title: Molecular mechanisms of junctional epidermolysis bullosa: Col 15 domain mutations decrease the thermal stability of collagen XVII.
abstract: Mutations in the collagen XVII gene, COL17A1, are associated with junctional epidermolysis bullosa. Most COL17A1 mutations lead to a premature termination codon (PTC), whereas only a few mutations result in amino acid substitutions or deletions. We describe here two novel glycine substitutions, G609D and G612R, and a splice site mutation resulting in a deletion of three Gly-X-Y amino acid triplets. In order to investigate the molecular pathomechanisms of non-PTC mutations, G609D and G612R and two previously known substitutions, G627V and G633, and deletion of the amino acids 779-787 were introduced into recombinant collagen XVII. The thermal stability of the mutated collagens was assessed using trypsin digestions at incremental temperatures. All the four glycine substitutions significantly destabilized the ectodomain of collagen XVII, which manifested as 16 degrees C-20 degrees C lower T(m) (midpoint of the helix-to-coil transition). These results were supported by secondary structure predictions, which suggested interruptions of the collagenous triple helix within the largest collagenous domain, Col15. In contrast, deletion of the three full Gly-X-Y triplets, amino acids 779-787, had no overall effect on the stability of the ectodomain, as the deletion was in register with the triplet structure and also generated compensatory changes in the NC15 domain.
authors.affiliation: Department of Dermatology, University of Oulu, Oulu, Finland.
authors.forename: Laura
authors.initials: L
authors.lastname: Väisänen
authors.forename: Cristina
authors.initials: C
authors.lastname: Has
authors.forename: Claus
authors.initials: C
authors.lastname: Franzke
authors.forename: Tiina
authors.initials: T
authors.lastname: Hurskainen
authors.forename: Marja-Leena
authors.initials: ML
authors.lastname: Tuomi
authors.forename: Leena
authors.initials: L
authors.lastname: Bruckner-Tuderman
authors.forename: Kaisa
authors.initials: K
authors.lastname: Tasanen
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics*; D003094:Collagen / Q000737:chemistry / Q000235:genetics*; D003241:Consanguinity; D004820:Epidermolysis Bullosa / Q000235:genetics*; D005260:Female; D006580:Genetic Carrier Screening; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008969:Molecular Sequence Data; D024041:Non-Fibrillar Collagens / Q000737:chemistry / Q000235:genetics*; D010375:Pedigree; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D013816:Thermodynamics; D000097805:Collagen Type XVII
chemical_list: D001324:Autoantigens; D003094:Collagen; D024041:Non-Fibrillar Collagens; D011994:Recombinant Proteins; D000097805:Collagen Type XVII; C085811:COL15A1 protein, human