Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII
Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII
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DOI：
10.1093/glycob/cwi032
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发表时间：
2005-05-01
期刊：
影响因子：
4.3
通讯作者：
Steenstrup, TD
中科院分区：
文献类型：
--
作者：
Bolt, G;Kristensen, C;Steenstrup, TD
N-糖基化通常是在新生蛋白质易位至内质网期间发生的共翻译过程。然而，在本研究中，我们证明了重组人凝血因子VII（FVII）在CHO-K1和293 A细胞中的翻译后N-糖基化。人FVII具有两个N-糖基化位点（N145和N322）。脉冲追踪标记的细胞内FVII迁移为两条带，分别对应于具有一个和两个N-聚糖的FVII。N-糖苷酶处理将这两条带转化为单一带，其与不含N-聚糖的突变的FVII共迁移。脉冲后，大多数标记的细胞内FVII有一个N-聚糖，但在1小时的追逐，绝大多数被加工成FVII与两个N-聚糖，证明FVII的翻译后N-糖基化。N-糖基化位点敲除突变体的脉冲追踪分析表明N145的共翻译糖基化，但N322的主要或完全翻译后糖基化。翻译后N-糖基化似乎发生在同一时间框架内的新生FVII折叠成分泌能力的构象，表明这两个过程之间的联系。我们认为，FVII的共翻译构象不利于N332的糖基化，而在翻译后折叠过程中获得了更有利的构象。这是第一个文件的翻译后N-糖基化的未修饰的蛋白质在哺乳动物细胞与完整的N-糖基化机制。因此，本研究表明，翻译后N-糖基化可以是糖蛋白正常加工的一部分。
N-glycosylation is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. In the present study, however, we demonstrate posttranslational N-glycosylation of recombinant human coagulation factor VII (FVII) in CHO-K1 and 293A cells. Human FVII has two N-glycosylation sites (N145 and N322). Pulse-chase labeled intracellular FVII migrated as two bands corresponding to FVII with one and two N-glycans, respectively. N-glycosidase treatment converted both of these band into a single band, which comigrated with mutated FVII without N-glycans. Immediately after pulse, most labeled intracellular FVII had one N-glycan, but during a 1-h chase, the vast majority was processed into FVII with two N-glycans, demonstrating posttranslational N-glycosylation of FVII. Pulse-chase analysis of N-glycosylation site knockout mutants demonstrated cotranslational glycosylation of N145 but primarily or exclusively posttranslational glycosylation of N322. The posttranslational N-glycosylation appeared to take place in the same time frame as the folding of nascent FVII into a secretion-competent conformation, indicating a link between the two processes. We propose that the cotranslational conformation(s) of FVII are unfavorable for glycosylation at N332, whereas a more favorable conformation is obtained during the posttranslational folding. This is the first documentation of posttranslational N-glycosylation of a non-modified protein in mammalian cells with an intact N-glycosylation machinery. Thus, the present study demonstrates that posttranslational N-glycosylation can be a part of the normal processing of glycoproteins.