Orthogonal-Group-Controlled Site-Selective I-Branching of Poly-N-acetyllactosamine Chains Reveals Unique Binding Specificities of Proteins towards I-Antigens

Abstract

Poly-N-acetyllactosamine (poly-LacNAc) is ubiquitously expressed on cell surface glycoconjugates, serving as the backbone of complex glycans and an extended scaffold that presents diverse glycan epitopes. The branching of poly-LacNAc, where internal galactose (Gal) residues have β1-6 linked N-acetylglucosamine (GlcNAc) attached, forms the blood group I-antigen, which is closely associated with various physiological and pathological processes including cancer progression. However, the underlying mechanisms remain unclear as many of the I-antigen sequences are undefined and inaccessible. In this study, we developed a highly efficient orthogonal-group-controlled approach to access site-selectively I-branched poly-LacNAc chains. The approach relies on three orthogonal protecting groups, each of them “caps” one internal Gal residue of poly-LacNAc. These groups can be readily “decapped” by specific enzymes or chemical reduction to expose desired sites for GCNT2-catalyzed I-branching. This approach enabled the rapid preparation of a diverse library of 41 linear and branched poly-LacNAc glycans from a single precursor. Glycan microarray analysis using these complex glycans revealed unique recognitions of I-branches by lectins, anti-I mAbs, and galectins. Surprisingly, oxidized forms of linear poly-LacNAc strongly bound to several glycan-binding proteins (GBPs). These findings help to bridge the gap in recognition of I-branching and open new avenues for therapeutic development by targeting galectins.