Monomer sequence governs the anti-inflammatory activity of chitooligosaccharides.

Carbohydrates, together with nucleic acids and proteins, constitute the three major information carriers, however, the role of glycan sequence in governing biological processes remain unresolved. This study prepared sequence-precise chitooligosaccharides using multiple chitin deacetylases (VcCOD, NodB, PesCDA, and ArCE4), leading to the first decoding of a relationship between monomer sequence and its anti-inflammatory activity. The results indicate that chitooligosaccharides with distinct sequences and the same degree of polymerization exert much different anti-inflammatory potency, and the oligomers containing two glucosamine (D) and one N-acetyl glucosamine (A) in the DDA sequence at the non-reducing end, including DDAA, DDAD, and DDAAA, exhibited higher inhibitory effects on NO release in the LPS-induced RAW264.7 than other tetramers and pentamers, respectively. Furthermore, in endotoxemia model, DDAA normalized serum ALT, UA, and CrK levels, and significantly inhibited the cytokines of IL-1β, IL-6, and IFN-γ. Notably, at a low dose of 40 mg/kg, DDAA demonstrated a more pronounced anti-inflammatory effect outperformed dexamethasone, with restoration of the length-to-crypt depth ratio of intestinal villi and the preservation of mitochondrial cristae integrity in intestine and kidney. Collectively, this study suggests that heterogeneous chitooligosaccharides carry sequence-encoded biological information, and the DDAA is identified as a promising anti-inflammatory candidate for inflammatory diseases.