Pinnaduwage P D, Bhavanandan V P, Davidson E A
Carbohydr Res. 1986 Aug 15;151:37-50. doi: 10.1016/s0008-6215(00)90328-8.
Two variants of B16 mouse melanoma cells, selected for their resistance to toxic levels of wheat germ agglutinin isolectin 1 (WGA-1) in serum-free medium, showed by chromosome analysis that they are still mouse cell-lines, continue to produce melanin, and are less tumorigenic in mice than the parent B16 cells. The variants showed a marked decrease in cell agglutination with the wheat germ lectin and a slight increase in cell agglutination with concanavalin A. The binding of 125I-labeled wheat germ agglutinin to the two variant lines was likewise decreased over a 10(3)-fold range of lectin concentrations. Terminal sialyl residues were critical in WGA-1 binding to the wild-type cells. The binding data indicated a decrease in high-affinity binding as well as a decrease in the total number of binding sites in the variants. Polyacrylamide gel electrophoresis, followed by affinity staining with 125I-wheat germ agglutinin, showed alterations in the wheat germ agglutinin-binding glycoproteins in the variants compared to those of the parent cell line. However, lactoperoxidase-catalyzed iodination revealed a similar cell-surface protein pattern among the three cell lines. Radioactive glycoproteins secreted or shed by the three cell lines grown in the presence of [3H]glucosamine in serum-free medium were fractionated on the basis of their interaction with WGA-Sepharose (2 mg/mL). The WGA-bound glycoproteins from the two variants had molecular weights of 92,000, 56,000, and 42,000. None of these components was detected in the parent cell-line. A major WGA-binding glycoprotein, which accounted for 37% of the total [3H]glucosamine incorporated, was isolated from the spent medium of the parent mouse melanoma cell-line. This glycoprotein was apparently absent in the WGA-1-resistant variants.
两种B16小鼠黑色素瘤细胞变体，是在无血清培养基中因对毒性水平的麦胚凝集素同工凝集素1（WGA-1）具有抗性而筛选出来的。染色体分析表明，它们仍是小鼠细胞系，继续产生黑色素，并且在小鼠体内的致瘤性比亲本B16细胞低。这些变体与麦胚凝集素的细胞凝集作用明显降低，而与伴刀豆球蛋白A的细胞凝集作用略有增加。在10³倍的凝集素浓度范围内，¹²⁵I标记的麦胚凝集素与这两种变体细胞系的结合同样减少。末端唾液酸残基在WGA-1与野生型细胞的结合中起关键作用。结合数据表明，变体中高亲和力结合减少，结合位点总数也减少。聚丙烯酰胺凝胶电泳，随后用¹²⁵I-麦胚凝集素进行亲和染色，结果显示与亲本细胞系相比，变体中麦胚凝集素结合糖蛋白发生了改变。然而，乳过氧化物酶催化的碘化反应显示这三种细胞系的细胞表面蛋白模式相似。在无血清培养基中于[³H]葡萄糖胺存在下培养的这三种细胞系分泌或脱落的放射性糖蛋白，根据它们与WGA-琼脂糖（2 mg/mL）的相互作用进行分级分离。来自两种变体的WGA结合糖蛋白的分子量分别为92,000、56,000和42,000。在亲本细胞系中未检测到这些成分中的任何一种。从亲本小鼠黑色素瘤细胞系的用过的培养基中分离出一种主要的WGA结合糖蛋白，它占掺入的总[³H]葡萄糖胺的37%。这种糖蛋白在对WGA-1有抗性的变体中显然不存在。
