Flagellin is the major protein of the bacterial flagellar filament. As a structural element, it is involved in chemotaxis in motile bacteria. In addition, plant cells and mammal immune system cells have receptors on their surface that are specific for this bacterial protein (FLS2 and ТLR5, respectively). These facts make the study of bacterial flagellin an important aspect in understanding the functioning of both free-living bacteria and those engaging in a symbiosis with macroorganisms.
This work is the first to show that the carbohydrate portion of bacterial flagellin can be represented by a polysaccharide. Previously, only monosaccharide and oligosaccharide carbohydrate fragments have been described as part of bacterial flagellins.
The flagellin of the Azospirillum polar flagellum had a high molecular mass (more than 80 kDa). Carbohydrate fragments accounted for more than 30% of the mass fraction in these molecules. Such a high molecular mass and a large amount of carbohydrates are not typical of the investigated flagellins of other bacteria. Mass spectrometry showed that the carbohydrate isolated by us from the polar flagellum flagellin of Azospirillum brasilense Sp7 had a molecular mass of 7700 Da and contained residues of D-galactose, L-rhamnose, L-fucose, and N-acetyl-D-glucosamine at an equimolar ratio. NMR analysis enabled the repeating unit structure of this polysaccharide to be defined. As the molecular mass of the flagellin decreased after deglycosylation, it was suggested that the flagellin composition included three or four polysaccharide chains.
Immunodiffusion and immunoblotting analyses demonstrated that the antigenic properties of the flagellin’s carbohydrate fragments were similar to those of the lipopolysaccharide of strain Sp7.
Possibly owing to the large amount of carbohydrates present in the molecule, the glycosylated flagellin of the A. brasilense Sp7 polar flagellum exhibited high thermal stability and high aggregation ability. In solution, it formed spherical or irregularly shaped aggregates, not a filament.
