title: A truncating mutation in the laminin-332α chain highlights the role of the LG45 proteolytic domain in regulating keratinocyte adhesion and migration.
abstract: BACKGROUND
  Altered function of laminin-332 (α3β3γ2) 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.
authors.affiliation: Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata-IRCCS, via dei Monti di Creta 104, 00167, Rome, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Di Zenzo
authors.forename: M
authors.initials: M
authors.lastname: El Hachem
authors.forename: A
authors.initials: A
authors.lastname: Diociaiuti
authors.forename: R
authors.initials: R
authors.lastname: Boldrini
authors.forename: V
authors.initials: V
authors.lastname: Calabresi
authors.forename: F
authors.initials: F
authors.lastname: Cianfarani
authors.forename: P
authors.initials: P
authors.lastname: Fortugno
authors.forename: E
authors.initials: E
authors.lastname: Piccinni
authors.forename: G
authors.initials: G
authors.lastname: Zambruno
authors.forename: D
authors.initials: D
authors.lastname: Castiglia
mesh_terms: D002448:Cell Adhesion / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000235:genetics*; D054443:Cell Migration Assays; D002465:Cell Movement / Q000235:genetics; D016109:Epidermolysis Bullosa, Junctional / Q000235:genetics*; D016368:Frameshift Mutation / Q000235:genetics*; D006801:Humans; D015603:Keratinocytes / Q000502:physiology; D007797:Laminin / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D000097794:Kalinin
chemical_list: D015815:Cell Adhesion Molecules; D007797:Laminin; D000097794:Kalinin; C095567:laminin alpha 3