A truncating mutation in the laminin-332α chain highlights the role of the LG45 proteolytic domain in regulating keratinocyte adhesion and migration.

BACKGROUND: Altered function of laminin-332 (α3ß3γ2) consequent to mutations in the LAMA3, LAMB3 and LAMC2 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 (LG45). The LG45 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 LG45-mediated biological functions using keratinocytes from the patient. METHODS: A mutational search in laminin-332 genes was performed by hetero-duplex analysis. LAMA3 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 LAMA3 mutation, p.Leu1648TrpfsX32, which truncates the last 45 amino acids of the carboxyl terminal LG5 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 LG5 highlights the critical functions of the LG45 proteolytic region in precursor laminin-332 secretion and keratinocyte adhesion and migration. Perturbation of LG45 function might explain the non-aggressive behaviour of carcinomas in this patient.

Nectinopathies: an emerging group of ectodermal dysplasia syndromes.

Cleft Lip/Palate-Ectodermal Dysplasia and Ectodermal Dysplasia-Syndactyly Syndrome are rare congenital disorders caused by recessive mutations in the PVRL1 and PVRL4 genes, respectively. These genes encode nectins 1 and 4, an emerging class of molecules acting in cooperation with cadherins to form cell-cell adhesion especially at adherens junctions. Their role in skin, hair and teeth biology and in the fine-tuning morphogenesis of craniofacial (lip/palate) and limbs is yet to be outlined prompting future research. We propose refer to these entities (nectin 1-ED and nectin 4-ED) as "nectinopathies", which are likely to be underestimated/underdiagnosed ED syndomes.

Induction of anti-carbohydrate antibodies by phage library-selected peptide mimics.

One of the prerequisites for the development of polysaccharide subunit vaccines is the induction of an efficient immune response to carbohydrate antigens like lipopolysaccharide (LPS) or capsular polysaccharide antigens of pathogens. In an attempt to overcome the problems that arise from the T-independent immune response induced by such antigens, selecting peptide sequences that mimic protective carbohydrate epitopes has been proposed. In this study, we investigate a new selection strategy for immunogenic peptide mimics using the phage-displayed peptide library technology. Two monoclonal antibodies (mAb) of the A isotype (mIgA), mIgA C5 and mIgA I3, specific for the O-antigen (O-Ag) part of the human pathogen Shigella flexneri serotype 5a LPS and protective against homologous infection were used to screen two phage-displayed nonapeptide libraries in pVIII. Using mIgA C5, 13 different specific clones were selected, and 6 using mIgA I3; 5 of the latter also interacted in enzyme-linked immunosorbent assay with the first mAb. All of the 19 clones selected were separately used to immunize mice, but only 2 of them, p100c (mIgA I3-specific) and p115 (interacting with both mIgA) were able to induce anti-O-Ag antibodies. The immune response was specific for the O-Ag of the S. flexneri serotype 5a, and also selectively recognized the corresponding bacterial strain. The amino acid sequences of p100c and p115 immunogenic peptide mimics were YKPLGALTH (flanked by two Cys residues) and KVPPWARTA, respectively. These results are the first example of immunogenic mimicry of carbohydrates by phage-displayed peptides, and indicate a new strategy of selection of immunogens for the development of anti-polysaccharide vaccines.

Selection of biologically active peptides by phage display of random peptide libraries.

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein-protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.