N‐glycosylation of blood coagulation factor XIII subunit B and its functional consequence

Abstract

Background The protective/inhibitory B subunits of coagulation factor XIII (FXIII‐B) is a ~80 kDa glycoprotein containing two N‐glycosylation sites. Neither the structure nor the functional role of the glycans on FXIII‐B has been explored. Objective To reveal the glycan structures linked to FXIII‐B, to design a method for deglycosylating the native protein, to find out if deglycosylation influences the dimeric structure of FXIII‐B and its clearance from the circulation. Methods Asparagine‐linked carbohydrates were released from human FXIIII‐B by PNGase F digestion. The released N‐linked oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis. Structural identification utilized glycan database search and exoglycosidase digestion based sequencing. The structure of deglycosylated FXIII‐B was investigated by gel filtration. The clearance of deglycosylated and native FXIII‐B from plasma was compared in FXIII‐B knock out mice. Results PNGase F completely removed N‐glycans from the denatured protein. Deglycosylation of the native protein was achieved by repeated digestion at elevated PNGase F concentration. The total N‐glycan profile of FXIII‐B featured nine individual structures; three were fucosylated and each structure contained at least one sialic acid. Deglycosylation did not change the native dimeric structure of FXIII‐B, but accelerated its clearance from the circulation of FXIII‐B knock out mice. Conclusion Characterization of the glycan moieties attached to FXIII‐B is reported for the first time. Complete deglycosylation of the native protein was achieved by a deglycosylation workflow. The associated glycan structure is not required for FXIII‐B dimer formation, but it very likely prolongs the half‐life of FXIII‐B in the plasma.

Strategies for Glycoengineering Therapeutic Proteins

“…Another category of blood-regulatory proteins whose activity critically depends on glycosylation are clotting factors that need to be administered therapeutically for people with deficiencies in these proteins, such as hemophilia patients. Deglycosylation diminishes the conformational stability, activity, and macromolecular interactions of coagulation factor VIII [FVIII ( Kosloski et al, 2009 )] and decreases the effectiveness of factor XIII-B [FXIII-B ( Hurjak et al, 2020 )]. Based on the importance of glycosylation in blood clotting, efforts to produce coagulation factors in low-cost hosts (e.g., in plant cells, Section 5.3.4 ) to increase availability for patients are cognizant of the importance of maintaining appropriate glycosylation; this topic is discussed extensively in a review article by Top and coauthors ( Top et al, 2019 ).…”

Liquid chromatography and capillary electrophoresis in glycomic and glycoproteomic analysis

“…The exoglycosidases cleaved specific terminal monosaccharide units from glycan structures and this caused migration time shifts of their peaks, which was used to elucidate the structures [ 178 ]. Hurjak et al [ 179 ] used the gel to analyze APTS-labeled N-glycans released from protective/inhibitory B subunits of human coagulation factor XIII.…”

Sweetening the Deal: Glycosylation and its Clinical Applications

“…In the autoimmunity realm, Fc glycosylation of anti-human IFN-alpha2b antibodies has been shown to influence their binding affinity, thus potentially affecting their efficacy in treating Systemic Lupus Erythematous [15]. In haematology, certain glycosylation patterns on Factor XIII-B prolonged its serum half-life, affecting the body ' s coagulative balance [16]. In endocrinology, Nglycosylation of Calcitonin receptor resulted in increased peptide hormone affinity [17].…”