Characterization of a 50-kDa component of epithelial basement membranes using GDA-J/F3 monoclonal antibody.
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.
Gayraud
B
B
Institut de Biologie et Chimie des Protéines, CNRS, 69367 Lyon Cedex 07, France.
Höpfner
B
B
Jassim
A
A
Aumailley
M
M
Bruckner-Tuderman
L
L
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001485:Basement Membrane / Q000276:immunology; D002352:Carrier Proteins / Q000032:analysis* / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000032:analysis; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004848:Epithelium / Q000276:immunology; D000939:Epitopes / Q000032:analysis; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D012867:Skin / Q000276:immunology*; D013094:Spermatozoa / Q000276:immunology*; D000097794:Kalinin
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D003094:Collagen; D010450:Endopeptidases; D000939:Epitopes; D008565:Membrane Proteins; D000097794:Kalinin