A clonal derivative of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosamine-phosphate transferase activity has altered asparagine-linked glycosylation.
A clonal derivative of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosamine-phosphate transferase activity has altered asparagine-linked glycosylation.
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具有增强的 N-乙酰氨基葡萄糖-磷酸转移酶活性的耐衣霉素中国仓鼠卵巢细胞的克隆衍生物改变了天冬酰胺连接的糖基化。
DOI：
10.1002/jcp.1041310303
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发表时间：
1987
影响因子：
5.6
通讯作者：
Krag,SS
中科院分区：
文献类型：
--
作者：
Waldman,BC;Oliver,C;Krag,SS
Criscuolo, b.a., and Krag， S.S. (1982) J. Cell biology .94:586 - 591)通过逐步选择将TM添加到培养基中分离出对抗生素tunicamycin （TM）耐药的中国仓鼠卵巢（CHO）细胞群体。TM通过阻断N -乙酰氨基葡萄糖- 1 -磷酸从糖核苷酸UDP - N -乙酰氨基葡萄糖- 1 -磷酸向类异戊二烯脂质载体磷酸酰基的转移，抑制天冬酰胺相关糖蛋白的生物合成。在27 μg TM/ml的条件下，从TM抗性群体中分离出4个克隆衍生物，发现它们在相同程度上过量产生N -乙酰氨基葡萄糖-磷酸转移酶活性（与野生型细胞相比约为15倍）。其中一个克隆3E11对TM的抗性是野生型细胞的550倍。在没有TM的情况下，抗性表型至少在2.5个月的生长期间保持不变。3E11细胞表现为染色体易位，但无均匀染色区（HSR）或双微小染色体。3E11细胞的N -乙酰氨基葡萄糖磷酸酯转移酶活性与细胞膜相关，并被TM抑制。在3E11细胞中富集了一种140,000道尔顿膜蛋白和至少四种其他膜蛋白。甘露糖基磷酸多醇合成酶和葡萄糖基磷酸多醇合成酶活性在3E11细胞制备的膜中没有升高。天冬酰胺连接的糖基化发生了改变，使得3E11细胞主要合成了一种截断的低聚糖，Man5GlcNAc2，这可能是由于相对于野生型细胞，甘露糖基磷酸酯醇的数量减少了。
A population of Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) had previously been isolated (Criscuolo, B.A., and Krag, S.S. (1982) J. Cell Biol.94:586–591) by a stepwise selection procedure using progressive increments of TM added to the medium. TM inhibits asparaginelinked glycoprotein biosynthesis by blocking the transfer of N‐acetylglucosamine‐1‐phosphate from the sugar nucleotide UDP‐N‐acetylglucosamine to the isoprenoid lipid carrier, dolichyl phosphate. Four clonal derivatives were isolated from the TM‐resistant population in the presence of 27 μg TM/ml and were found to overproduce the N‐acetylglucosamine‐phosphate transferase activity to the same extent (approximately 15‐fold compared to wild‐type cells). One of these clones, 3E11, was > 550‐fold more resistant to TM than wild‐type cells. The resistance phenotype remained during at least 2.5 months of growth in the absence of TM. 3E11 cells exhibited chromosomal translocations, but no homogeneously staining regions (HSR) or double minute chromosomes. The N‐acetylglucosamine‐phosphate transferase activity in 3E11 cells was membrane‐associated and was inhibited by TM. A 140,000‐dalton membrane protein and at least four other membrane proteins were enriched in 3E11 cells. Mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities were not elevated in membranes prepared from 3E11 cells. Asparagine‐linked glycosylation was altered such that 3E11 cells synthesized primarily a truncated oligosaccharide, Man5GlcNAc2, perhaps due to the reduced amount of mannosylphosphoryldolichol relative to wild‐type cells.