrin alpha6beta4 and plectin, which persist throughout the pregnancy. Based on this observation, we implemented 25 prenatal diagnoses in kindred at risk for PA-EB by immunomapping, which identified three PA-JEB-affected fetuses and 22 healthy ones. In 19 cases, including the three PA-JEB pregnancies that were prematurely terminated, the results were confirmed by chorionic villous DNA-based tests, which also led to the identification of seven previously unreported mutations in the alpha6beta4 integrin genes. Our prediction was further sustained by the birth of 22 healthy babies. These results validate chorionic villi immunofluorescence examination as a tool for prenatal diagnosis of PA-JEB and PA-EBS and indicate that this procedure could be devised for EB with muscular dystrophy, which is also associated with genetic mutations in plectin.

Characterization and expression analysis of the Spink5 gene, the mouse ortholog of the defective gene in Netherton syndrome.

The human SPINK5 gene, encoding the putative 15-domain serine protease inhibitor LEKTI, was identified as the defective gene in the severe autosomal recessive ichthyosiform skin disorder known as Netherton syndrome and as a candidate susceptibility gene for atopic disease. Here we report mapping of the murine Spink5 gene to chromosome 18 and its characterization. We show that, unlike in humans, transcription of the mouse Spink5 gene generates two mRNAs that differ in the 3' untranslated region. The encoded protein, which is detected in differentiated primary cultured keratinocytes and mouse skin as an approximately 130-kDa glycosylated precursor, displays approximately 60% identity with its human counterpart but lacks the human LEKTI domain 6. As in the human, mouse Lekti represents a marker of epithelial differentiation, strongly expressed in the granular layer of the epidermis, in suprabasal layers of stratified epithelia, and in thymic Hassall's bodies. Our data indicate that mouse Spink5/Lekti, like its human counterpart, is involved in the control of epithelial tissue homeostasis, but also highlight specific features of the murine gene and protein.

LEKTI proteolytic processing in human primary keratinocytes, tissue distribution and defective expression in Netherton syndrome.

SPINK5, encoding the putative multi-domain serine protease inhibitor LEKTI, was recently identified as the defective gene in the severe autosomal recessive ichthyosiform skin condition, Netherton syndrome (NS). Using monoclonal and polyclonal antibodies, we show that LEKTI is a marker of epithelial differentiation, strongly expressed in the granular and uppermost spinous layers of the epidermis, and in differentiated layers of stratified epithelia. LEKTI expression was also demonstrated in normal differentiated human primary keratinocytes (HK) through detection of a 145 kDa full-length protein and a shorter isoform of 125 kDa. Both proteins are N-glycosylated and rapidly processed in a post-endoplasmic reticulum compartment into at least three C-terminal fragments of 42, 65 and 68 kDa, also identified in conditioned media. Processing of the 145 and 125 kDa precursors was prevented in HK by treatment with a furin inhibitor. In addition, in vitro cleavage of the recombinant 145 kDa precursor by furin generated C-terminal fragments of 65 and 68 kDa, further supporting the involvement of furin in LEKTI processing. In contrast, LEKTI precursors and proteolytic fragments were not detected in differentiated HK from NS patients. Defective expression of LEKTI in skin sections was a constant feature in NS patients, whilst an extended reactivity pattern was observed in samples from other keratinizing disorders, demonstrating that loss of LEKTI expression in the epidermis is a diagnostic feature of NS. The identification of novel processed forms of LEKTI provides the basis for future functional and structural studies of fragments with physiological relevance.