Free Glycans Research Articles

Evaluation of ion mobility for the separation of glycoconjugate isomers due to different types of sialic acid linkage, at the intact glycoprotein, glycopeptide and glycan level

The study of protein glycosylation can be regarded as an intricate but very important task, making glycomics one of the most challenging and interesting, albeit under-researched, type of “omics” science. Complexity escalates remarkably when considering that carbohydrates can form severely branched structures with many different constituents, which often leads to the formation of multiple isomers. In this regard, ion mobility (IM) spectrometry has recently demonstrated its power for the separation of isomeric compounds. In the present work, the potential of traveling wave IM (TWIMS) for the separation of isomeric glycoconjugates was evaluated, using mouse transferrin (mTf) as model glycoprotein. Particularly, we aim to assess the performance of this platform for the separation of isomeric glycoconjugates due to the type of sialic acid linkage, at the intact glycoprotein, glycopeptide and glycan level. Straightforward separation of isomers was achieved with the analysis of released glycans, as opposed to the glycopeptides which showed a more complex pattern. Finally, the developed methodology was applied to serum samples of mice, to investigate its robustness when analyzing real complex samples. Biological significanceIon mobility mass spectrometry is a promising analytical technique for the separation of glycoconjugate isomers due to type of sialic acid linkage. The impact of such a small modification in the glycan structure is more evident in smaller analytes, reason why the analysis of free glycans was easier compared to the intact protein or the glycopeptides. The established methodology could be regarded as starting point in the separation of highly decorated glycoconjugates. This is an important topic nowadays, as differences in the abundance of some glycan isomers could be the key for the early diagnosis, control or differentiation of certain diseases, such as inflammation or cancer.

Solid-Phase Chemical Modification for Sialic Acid Linkage Analysis: Application to Glycoproteins of Host Cells Used in Influenza Virus Propagation.

Differentiation between the sialyl linkages is often critical to understanding biological consequence. Here we present a facile method for determining these linkages in glycans. Analysis of sialic acids is challenging due to their labile nature during sample preparation and ionization. Derivatization is often required via chemical reaction. Amidation derivatizes all sialic acids regardless of linkage, while esterification enables differentiation between α2,3-linked and α2,6-linked sialic acids. Reactions have been primarily performed on free glycans in solution but have been recently adapted to solid-phase providing unique advantages such as simplified sample preparation, improved yield, and high throughput applications. Here, we immobilized glycoproteins on resin via reductive amination, modified α2,6-linked sialic acids through ethyl esterification, and α2,3-linked sialic acids via amidation. N-glycans and O-glycans were released via enzyme and chemical reactions. The method was applied for analysis of three different MDCK cell lines used for influenza propagation and where distributions of α2,3 and α2,6 sialic acids are critical for cell performance. Linkage specific distribution of these sialic acids was quantitatively determined and unique for each cell line. Our study demonstrates that protein sialylation can be reliably and quantitatively characterized in terms of sialic acid linkage of each glycan using the solid-phase esterification/amidation strategy.

Biotinylated N-Acetyllactosamine- and N,N-Diacetyllactosamine-Based Oligosaccharides as Novel Ligands for Human Galectin-3.

Galectin inhibitor design is an emerging research field due to the involvement of galectins in cancer. Galectin-3, in particular, plays an important role in tumor progression. To generate inhibitors, modifications of the glycan structure can be introduced. Conjugation of hydrophobic compounds to saccharides has proven to be promising as increased binding of galectin-3 can be observed. In the present study, we report on neo-glycans carrying hydrophobic biotin as novel ligands for human galectin-3. We modified N-acetyllactosamine- and N,N-diacetyllactosamine-based tetrasaccharides at the C6-position of the terminal saccharide unit using selective enzymatic oxidation and subsequent chemical conjugation of biotinamidohexanoic acid hydrazide. These neo-glycans were much better bound by galectin-3 than the unmodified counterparts. High selectivity for galectin-3 over galectin-1 was also proven. We generated multivalent neo-glycoproteins by conjugation of neo-glycans to bovine serum albumin showing high affinity for galectin-3. Compared to non-biotinylated neo-glycoproteins, we achieved high binding levels of galectin-3 with a lesser amount of conjugated neo-glycans. Multivalent ligand presentation of neo-glycoproteins significantly increased the inhibitory potency towards galectin-3 binding to asialofetuin when compared to free monovalent glycans. Our findings show the positive impact of 6-biotinylation of tetrasaccharides on galectin-3 binding, which broadens the recent design approaches for producing high-affinity ligands.

