Automated measurement of permethylated serum N-glycans by MALDI–linear ion trap mass spectrometry

The use of N-glycan mass spectrometry for clinical diagnostics requires the development of robust high-throughput profiling methods. Still, structural assignment of glycans requires additional information such as MS² fragmentation or exoglycosidase digestions. We present a setting which combines a MALDI ionization source with a linear ion trap analyzer. This instrumentation allows automated measurement of samples thanks to the crystal positioning system, combined with MSⁿ sequencing options. 2,5-Dihydroxybenzoic acid, commonly used for the analysis of glycans, failed to produce the required reproducibility due to its non-homogeneous crystallization properties. In contrast, α-cyano-4-hydroxycinnamic acid provided a homogeneous crystallization pattern and reproducibility of the measurements. Using serum N-glycans as a test sample, we focused on the automation of data collection by optimizing the instrument settings. Glycan structures were confirmed by MS² analysis. Although sample processing still needs optimization, this method provides a reproducible and high-throughput approach for measurement of N-glycans using a MALDI–linear ion trap instrument.     

Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway

Background

Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses.

Results

The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Le^x and H type 2 antennae in sialylated complex-type N-glycans.

Conclusion

The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.     

Differential Anti-Glycan Antibody Responses in Schistosoma mansoni-Infected Children and Adults Studied by Shotgun Glycan Microarray

Background

Schistosomiasis (bilharzia) is a chronic and potentially deadly parasitic disease that affects millions of people in (sub)tropical areas. An important partial immunity to Schistosoma infections does develop in disease endemic areas, but this takes many years of exposure and maturation of the immune system. Therefore, children are far more susceptible to re-infection after treatment than older children and adults. This age-dependent immunity or susceptibility to re-infection has been shown to be associated with specific antibody and T cell responses. Many antibodies generated during Schistosoma infection are directed against the numerous glycans expressed by Schistosoma. The nature of glycan epitopes recognized by antibodies in natural schistosomiasis infection serum is largely unknown.

Methodology/Principal Findings

The binding of serum antibodies to glycans can be analyzed efficiently and quantitatively using glycan microarray approaches. Very small amounts of a large number of glycans are presented on a solid surface allowing binding properties of various glycan binding proteins to be tested. We have generated a so-called shotgun glycan microarray containing natural N-glycan and lipid-glycan fractions derived from 4 different life stages of S. mansoni and applied this array to the analysis of IgG and IgM antibodies in sera from children and adults living in an endemic area. This resulted in the identification of differential glycan recognition profiles characteristic for the two different age groups, possibly reflecting differences in age or differences in length of exposure or infection.

Conclusions/Significance

Using the shotgun glycan microarray approach to study antibody response profiles against schistosome-derived glycan elements, we have defined groups of infected individuals as well as glycan element clusters to which antibody responses are directed in S. mansoni infections. These findings are significant for further exploration of Schistosoma glycan antigens in relation to immunity.     

Structural Characterization by Multistage Mass Spectrometry (MSn) of Human Milk Glycans Recognized by Human Rotaviruses

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.², describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.     

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

We investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (α1,3- and α1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF³, and GnGnF⁶ CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core α1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies.     

Twoplex 12/13C6 aniline stable isotope and linkage‐specific sialic acid labeling 2D‐LC‐MS workflow for quantitative N‐glycomics

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease‐associated glycan alterations to the quantitative characterization of N‐glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI‐TOF‐MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run‐to‐run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC‐MS^E workflow for the fractionation and relative quantitation of twoplex isotopically labeled N‐linked oligosaccharides using neutral ¹²C₆ and ¹³C₆ aniline (Δmass = 6 Da). Additional linkage‐specific derivatization of sialic acids using 4‐(4,6‐dimethoxy‐1,3,5‐trizain‐2‐yl)‐4‐methylmorpholinium chloride offered simultaneous and advanced in‐depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N‐glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex ¹²C₆/¹³C₆ aniline 2D LC‐MS platform is ideally suited for differential glycomic analysis of structurally complex N‐glycan pools due to combination and analysis of samples in a single LC‐MS injection and the associated minimization in technical variation.     

Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry

The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.     

Site specific <Emphasis Type="Italic">N</Emphasis>-glycan profiling of NeuAc(α2-6)-Gal/GalNAc-binding bark <Emphasis Type="Italic">Sambucus nigra</Emphasis> agglutinin using LC–MS<Superscript>n</Superscript> revealed differential glycosylation

