Title : Site-specific N-glycosylation analysis of human
factor XI : Identification of a noncanonical NXC
glycosite
Abstract :
- Human factor XI ( hFXI ) is a 160-kDa disulphide-linked homodimer zymogen involved in the coagulation cascade
- Its deficiency results in bleeding diathesis referred to as hemophilia C. hFXI bears five N-glycosylation consensus sites per monomer, N72 , N108 , N335 on the heavy chain and N432 , N473 on the light chain
- This study reports the first in-depth glycosylation analysis of hFXI based on advanced MS approaches
- Hydrophilic interaction LC and MS characterization and quantification of the N-glycans showed that the two major forms are complex biantennary mono-α2,6-sialylated (A2 S1 , 20%) and bis-α2,6-sialylated structures ( A2-S2 , 66%)
- Minor triantennary structures ( A3-S3 F, ∼1.5%; A3-S3 , ∼2%) were also identified
- MS analyses of intact hFXI revealed full occupation of two of the three heavy-chain glycosites and almost full-site occupancy of the light chain
- Analysis of hFXI glycopeptides by LC-MS/MS enabled site-specific glycan profiling and occupancy
- It was evidenced that N335 was not glycosylated and that N72 and N108 were fully occupied, whereas N432 and N473 were occupied at about 92 and 95%, respectively
- We also identified a new glycosite of the noncanonical format NXC at N145 , occupied at around 5%
- These data provide valuable structural information useful to understand the potential roles of N-glycosylation on hFXI function and could serve as a structural reference