Hirano T, Yamada H, Miyazaki T
J Biochem. 1985 Jul;98(1):199-208. doi: 10.1093/oxfordjournals.jbchem.a135259.
In the cell adhesion of aggregation-competent Dictyostelium cells, the requirement for the carbohydrate moiety of the glycoprotein appeared to be indirect in that it acts to protect the protein moiety from proteolytic degradation; however, the effect was limited to the tunicamycin (TM)-sensitive carbohydrate moiety (Hirano, T., et al. (1983) J. Biochem. 93, 1249-1257). In the present study, we showed that the EDTA-stable adhesion of aggregation-competent Dictyostelium cells was abolished by the treatment of intact cells with jack bean alpha-mannosidase, whereas neuraminidase, beta-galactosidase, beta-N-acetylhexosaminidase, or alpha-L-fucosidase had no effect. The EDTA-stable cohesiveness of TM-treated cells in the presence of leupeptin (TM/LP cells) was also abolished by the treatment of the cells with alpha-mannosidase. The effect of alpha-mannosidase was not prevented in the presence of LP. The N-glycoside-deficient contact site A (an adhesion-mediating glycoprotein) was obtained from TM/LP cells and was shown to have a molecular weight of 70,000. This protein (p 70) was shown to still have carbohydrates as detected by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) and subsequent staining of the gel with periodic acid-silver stain. Moreover, p 70 reacted with anti-gp 68, which has a specificity against alpha-mannosyl residues of carbohydrate chains. However, p 70 treated with alpha-mannosidase showed decreased reactivity with anti-gp 68. The monovalent antibody fragment of anti-contact site A or anti-p 70 inhibited EDTA-stable cell adhesion of both control and TM/LP cells. These results indicated that TM-resistant mannosyl residues of contact site A are directly involved in EDTA-stable adhesion of aggregation-competent cells. This is the first report of the direct involvement of the carbohydrate moiety in cell adhesion of aggregation-competent Dictyostelium cells. A schematic model is presented of the role of the carbohydrate moiety in EDTA-stable cell adhesion, including the direct effect of carbohydrates.
在具有聚集能力的盘基网柄菌细胞的细胞黏附中，糖蛋白碳水化合物部分的需求似乎是间接的，因为它起到保护蛋白质部分免受蛋白水解降解的作用；然而，这种作用仅限于对衣霉素（TM）敏感的碳水化合物部分（Hirano, T., 等人 (1983) J. Biochem. 93, 1249 - 1257）。在本研究中，我们发现用刀豆α-甘露糖苷酶处理完整细胞后，具有聚集能力的盘基网柄菌细胞的EDTA稳定黏附被消除，而神经氨酸酶、β-半乳糖苷酶、β-N-乙酰己糖胺酶或α-L-岩藻糖苷酶则没有作用。在用亮抑酶肽处理的情况下（TM/LP细胞），经α-甘露糖苷酶处理的TM处理细胞的EDTA稳定黏聚性也被消除。在存在亮抑酶肽的情况下，α-甘露糖苷酶的作用并未受到抑制。从TM/LP细胞中获得了缺乏N-糖苷的接触位点A（一种介导黏附的糖蛋白），其分子量为70,000。在十二烷基硫酸钠（SDS）存在下进行聚丙烯酰胺凝胶电泳（PAGE），随后用高碘酸-银染色检测，该蛋白（p70）仍含有碳水化合物。此外，p70与抗gp68反应，抗gp68对碳水化合物链的α-甘露糖基残基具有特异性。然而，用α-甘露糖苷酶处理后的p70与抗gp68的反应性降低。抗接触位点A或抗p70的单价抗体片段抑制了对照细胞和TM/LP细胞的EDTA稳定细胞黏附。这些结果表明，接触位点A的耐TM甘露糖基残基直接参与了具有聚集能力细胞的EDTA稳定黏附。这是关于碳水化合物部分直接参与具有聚集能力的盘基网柄菌细胞黏附的首次报道。本文提出了一个碳水化合物部分在EDTA稳定细胞黏附中作用的示意图模型，包括碳水化合物的直接作用。
