A novel endo-α-(1,3)-L-fucanase of the GH107 family from Winogradskyella eximia reveals source-dependent anticoagulant fucoidan oligosaccharides.

Fucoidans are sulfated polysaccharides from brown algae whose diverse biological activities depend critically on their structural features. In particular, 2,4-di-sulfation is considered one of the key determinants of anticoagulant effects. Endo-fucanases of glycoside hydrolase family 107 (GH107) are valuable tools for the structural elucidation and controlled depolymerization of fucoidans into bioactive oligosaccharides. However, the specificity of endo-α-(1,3)-L-fucanases toward defined sulfation patterns remains insufficiently characterized. In the present work, three novel GH107 endo-fucanases (Wef1, Wef2, and Wef3) from the fucoidan-degrading gene cluster of Winogradskyella eximia KMM 3944T were identified. Sequence similarity network (SSN) clustering and virtual screening of 2,4-di-sulfated L-fucose (Fuc2,4S)-binding sites predicted preference of Wef3 for α-(1,3)-glycosidic bonds with selectivity toward Fuc2,4S residues. Specificity analysis confirmed that Wef3 preferentially hydrolyzes α-(1,3)-linkages between Fuc2,4S-Fuc2,4S and/or Fuc4S-Fuc4S in α-(1,3)-L-fucoidans, while accommodating C2-branched residues in defined sugar-binding subsites. Wef3 was also found to cleave the 2,3-di-sulfated α-(1,3;1,4)-decasaccharide, indicating catalytic promiscuity toward oligosaccharides with defined sulfation patterns. Recombinant Wef3 was used to depolymerize α-(1,3)-L-fucoidans from Saccharina cichorioides (ScF) and Saccharina bongardiana (SbF). The resulting reaction products were fractionated and evaluated for their anticoagulant and platelet-modulating effects in vitro. The oligosaccharide fraction LMP-SbF, compared with LMP-ScF, showed pronounced anticoagulant effects, primarily consistent with its higher Fuc2,4S content. Platelet activation similarly correlated with the 2,4-di-sulfation pattern and higher molecular weight. Native ScF and SbF significantly reduced ADP- and ristocetin-induced platelet aggregation. The obtained data expand the repertoire of structurally-specific fucanases for fucoidan structure analysis and bioactive oligosaccharide production, highlighting the importance of the fucoidan source.