Straightforward Synthesis of N-Glycan Polymers from Free Glycans via Cyanoxyl Free Radical-Mediated Polymerization.

We report a straightforward synthesis of N-glycan polymers from free glycans via glycosylamine intermediates followed by acrylation and polymerization via cyanoxyl-mediated free radical polymerization (CMFRP) in one-pot fashion. No protection and deprotection were used in either glycomonomer or glycopolymer synthesis. A typical synthetic procedure for N-glycan polymers from free monosaccharide and disaccharide, Glc, Gal, Man, GlcNAc, and Lac, was demonstrated. In addition, enzymatic sialylation of the Lac-containing N-glycan polymers and their anti-influenza virus hemagglutination activities were investigated.

Human milk and mucosal lacto- and galacto-N-biose synthesis by transgalactosylation and their prebiotic potential in Lactobacillus species.

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69±4 and 71±1% of o-nitrophenyl-β-D-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7±0.2g/l and for GNB was 10.8±0.3g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants.

Fast Characterization of Fc-Containing Proteins by Middle-Down Mass Spectrometry Following IdeS Digestion

The rapid growth of biotherapeutics (monoclonal antibodies and antibody derivatives) demands improved techniques for their quality control and batch-to-batch study. Traditionally, the top-down and bottom-up mass spectrometric approaches may be good options, but they have their share of drawbacks. The middle-down technology has unique superiorities for fast characterization of very large proteins. In this article, we report a systematic approach to characterize Fc-containing proteins (antibody, Fc fusion protein, and antibody–drug conjugate) by middle-down mass spectrometry following IdeS proteolytic digestion. We characterized the main protein modifications including glycosylation, glycation, and other modifications with a relatively small proportion, and results were consistent with free glycan profiling analysis. Meanwhile, the O-acetylated sialic acid modification of cetuximab was reported for the first time; its potential roles in biotherapeutics need further study. The middle-down technology following IdeS proteolytic digestion could be used not only for analyzing the modifications of Fc-containing proteins at the subunit level but also for characterizing the potential modifications at early development stages.

Human DC-SIGN binds specific human milk glycans.

Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBP) expressed by dendritic cells (DCs) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and sialic acid-binding immunoglobulin-like lectins (Siglecs) expressed by DCs for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglec-5 and Siglec-9 showed weak binding to a few glycans. By contrast, most hGBP bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2'-fucosyl-lactose (2'-FL) and 3-fucosyl-lactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2'-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2'-FL had an IC50 of ∼1mM for DC-SIGN, which is within the physiological concentration of 2'-FL in human milk. These results demonstrate that DC-SIGN among the many hGBP expressed by DCs binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant.

Complement Factor H and Simian Virus 40 bind the GM1 ganglioside in distinct conformations.

Mammalian cell surfaces are decorated with a variety of glycan chains that orchestrate development and defense and are exploited by pathogens for cellular attachment and entry. While glycosidic linkages are, in principle, flexible, the conformational space that a given glycan can sample is subject to spatial and electrostatic restrictions imposed by its overall chemical structure. Here, we show how the glycan moiety of the GM1 ganglioside, a branched, monosialylated pentasaccharide that serves as a ligand for various proteins, undergoes differential conformational selection in its interactions with different lectins. Using STD NMR and X-ray crystallography, we found that the innate immune regulator complement Factor H (FH) binds a previously not reported GM1 conformation that is not compatible with the GM1-binding sites of other structurally characterized GM1-binding lectins such as the Simian Virus 40 (SV40) capsid. Molecular dynamics simulations of the free glycan in explicit solvent on the 10 μs timescale reveal that the FH-bound conformation nevertheless corresponds to a minimum in the Gibbs free energy plot. In contrast to the GM1 conformation recognized by SV40, the FH-bound GM1 conformation is associated with poor NOE restraints, explaining how it escaped(1)H-(1)H NOE-restrained modeling in the past and highlighting the necessity for ensemble representations of glycan structures.

Exonuclease TREX1 also Has a Sweet Tooth

TREX1 regulates innate immune responses by counteracting DNA accumulation in the cytosol. In this issue of Immunity, Hasan etal. (2015) show that TREX1 also safeguards the cell against free glycan build-up in the endoplasmic reticulum, thereby preventing glycan-induced inflammation.

