Chemistry of natural glycan microarrays

doi:10.1016/j.cbpa.2014.01.001

Current Opinion in Chemical Biology

Volume 18, February 2014, Pages 70-77

https://doi.org/10.1016/j.cbpa.2014.01.001 Get rights and content

Highlights

•
Natural glycan microarray is a crucial technology for protein/glycan interactions.
•
Expanding natural glycan libraries is the most important task for the field.
•
Chemo-enzymatic synthesis applied to natural glycans will expand glycan array diversity.
•
Presentation of glycans could affect sensitivity of detection of GBP binding.

Glycan microarrays have become indispensable tools for studying protein–glycan interactions. Along with chemo-enzymatic synthesis, glycans isolated from natural sources have played important roles in array development and will continue to be a major source of glycans. N-glycans and O-glycans from glycoproteins, and glycans from glycosphingolipids (GSLs) can be released from corresponding glycoconjugates with relatively mature methods, although isolation of large numbers and quantities of glycans is still very challenging. Glycosylphosphatidylinositol (GPI) anchors and glycosaminoglycans (GAGs) are less represented on current glycan microarrays. Glycan microarray development has been greatly facilitated by bifunctional fluorescent linkers, which can be applied in a ‘Shotgun Glycomics’ approach to incorporate isolated natural glycans. Glycan presentation on microarrays may affect glycan binding by GBPs, often through multivalent recognition by the GBP.

Introduction

Glycoproteins, proteoglycans, and glycolipids within the glycocalyx, defined as the assortment of complex glycoconjugates on the plasma membrane and associated with the surface of an animal cell, are involved in myriad molecular interactions. Functional glycomics is the systematic study of the structurally and functionally important glycans, which often involve glycan-binding proteins (GBPs) that recognize specific glycan sequences and thereby ‘decode’ the complex structural information in glycans. A major technical breakthrough in glycosciences that facilitated decoding glycan functions was the development of printed glycan microarrays, in which many defined glycan structures are simultaneously presented to GBPs or microorganisms including viruses and bacteria. While the applications of glycan microarrays have been extensively reviewed in the last decade [1•, 2, 3, 4, 5], this article provides some highlights and perspectives of the chemistry, current challenges, and promises of natural glycan microarray technology.

Section snippets

Glycan libraries for glycan microarrays

Glycan microarrays are essentially a simultaneous presentation of a library of defined glycans in a resolvable pattern for the purpose of defining binding specificities of GBPs. GBP specificities are determined by comparing binding to all glycans presented on the microarray, including bound and unbound glycans and deciphering the key glycan determinants associated with the highest degree of binding [4]. Therefore, the number and diversity of defined glycans on a microarray are paramount for

Presentation of glycans on glycan microarrays

The analyses of hundreds of GBPs on the CFG defined glycan microarray over the past decade have generated interesting and paradigm-changing data (www.functionalglycomics.org under ‘CFG paradigm pages’ and ‘CFG Library’). For example, previously influenza viruses were thought to simply distinguish α2-6 from α2-3 sialylated glycans, but glycan microarray studies indicated that each virus strain has specific and unique glycan specificity, and that sialic acid and its linkages are necessary, but

Conclusions

Defined glycan microarrays are generally used to determine the glycan-binding specificity of GBPs, and the effectiveness of any array is more dependent on the number and diversity of the glycans on the array than other parameters such as density or presentation. Since little is known about the presentation of glycans in nature, it is not possible to define a single best presentation or density of glycans on an array. Nevertheless, it will certainly be important to carry out well-designed

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

This work was supported by P41 BTRC grant (P41GM10369) (to R.D.C), a EUREKA Grant (GM085448) (to D.F.S), and a Defense Advanced Research Projects Agency Grant HR0011-10-00.

