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

Abstract

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.

Analysis of the individual and aggregate genetic contributions of previously identified serine peptidase inhibitor Kazal type 5 (SPINK5), kallikrein-related peptidase 7 (KLK7), and filaggrin (FLG) polymorphisms to eczema risk

“…One of the proteases that has been suggested to be implicated in profilaggrin processing is the stratum corneum chymotryptic enzyme (SSCE) 31,32 , which is possibly regulated by the serine protease inhibitor LETKI, encoded by SPINK5 [32][33][34] . Interestingly, an insertion in the 3′ untranslated region of the kallikrein 7 gene (KLK7) encoding SCCE 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 35 has been reported to be associated with eczema.…”

Prenatal diagnosis of Comel-Netherton syndrome with PGD, case report and review article

“…After the first report of mutations in C-NS [19], more than 20 SPINK5 mutations have been identified and reported [23,24].The recent discovery of SPINK5 on chromosomal region 5q31 as the gene responsible for C-NS now enables causative mutations to be identified in these families. However, SPINK5 comprises 33 exons transcribed into a 3.7 kb coding mRNA, and is not amenable to quick mutation screening.…”

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

“…4 Keratinocytes were maintained in low calcium KGM (Invitrogen, Carlsbad, CA, USA) up to confluence, and then, terminal differentiation was induced by raising the calcium concentration to 1.2 mM for 5 days. 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.…”

“…2 Netherton syndrome is caused by loss-of-function mutations in the SPINK5 gene encoding lymphoepithelial Kazal-type -related inhibitor (LEKTI), a 15-domain serine proteases inhibitor with a crucial role in regulating skin desquamation and maintaining epidermal barrier function. 3,4 Differentiated keratinocytes synthesize three different LEKTI precursors, which are rapidly processed and then secreted into several shorter bioactive fragments. 5,6 The SPINK5 mutation database in NS comprises 62 different mutations, [7][8][9][10] and the genotype-phenotype correlation suggests that downstream mutations may partly allow for residual expression of functional LEKTI fragments, which results in less severe phenotypes.…”