Abstract
Carbohydrate-lectin interactions are relatively weak. As they play an important role in biological recognition processes, multivalent glycan ligands are designed to enhance binding affinity and inhibitory potency. We here report on novel neo-glycoproteins based on bovine serum albumin as scaffold for multivalent presentation of ligands for galectins. We prepared two kinds of tetrasaccharides (N-acetyllactosamine and N,N-diacetyllactosamine terminated) by multi-step chemo-enzymatic synthesis utilizing recombinant glycosyltransferases. Subsequent conjugation of these glycans to lysine groups of bovine serum albumin via squaric acid diethyl ester yielded a set of 22 different neo-glycoproteins with tuned ligand density. The neo-glycoproteins were analyzed by biochemical and chromatographic methods proving various modification degrees. The neo-glycoproteins were used for binding and inhibition studies with human galectin-3 showing high affinity. Binding strength and inhibition potency are closely related to modification density and show binding enhancement by multivalent ligand presentation. At galectin-3 concentrations comparable to serum levels of cancer patients, we detect the highest avidities. Selectivity of N,N-diacetyllactosamine terminated structures towards galectin-3 in comparison to galectin-1 is demonstrated. Moreover, we also see strong inhibitory potency of our scaffolds towards galectin-3 binding. These novel neo-glycoproteins may therefore serve as selective and strong galectin-3 ligands in cancer related biomedical research.
Highlights
Carbohydrates play an important role in many biological processes as they act as recognition motifs for lectins and receptors triggering cellular signals and signal cascades [1,2,3,4,5,6,7,8]
We present neo-glycoproteins with varying glycosylation density based on bovine serum albumin (BSA) and their application in galectin binding studies
The oligomers LacNAc-LacNAc and LacDiNAc-LacNAc were synthesized de novo chemo-enzymatically
Summary
Carbohydrates play an important role in many biological processes as they act as recognition motifs for lectins and receptors triggering cellular signals and signal cascades [1,2,3,4,5,6,7,8]. Neo-glycoproteins are in general synthesized by chemical conjugation of glycans to native proteins [35,36] They can be designed for lectin binding studies by attachment of various types of glycan epitopes and by their glycosylation density [37,38,39]. Galectins, -galactoside binding lectins, mediate most of their functions by galectin–glycan interactions [4,40,41] They play an important role in a variety of biological processes including cancer progression and immune response [42,43,44,45,46,47,48]. We concluded that multivalent presentation of a tetrasaccharide glycan structure with a LacDiNAc epitope on a suitable scaffold should lead to increased inhibition of Gal-3 binding to glycoproteins. The multivalent neo-glycoproteins were evaluated in binding studies with human Gal-3 and Gal-1 to determine their binding and affinity properties as well as inhibitory potential
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