Lack of K140 immunoreactivity in junctional epidermolysis bullosa skin and keratinocytes associates with misfolded laminin epidermal growth factor-like motif 2 of the ß3 short arm.

Recessive mutations in the LAMA3, LAMB3 and LAMC2 genes that encode laminin-332 (LM332) (α3a, ß3 and γ2 chains, respectively) cause different junctional epidermolysis bullosa (JEB) subtypes. Biallelic truncating mutations in any of these three genes usually lead to lack of protein expression resulting in the severe generalized JEB subtype, while missense or splice-site mutations in at least one allele lead to reduced expression typical of JEB generalized intermediate (JEB-gen intermed) or localized. Here, we molecularly characterized an adult patient with JEB showing negative skin staining for the anti-ß3 chain monoclonal antibody K140. This antibody recognizes an as yet unidentified epitope within the laminin ß3 short arm. The patient harbours a homozygous splice-site mutation resulting in highly aberrant transcripts with partial skipping of the LAMB3 exon that encodes the laminin epidermal growth factor-like motif 2 of the ß3 short arm (ß3-LE2). At the protein level, mutation consequences predict a misfolded ß3-LE2 motif and, indeed, we found that LM332 is correctly assembled but retained in the endoplasmic reticulum (ER) where it colocalizes with the lumenal ER chaperone protein BiP, leading to dramatically reduced secretion. Lack of K140 reactivity to mutant LM332 was confirmed by immunoprecipitation and Western blot analyses. Our findings not only identify the ß3-LE2 subdomain as the region recognized by K140, but also show that misfolding of LM332 structural motifs and subsequent protein retention in the ER is a common pathomechanism in JEB-gen intermed. In addition to its usefulness in antigen mapping diagnosis of JEB subtypes, this knowledge is relevant to the design of therapeutic strategies aimed at releasing ER-retained LM332 in JEB.