Cytosolic Nuclease TREX1 Regulates Oligosaccharyltransferase Activity Independent of Nuclease Activity to Suppress Immune Activation

TREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mutations are associated with autoimmune and autoinflammatory diseases. Biallelic mutations abrogating DNase activity cause autoimmunity by allowing immunogenic self-DNA to accumulate, but it is unknown how dominant frameshift (fs) mutations that encode DNase-active but mislocalized proteins cause disease. We found that the TREX1 C terminus suppressed immune activation by interacting with the ER oligosaccharyltransferase (OST) complex and stabilizing its catalytic integrity. C-terminal truncation of TREX1 by fs mutations dysregulated the OST complex, leading to free glycan release from dolichol carriers, as well as immune activation andautoantibody production. A connection between OST dysregulation and immune disorders was demonstrated in Trex1(-/-) mice, TREX1-V235fs patient lymphoblasts, and TREX1-V235fs knock-in mice. Inhibiting OST with aclacinomycin corrects the glycan and immune defects associated with Trex1 deficiency or fs mutation. This function of the TREX1 C terminus suggests a potential therapeutic option for TREX1-fs mutant-associated diseases.

A Milk‐Oriented Microbiota (MOM) in Infants: Lessons from Your Other MOM

Human milk contains numerous components that shape the microbial content of the developing infant gastrointestinal tract. A prominent feature of milk is an array of complex free glycans and glycoconjugates that serve a passive immune function by deflecting pathogens while simultaneously enriching a protective microbiota often dominated by bifidobacteria. Human milk glycan structures are also host‐specific and these structural variations differentially influence the composition and function of the microbiota populations enriched. Infant‐borne bifidobacteria utilize human milk glycans as a carbon source for growth however the specific deconstruction mechanisms vary by glycan‐type. These bifidobacteria contain glycosidases and transport systems required to utilize milk glycans either by internalizing large glycans or degrading them extracellularly followed by import and catabolism of specific components. Growth on milk glycans also enhances bifidobacterial interaction with the host through direct and indirect routes—increasing bifidobacterial binding to epithelial cells and producing short chain fatty acids that beneficially modulate host immunity and barrier function. In aggregate, these studies suggest a co‐evolutionary relationship between mammalian milk glycans, infant‐borne bifidobacteria and the infant host that has enabled a programmed enrichment of a protective microbiota during a critical stage of infant development. Importantly, analysis of this natural system serves as a key model for design of probiotic and prebiotic‐based manipulation of the gut microbiota in a range of health settings.

Generation and degradation of free asparagine-linked glycans.

Asparagine (N)-linked protein glycosylation, which takes place in the eukaryotic endoplasmic reticulum (ER), is important for protein folding, quality control and the intracellular trafficking of secretory and membrane proteins. It is known that, during N-glycosylation, considerable amounts of lipid-linked oligosaccharides (LLOs), the glycan donor substrates for N-glycosylation, are hydrolyzed to form free N-glycans (FNGs) by unidentified mechanisms. FNGs are also generated in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins during ER-associated degradation. FNGs derived from LLOs and misfolded glycoproteins are eventually merged into one pool in the cytosol and the various glycan structures are processed to a near homogenous glycoform. This article summarizes the current state of our knowledge concerning the formation and catabolism of FNGs.

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses

Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MS(n) analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MS(n) are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures.

Significant other half of a glycoconjugate: contributions of scaffolds to lectin-glycoconjugate interactions.

The glycan epitopes of natural and synthetic glycoconjugates exist as covalent attachments of well-defined inner structures or scaffolds. Macromolecules such as proteins, peptides, lipids, and saccharides and synthetic structures serve as scaffolds of glycoconjugates. It is generally perceived that the biological activities of glycoconjugates are determined mainly by the attached glycans, while the seemingly inert inner scaffolds play a passive role by providing physical support to the attached glycan epitopes. However, our data show that scaffolds actively influence lectin recognition and can potentially modulate lectin-mediated signaling properties of glycoconjugates. Through in vitro experiments, we found that the scaffolds significantly altered the thermodynamic binding properties of the covalently attached glycan epitopes. When a free glycan was attached to a scaffold, its lectin binding entropy became more positive. The level of positive entropic gain was dependent on the types of scaffolds tested. For example, protein scaffolds of glycoproteins were found to generate more positive entropy of binding than synthetic scaffolds. Certain scaffolds were found to have limiting effects on glycoconjugate affinity. We also found that scaffold-bearing glycans with a similar affinity or an identical valence demonstrated different kinetics of lattice formation with lectins, when the scaffold structures were different. Our data support the view that scaffolds of glycoconjugates (i) help the covalently attached glycans become more spontaneous in lectin binding and (ii) help diversify the lattice forming or cross-linking properties of glycoconjugates.

