Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII

• Published in: Glycobiology (Oxford) Vol. 15; no. 5; pp. 541 - 547
• Main Authors: Bolt, Gert, Kristensen, Claus, Steenstrup, Thomas Dock
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.05.2005; Oxford Publishing Limited (England)
• Subjects: Animals; Cell Line; Chinese hamster ovary; CHO; CHO Cells; connecting region; Cricetinae; DMEM; Dulbecco’s modified Eagle medium; EGF; endoplasmic reticulum; epidermal growth factor; factor VII; Factor VII - chemistry; Factor VII - metabolism; gamma-carboxy glutamic acid; Gla; Glycosylation; HEK; human embryonal kidney; Humans; mammalian cells; Mutation; oligosaccharyltransferase; OST; PAM-3; peptide: N-glycosidase F; peptidylglycine α-midating monooxygenase-3; PNGase F; posttranslational N-glycosylation; protein folding; Protein Processing, Post-Translational; Protein Structure, Tertiary; pulse-chase; SDS–PAGE; sodium dodecyl sulfate–polyacrylamide gel electrophoresis
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwi032

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.