Cytovillin and other microvillar proteins of human choriocarcinoma cells

Abstract

Microvilli were isolated from cultured human JEG-3 choriocarcinoma cells using a gentle shearing method. The protein components of the isolated microvilli were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The major Mr 42,000 and Mr 100,000 polypeptide bands reacted with anti-actin and anti-alpha-actinin antisera, respectively. Extraction of the isolated JEG-3 microvilli with Triton X-100 left an insoluble cytoskeletal residue containing mainly actin, alpha-actin, and polypeptides of Mr 200,000, 55,000 and 35,000. The Mr 35,000 polypeptide remained insoluble only at high concentrations of free Ca2+. Immunoblotting analysis of the JEG-3 microvilli indicated that they were devoid of tropomyosin, although the total JEG-3 protein lysates gave a strong positive reaction with anti-tropomyosin antiserum. The different subcellular localization of cytovillin and tropomyosin was also shown by indirect immunofluorescence microscopy. Cytovillin, an Mr 75,000 microvillus-specific membrane protein of JEG-3 cells, existed in an oligomeric form (dimer or trimer) as shown by gel filtration of Triton X-100 solubilized microvillar proteins and by native polyacrylamide gel electrophoresis of purified cytovillin. Disulfide bridges were not involved in the aggregation, because the mobility of cytovillin was similar under reducing and nonreducing conditions in SDS-PAGE. Cytovillin was shown to be closely related to ezrin, a minor component of chicken intestinal brush border microvilli.