Characterization of molecular mechanisms underlying mutations in dystrophic epidermolysis bullosa using site-directed mutagenesis.
Type VII collagen (C7) is a major component of anchoring fibrils, structures that mediate epidermal-dermal adherence. Mutations in gene COL7A1 encoding for C7 cause dystrophic epidermolysis bullosa (DEB), a genetic mechano-bullous disease. The biological consequences of specific COL7A1 mutations and the molecular mechanisms leading to DEB clinical phenotypes are unknown. In an attempt to establish genotype-phenotype relationships, we generated four individual substitution mutations that have been associated with recessive DEB, G2049E, R2063W, G2569R, and G2575R, and purified the recombinant mutant proteins. All mutant proteins were synthesized and secreted as a 290-kDa mutant C7 alpha chain at levels similar to wild type C7. The G2569R and G2575R glycine substitution mutations resulted in mutant C7 with increased sensitivity to protease degradation and decreased ability to form trimers. Limited proteolytic digestion of mutant G2049E and R2063W proteins yielded aberrant fragments and a triple helix with reduced stability. These two mutations next to the 39-amino acid helical interruption hinge region caused local destabilization of the triple-helix that exposed an additional highly sensitive proteolytic site within the region of the mutation. Our functional studies demonstrated that C7 is a potent pro-motility matrix for skin human keratinocyte migration and that this activity resides within the triple helical domain. Furthermore, G2049E and R2063W mutations reduced the ability of C7 to support fibroblast adhesion and keratinocyte migration. We conclude that known recessive DEB C7 mutations perturb critical functions of the C7 molecule and likely contribute to the clinical phenotypes of DEB patients.
Woodley
David T
DT
Department of Dermatology, The Keck School of Medicine at the University of Southern California, Los Angeles, CA 90033, USA. dwoodley@usc.edu
Hou
Yingping
Y
Martin
Sabrina
S
Li
Wei
W
Chen
Mei
M
D002448:Cell Adhesion; D002460:Cell Line; D002465:Cell Movement; D024143:Collagen Type VII / Q000737:chemistry / Q000235:genetics*; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics* / Q000473:pathology; D005347:Fibroblasts / Q000166:cytology; D005808:Genes, Recessive; D005998:Glycine / Q000737:chemistry; D006801:Humans; D015603:Keratinocytes / Q000166:cytology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D024143:Collagen Type VII; D011994:Recombinant Proteins; D005998:Glycine