Jump to Main Content
Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII
- Bolt, Gert, Kristensen, Claus, Steenstrup, Thomas Dock
- Glycobiology 2005 v.15 no.5 pp. 541-547
- N-glycoside hydrolases, coagulation, endoplasmic reticulum, factor VII, glycoproteins, glycosylation, humans, knockout mutants
- 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.