Lack of K140 immunoreactivity in junctional epidermolysis bullosa skin and keratinocytes associates with misfolded laminin EGF-like (LE) motif 2 of the β3 short arm

Abstract

Recessive mutations in the LAMA3A, LAMB3 and LAMC2 genes coding for laminin-332 (α3aβ3γ2) chains cause different junctional epidermolysis bullosa (JEB) subtypes. Biallelic truncating mutations in any of the three genes usually lead to lack of protein expression resulting in the severe generalized JEB subtype, while missense or splicing mutations in at least one allele lead to reduced expression typical of generalized intermediate or localized JEB. Here, we molecularly characterized an adult JEB patient showing negative skin staining for the anti-β3 chain mAb K140. This antibody recognizes an as yet unidentified epitope within the β3 short arm. The patient harbours a homozygous splicing mutation resulting in major aberrant transcripts with partial skipping of the LAMB3 exon coding for the laminin EGF-like (LE) motif 2 of the β3 short arm (β3-LE2). At protein level, mutation consequences predict a misfolded β3-LE2 motif and, indeed, we found that laminin-332 is correctly assembled but retained in the endoplasmic reticulum (ER) where it co-localizes with the lumenal ER chaperone BiP, leading to dramatically reduced secretion. Lack of K140 reactivity to mutant laminin-332 was confirmed by immunoprecipitation and Western blot analyses. Our findings identify the β3-LE2 subdomain as the region recognized by K140 mAb. In addition, they further show that misfolding of laminin-332 structural motifs and subsequent ER protein retention is a common pathomechanism in JEB generalized intermediate. In addition to be useful for antigen mapping diagnosis of JEB subtypes, this knowledge is relevant to the design of therapeutic strategies aimed at releasing ER-retained laminin-332 in JEB.

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