Ishikawa Tomo-O, Urushihara Hideko, Yanagisawa Kaichiro
Institute of Biological Sciences University of Tsukuba Ibaraki 305 Japan.
Dev Growth Differ. 1991 Apr;33(2):131-137. doi: 10.1111/j.1440-169X.1991.00131.x.
In order to analyze the molecular mechanism of sexual cell fusion between cells of HM1 and NC4 (opposite mating type strains in Dictyostelium discoideum), monoclonal antibodies were raised against partially-purified gp 70, a fusion-related protein of HM1 cells. The antibodies were screened for activity to inhibit cell fusion and 9 hybridoma clones were obtained. One of the fusion-blocking monoclonal antibodies, mAb1G7, was used for further analysis. It recognized nearly ten bands in an immunoblot of fusion competent HM1 cells, but no bands when HM1 membrane proteins had been deglycosylated. These results suggest the importance of carbohydrates in the cell fusion process. To confirm this possibility, effects of sugars or lectins on cell fusion were examined. Although inhibition by the sugars was incomplete, Con A, WGA, LCA, strongly inhibited cell fusion. Furthermore, tunicamycin inhibited the acquisition of fusion competence in HM1 cells, indicating the importance of N-linked glycosylation of proteins in cell fusion. All above results suggest that N-linked carbohydrates on HM1 cell surface are involved in the sexual cell fusion of D. discoideum.
为了分析盘基网柄菌中HM1和NC4（相反交配型菌株）细胞间有性细胞融合的分子机制，制备了针对HM1细胞中部分纯化的融合相关蛋白gp 70的单克隆抗体。筛选这些抗体抑制细胞融合的活性，获得了9个杂交瘤克隆。其中一种融合阻断单克隆抗体mAb1G7用于进一步分析。它在有融合能力的HM1细胞的免疫印迹中识别出近十条带，但当HM1膜蛋白去糖基化时则无条带。这些结果表明碳水化合物在细胞融合过程中的重要性。为了证实这种可能性，检测了糖或凝集素对细胞融合的影响。虽然糖的抑制作用不完全，但伴刀豆球蛋白A、小麦胚凝集素、扁豆凝集素强烈抑制细胞融合。此外，衣霉素抑制HM1细胞融合能力的获得，表明蛋白质的N-连接糖基化在细胞融合中的重要性。上述所有结果表明，HM1细胞表面的N-连接碳水化合物参与了盘基网柄菌的有性细胞融合。
