Growth suppression of tumor cells by controlling the galactosylation of cell surface N-glycans

我们以前的研究表明，逆转β-1，4-半乳糖基转移酶（β-1，4-GalT）II或V基因在癌细胞中的表达可抑制肿瘤生长，这表明表面碳水化合物在细胞生长中的重要性。由于正常细胞在较高细胞密度的细胞相互作用后停止增殖，我们研究了细胞表面糖基化，特别是N-糖基化是否因不同细胞密度而改变。通过刮刀收获以20%、50%和100%密度生长的小鼠NIH 3 T3细胞，并对膜制备物进行凝集素印迹分析。CBB染色结果显示，在生长过程中，除50 K和250 K蛋白的表达量增加外，大部分组成蛋白的表达量基本不变。当印迹与结合β-1，4-半乳糖基化寡糖的RCA-Ⅰ孵育时，凝集素的反应性在80 K、110 K、120 K和160 K带中增加，特别是在230 K带中，与细胞密度成比例。使用L-PHA观察到对这些蛋白条带的类似但弱得多的反应性。北方印迹分析显示，β-1，4-GalT II基因的表达仅在100%细胞密度下显著增加，表明β-1，4-GalT II编码的半乳糖残基对正常细胞的生长抑制是重要的。通过使用无唾液酸转铁蛋白-琼脂糖柱，从高密度细胞中获得作为半乳糖结合蛋白的31 K蛋白，表明该蛋白参与结合在较高密度细胞上表达的半乳糖残基。

Our previous study showed that reverting the expression of the β-1,4-galactosyltransferase (β-1,4-GalT) II or V gene in cancer cells results in the suppression of tumor growth, suggesting the importance of the surface carbohydrates in cell growth. Since normal cells cease to proliferate upon interaction of cells at higher cell density, we investigated whether or not cell surface glycosylation, particularly N-glycosylation, change by different cell density. Mouse NIH3T3 cells grown at 20%, 50% and 100% in density were harvested by scraper, and membrane preparations were subjected to lectin blot analysis. CBB-staining showed that the expression of most constitutional proteins unchanges during growth except for 50 K and 250 K proteins whose amounts increase. When the blot was incubated with RCA-I, that binds to β-1,4-galactosylated oligosaccharides, the reactivity of lectin increased in 80 K,110 K,120 K and 160 K bands and particularly in 230 K band in proportion to the cell density. A similar but much weaker reactivity towards these protein bands was observed using L-PHA. Northern blot analysis showed that the expression of the β-1,4-GalT II gene only increases significantly at 100% cell density, suggesting that the galactose residues coded by β-1,4-GalT II is important for growth inhibition of normal cells. By using an asialo-transferrin-Sepharose column, 31 K protein was obtained as galactose-binding protein from highly dense cells, suggesting the involvement of this protein in binding to galactose residues expressed on cells at higher density.