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.
authors.affiliation: Analytical Department, LFB Biotechnologies, Courtaboeuf, France.
authors.forename: Valegh
authors.initials: V
authors.lastname: Faid
authors.forename: Nouha
authors.initials: N
authors.lastname: Denguir
authors.forename: Violaine
authors.initials: V
authors.lastname: Chapuis
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bihoreau
authors.forename: Guillaume
authors.initials: G
authors.lastname: Chevreux
mesh_terms: D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid; D005172:Factor XI / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D011134:Polysaccharides; D005172:Factor XI
keywords: Coagulation factor XI; Glycoproteomics; Glycosylation; Mass spectrometry; NXC glycosite