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Site‐specific N ‐glycosylation analysis of human factor XI: Identification of a noncanonical NXC glycosite
Author(s) -
Faid Valegh,
Denguir Nouha,
Chapuis Violaine,
Bihoreau Nicolas,
Chevreux Guillaume
Publication year - 2014
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201400038
Subject(s) - glycosylation , glycan , chemistry , glycoproteomics , glycoprotein , n linked glycosylation , biochemistry , immunoglobulin light chain , biology , antibody , genetics
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, N 72 , N 108 , N 335 on the heavy chain and N 432 , N 473 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 (A 2 S 1 , 20%) and bis‐α2,6‐sialylated structures (A 2 S 2 , 66%). Minor triantennary structures (A 3 S 3 F, ∼1.5%; A 3 S 3 , ∼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 N 335 was not glycosylated and that N 72 and N 108 were fully occupied, whereas N 432 and N 473 were occupied at about 92 and 95%, respectively. We also identified a new glycosite of the noncanonical format NXC at N 145 , 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.