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

我们之前的研究表明，逆转癌细胞中β-1,4-半乳糖转移酶（β-1,4- galt） II或V基因的表达可抑制肿瘤生长，提示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的细胞密度下停止增殖，我们研究了细胞表面糖基化，特别是n -糖基化是否会随着细胞密度的不同而改变。采用刮刀法收获密度为20%、50%和100%的小鼠NIH3T3细胞，制备膜后进行凝集素印迹分析。cbb染色结果显示，除50 K和250 K蛋白表达量增加外，其余组成蛋白在生长过程中表达量基本不变。与β-1,4-半乳糖化寡糖结合的rca -1孵育后，凝集素在80k、110k、120k和160k波段的反应性随细胞密度的增加而增加，其中在230k波段的反应性增加最为明显。L-PHA对这些蛋白带的反应性相似，但弱得多。Northern blot分析显示，β-1,4- galt II基因的表达仅在100%细胞密度时显著增加，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有重要作用。利用asialo- transferin - sepharose色谱柱，从高密度细胞中获得了31 K蛋白作为半乳糖结合蛋白，表明该蛋白与高密度细胞上表达的半乳糖残基结合。

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.