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Glycan-mediated molecular interactions in bacterial pathogenesis
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Glycans are expressed on the surface of nearly all host and bacterial cells. Not surprisingly, glycan-mediated molecular interactions play a vital role in bacterial pathogenesis and host responses against pathogens. Glycan-mediated host–pathogen interactions can benefit the pathogen, host, or both. Here, we discuss (i) bacterial glycans that play a critical role in bacterial colonization and/or immune evasion, (ii) host glycans that are utilized by bacteria for pathogenesis, and (iii) bacterial and host glycans involved in immune responses against pathogens. We further discuss (iv) opportunities and challenges for transforming these research findings into more effective antibacterial strategies, and (v) technological advances in glycoscience that have helped to accelerate progress in research. These studies collectively offer valuable insights into new perspectives on antibacterial strategies that may effectively tackle the drug-resistant pathogens that are rapidly spreading globally.
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Absolute quantitation of IgG-1 Fc-glycosylation, which is crucial for the clinical practice of glyco-biomarkers and quality control of biopharmaceuticals, has been hindered by the lack of glycopeptide standards. In this study, eleven high abundant IgG-1 Fc-glycopeptides with definite peptide sequences and glycoforms were purified from commercial IgG protein by using two-dimensional hydrophilic interaction liquid chromatographic system. Based on the acquired glycopeptide standards, an absolute quantitation strategy was developed to determine the concentrations of 11 target IgG-1 glycopeptides from pooled human sera. A wide range of Fc-glycopeptide concentrations from 0.60 to 17.61 nmol mL⁻¹ was achieved with excellent accuracy and reproducibility from pooled human sera IgG-1. Compared to conventional relative quantitation, this strategy provides more accurate distribution profiles of 11 high abundant Fc-glycopeptides and degree of glycosylation from pooled human sera IgG-1.
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To aid in generating complex and diverse natural glycan libraries for functional glycomics, more efficient and reliable methods are needed to derivatize glycans. Here we present our development of a reversible, cleavable bifunctional linker 3-(methoxyamino)propylamine (MAPA). As the fluorenylmethyloxycarbonate (Fmoc) version (F-MAPA), it is highly fluorescent and efficiently derivatizes free reducing glycans to generate closed-ring derivatives that preserve the structural integrity of glycans. A library of glycans were derivatized and used to generate a covalent glycan microarray using N-hydroxysuccinimide derivatization. The array was successfully interrogated by a variety of lectins and antibodies, demonstrating the importance of closed-ring chemistry. The glycan derivatization was also performed at large scale using milligram quantities of glycans and excess F-MAPA, and the reaction system was successfully recycled up to five times, without an apparent decrease in conjugation efficiency. The MAPA-glycan is also easy to link to protein to generate neoglycoproteins with equivalent glycan densities. Importantly, the MAPA linker can be reversibly cleaved to regenerate free reducing glycans for detailed structural analysis (catch-and-release), often critical for functional studies of undefined glycans from natural sources. The high conjugation efficiency, bright fluorescence, and reversible cleavage of the linker enable access to natural glycans for functional glycomics.
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View full text

Chemistry of natural glycan microarrays

Highlights

Introduction

Section snippets

Glycan libraries for glycan microarrays

Presentation of glycans on glycan microarrays

Conclusions

References and recommended reading

Acknowledgements

Methods Enzymol

Methods Mol Biol

Glycoconj J

J Biol Chem

Methods Enzymol

Anal Chem

Proteomics

Curr Protoc Mol Biol

Anal Biochem

Proc Natl Acad Sci USA

Anal Biochem

Anal Biochem

PLoS ONE

Angew Chem Int Ed Engl

Methods Mol Biol

Nat Rev Mol Cell Biol

Chem Commun

Biosens Bioelectron

J Am Chem Soc

Glycan microarrays for decoding the glycome

Annu Rev Biochem

Carbohydrate microarray technology for functional glycomics

Curr Chem Biol

Glycan arrays: recent advances and future challenges

Curr Opin Chem Biol

The repertoire of glycan determinants in the human glycome

Mol Biosyst

Toward automated oligosaccharide synthesis

Angew Chem Int Ed

A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans

Science

General microarray technique for immobilization and screening of natural glycans

Anal Chem

Generation of a natural glycan microarray using 9-fluorenylmethyl chloroformate (FmocCl) as a cleavable fluorescent tag

Anal Biochem

Shotgun glycomics: a microarray strategy for functional glycomics

Nat Methods

Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays

ACS Chem Biol

Novel fluorescent glycan microarray strategy reveals ligands for galectins

Chem Biol

Versatile fluorescent derivatization of glycans for glycomic analysis

Nat Methods

Analysis of O-glycans as 9-fluorenylmethyl derivatives and its application to the studies on glycan array

Anal Chem

Novel cleavage of reductively aminated glycan-tags by N-bromosuccinimide to regenerate free, reducing glycans

ACS Chem Biol

Differential anti-glycan antibody responses in Schistosoma mansoni-infected children and adults studied by shotgun glycan microarray

PLoS Negl Trop Dis

Glycan microarray analysis of P-type lectins reveals distinct phosphomannose glycan recognition

J Biol Chem

A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses

J Biol Chem

The glycan-binding properties of the cation-independent mannose 6-phosphate receptor are evolutionary conserved in vertebrates

Glycobiology