Monoclonal antibody to rat CD63 detects different molecular forms in rat tissue

From mice immunized with rat endothelial cell membranes, we isolated several hybridomas secreting monoclonal antibodies (MAbs) to a 45-kDa glycoprotein expressed on the surface of cultured cells. One of these antibodies, 523-14A, was purified and used for immunoaffinity chromatography, Western blotting, and immunohistochemistry. The glycoprotein containing the antigen for MAb 523-14A, gp45, was isolated from rat lung endothelial cell membranes using wheat germ agglutinin and antibody affinity chromatography sequentially. Mass spectrometry of tryptic peptides from gel purified bands identified gp45 as rat CD63, a member of the transmembrane-4 superfamily. Western blot analyses of tissues from F344 rats showed that kidney, spleen, uterus, and ovaries expressed CD63 at high levels. Thymus, salivary gland, testicles, intestines, pancreas, and adrenals expressed lower amounts. Tissue cell types expressing CD63 were also examined and the results showed that, in addition to the expected expression on lymphoid cells, CD63 was expressed on many epithelial and muscle cells as well. The mobility of CD63 on SDS-PAGE varied widely, indicative of molecular masses ranging from 45 kDa in some tissues to nearly 60 kDa in others.