Using the
monoclonal antibody GDA-J/F3, a 50-kDa noncollagenous component of human skin basement membrane zone was identified. Immunofluorescence stainings of normal human skin with the GDA-J/F3 antibody showed a linear fluorescence decorating the basement membrane zone. With immunoelectron microscopy, the
epitope was localized to the insertion points of the anchoring fibrils into the lamina densa. The
antigen is distinct from
collagen VII, from the main structural
protein of the anchoring fibrils, and from several other structural molecules of the basement membrane zone, because the GDA-J/F3 antibody did not react with purified basement membrane components in vitro. In serum-free cultures, the
antigen was synthesized and secreted by normal and transformed human keratinocytes and to a lesser extent by normal human skin fibroblasts. Immunoprecipitation of radiolabeled epithelial cell-
conditioned medium with the GDA-J/F3 antibody yielded two
polypeptides that migrated on SDS-
polyacrylamide gel electrophoresis with apparent molecular masses of 46 and 50 kDa under nonreducing conditions. Using reducing
gels, only the 50-kDa
polypeptide was observed. The
antigen was resistant to digestion with bacterial
collagenase but sensitive to
trypsin and
pepsin. It also bound to
heparin and
DEAE cellulose at low ionic strength and alkaline pH. These findings indicate that the GDA-J/F3
antigen is a small globular disulphide-bonded
protein with a potential to interact with basement membrane
proteoglycans. Integration of the GDA-J/F3
antigen into the histoarchitecture of the dermo-epidermal junction is dependent on the presence of
collagen VII, because the GDA-J/
F3 epitope was missing in several patients with a genetic blistering disorder of the skin,
epidermolysis bullosa dystrophica, who lacked
collagen VII and anchoring fibrils.