The bark of Sambucus nigra contains a complex mixture of glycoproteins that are characterized as chimeric lectins known as type II ribosome inactivating proteins and holo lectins. These type II ribosome inactivating proteins possess RNA N-glycosidase activity in subunit A and lectin activity associated with subunit B exhibiting distinct sugar specificities to NeuAc(α2-6)-Gal/GalNAc and Gal/GalNAc. In the present study we have determined the N-glycosylation pattern of type II ribosome inactivating protein specific to NeuAc(α2-6)-Gal/GalNAc (Sambucus nigra agglutinin I) by subjecting it to digestion with multiple proteases. The resulting mixture of peptides and N-glycopeptides were analyzed on liquid chromatography coupled to electro spray ionization-iontrap mass spectrometry in MSⁿ mode. MS² of precursor ions was carried out using CID which provided information on glycan sequence. In subsequent MS³ of Y₁/Y_1α ions (peptide + HexNAc)⁺ⁿ of corresponding N-glycopeptides, resulted in the fragmentation of peptide backbone confirming the site of attachment. We observed microheterogeneity in each glycan occupied site with subunit A possessing four N-glycans out of six sites with complex and paucimannose types while subunit B comprises occupancy of two sites with a paucimannose and a high mannose type. The differential N-glycosylation of subunits in SNA is discussed in the context of other type II RIPs glycans.     

Analysis of Site-specific Glycosylation of Renal and Hepatic γ-Glutamyl Transpeptidase from Normal Human Tissue

The cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.     

Serum Glycan Markers for Evaluation of Disease Activity and Prediction of Clinical Course in Patients with Ulcerative Colitis

Background

The aims of this study were to determine the change of whole-serum N-glycan profile in ulcerative colitis (UC) patients and to investigate its clinical utility.

Methods

We collected serum from 75 UC patients at the time of admission and the same number of age/sex-matched healthy volunteers. Serum glycan profile was measured by comprehensive quantitative high-throughput glycome analysis and was compared with disease activity and prognosis.

Results

Out of 61 glycans detected, 24 were differentially expressed in UC patients. Pathway analysis demonstrated that highly sialylated multi-branched glycans and agalactosyl bi-antennary glycans were elevated in UC patients; in addition, the glycan ratio m/z 2378/1914, which also increased in UC, showed the highest Area under Receiver Operating Characteristic curve (0.923) for the diagnosis of UC. Highly sialylated multi-branched glycans and the glycan ratio m/z 2378/1914 were higher in the patients with total colitis, Clinical Activity Index >10, Mayo endoscopic score 3, or a steroid-refractory status. In particular, the glycan ratio m/z 2378/1914 (above median) was an independent prognostic factor for the need for an operation (hazard ratio, 2.67; 95% confidence interval, 1.04–7.84).

Conclusions

Whole-serum glycan profiles revealed that the glycan ratio m/z 2378/1914 and highly sialylated multi-branched glycans increase in UC patients, and are correlated with disease activity. The glycan ratio m/z 2378/1914 was an independent predictive factor of the prognosis of UC.     

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)^1,2. However, understanding the biosynthesis and function of these components remains challenging.Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall³. Furthermore, their composition is also modified during development¹, or in response to environmental cues⁴.In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis⁵. However, relatively few of the biosynthetic genes have been functionally characterized ^4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types⁶. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained⁷. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.Monoclonal antibodies (mAbs)^8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously^9,10,11.Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 & 2)^10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems¹² and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.     

Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host–parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDI-TOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.     

Improved Method for Drawing of a Glycan Map,and the First Page of Glycan Atlas,Which Is a Compilation of Glycan Maps for a Whole Organism

Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC) is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA-) glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.     

Glycan structures and antiviral effect of the structural subunit RvH2 of Rapana hemocyanin

Molluscan hemocyanins are very large biological macromolecules and they act as oxygen-transporting glycoproteins. Most of them are glycoproteins with molecular mass around 9000 kDa. The oligosaccharide structures of the structural subunit RvH2 of Rapana venosa hemocyanin (RvH) were studied by sequence analysis of glycans using MALDI-TOF-MS and tandem mass spectrometry on a Q-Trap mass spectrometer after enzymatical liberation of the N-glycans from the polypeptides. Our study revealed a highly heterogeneous mixture of glycans of the compositions Hex_0-9 HexNAc_2-4 Hex_0-3 Pent_0-3 Fuc_0-3. A novel type of N-glycan, with an internal fucose residue connecting one GalNAc(β1-2) and one hexuronic acid, was detected, as also occurs in subunit RvH1. A glycan with the same structure but with two deoxyhexose residues was observed as a doubly charged ion. Antiviral effects of the native molecules of RvH and also of Helix lucorum hemocyanin (HlH), of their structural subunits, and of the glycosylated functional unit RvH2-e and the non-glycosylated unit RvH2-c on HSV virus type 1 were investigated. Only glycosylated FU RvH2-e exhibits this antiviral activity. The carbohydrate chains of the FU are likely to interact with specific regions of glycoproteins of HSV, through van der Waals interactions in general or with certain amino acid residues in particular. Several clusters of these residues can be identified on the surface of RvH2-e.     

