A glycosaminoglycan microarray identifies galectin-4 binding to sulfated glycosaminoglycans.

Lectin-glycosaminoglycan (GAG) interactions remain incompletely understood despite their potential roles in cell signaling and immune regulation. In this study, we performed a comprehensive profiling of the binding specificity of 49 types of human endogenous lectins-including SIGLECs, C-type lectins, and galectins-for GAGs using GAG microarrays. GAGs containing 6-O-sulfation, such as heparin (HP), chondroitin sulfate C (CSC), and chondroitin sulfate E (CSE), exhibited broad binding to multiple SIGLECs and C-type lectins, whereas non-sulfated or low-sulfated GAGs exhibited minimal interactions, indicating a strong dependence on sulfation patterns. In contrast, most galectins displayed little or no detectable binding to GAG. Notably, galectin-4 (Gal-4) uniquely exhibited significant affinity for 6-O-sulfated GAGs, particularly HP. Surface plasmon resonance analysis revealed high-affinity binding of Gal-4 to HP (Kd = 4.70 × 10-8 M), substantially stronger than its carbohydrate recognition domains, indicating cooperative contributions of both N- and C-terminal CRDs of Gal-4. Molecular dynamics simulations further supported a binding mode involving both N- and C-terminal domains. Consistent with these findings, Gal-4 bound to endogenous HP-positive mast cells, and this interaction was competitively inhibited by HP. Together, these results identify Gal-4 as a unique galectin with a noncanonical capacity to recognize sulfated GAGs, revealing an alternative glycan-recognition mechanism beyond the conventional β-galactoside paradigm.