Using glycan microarrays to understand immunity

Abstract

Host immunity represents a complex array of factors that evolved to provide protection against potential pathogens. While many factors regulate host immunity, glycan binding proteins (GBPs) appear to play a fundamental role in orchestrating this process. In addition, GBPs also reside at the key interface between host and pathogen. While early studies sought to understand GBP glycan binding specificity, limitations in the availability of test glycans made it difficult to elucidate a detailed understanding of glycan recognition. Recent developments in glycan microarray technology revolutionized analysis of GBP glycan interactions with significant implications in understanding the role of GBPs in host immunity. In this review, we explore different glycan microarray formats with a focus on the impact of these arrays on understanding the binding specificity and function of GBPs involved in immunity.

Figure 1

Utilization of defined glycan microarrays to elucidate GBP specificity. Libraries of well-characterized glycans generated by release of defined glycans from glycoproteins, other natural sources or by chemical or chemoenzymatic synthesis are used to populate well-defined glycan microarrays. Structures reflect naturally occurring glycans and modifications of glycans not typically found in nature. Glycan libraries undergo derivatization with a functional coupling moiety, followed by printing in a microarray format to generate the glycan microarray. GBPs are incubated with the glycan microarrays over different concentrations and detected by fluorescence emission if directly labeled or by a similarly labeled suitable secondary detecting agent. While many approaches can be taken to analyze glycan array data, examination of GBP binding over a variety of concentrations for individual glycans is shown.

Figure 2

Using shotgun glycan microarrays to identify native glycan ligands for GBPs. Glycans released from natural sources, such as red blood cells or neutrophils shown in the schematic, are derivatized with a functional linker, subjected to multidimensional chromatography and printed in a microarray format. Following interrogation of the library with GBP, glycan fractions isolated following multidimensional chromatography that correspond to GBP bound ligands are subjected to mass spec glycan sequencing to identify native glycans recognized by GBP.

Figure 3

Using microbial glycans to examine host pathogen interactions. Glycans, in particular lipopolysaccharide O antigens, released from well-characterized microbes, undergo derivatization and printing in a microarray format. GBPs with putative pathogen recognition activity or sera are incubated with the microarray to examine potential interactions with microbial glycans. Positive interactions are confirmed by examining GBP or sera with intact microbe.