Extracellular vesicles (EVs) are a diverse population of complex biological particles with diameters ranging from approximately 20 to 1000 nm. Tremendous interest in EVs has been generated following ...a number of recent, high-profile reports describing their potential utility in diagnostic, prognostic, drug delivery, and therapeutic roles. Subpopulations, such as exosomes, are now known to directly participate in cell-cell communication and direct material transfer. Glycomics, the 'omic' portion of the glycobiology field, has only begun to catalog the surface oligosaccharide and polysaccharide structures and also the carbohydrate-binding proteins found on and inside EVs. The EV glycome undoubtedly contains vital clues essential to better understanding the function, biogenesis, release and transfer of vesicles, however getting at this information is technically challenging and made even more so because of the small physical size of the vesicles and the typically minute yield from physiological-scale biological samples. Vesicle micro-heterogeneity which may be related to specific vesicle origins and functions presents a further challenge. A number of primary studies carried out over the past decade have turned up specific and valuable clues regarding the composition and roles of glycan structures and also glycan binding proteins involved EV biogenesis and transfer. This review explores some of the major EV glycobiological research carried out to date and discusses the potential implications of these findings across the life sciences.
Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been ...characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.
Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in ...detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.
The use of glycan microarrays to study carbohydrate interactions of bacterial cells is of great interest owing to the key roles these interactions play in bacterial colonization and infection of a ...host. In this chapter, the methods to fluorescently stain Gram-positive or Gram-negative bacteria and profiling them for glycan interactions using glycan microarrays are described in detail. The application of the Student's t-test to glycan microarray data using an example data set comparing glycan microarray binding of an Acinetobacter baumannii wild type and mutant strain is also described in step-by-step detail.
Mammalian cell surface lectins mediate many important biological interactions which regulate physiological processes and therefore profiling mammalian cells on glycan microarray is of interest. ...However, many whole mammalian cells are not compatible with glycomics microarray formats and instead cell-derived micelles are prepared and profiled instead of whole cells as they can accurately represent the parental cell glycome. In this chapter, we describe the preparation of cell-derived micelles from mammalian cells, their labeling using a membrane-incorporating dye, and their profiling on a glycan microarray platform.
Glycan microarrays are widely used to elucidate carbohydrate binding specificity and affinity of various analytes including proteins, microorganisms, cells, and tissues. Glycan microarrays comprise a ...wide variety of platforms, differing in surface chemistry, presentation of carbohydrates, carbohydrate valency, and detection strategies, all of which impact on analyte performance. This chapter describes detailed methods for printing neoglycoprotein and glycoprotein microarrays on hydrogel-coated slides and incubation of these glycan microarrays with fluorescently labeled lectins.
Half maximal inhibitory concentration (IC
) is a measurement often used to compare the efficiency of various carbohydrates and their derivatives for inhibition of lectin binding to particular ...ligands. IC
values can be calculated using experimental data from various platforms including enzyme-linked immunosorbent assay- (ELISA-)type microtiter plate assays, isothermal titration calorimetry (ITC), or glycan microarrays. In this chapter, we describe methods to fluorescently label a lectin, to carry out a lectin binding inhibition experiment on glycan microarrays, and to calculate the IC
value of a binding inhibitory molecule using GraphPad Prism software. In the example used to illustrate the method in this chapter, IC
calculation is demonstrated for inhibition of Maackia amurensis agglutinin (MAA) binding to 3'sialyl-N-acetyllactosamine (3SLN) using free lactose.
Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs ...isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) and α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)-Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. The role of these newly identified components in the interaction with the host remains to be unraveled.
Carbohydrates participate in almost every aspect of biology from protein sorting to modulating cell differentiation and cell–cell interactions. To date, the majority of data gathered on glycan ...expression has been obtained via analysis with either anti-glycan antibodies or lectins. A detailed understanding of the specificities of these reagents is critical to the analysis of carbohydrates in biological systems. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins (GBPs). In this study, six different glycan microarray platforms with different modes of glycan presentation were compared using five well-known lectins; concanavalin A, Helix pomatia agglutinin, Maackia amurensis lectin I, Sambucus nigra agglutinin and wheat germ agglutinin. A new method (universal threshold) was developed to facilitate systematic comparisons across distinct array platforms. The strongest binders of each lectin were identified using the universal threshold across all platforms while identification of weaker binders was influenced by platform-specific factors including presentation of determinants, array composition and self-reported thresholding methods. This work compiles a rich dataset for comparative analysis of glycan array platforms and has important implications for the implementation of microarrays in the characterization of GBPs.
Microtiter plate colorimetric assays are widely used for analysis of carbohydrates and glycoconjugates. However, mucins are often not easily detected, as they have low neutral sugar content. We have ...adapted and optimised the periodic acid–Schiff’s reagent (PAS) staining for microtiter plate assay by examining five factors: concentration and volume of periodic acid, oxidation time, volume of Schiff’s reagent, and color development time. This assay requires just 25
μl of sample, utilises standardised Schiff’s reagent, and has decreased assay time (140
min to completion). Seventeen monosaccharides (acidic, neutral, basic, phosphorylated, and deoxy) and four disaccharides were assessed. PAS-positive carbohydrates (amino,
N-acetylamino, deoxy, and certain neutral monosaccharides, and sialic acids) responded linearly within a 10–100
nmol range approximately, which varied for each carbohydrate. The assay response for fetuin and porcine gastric mucin (PGM) was linear up to 150
μg (highest concentration tested), with no response from nonglycosylated protein. A lower response for asialofetuin was observed, but desialylated PGM preparations were similar or higher in response than their sialylated counterparts. The simplicity and low sample consumption of this method make it an excellent choice for screening or quantitation of chromatographic fractions containing carbohydrates and glycoconjugates, especially in the case of mucins.
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