IMMUNOHISTOCHEMICAL AND MUTATION ANALYSES DEMONSTRATE THAT PROCOLLAGEN-VII IS PROCESSED TO COLLAGEN-VII THROUGH REMOVAL OF THE NC-2 DOMAIN

Collagen VII is the major structural constituent of anchoring fibrils in the skin. It is synthesized as a procollagen that is larger than th e collagen deposited in the tissue, In this study, we investigated the conversion of procollagen VII to collagen VII in human skin and in cu taneous cells in vitro and identified the propeptide using domain-spec ific antibodies. For this purpose, two bacterial fusion proteins conta ining unique sequences of the carboxy-terminal globular NC-2 domain of procollagen VII were prepared, and polyclonal antibodies raised again st them. Immunoblotting showed that the anti-NC-2 antibodies reacted w ith 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 kerat inocytes, but the basement membrane zone of normal skin remained negat ive. The staining could not be rendered positive by chemical or enzyma tic unmasking of potential hidden epitopes in the skin, indicating tha t most of the NC-2 domain is absent from normal skin. In contrast, a p ositive staining with NC-2 antibodies was observed in the skin of a pa tient with dystrophic epidermolysis bullosa, who carried a 14-bp delet ion 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 putat ive cleavage site for the physiological processing enzyme, procollagen C-proteinase. The results indicate that in normal human skin, the rem oval of the NC-2 domain from procollagen VII precedes its deposition a t the dermal-epidermal junction. Furthermore, they suggest that an abe rration in the procollagen VII cleavage interferes with the normal fib rillogenesis of the anchoring fibrils.