Enzymatic tailoring of anionic glycans for characterizing lectin and antibody specificities in a microarray format

Despite many studies on binding patterns of commercial lectins and other glycan binding proteins, unexpected recognition motifs are still revealed, highlighting the necessity to (re-)investigate these specificities deeply, as they are used for rapid screening of glycosylation patterns or diagnostic histochemistry. The current work is an extension of previous defined glycan microarrays, displaying a library of natural anionic structures not only including N-acetylneuraminic acid but also N-glycolylneuraminic acid as well as sulfated galactose, N-acetylglucosamine or glucuronic acid based on using recombinant glycosyl- and sulfo-transferases. Moreover, anionic modifications on linear tetrasaccharides versus on typical biantennary N-glycan core structures were compared regarding binding pattern and intensities. As in our previous studies, 2-amino-N-(2-amino-ethyl)-benzamide (AEAB)-labeled glycans were probed with various plant lectins, C-type lectins, sialic-acid specific lectins, different antibodies, e.g. anti-NeuGc and recombinant prokaryotic lectins (RPLs). Indeed, expected binding patterns were observed; however, some proteins revealed more narrow specificities. The L2 anti-HNK-1 elicits its specificity for GlcAβ1-3Galβ1-4GlcNAc without the necessity of sulfation also for linear glycans. For anti-Le^X (clone L5) it is known that sialic acid masking of the epitope is not tolerated, while here we demonstrate that anti-Le^A antibody (clone T174) can interact with α2,3 Neu5Ac and Neu5Gc capped epitopes. HECA-452 recognizes sialyl Le^X and sialyl Le^A, but only with Neu5Ac attached. This anionic glycan array contains most common anionic glycan modifications, can be flexibly modified and reveals commonly-overlooked specificities providing insights into sialic acid-specific interactions, including the often neglected N-glycolylneuraminic acid, thereby applying these findings also to murine or other deuterostome models.