Tissue-specific distribution of a novel component of epithelial basement membranes

Abstract

Monoclonal antibodies against basement membrane (BM) were generated using the matrix deposited by cultured rabbit corneal epithelial cells as immunogen. BM antibodies were identified by immunofluorescent staining of frozen tissue sections and of extracellular matrix of living cultured cells. BM localization was confirmed by immunoelectron microscopy. Antibody AE26 immunoprecipitates a 140,000 M r component from radiolabeled corneal epithelial cells and recognizes this component plus a 95,000 M r band on Western blots. The antigen resists extraction by high and low salt and by nonionic detergents, but is solubilized in 4 M urea/1% mercaptoethanol. On isoelectric focusing and nonequilibrium pH gradient gels, AE26 antigen migrates to the acidic region (p I < 3). The molecule is destroyed by trypsin, but is insensitive to bacterial collagenase. In frozen tissue sections, AE26 stains only BM of stratified epithelia plus trachea, ureter, lung, and intestine, but no other epithelial or nonepithelial BM. AE26 antigen is detected on Western blots of cornea, skin, and lung extracts, but not liver, kidney, or muscle, indicating that this is not due to masking of the epitope. This tissue distribution is different from any previously described BM molecule. Although we have not ruled out the possibility that AE26 recognizes a modification or fragment of a known BM component (particularly entactin), the acidic p I, collagenase resistance, and unusual tissue specificity suggest that AE26 recognizes a new BM protein. The BM heterogeneity demonstrated by AE26 may play a structural role or provide positional signals to the overlying epithelium.