Glycan Node Analysis of Plasma-Derived Extracellular Vesicles

Abstract

Blood plasma is a readily accessible source of extracellular vesicles (EVs), i.e., cell-secreted nanosized carriers that contain various biomolecules, including glycans. Previous studies have demonstrated that glycans play a major role in physiological and pathological processes, and certain plasma glycans have been associated with disease conditions. However, glycome studies have been limited by a lack of analytical techniques with the throughput capacity necessary to study hundreds of clinical samples. This study is the first to characterize the EV plasma glycome based on all major glycan classes. The results based on glycan node analysis revealed, as expected, that plasma-derived EVs have distinct glycan features from donor-matched whole plasma. Specifically, glycan nodes corresponding to those observed in chondroitin sulfate, dermatan sulfate, type I keratan sulfate, and type II keratan sulfate were enriched on EVs. The identification of specific differences in glycan features in plasma vs. plasma-derived EVs is relevant for understanding the physiological role of EVs and as a reference for future diagnostic studies. Additionally, the results indicate that EV glycan nodes do not substantially differ among a small set of healthy donors. These results lay the framework for the further evaluation of all EV glycan classes as diagnostic markers, therapeutic targets, and biologically active components in health and disease.

Glycosylation of Cancer Extracellular Vesicles: Capture Strategies, Functional Roles and Potential Clinical Applications

“…Nevertheless, the N -glycome of exosomes from hepatocellular carcinoma patient samples was characterized using a reverse capture strategy, and the majority of the N -glycans found in EVs from patients with HCC were modified with sialic acids or fucoses, in contrast to the N -glycans identified in EV from healthy samples [ 151 ]. Walker et al also reported significant differences between the glycan profiles identified directly in the plasma or the plasma-derived EVs from the same individuals [ 217 ]. Interestingly, a new integrated analytical platform, termed the integrated magnetic analysis of glycans in extracellular vesicles (iMAGE), was developed to directly analyze the EV glycosylation profile in biological samples.…”

Oxidized-Desialylated Low-Density Lipoprotein Inhibits the Antitumor Functions of Lymphokine Activated Killer Cells

“…Native LDL was desialylated with neuraminidase enzyme and added to the LAK cells at day 5 at 50 μg/ml and incubated for 72 hours. Our glycan node analysis procedure [33][34][35][36][37][38] was used to verify LDL desialylation prior to adding the LDL samples to the LAK cells. An increase in the relative abundance of terminal galactose, a decrease in 3-linked galactose, and a near-complete loss of 6-linked galactose revealed a major decrease in the number of terminal sialic acid residues present on LDL (Supplementary Fig.…”

“…Samples were reconstituted in 450 μl of 10 mM HEPES in 0.15 M NaCl pH 7 buffer each time for a total of 4 spin throughs. To verify complete desialylation of LDL, the glycan node analysis method [33][34][35][36][37][38] was used. Glycan node analysis is a procedure based on glycan methylation analysis by which pooled glycans within whole biological samples are deconstructed in a way that conserves their monosaccharide and linkage information [33].…”

“…Then samples were added to sodium hydroxide beads for 11 minutes to drive permethylation. The rest of the steps for glycan linkage analysis of intact, complex biospecimens are described elsewhere [33][34][35][36][37][38]. An increase in the relative abundance of terminal galactose and a near-complete loss of 6-linked galactose signals (which arise solely due to terminal sialylation of glycans) were used to verify desialylation of LDL [33].…”

A Simple and Quick Method for Loading Proteins in Extracellular Vesicles

“…In recent decades, extracellular vesicles (EVs) have generated considerable interest due to their involvement in physiological and pathological intercellular communication [ 1 , 2 , 3 , 4 ]. EVs are composed of an external bilayer formed by lipids [ 5 ], proteins [ 6 ], and glycans [ 7 ] that enclose an aqueous core with additional bimolecular cargo, such as nucleic acids [ 8 ]. EVs are released by all cells and are classified based on biogenesis, but subtypes also differ in terms of size and composition.…”