PMID: 15616124

 

    Legend: Sugar

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

Abstract :

1. N-glycosylation is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum
2. In the present study, however, we demonstrate posttranslational N-glycosylation of recombinant human coagulation factor VII ( FVII ) in CHO-K1 and 293A cells
3. Human FVII has two N-glycosylation sites ( N145 and N322 )
4. Pulse-chase labeled intracellular FVII migrated as two bands corresponding to FVII with one and two N-glycans , respectively
5. N-glycosidase treatment converted both of these band into a single band, which comigrated with mutated FVII without N-glycans
6. 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
7. Pulse-chase analysis of N-glycosylation site knockout mutants demonstrated cotranslational glycosylation of N145 but primarily or exclusively posttranslational glycosylation of N322
8. 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
9. 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
10. This is the first documentation of posttranslational N-glycosylation of a non-modified protein in mammalian cells with an intact N-glycosylation machinery
11. Thus, the present study demonstrates that posttranslational N-glycosylation can be a part of the normal processing of glycoproteins