Tailored Multivalent Neo-Glycoproteins: Synthesis, Evaluation, and Application of a Library of Galectin-3-Binding Glycan Ligands

TLDR

Galectin‑3 is a β‑galactoside‑binding lectin that serves as a tumor biomarker and promotes angiogenesis and metastasis, making it a promising target for early cancer diagnosis and therapy. This study synthesizes a library of tailored multivalent neo‑glycoproteins and evaluates their Gal‑3 binding properties. By combining glycosyltransferases and chemo‑enzymatic reactions, the authors produced LacNAc‑based oligosaccharides with five distinct terminal epitopes and conjugated them to bovine serum albumin via dialkyl squarate coupling to lysine residues, yielding defined multivalent neo‑glycoproteins. Binding assays revealed that neo‑glycoproteins bearing LacdiNAc epitopes captured Gal‑3 from human serum with high affinity, that biotinylated LacNAc motifs enabled ELISA detection, and that these proteins outperform antibody‑based capture for detecting functionally intact Gal‑3, offering new insights and potential cancer‑research applications.

Abstract

Galectin-3 (Gal-3), a member of the β-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N′,N″-diacetyllactosamine (GalNAcβ1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.