Publications
86 results found
Lundstrøm J, Gillon E, Chazalet V, et al., 2024, Elucidating the glycan-binding specificity and structure of Cucumis melo agglutinin, a new R-type lectin., Beilstein J Org Chem, Vol: 20, Pages: 306-320, ISSN: 1860-5397
Plant lectins have garnered attention for their roles as laboratory probes and potential therapeutics. Here, we report the discovery and characterization of Cucumis melo agglutinin (CMA1), a new R-type lectin from melon. Our findings reveal CMA1's unique glycan-binding profile, mechanistically explained by its 3D structure, augmenting our understanding of R-type lectins. We expressed CMA1 recombinantly and assessed its binding specificity using multiple glycan arrays, covering 1,046 unique sequences. This resulted in a complex binding profile, strongly preferring C2-substituted, beta-linked galactose (both GalNAc and Fuca1-2Gal), which we contrasted with the established R-type lectin Ricinus communis agglutinin 1 (RCA1). We also report binding of specific glycosaminoglycan subtypes and a general enhancement of binding by sulfation. Further validation using agglutination, thermal shift assays, and surface plasmon resonance confirmed and quantified this binding specificity in solution. Finally, we solved the high-resolution structure of the CMA1 N-terminal domain using X-ray crystallography, supporting our functional findings at the molecular level. Our study provides a comprehensive understanding of CMA1, laying the groundwork for further exploration of its biological and therapeutic potential.
Clark LC, Atkin KE, Whelan F, et al., 2023, Staphylococcal Periscope proteins Aap, SasG, and Pls project noncanonical legume-like lectin adhesin domains from the bacterial surface, Journal of Biological Chemistry, Vol: 299, Pages: 1-11, ISSN: 0021-9258
Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG, and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonization; they comprise a repetitive region ("B region") and an N-terminal host colonization domain within the "A region," predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG, and Pls) form elongated "stalks" that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap, and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain had been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, while the Aap, SasG, and Pls lectin domains bind a metal ion, they lack the nonproline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of noncanonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.
Sorin MN, Di Maio A, Silva LM, et al., 2023, Structural and functional analysis of natural capsid variants reveals sialic-acid independent entry of BK polyomavirus, Cell Reports, Vol: 42, Pages: 1-18, ISSN: 2211-1247
BK Polyomavirus (BKPyV) is an opportunistic pathogen that causes nephropathy in kidney transplant recipients. The BKPyV major capsid protein, VP1, engages gangliosides, lipid-linked sialylated glycans at the cell surface, to gain entry into cells. Here, we characterise the influence of VP1 mutations observed in patients with persistent post-transplant BKPyV replication on ganglioside binding, VP1 protein structure, and the tropism of the virus in two renal cell lines: 293TT and immortalised renal tubular epithelial (RS) cells. Infectious entry of single mutants E73Q, E73A and the triple mutant A72V-E73Q-E82Q (VQQ) remained sialic acid-dependent. These three variants acquired binding to a-series gangliosides, including GD1a, although only E73Q was able to infect GD1a-supplemented LNCaP or GM95 cells. Crystal structures of the three mutants showed a clear shift of the BC2 loop in mutants E73A and VQQ that correlated with the inability of these VP1 variants to infect ganglioside complemented cells. On the other hand, the double mutant K69N-E82Q lost the ability to bind sialic acid, with the K69N mutation leading to a steric clash which precludes sialic acid binding. Nevertheless, this mutant retained significant infectivity in 293TT cells that was not dependent on heparan sulphate proteoglycans, implying that an unknown sialic acid-independent entry receptor for BKPyV exists.
Shahin M, Sheppard D, Raynaud C, et al., 2023, Characterization of a glycan-binding complex of minor pilins completes theanalysis of Streptococcus sanguinis type 4 pili subunits, Proceedings of the National Academy of Sciences of USA, Vol: 120, ISSN: 0027-8424
Type 4 filaments (T4F)—of which type 4 pili (T4P) are the archetype—are a superfamily of nanomachines nearly ubiquitous in prokaryotes. T4F are polymers of one major pilin, which also contain minor pilins whose roles are often poorly understood. Here, we complete the structure/function analysis of the full set of T4P pilins in the opportunistic bacterial pathogen Streptococcus sanguinis. We determined the structure of the minor pilin PilA, which is unexpectedly similar to one of the subunits of a tip-located complex of four minor pilins, widely conserved in T4F. We found that PilA interacts and dramatically stabilizes the minor pilin PilC. We determined the structure of PilC, showing that it is a modular pilin with a lectin module binding a subset of glycans prevalent in the human glycome, the host of S. sanguinis. Altogether, our findings support a model whereby the minor pilins in S. sanguinis T4P form a tip-located complex promoting adhesion to various host receptors. This has general implications for T4F.
