Effect of galactose side-chain modification on immunomodulatory activity of tamarind xyloglucan oligosaccharides

Xyloglucan, an abundant polysaccharide bearing galactose side-chains within the cell wall of cotyledons from leguminous seeds, exhibits superior immunomodulatory properties. However, the contribution of its galactose side-chains remains unclear. This study aimed to elucidate the role of the galactose side-chains in the immunomodulatory activity by comparing the effects of oligosaccharides with different degrees of galactosyl substitution on RAW 264.7 mouse macrophages. Three structurally distinct xyloglucan fractions were prepared from tamarind seed: native high-molecular-weight xyloglucan (XG), low-molecular-weight xyloglucan oligosaccharides (XGOS) and partially degalactosylated XGOS (De-XGOS). All three components effectively promote macrophage proliferation and activation. Compared with De-XGOS, XGOS, which retained more galactose side-chains, generally exhibited stronger immunomodulatory responses in several functional assays. Furthermore, by stimulating the activation of the TLR4 signaling pathway, it triggered the expression of NF-κB pathway proteins, thereby elevating reactive oxygen species (ROS) levels to 1.71-fold and promoting the secretion of cytokines such as IL-1β, IL-6, and TNF-α. Pretreatment with the TLR4 inhibitor TAK-242 significantly attenuated XGOS-induced NO and TNF-α production, supporting the involvement of TLR4 signaling in macrophage activation. Molecular docking further suggested potential interactions between representative xyloglucan oligosaccharide structures and TLR4/MD2. In addition, the conditioned medium from XGOS-treated macrophages inhibited 4 T1 breast cancer cell migration and invasion by 38.90% and 33.33%, respectively. This study provides mechanistic insights into the structure-function relationship of xyloglucan oligosaccharides and suggests that galactose side-chain retention may contribute to their immunomodulatory activity.