Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII.
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.
Bolt
Gert
G
Mammalian Cell Technology, Novo Nordisk A/S, Novo Allé, 2880 Bagsvaerd, Denmark. bolt@novonordisk.com
Kristensen
Claus
C
Steenstrup
Thomas Dock
TD
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D005167:Factor VII / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary
D005167:Factor VII