Chitinase-3-like protein 1 decodes chitosan acetylation patterns into toll-like receptor 2 signaling through heparan sulfate

Abstract Chitinase-3-like protein 1 (CHI3L1), which is associated with a wide range of inflammatory diseases, lacks chitinase activity but retains the ability to bind chitin and chitosan. Chitin is a major component of fungal cell walls, whereas chitosan is used in biomedicine. In addition to chitosan, CHI3L1 has been proposed to interact with heparan sulfate (HS), a highly sulfated glycosaminoglycan on mammalian cell surfaces. Here, we investigated how interactions with chitosan and HS regulate the pro-inflammatory activity of CHI3L1. Mapping of the chitin-binding cleft revealed preferential binding of CHI3L1 to chitosans with a regular acetylation pattern that, together with CHI3L1, promoted toll-like receptor 2 signaling. We further identified a dominant HS-binding site that recognizes a distinct HS sulfation code containing a coherent motif of N- and 6-O-sulfations. Mutation of this HS-binding site or impaired HS biosynthesis prevented CHI3L1 accumulation at the cell surface and abolished CHI3L1-mediated cell activation. Together, our findings establish HS as a critical co-receptor for CHI3L1 and reveal a pro-inflammatory cross-talk between HS, CHI3L1, and chitosan that may contribute to host defense against fungal pathogens and responses to chitosan-based biomaterials. These findings identify HS- and chitosan-dependent CHI3L1 signaling as a potential target for modulating inflammatory responses.