Combining glycocluster synthesis with protein engineering: an approach to probe into the significance of linker length in a tandem-repeat-type lectin (galectin-4)

• Synthesis of glycoclusters presenting lactose and 2′-fucosyl lactose on different scaffolds.
• Engineering of galectin-4 mutants to probe structure–activity relationships with glycoclusters.
• The impact of structural alterations by combining application of glycoclusters and mutants.

Complementarity in lectin–glycan interactions in situ is assumed to involve spatial features in both the lectin and the glycan, giving a functional meaning to structural aspects of the lectin beyond its carbohydrate-binding site. In combining protein engineering with glycocluster synthesis, it is shown that the natural linker length of a tandem-repeat-type human lectin (galectin-4) determines binding properties in two binding assays (using surface-presented glycoprotein and cell surface assays). The types of glycocluster tested included bivalent lactosides based on tertiary amides of terephthalic, isophthalic, 2,6-naphthalic and oxalic acids as well as bivalent H(type 2) trisaccharides grafted on secondary/tertiary terephthalamides and two triazole-linker-containing cores. The presented data reveal a marked change in susceptibility to the test compounds when turning the tandem-repeat-type to a proto-type-like display. The testing of glycoclusters is suggested as a general strategy to help to delineate the significance of distinct structural features of lectins beyond their contact sites to the glycan.

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