Recent Advances in Molecular Recognition Based on Nanoengineered Platforms

Nanoparticles and nanoengineered platforms have great potential for technologies involving biomoleuclar detection or cell-related biosensing, and have provided effective chemical interfaces for molecular recognition. Typically, chemists work on the modification of synthetic polymers or macromolecules, which they link to the nanoparticles by covalent or noncovalent approaches. The motivation for chemical modification is to enhance the selectivity and sensitivity, and to improve the biocompatibility for the in vivo applications. In this Account, we present recent advances in the development and application of chemical interfaces for molecular recognition for nanoparticles and nanoengineered platforms, in particular single-walled carbon nanotubes (SWNTs). We discuss emerging approaches for recognizing small molecules, glycosylated proteins, and serum biomarkers. For example, we compare and discuss detection methods for ATP, NO, H2O2, and monosaccharides for recent nanomaterials. Fluorometric detection appears to have great potential for quantifying concentration gradients and determining their location in living cells. For macromolecular detection, new methods for glycoprofiling using such interfaces appear promising, and benefit specifically from the potential elimination of cumbersome labeling and liberation steps during conventional analysis of glycans, augmenting the currently used mass spectrometry (MS), capillary electrophoresis (CE), and liquid chromatography (LC) methods. In particular, we demonstrated the great potential of fluorescent SWNTs for glycan-lectin interactions sensing. In this case, SWNTs are noncovalently functionalized to introduce a chelated nickel group. This group provides a docking site for the His-tagged lectin and acts as the signal modulator. As the nickel proximity to the SWNT surface changes, the fluorescent signal is increased or attenuated. When a free glycan or glycosylated probe interacts with the lectin, the signal increases and they are able to obtain loading curves similar to surface plasmon resonance measurements. They demonstrate the sensitivity and specificity of this platform with two higher-affined glycan-lectin pairs: fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. Lastly, we discuss how developments in protein biomarker detection in general are benefiting specifically from label-free molecular recognition. Electrical field effect transistors, chemi-resistive and fluorometric nanosensors based on various nanomaterials have demonstrated substantial progress in recent years in addressing this challenging problem. In this Account, we compare the balance between sensitivity, selectivity, and nonspecific adsorption for various applications. In particular, our group has utilized SWNTs as fluorescence sensors for label-free protein-protein interaction measurements. In this assay, we have encapsulated each nanotube in a biocompatible polymer, chitosan, which has been further modified to conjugate nitrilotriacetic acid (NTA) groups. After Ni(2+) chelation, NTA Ni(2+) complexes bind to his-tagged proteins, resulting in a local environment change of the SWNT array, leading to optical fluorescence modulation with detection limit down to 100 nM. We have further engineered the platform to monitor single protein binding events, with an even lower detection limit down to 10 pM.

2′-fucosyllactose: an abundant, genetically determined soluble glycan present in human milk

Lactose is the preeminent soluble glycan in milk and a significant source of energy for most newborn mammals. Elongation of lactose with additional monosaccharides gives rise to a varied repertoire of free soluble glycans such as 2'-fucosyllactose (2'-FL), which is the most abundant oligosaccharide in human milk. In infants, 2'-FL is resistant to digestion and reaches the colon where it is partially fermented, behaving as soluble prebiotic fiber. Evidence also suggests that portions of small soluble milk glycans, including 2'-FL, are absorbed, thus raising the possibility of systemic biological effects. 2'-FL bears an epitope of the Secretor histo-blood group system; approximately 70-80% of all milk samples contain 2'-FL, since its synthesis depends on a fucosyltransferase that is not uniformly expressed. The fact that some infants are not exposed to 2'-FL has helped researchers to retrospectively probe for biological activities of this glycan. This review summarizes the attributes of 2'-FL in terms of its occurrence in mammalian phylogeny, its postulated biological activities, and its variability in human milk.

Aiming at the sweet side of cancer: Aberrant glycosylation as possible target for personalized-medicine

One of the frontiers in cancer personalized-medicine aims at glycosylation. Cells are covered with a dense sugar coat of glycolipids, glycoproteins and free glycans. In cancer, the characteristic cell surface glycosylation is frequently transformed due to altered expression of glycan-modifying enzymes. This often leads to aberrant expression of sialic acids (Sia) that cap glycan-chains. Additionally, dietary intake of the non-human Sia N-glycolylneuraminic acid (Neu5Gc) leads to natural metabolic-glycoengineering of human carcinomas that accumulate and express Neu5Gc. This Sia provokes a polyclonal anti-Neu5Gc xeno-autoantibodies response that can exacerbate cancer. This review highlights cancer-associated changes in Sia expression and their potential for personalized-theranostics.

