Glycobiology Advance Access originally published online on December 22, 2004
Glycobiology 2005 15(5):541-547; doi:10.1093/glycob/cwi032
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Glycobiology vol. 15 no. 5 © Oxford University Press 2004; all rights reserved.
Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII
Mammalian Cell Technology, Novo Nordisk A/S, Novo Allé, 2880 Bagsværd, Denmark
1 To whom correspondence should be addressed; e-mail: bolt{at}novonordisk.com
Received on October 22, 2004; revised on December 15, 2004; accepted on December 15, 2004
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.
Key words: factor VII / mammalian cells / posttranslational N-glycosylation / protein folding / pulse-chase
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