Monoclonal antibodies against caldesmon, a Ca++/calmodulin- and actin-binding protein of smooth muscle and nonmuscle cells.

Abstract

Monoclonal antibodies, C2, C9, C18, C21 and C23, against chicken gizzard caldesmon have been prepared and characterized. These antibodies reacted with gizzard caldesmon (150 KDa) by enzyme-linked immunosorbent assay and protein immunoblotting. Immunofluorescence microscopy with these antibodies on cultured gizzard cells showed strong stress fiber and membrane ruffle stainings. Surprisingly, in addition to these cytoplasmic staining patterns, the C23 antibody also stained nuclei in these cells. Preabsorption of C23 with purified caldesmon abolished the staining of stress fibers and membrane ruffles as well as the staining of nuclei, suggesting that a common epitope existed in both gizzard caldesmon and the nuclear protein. Western blot analysis on the cell extract of chicken embryo fibroblast (CEF) showed that antibodies C2, C9, C18 and C21 recognized a nonmuscle caldesmon (66 KDa), whereas C23 reacted with a protein (60 KDa) different from nonmuscle caldesmon. Antibody C21 also crossreacted with a nonmuscle caldesmon (80 KDa) in normal rat kidney (NRK) cells, with a nonmuscle caldesmon (78 KDa) in human cells, and with a nonmuscle caldesmon (72 KDa) in gerbil fibroma cells. This antibody had broad-species specificity. Immunofluorescent staining of CEF cells with antibodies C2, C9, C18 and C21 showed some stress fibers and ruffles, but mostly diffuse staining. Antibody C23 crossreacted with 62 KDa and 55 KDa proteins in NRK cells, 63 KDa and 55 KDa proteins in gerbil fibroma cells and 66 KDa and 56 KDa proteins in human bladder carcinoma cells. These polypeptides were identified as nuclear lamins A and C by an anti-lamin antibody in immunoblots and two-dimensional gel analysis. Like the nuclear lamins, the C23 antigens also underwent a reversible disassembly during mitosis, as detected by double-label immunofluorescence with C23 antibody and a polyclonal anti-tubulin antibody. Tropomyosin-enriched microfilaments isolated from fibroblastic and epithelial types of NRK cells by monoclonal anti-tropomyosin antibody contained an 80 KDa protein, which had the heat-resistant property of caldesmon. The polyclonal antiserum generated against this 80 KDa protein showed a crossreactivity with purified gizzard caldesmon and vice versa. The amount of this nonmuscle caldesmon associated with the microfilaments of Kirstein virus-transformed NRK cells was greatly decreased.