Letter to the Glycoforum: Improved Protocols For Preparing Lipid-Linked And Related Saccharides For Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)

Our laboratory has used Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) to analyze many saccharides related to the dolichol pathway (Gao and Lehrman, 2002, 2003; Lehrman, 2007). This includes the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol, LLO intermediates, mannose-P-dolichol, glucose-P-dolichol, N-glycans, free glycans, sugar mono- and bis-phosphates, and nucleotide-sugars. Some examples of the variety of saccharides that can be studied are given in (Gao and Lehrman, 2002; Gao et al., 2011; Gao et al., 2008). In addition to these studies with mammalian cultured cells and tissues, we used FACE to identify saccharides from an assortment of sources including Danio rerio (Cline et al., 2012), Saccharomyces cerevisiae (Rush et al., 2009), Trypanosoma brucei (Jelk et al., 2013), and Arabidopsis thaliana (Amtmann, A., unpublished). Since publication of a comprehensive method (Gao and Lehrman, 2006) we identified problematic steps during the preparation of samples, prior to gel electrophoresis. Here, as Supplemental Information, we provide a revised protocol (in lab bench-ready format) for preparing dolichol pathway samples for FACE. One vexing problem in particular is solved with this new protocol. Previously, it was necessary at different stages to dry proteinacious cellular residues containing saccharides of interest. Great care was necessary to avoid overdrying, to prevent these residues from forming tough unworkable aggregates refractory to extraction. The extractions were further hampered by variable losses of residue at each step. These and other technical problems have now been solved with a single-step extraction method2 in which three distinct phases are generated (each enriched with a different group of saccharides), and by eliminating the need to dry proteinacious cellular residues. Various members of our group have successfully used different aspects of this new protocol, which we conclude is a significant improvement over our earlier methods. We thank Dan Beriault for very helpful comments on the text. Supported by NIH grant GM038545.

EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein

Many bacteria have evolved ways to interact with glycosylation functions of the immune system of their hosts. Streptococcus pyogenes [GAS (group A Streptococcus)] secretes the enzyme EndoS that cleaves glycans on human IgG and impairs the effector functions of the antibody. The ndoS gene, encoding EndoS, has, until now, been thought to be conserved throughout the serotypes. However, in the present study, we identify EndoS2, an endoglycosidase in serotype M49 GAS strains. We characterized EndoS2 and the corresponding ndoS2 gene using sequencing, bioinformatics, phylogenetic analysis, recombinant expression and LC–MS analysis of glycosidic activity. This revealed that EndoS2 is present exclusively, and highly conserved, in serotype M49 of GAS and is only 37% identical with EndoS. EndoS2 showed endo-β-N-acetylglucosaminidase activity on all N-linked glycans of IgG and on biantennary and sialylated glycans of AGP (α1-acid glycoprotein). The enzyme was found to act only on native IgG and AGP and to be specific for free biantennary glycans with or without terminal sialylation. GAS M49 expression of EndoS2 was monitored in relation to carbohydrates present in the culture medium and was linked to the presence of sucrose. We conclude that EndoS2 is a unique endoglycosidase in serotype M49 and differs from EndoS of other GAS strains by targeting both IgG and AGP. EndoS2 expands the repertoire of GAS effectors that modify key glycosylated molecules of host defence.

Novel Cleavage of Reductively Aminated Glycan-Tags by N-Bromosuccinimide to Regenerate Free, Reducing Glycans

Glycans that are fluorescently tagged by reductive amination have been useful for functional glycomic studies. However, the existing tags can introduce unwanted properties to the glycans and complicate structural and functional studies. Here, we describe a facile method using N-bromosuccinimide (NBS) to remove the tags and efficiently regenerate free reducing glycans. The regenerated free reducing glycans can be easily analyzed by routine mass spectrometry or retagged with different tags for further studies. This new method can be used to efficiently remove a variety of fluorescent tags installed by reductive amination, including 2-aminobenzoic acid and 2-aminopyridine. NBS treatment essentially transforms the commonly used 2-aminobenzoic linkage to a cleavable linkage. It can be used to cleave printed glycans from microarrays and cleave neoglycopeptides containing a 2-aminobenzoic linker.