Novel Fluorescent Glycan Microarray Strategy Reveals Ligands for Galectins

Abstract

Summary Galectin-1 (Gal-1) and galectin-3 (Gal-3) are widely expressed galectins with immunoregulatory functions in animals. To explore their glycan specificity, we developed microarrays of naturally occurring glycans using a novel bifunctional fluorescent linker, 2-amino-N-(2-aminoethyl)-benzamide (AEAB), directly conjugated through its arylamine group by reductive amination to free glycans to form glycan-AEABs (GAEABs). Glycans from natural sources were used to prepare over 200 GAEABs, which were purified by multidimensional HPLC and covalently immobilized onto NHS-activated glass slides via their free alkylamine. Fluorescence-based screening demonstrated that Gal-1 recognizes a wide variety of complex N-glycans, whereas Gal-3 primarily recognizes poly-N-acetyllactosamine-containing glycans independent of N-glycan presentation. GAEABs provide a general solution to glycan microarray preparation from natural sources for defining the specificity of glycan-binding proteins.

A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

“…Preparation of Sialylated GAEABs-We chose a synthetic strategy that combined two earlier developments, the bifunctional fluorescent tag AEAB (27) and the one-pot three-enzyme sialylation reaction (19,20,25) (Fig. 1).…”

“…Using GAEABs with terminal galactose residues as acceptors for sialyltransferases, we explored a one-pot three-enzyme sialylation system to synthesize terminally sialylated glycans with various sialic acid modifications. The GAEAB acceptors included lactose, LNnT, LNT, and NA2, which were prepared from natural glycans as described previously (27) and purified prior to the multienzyme sialylation. Aliquots (50 g to 1 mg) of the GAEABs were subjected to one-pot three-enzyme enzymatic sialylation in a combinatorial fashion (16).…”

“…3c). Each glycan was quantified based on its fluorescence and printed in replicates of n ϭ 4 on NHS-activated glass slides as described previously (27) to provide a sialylated glycan microarray (SGM). The selection of this set of sialic acid structures is based on structures of known importance, natural occurrence, and higher abundance, along with current chemical accessibility.…”

“…Glycan-AEAB Conjugation, Sialylation, and PurificationFree reducing glycans were conjugated with AEAB as described previously (27). Briefly, 1-10 mg of glycan was mixed with 50 -250 l of AEAB hydrochloride salt solution freshly prepared at 0.35 M in DMSO/AcOH (7:3, v/v) followed by an equal volume of sodium cyanoborohydride solution freshly prepared in the same solvent.…”

“…Asialo, biantennary N-glycan (Gal␤1-4GlcNAc␤1-2Man␤1-6(Gal␤1-4GlcNAc␤1-2Man␤1-3) Man␤1-4GlcNAc␤1-4GlcNAc; NA2) was prepared from a chicken egg yolk glycopeptide (26) by mild acid hydrolysis and peptide:N-glycosidase F digestion. The bifunctional linker, 2-amino(N-aminoethyl) benzamide (AEAB), was prepared as described previously (27). HPLC solvents were purchased from Fisher.…”