Immunohistochemical and mutation analyses demonstrate that procollagen VII is processed to collagen VII through removal of the NC-2 domain.

Abstract

Abstract. Collagen VII is the major structural constituent of anchoring fibrils in the skin. It is synthesized as a procollagen that is larger than the collagen deposited in the tissue. In this study, we investigated the conversion of procollagen VII to collagen VII in human skin and in cutaneous cells in vitro and identified the propeptide using domain-specific antibodies. For this purpose, two bacterial fusion proteins containing unique sequences of the carboxy-terminal globular NC-2 domain of procollagen VII were prepared, and polyclonal antibodies raised against them. Immunoblotting showed that the anti-NC-2 antibodies reacted with procollagen VII isolated from cultured keratinocytes, but not with collagen VII extracted from the skin. Immunohistochemical experiments with the NC-2 antibodies revealed a strong reaction in cultured keratinocytes, but the basement membrane zone of normal skin remained negative. The staining could not be rendered positive by chemical or enzymatic unmasking of potential hidden epitopes in the skin, indicating that most of the NC-2 domain is absent from normal skin. In contrast, a positive staining with NC-2 antibodies was observed in the skin of a patient with dystrophic epidermolysis bullosa, who carried a 14-bp deletion at one of the intron-exon junctions of the collagen VII gene. This aberration led to an in-frame skipping of exon 115 from the mRNA and eliminated 29 amino acids from the NC-2 domain which include the putative cleavage site for the physiological processing enzyme, procollagen C-proteinase. The resuits indicate that in normal human skin, the removal of the NC-2 domain from procollagen VII precedes its deposition at the dermal-epidermal junction. Furthermore, they suggest that an aberration in the procollagen VII cleavage interferes with the normal fibrillogenesis of the anchoring fibrils.CHORIN6 fibrils extend from the lamina densa of the epidermal basement membrane to the papillary connective tissue, thus ensuring tight cohesion between the basement membrane and the dermis in the skin. The main structural protein of the fibrils is collagen VII, a homotrimeric collagen containing three identical al(VII) chains (4,5,31). This collagen is synthesized mainly by epidermal keratinocytes (20,30). The primary synthetic product is larger than the collagen deposited in the tissue, and has been called procollagen VII in analogy to other members of the collagen protein family (5,18,20). repeat sequence, a large globular NH2-terminal NC-1 domain, and a small globular COOH-terminal NC-2 domain (24,25). Based on the cDNA-derived amino acid sequence, the following molecular masses have been calculated for the three procollagen VII domains: NC-1, 133 kD; collagenous domain, 145 kD; and NC-2, ~18 kD (7,12). Rotary shadowing electron micrographs of anchoring fibrils from tissue extracts suggested that NC-2 cleavage is necessary for correct assembly of collagen VII (24,25). During fibrillogenesis, collagen VII molecules form antiparallel tail-to-tail dimers with a small carboxy-term...

Tissue-specific expression and long-term deposition of human collagen VII in the skin of transgenic mice: implications for gene therapy

“…The secreted protein diffuses into the medium. 5,14 In agreement with the observations on the skin, the fibroblasts of normal littermates remained negative with antibodies to human collagen VII (Figure 4cii).…”

“…Procollagen VII molecules associate via their carboxyl termini following which the NC-2 domains are cleaved off. 5 The resulting collagen VII molecules further condense to form the was present at the skin basement membrane zone of the transgenic mice. Dermal extracts from 19-month-old transgenic mice contained mature human collagen VII protein, and fibroblasts derived from skin biopsies of these mice actively synthesized human collagen VII.…”

The bone morphogenetic protein 1/Tolloid-like metalloproteinases

“…Initial dimer formation and stabilization through disulfide bonding seems to require retention of the type VII procollagen C-propeptide, also known as the NC2 domain (Chen et al, 2001;Lunstrum et al, 1987). However, proteolytic removal of NC2 seems necessary to anchoring fibril formation, as a mutation that eliminates the cleavage site results in the severe blistering disease dystrophic epidermolysis bullosa (Bruckner-Tuderman et al, 1995). This mutation removes 29 amino acids containing a potential cleavage site with features found in known substrates of BMP1/TLD-like proteinases (Figs.…”

Proteinases of the Bone Morphogenetic Protein-1 Family Convert Procollagen VII to Mature Anchoring Fibril Collagen

“…In normal human skin, the C-propeptide is completely removed and is not found at the dermal-epidermal junction. However, in dystrophic epidermolysis bullosa (DEB), a genetic disease caused by mutations of collagen VII (30,31), procollagen can be retained in the skin of the patient, implying that deficient C-propeptide processing is associated with functional abnormalities of the anchoring fibrils (32). The importance of the NC-2 domain for antiparallel dimer formation was recently demonstrated by Chen et al (33).…”