Dennis J W, Laferté S, Fukuda M, Dell A, Carver J P
Eur J Biochem. 1986 Dec 1;161(2):359-73. doi: 10.1111/j.1432-1033.1986.tb10455.x.
MDW4, a wheat germ agglutinin-resistant mutant of the metastatic murine tumor MDAY D2 has previously been shown to be poorly metastatic when injected intravenously and non-metastatic when injected subcutaneously into syngeneic mice. W4EB8, a Bandeiraea simplicifolia (BSII) lectin-selected subline of MDW4 has previously been shown to be intermediate between that of MDAY-D2 and MDW4 cell for sensitivity to lectin and metastatic phenotype when injected intravenously into mice. The Asn-linked oligosaccharides from MDAY-D2, MDW4 and W4EB8 cells were released enzymatically with peptide N-glycosidase, reduced with tritiated sodium borohydride and fractionated by Concanavalin-A--Sepharose affinity chromatography and high-performance liquid chromatography (HPLC). Structures of the major fractions were determined by a combination of glycosidase digestion and sizing, gas-liquid chromatography/mass spectrometry and by proton nuclear magnetic resonance. Wild-type and mutant cells processed high-mannose-type structures to biantennary (GlcNAc)2(Man)3(GlcNAc)2. In MDAY-D2 cells this structure was processed further to sialylated tetra-antennary complex with polylactosamine-containing antennae terminating in either sialic acid or alpha 1-3-linked galactose. MDW4 cells had four or five times more (GlcNAc)2(Man)3(GlcNAc)2 than MDAY-D2 cells and a major component of tri-antennary (GlcNAc)3(Man)3(GlcNAc)2 (i.e. 2,2,6-substituted tri-mannosyl core) that was not found in wild-type cells. The partial revertant, W4EB8 had intermediate levels of mutant (GlcNAc)3(Man)3(GlcNAc)2 and sialylated complex-type carbohydrates. The results indicate that a shift in expression from incomplete complex type to sialylated tri/tetra-antennary complex-type carbohydrates in tumor cell may enhance the metastatic potential of tumor cells in the experimental metastasis assay. In addition, somatic cell hybridization analysis indicated that the defect in MDW4 cells was identical to that of the Chinese hamster ovary mutant Lec8: a deficiency in UDP-galactose transport into the golgi.
MDW4是转移性小鼠肿瘤MDAY D2的一种抗小麦胚凝集素突变体，先前已表明，将其静脉注射时转移能力较弱，而将其皮下注射到同基因小鼠体内时则无转移能力。W4EB8是MDW4经单叶豆（BSII）凝集素筛选的亚系，先前已表明，将其静脉注射到小鼠体内时，其对凝集素的敏感性和转移表型介于MDAY-D2细胞和MDW4细胞之间。用肽N-糖苷酶酶解释放MDAY-D2、MDW4和W4EB8细胞中与天冬酰胺连接的寡糖，用氚化硼氢化钠还原，并用伴刀豆球蛋白A-琼脂糖亲和色谱和高效液相色谱（HPLC）进行分级分离。通过糖苷酶消化和大小测定、气-液色谱/质谱联用以及质子核磁共振联用确定主要级分的结构。野生型和突变型细胞将高甘露糖型结构加工成双触角型(GlcNAc)2(Man)3(GlcNAc)2。在MDAY-D2细胞中，这种结构进一步加工成唾液酸化的四触角型复合糖，含多乳糖胺的触角末端为唾液酸或α1-3连接的半乳糖。MDW4细胞中(GlcNAc)2(Man)3(GlcNAc)2的含量比MDAY-D2细胞多四到五倍，并且有三触角型(GlcNAc)3(Man)3(GlcNAc)2（即2,2,6-取代的三甘露糖基核心）这一主要成分，而在野生型细胞中未发现该成分。部分回复突变体W4EB8中突变型(GlcNAc)3(Man)3(GlcNAc)2和唾液酸化复合糖型碳水化合物的水平处于中间状态。结果表明，肿瘤细胞中表达从不完全复合糖型向唾液酸化三/四触角型复合糖型碳水化合物的转变可能会增强肿瘤细胞在实验性转移试验中的转移潜能。此外，体细胞杂交分析表明，MDW4细胞中的缺陷与中国仓鼠卵巢突变体Lec8相同：缺乏UDP-半乳糖转运至高尔基体的能力。
