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A truncating mutation in the laminin‐332α chain highlights the role of the LG 45 proteolytic domain in regulating keratinocyte adhesion and migration
Author(s) -
Di Zenzo G.,
El Hachem M.,
Diociaiuti A.,
Boldrini R.,
Calabresi V.,
Cianfarani F.,
Fortugno P.,
Piccinni E.,
Zambruno G.,
Castiglia D.
Publication year - 2014
Publication title -
british journal of dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.304
H-Index - 179
eISSN - 1365-2133
pISSN - 0007-0963
DOI - 10.1111/bjd.12816
Subject(s) - laminin , keratinocyte , extracellular matrix , junctional epidermolysis bullosa (veterinary medicine) , cell adhesion , biology , microbiology and biotechnology , basement membrane , epidermolysis bullosa , extracellular , mutant , gene , biochemistry , in vitro , genetics , cell
Summary Background Altered function of laminin‐332 (α3β3γ2) consequent to mutations in the LAMA 3, LAMB 3 and LAMC 2 genes causes junctional epidermolysis bullosa non‐Herlitz ( JEB ‐ nH ). JEB‐ nH patients suffer from skin blistering and have an increased risk of developing aggressive skin carcinomas in adulthood. Laminin‐332 is proteolytically processed and its extracellular mature form lacks the α3 chain C‐terminal globules 4 and 5 ( LG 45). The LG 45 tandem has cell adhesion and protumorigenic properties. However, mutations that affect this domain are very rare and their functional effects in patients have not been explored to date. Objective To characterize molecularly an adult patient with JEB ‐ nH and altered laminin‐332 expression presenting multiple skin carcinomas, and to analyse LG 45‐mediated biological functions using keratinocytes from the patient. Methods A mutational search in laminin‐332 genes was performed by hetero‐duplex analysis. LAMA 3 mRNA and laminin‐332 protein levels in patient keratinocytes were investigated by real‐time reverse transcriptase polymerase chain reaction and radioimmunoprecipitation assay, respectively. Keratinocyte migration was examined by scratch and Boyden chamber assays. Results We identified a homozygous LAMA 3 mutation, p.Leu1648Trp fs X32, which truncates the last 45 amino acids of the carboxyl terminal LG 5 subdomain. Gene expression studies revealed that the mutant transcripts were stable and even increased, precursor laminin‐332 molecules were retained intracellularly and the amount of mature extracellular heterotrimers was reduced to about 50%. Finally, the patient's keratinocytes migrated faster than normal keratinocytes. Conclusions Structural disruption of LG 5 highlights the critical functions of the LG 45 proteolytic region in precursor laminin‐332 secretion and keratinocyte adhesion and migration. Perturbation of LG 45 function might explain the non‐aggressive behaviour of carcinomas in this patient.