Macrophage-stimulating activities of a novel low molecular weight saccharide fragment prepared from ascophyllan with alginate lyase

Abstract

• Sulphated polysaccharide ascophyllan can be degraded by alginate lyase, EC 4.2.2.3. • A novel low molecular mass fragment LMWAs-L is purified from enzymolysis products. • LMWAs-L is a low fucose- and sulphate-containing saccharide fragment. • LMWAs-L exhibits significant macrophage-stimulating activities in vitro . • The proposal structure of LMWAs-L was established. In this study, the polysaccharide ascophyllan, isolated from Ascophyllum nodosum, was degraded using alginate lyase (EC 4.2.2.3) to prepare low-molecular-weight fragments. LMWAs-L, an extremely low-molecular-weight saccharide (6.71 kDa) with low fucose- and sulphate-contents, was purified by gel filtration chromatography. LMWAs-L exhibited significant macrophage-stimulating activities, similar to ascophyllan, through intracellular signalling pathways. Methylation and GC/MS analyses indicated that the main glycosidic linkages of LMWAs-L were 1,2-linked-Glc p A, followed by T -linked-Glc p , 1,3,6-linked-Gal p , 1,3-linked-Fuc p , 1,4-linked-Xyl p , and 1,4-linked-Man p A at a molar ratio of 3.37: 1.13: 1.11: 1.00: 0.96: 0.61. The proposal repeating structure of LMWAs-L was shown to be →2)-α-D-Glc p A-(1 → 2)-α-D-Glc p A-(1 → 6)-α-D-Gal p -(1 → 2)-α-D-Glc p A-(1→ as main chain, branched by T -α-D-Glc p -(1 → 4)-β-D-Xyl p -(1 → 3)-α-L-Fuc p 4S-(1→ at the O -3 of 3,6)-α-D-Gal p -(1→ residues. 4-deoxy-L- erythro -hex-4-enuronosyluronate-4-β-D-Man p A-(1→ and →4)-β-D-Man p A red residues were attached to the ends of main chain as non-reducing end residue and reducing end residue, respectively. Our results suggest that the fragment LMWAs-L contain an essential structural element of ascophyllan that is responsible for the macrophage-stimulating activities.