Ralton J E, Jackson H J, Zanoni M, Gleeson P A
Department of Pathology and Immunology, Monash University Medical School, Melbourne, Australia.
Eur J Biochem. 1989 Dec 22;186(3):637-47. doi: 10.1111/j.1432-1033.1989.tb15254.x.
The murine transferrin receptor is a disulphide-linked dimer with three N-glycosylation sites. We have investigated the structural and functional properties of the transferrin receptor from murine plasmacytoma cells (NS-1 cells) treated with the glycosylation inhibitor, tunicamycin and the glycosylation-processing inhibitors, swainsonine and castanospermine. 1. Tunicamycin (1 microgram/ml) inhibited mannose incorporation in NS-1 cells by greater than 90%, but also inhibited methionine incorporation by up to 50%. Both swainsonine (1 microgram/ml) and castanospermine (50 micrograms/ml) resulted in mannose incorporation greater than 100% of untreated cells and neither drug affected methionine incorporation. 2. Incubation of NS-1 cells with tunicamycin resulted in a shift in the apparent molecular mass of the transferrin receptor from 96 kDa and 94 kDa to approximately 82 kDa. 3. Peptide N-glycosidase F digestion of the receptor from untreated cells resulted in the fully deglycosylated 82 kDa component as well as an 87 kDa component which represents partially deglycosylated receptor resistant to peptide N-glycosidase F digestion. 4. The receptor from swainsonine-treated cells was equally sensitive to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H (endo H; resulting in both 87-kDa and 82-kDa components), whereas the receptor from castanospermine-treated cells was only partially sensitive to endo H. 5. Analysis of mannose- and fucose-labelled cellular glycopeptides by concanavalin-A--Sepharose chromatography showed that swainsonine (1 microgram/ml) treatment resulted in approximately 90% inhibition of the synthesis of complex N-glycans and an accumulation of fucosylated hybrid structures. In contrast, castanospermine (100 micrograms/ml) treatment resulted in only partial inhibition (60%) of the synthesis of complex N-glycans. 6. Analysis of the receptor from tunicamycin, swainsonine and castanospermine treated cells under nonreducing conditions showed a single component corresponding to the dimer, indicating that dimerisation of newly synthesised murine receptor is independent of carbohydrate. 7. The non-glycosylated receptor from tunicamycin-treated cells appears to bind transferrin as demonstrated by interaction with transferrin-Sepharose. 8. Surface expression of the receptor was not significantly altered in the presence of either swainsonine or castanospermine as judged by flow cytometry.
小鼠转铁蛋白受体是一种由二硫键连接的二聚体，具有三个N - 糖基化位点。我们研究了用糖基化抑制剂衣霉素、糖基化加工抑制剂苦马豆素和蓖麻毒蛋白处理的小鼠浆细胞瘤细胞（NS - 1细胞）中转铁蛋白受体的结构和功能特性。1. 衣霉素（1微克/毫升）抑制NS - 1细胞中甘露糖掺入超过90%，但也抑制蛋氨酸掺入高达50%。苦马豆素（1微克/毫升）和蓖麻毒蛋白（50微克/毫升）均使甘露糖掺入量高于未处理细胞的100%，且两种药物均不影响蛋氨酸掺入。2. 用衣霉素孵育NS - 1细胞导致转铁蛋白受体的表观分子量从96 kDa和94 kDa转变为约82 kDa。3. 用肽N - 糖苷酶F消化未处理细胞的受体，产生完全去糖基化的82 kDa组分以及一个87 kDa组分，后者代表对肽N - 糖苷酶F消化有抗性的部分去糖基化受体。4. 来自苦马豆素处理细胞的受体对肽N - 糖苷酶F和内切β - N - 乙酰葡糖胺糖苷酶H（内切H；产生87 kDa和82 kDa组分）同样敏感，而来自蓖麻毒蛋白处理细胞的受体对内切H仅部分敏感。5. 通过伴刀豆球蛋白A - 琼脂糖层析分析甘露糖和岩藻糖标记的细胞糖肽表明，苦马豆素（1微克/毫升）处理导致复合N - 聚糖合成受到约90%的抑制以及岩藻糖基化杂合结构的积累。相比之下，蓖麻毒蛋白（100微克/毫升）处理仅导致复合N - 聚糖合成受到部分抑制（60%）。6. 在非还原条件下分析衣霉素、苦马豆素和蓖麻毒蛋白处理细胞的受体，显示对应二聚体的单一成分，表明新合成的小鼠受体的二聚化与碳水化合物无关。7. 如与转铁蛋白 - 琼脂糖相互作用所示，来自衣霉素处理细胞的非糖基化受体似乎能结合转铁蛋白。8. 通过流式细胞术判断，在存在苦马豆素或蓖麻毒蛋白的情况下，受体的表面表达没有显著改变。
