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

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.

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.

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.

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.

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.