GlycReSoft: A Software Package for Automated Recognition of Glycans from LC/MS Data

Glycosylation modifies the physicochemical properties and protein binding functions of glycoconjugates. These modifications are biosynthesized in the endoplasmic reticulum and Golgi apparatus by a series of enzymatic transformations that are under complex control. As a result, mature glycans on a given site are heterogeneous mixtures of glycoforms. This gives rise to a spectrum of adhesive properties that strongly influences interactions with binding partners and resultant biological effects. In order to understand the roles glycosylation plays in normal and disease processes, efficient structural analysis tools are necessary. In the field of glycomics, liquid chromatography/mass spectrometry (LC/MS) is used to profile the glycans present in a given sample. This technology enables comparison of glycan compositions and abundances among different biological samples, i.e. normal versus disease, normal versus mutant, etc. Manual analysis of the glycan profiling LC/MS data is extremely time-consuming and efficient software tools are needed to eliminate this bottleneck. In this work, we have developed a tool to computationally model LC/MS data to enable efficient profiling of glycans. Using LC/MS data deconvoluted by Decon2LS/DeconTools, we built a list of unique neutral masses corresponding to candidate glycan compositions summarized over their various charge states, adducts and range of elution times. Our work aims to provide confident identification of true compounds in complex data sets that are not amenable to manual interpretation. This capability is an essential part of glycomics work flows. We demonstrate this tool, GlycReSoft, using an LC/MS dataset on tissue derived heparan sulfate oligosaccharides. The software, code and a test data set are publically archived under an open source license.     

Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization–mass spectrometry

One of the most widely used methods for glycan analysis is fluorescent labeling of released glycans followed by hydrophilic interaction chromatography–(ultra-)high-performance liquid chromatography [HILIC–(U)HPLC]. Here, we compare the data obtained by (U)HPLC–fluorescence (FLR) coupled to electrospray ionization–mass spectrometry (ESI–MS) for procainamide and 2-aminobenzamide (2-AB)-labeled N-glycans released from human immunoglobulin G (IgG). Fluorescence profiles from procainamide show comparable chromatographic separation to those obtained for 2-AB but gave higher fluorescence intensity as well as significantly improved ESI efficiency (up to 30 times that of 2-AB). Thus, labeling with procainamide increases the ability to identify minor glycan species that may have significant biological activity.     

Cell Surface-Specific N-Glycan Profiling in Breast Cancer

Aberrant changes in specific glycans have been shown to be associated with immunosurveillance, tumorigenesis, tumor progression and metastasis. In this study, the N-glycan profiling of membrane proteins from human breast cancer cell lines and tissues was detected using modified DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). The N-glycan profiles of membrane proteins were analyzed from 7 breast cancer cell lines and MCF 10A, as well as from 100 pairs of breast cancer and corresponding adjacent tissues. The results showed that, compared with the matched adjacent normal tissue samples, two biantennary N-glycans (NA2 and NA2FB) were significantly decreased (p <0.0001) in the breast cancer tissue samples, while the triantennary glycan (NA3FB) and a high-mannose glycan (M8) were dramatically increased (p = 0.001 and p <0.0001, respectively). Moreover, the alterations in these specific N-glycans occurred through the oncogenesis and progression of breast cancer. These results suggested that the modified method based on DSA-FACE is a high-throughput detection technology that is suited for analyzing cell surface N-glycans. These cell surface-specific N-glycans may be helpful in recognizing the mechanisms of tumor cell immunologic escape and could be potential targets for new breast cancer drugs.     

Insights into substrate recognition and specificity for IgG by Endoglycosidase S2

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins’ catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.     

IgG glycan patterns are associated with type 2 diabetes in independent European populations

BackgroundType 2 diabetes results from interplay between genetic and acquired factors. Glycans on proteins reflect genetic, metabolic and environmental factors. However, associations of IgG glycans with type 2 diabetes have not been described. We compared IgG N-glycan patterns in type 2 diabetes with healthy subjects.MethodsIn the DiaGene study, a population-based case-control study, (1886 cases and 854 controls) 58 IgG glycan traits were analyzed. Findings were replicated in the population-based CROATIA-Korcula-CROATIA-Vis-ORCADES studies (162 cases and 3162 controls), and meta-analyzed. AUCs of ROC-curves were calculated using 10-fold cross-validation for clinical characteristics, IgG glycans and their combination.ResultsAfter correction for extensive clinical covariates, 5 IgG glycans and 13 derived traits significantly associated with type 2 diabetes in meta-analysis (after Bonferroni correction). Adding IgG glycans to age and sex increased the AUC from 0.542 to 0.734. Adding them to the extensive model did not substantially improve the AUC. The AUC for IgG glycans alone was 0.729.ConclusionsSeveral IgG glycans and traits firmly associate with type 2 diabetes, reflecting a pro-inflammatory and biologically-aged state. IgG glycans showed limited improvement of AUCs. However, IgG glycans showed good prediction alone, indicating they may capture information of combined covariates. The associations found may yield insights in type 2 diabetes pathophysiology.General significanceThis work shows that IgG glycomic changes have biomarker potential and may yield important insights into pathophysiology of complex public health diseases, illustrated here for the first time in type 2 diabetes.