Nadja Hammami-Hauasli

Two Forms of Collagen XVII in Keratinocytes A FULL-LENGTH TRANSMEMBRANE PROTEIN AND A SOLUBLE ECTODOMAIN

The cDNA sequence of human collagen XVII predicts an unusual type II transmembrane protein, but a biochemical characterization of this structure has not been accomplished yet. Using domain-specific antibodies against recombinant collagen XVII fragments, we identified two molecular forms of the collagen in human skin and epithelial cells. Full-length collagen XVII appeared as a homotrimeric transmembrane molecule of three 180-kDa α1(XVII) chains. The globular intracellular domain was disulfide-linked, and theN-glycosylated extracellular domain of three 120-kDa polypeptides was triple-helical at physiological temperatures. A second, soluble form of collagen XVII in keratinocyte culture media was recognized with antibodies to the ectodomain, but not the endodomain. The soluble form exhibited molecular properties of the collagen XVII ectodomain: a triple-helical, N-glycosylated molecule of three 120-kDa polypeptides. Northern blot analysis with probes spanning either the distal 5′or the distal 3′ end of the collagen XVII cDNA revealed an identical 6-kb mRNA, suggesting that both the 180- and 120-kDa polypeptides were translated from the same mRNA, and that the 120-kDa polypeptide was generated post-translationally. In concert, keratinocytes harboring a homozygous nonsense mutation in theCOL17A1 gene synthesized neither the 180-kDa α1(XVII) chain nor the 120-kDa polypeptide. Finally, treatment of normal keratinocytes with a synthetic inhibitor of furin proprotein convertases, decanoyl-RVKR-chloromethyl ketone, prevented the generation of the 120-kDa polypeptide. These data strongly suggest that the soluble 120-kDa polypeptide represents a specifically cleaved ectodomain of collagen XVII, generated through furin-mediated proteolytic processing. Thus, collagen XVII is not only an unusual type II transmembrane collagen, but the first collagen with a specifically processed, soluble triple-helical ectodomain.

Biology of anchoring fibrils: lessons from dystrophic epidermolysis bullosa.

Anchoring fibrils are adhesive suprastructures that ensure the connection of the epidermal basement membrane with the dermal extracellular matrix. The fibrils represent polymers of collagen VII, the major structural fibril component, but may also contain other proteins. Remarkable progress has been made in the last few years in understanding the functions of skin basement membrane components including the anchoring fibrils. Novel insights into the biology of the anchoring fibrils have been gained from experimental studies on dystrophic epidermolysis bullosa (DEB), a group of inherited blistering disorders caused by mutations in the gene for collagen VII, COL7A1. Mutation analyses of DEB families have disclosed more than 100 COL7A1 gene defects so far, but the unusual complexity of the mutation constellations and their biological consequences are only beginning to emerge. In analogy to heritable disorders of other collagen genes, predictable phenotypes of COL7A1 mutations causing premature termination codons or dominant negative interference have been observed. However, collagen VII seems to represent a remarkable exception among collagens in that many mutations, including heterozygous glycine substitutions and deletions, lead to minimal phenotypes, or to no phenotype at all. In contrast to fibrillar collagens, structural abnormalities of collagen VII molecules in anchoring fibrils appear to be tolerated to a certain extent. However, the mild DEB phenotypes can be severely modulated by a second aberration in individuals compound heterozygous for two different COL7A1 mutations. Therefore, not only definition of mutation(s) but also cell biological, protein chemical and suprastructural studies of the mutated molecules yield novel insight into the molecular pathomechanisms underlying disease.

Three Novel Homozygous Point Mutations and a New Polymorphism in the COL17A1 Gene: Relation to Biological and Clinical Phenotypes of Junctional Epidermolysis Bullosa

Junctional epidermolysis bullosa (JEB) is a clinically and biologically heterogeneous genodermatosis, characterized by trauma-induced blistering and healing without scarring but sometimes with skin atrophy. We investigated three unrelated patients with different JEB phenotypes. Patients 1 and 2 had generalized atrophic benign epidermolysis bullosa (GABEB), with features including skin atrophy and alopecia. Patient 3 had the localisata variant of JEB, with predominantly acral blistering and normal hair. All patients carried novel homozygous point mutations (Q1016X, R1226X, and R1303Q) in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane component; and, therefore, not only GABEB but also the localisata JEB can be a collagen XVII disorder. The nonsense mutations led to drastically reduced collagen XVII mRNA and protein levels. In contrast, the missense mutation allowed expression of abnormal collagen XVII, and epidermal extracts from that patient contained polypeptides of normal size, as well as larger aggregates. The homozygous nonsense mutations in the COL17A1 gene were consistent with the absence of the collagen from the skin and with the GABEB phenotype, whereas homozygosity for the missense mutation resulted in expression of aberrant collagen XVII and, clinically, in localisata JEB.

Deletions within COL7A1 Exons Distant from Consensus Splice Sites Alter Splicing and Produce Shortened Polypeptides in Dominant Dystrophic Epidermolysis Bullosa

We describe two familial cases of dominant dystrophic epidermolysis bullosa (DDEB) that are heterozygous for deletions in COL7A1 that alter splicing, despite intact consensus splice-site sequences. One patient shows a 28-bp genomic deletion (6081del28) in exon 73 associated with the activation of a cryptic donor splice site within this exon; the combination of both defects restores the phase and replaces the last 11 Gly-X-Y repeats of exon 73 by a noncollagenous sequence, Glu-Ser-Leu. The second patient demonstrates a 27-bp deletion in exon 87 (6847del27), causing in-frame skipping of this exon; consensus splice sites, putative branch sites, and introns flanking exons 73 and 87 showed a normal sequence. Keratinocytes from the probands synthesized normal and shortened type VII collagen polypeptides and showed intracellular accumulation of type VII procollagen molecules. This first report of genomic deletions in COL7A1 in DDEB suggests a role for exonic sequences in the control of splicing of COL7A1 pre-mRNA and provides evidence that shortened type VII collagen polypeptides can alter, in a dominant manner, anchoring-fibril formation and can cause DDEB of differing severity.