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.

The neonatal skin blistering disorder transient bullous dermolysis of the newborn (TBDN) heals spontaneously or improves dramatically within the first months and years of life. TBDN is characterized by subepidermal blisters, reduced or abnormal anchoring fibrils at the dermo-epidermal junction, and electron-dense inclusions in keratinocytes. These features are partly similar to those in dystrophic epidermolysis bullosa, which is caused by defects in COL7A1 gene encoding collagen VII, the main anchoring fibril protein. TBDN has been grouped separately from dystrophic epidermolysis bullosa based on the pronounced morphologic features and the self-limiting course of the disorder; however, it remains unclear whether it represents a distinct clinical entity with a single etiology. We now report a TBDN patient who is compound heterozygous for a recessive and a dominant glycine substitution mutation in COL7A1. Two point mutations caused amino acid substitutions G1519D and G2251E in the triple helical domain of collagen VII. In the heterozygous state G1519D was silent, and G2251E led to nail dystrophy, but not to skin blistering. In the proband, compound heterozygosity for the mutations caused massive, transitory retention of collagen VII in the epidermis, its reduced deposition at the basement membrane zone, and extensive dermo-epidermal separation at birth. Accordingly, TBDN keratinocytes in vitro accumulated collagen VII intracellularly in the rough endoplasmic reticulum.

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COL7A1 gene mutations cause dystrophic epidermolysis bullosa, a skin blistering disorder. The phenotypes result from defects of collagen VII, the major component of the anchoring fibrils at the dermo-epidermal junction; however, the molecular mechanisms underlying the phenotypes remain elusive. We investigated naturally occurring COL7A1 mutations and showed that some, but not all, glycine substitutions in collagen VII interfered with biosynthesis of the protein in a dominant-negative manner. Three point mutations in exon 73 caused glycine substitutions G2006D, G2034R, and G2015E in the triple helical domain of collagen VII and interfered with its folding and secretion. Confocal laser scanning studies and semiquantitative immunoblotting determined that dystrophic epidermolysis bullosa keratinocytes retained up to 2.5-fold more procollagen VII within the rough endoplasmic reticulum than controls. Limited proteolytic digestions of mutant procollagen VII produced aberrant fragments and revealed reduced stability of the triple helix. In contrast, the glycine substitution G1519D in another segment of the triple helix affected neither procollagen VII secretion nor anchoring fibril function and remained phenotypically silent. These data demonstrate that collagen VII presents a remarkable exception among collagens in that not all glycine substitutions within the triple helix exert dominant-negative interference and that the biological consequences of the substitutions probably depend on their position within the triple helix.Anchoring fibrils attach the epidermal basement membrane of the skin to the underlying dermal connective tissue (1, 2). They represent polymers of collagen VII, a large homotrimeric protein with a central triple helix and flanking amino-and carboxyl-terminal globular domains. Epidermal keratinocytes synthesize and secrete collagen VII as a triple helical precursor (procollagen VII) into the extracellular matrix. After secretion, procollagen VII undergoes proteolytic trimming to collagen VII (3) and assembles to polymers (1). This is a multistep process during which collagen VII monomers first form disulfidebonded antiparallel dimers and then laterally aggregate into anchoring fibrils, which interact with laminin 5 to secure the dermo-epidermal adhesion (1, 4). Further stabilization of anchoring fibrils and presumably of intermolecular aggregates is achieved through cross-linking by transglutaminase-2 (5). Anchoring fibrils are functionally deficient in hereditary dystrophic epidermolysis bullosa (DEB), 1 a heterogeneous group of bullous skin disorders (for reviews, see Refs. 6 and 7) with mechanically induced blistering and scarring of the skin. In the most severe forms of the disease, both collagen VII protein and anchoring fibrils are absent from the skin (8), whereas in milder forms, collagen VII is expressed, but the morphology of the anchoring fibrils may be altered (7, 9).Mutations in COL7A1 encoding collagen VII have been disclosed in both recessive and dominant DEB subtypes (10 -17). In reces...

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 ␣1(XVII) chains. The globular intracellular domain was disulfide-linked, and the N-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 5or 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 the COL17A1 gene synthesized neither the 180-kDa ␣1(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.Collagen XVII, also known as the 180-kDa bullous pemphigoid antigen or BP180, is a structural component of the hemidesmosomes in epithelial cells (1). The cDNA sequence predicts a type II integral transmembrane protein of 1497 amino acids, with an NH 2 -terminal intracellular domain of 466 amino acids, a transmembrane domain of 23 residues and a COOH-terminal extracellular domain of 1008 amino acid residues (2). Because of 15 collagenous subdomains characterized by -Gly-X-Y-repeat sequences within the ectodomain, the molecule was designated collagen XVII (3, 4). Traditionally, collagens are defined as triple-helical proteins with -Gly-X-Y-repeat sequences and with a function as a structural protein of the extracellular matrix (5). Among the more than 20 homo-and heterotrimeric collagens, types XVII and XIII represent the only putative transmembrane collagens (for review, see Ref. 6). However, probably as a result of their low level of expression in tissue and inaccessibility to standard biochemical analyses, the structures of these collagens were deduced from the cDNA sequences rather than from protein chemical data. T...