Generation and characterization of β1,2-gluco-oligosaccharide probes from Brucella abortus cyclic β-glucan and their recognition by C-type lectins of the immune system.

Hongtao Zhang,Robert A Childs,Andrés E Ciocchini,Daniel A Mitchell,Ten Feizi,Yan Liu,Yibing Zhang,Barbara Mulloy,Angelina S Palma,Wengang Chai,Wilfried Weigel,Leticia S Guidolin

doi:10.1093/glycob/cww041

Abstract

The β1,2-glucans produced by bacteria are important in invasion, survival and immunomodulation in infected hosts be they mammals or plants. However, there has been a lack of information on proteins which recognize these molecules. This is partly due to the extremely limited availability of the sequence-defined oligosaccharides and derived probes for use in the study of their interactions. Here we have used the cyclic β1,2-glucan (CβG) of the bacterial pathogen Brucella abortus, after removal of succinyl side chains, to prepare linearized oligosaccharides which were used to generate microarrays. We describe optimized conditions for partial depolymerization of the cyclic glucan by acid hydrolysis and conversion of the β1,2-gluco-oligosaccharides, with degrees of polymerization 2–13, to neoglycolipids for the purpose of generating microarrays. By microarray analyses, we show that the C-type lectin receptor DC-SIGNR, like the closely related DC-SIGN we investigated earlier, binds to the β1,2-gluco-oligosaccharides, as does the soluble immune effector serum mannose-binding protein. Exploratory studies with DC-SIGN are suggestive of the recognition also of the intact CβG by this receptor. These findings open the way to unravelling mechanisms of immunomodulation mediated by β1,2-glucans in mammalian systems.

Highlights

Glucan polysaccharides are of biomedical interest because of their involvement in mechanisms of pathogen recognition and modulation of the immune system (Brown and Gordon 2003; Chen and Seviour 2007)
With the advent of oligosaccharide microarray technologies (Fukui et al 2002; Blixt et al 2004; Feizi and Chai 2004; Rillahan and Paulson 2011; Palma et al 2014), it is possible to explore interactions with proteins using oligosaccharide probes generated from a range of oligosaccharide sequences that can be prepared after partial depolymerization of the polysaccharides (Pedersen et al 2012; Palma et al 2015)
Dectin-1 belongs to the family of C-type lectin-like proteins; it lacks the canonical amino acid residues for ligating calcium, required for carbohydrate binding in classical C-type lectins (Drickamer and Taylor, 2015)

Summary

Introduction

Glucan polysaccharides are of biomedical interest because of their involvement in mechanisms of pathogen recognition and modulation of the immune system (Brown and Gordon 2003; Chen and Seviour 2007). The microarray system based on the neoglycolipid (NGL) technology (Chai et al 2003) for preparing lipid-linked oligosaccharide probes for immobilization and binding studies, lends itself well to analyses of glucan sequences as recognition structures within polysaccharides. This is the basis of the “designer” microarray approach (Palma et al 2006, 2014; Gao et al 2014) whereby microarrays are generated from oligosaccharides released from the targeted macromolecules; oligosaccharides bound by recognition proteins may be isolated for characterization. Designer microarrays (Palma et al 2006) generated from oligosaccharide fractions derived from fungal-type glucans (Brown and Gordon, 2001; Brown et al 2003), established that (i) dectin-1 is a calcium-independent carbohydratebinding protein and (ii) linear β1,3-linked glucose sequences with degrees of polymerization (DP) 10 or longer are required for detection of binding

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Results

Conclusion