Niu M, McGrath M, Sammon D, et al., 2023, Structure of the transmembrane protein 2 (TMEM2) ectodomain and its apparent lack of hyaluronidase activity, Wellcome Open Research, Vol: 8, Pages: 1-19, ISSN: 2398-502X
Background: Hyaluronic acid (HA) is a major polysaccharide component of the extracellular matrix. HA has essential functions in tissue architecture and the regulation of cell behaviour. HA turnover needs to be finely balanced. Increased HA degradation is associated with cancer, inflammation, and other pathological situations. Transmembrane protein 2 (TMEM2) is a cell surface protein that has been reported to degrade HA into ~5 kDa fragments and play an essential role in systemic HA turnover. Methods: We produced the soluble TMEM2 ectodomain (residues 106-1383; sTMEM2) in human embryonic kidney cells (HEK293) and determined its structure using X-ray crystallography. We tested sTMEM2 hyaluronidase activity using fluorescently labelled HA and size fractionation of reaction products. We tested HA binding in solution and using a glycan microarray. Results: Our crystal structure of sTMEM2 confirms a remarkably accurate prediction by AlphaFold. sTMEM2 contains a parallel β-helix typical of other polysaccharide-degrading enzymes, but an active site cannot be assigned with confidence. A lectin-like domain is inserted into the β-helix and predicted to be functional in carbohydrate binding. A second lectin-like domain at the C-terminus is unlikely to bind carbohydrates. We did not observe HA binding in two assay formats, suggesting a modest affinity at best. Unexpectedly, we were unable to observe any HA degradation by sTMEM2. Our negative results set an upper limit for k cat of approximately 10 -5 min -1. Conclusions: Although sTMEM2 contains domain types consistent with its suggested role in TMEM2 degradation, its hyaluronidase activity was undetectable. HA degradation by TMEM2 may require additional proteins and/or localisation at the cell surface.
Akune Y, Arpinar S, Silva LM, et al., 2022, CarbArrayART-An update on the software tool for carbohydrate microarray data, storage, processing, presentation and reporting, Glycobiology, Vol: 30, Pages: 1031-1031, ISSN: 0959-6658
Glycan microarrays are essential tools in glycobiology and are being widely used for assignment of glycan ligands in diverse glycan recognition systems. We have developed a new software, called Carbohydrate microArray Analysis and Reporting Tool (CarbArrayART), to address the need for a distributable application for glycan microarray data management. The main features of CarbArrayART include: (i) Storage of quantified array data from different array layouts with scan data and array-specific metadata, such as lists of arrayed glycans, array geometry, information on glycan-binding samples, and experimental protocols. (ii) Presentation of microarray data as charts, tables, and heatmaps derived from the average fluorescence intensity values that are calculated based on the imaging scan data and array geometry, as well as filtering and sorting functions according to monosaccharide content and glycan sequences. (iii) Data export for reporting in Word, PDF, and Excel formats, together with metadata that are compliant with the guidelines of MIRAGE (Minimum Information Required for A Glycomics Experiment). CarbArrayART is designed for routine use in recording, storage, and management of any slide-based glycan microarray experiment. In conjunction with the MIRAGE guidelines, CarbArrayART addresses issues that are critical for glycobiology, namely, clarity of data for evaluation of reproducibility and validity.
Correia VG, Trovao F, Pinheiro BA, et al., 2021, Mapping Molecular Recognition of β1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont <i>Bacteroides ovatus</i>, MICROBIOLOGY SPECTRUM, Vol: 9, ISSN: 2165-0497
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- Citations: 3
McAllister N, Liu Y, Silva LM, et al., 2021, Chikungunya Virus Strains from Each Genetic Clade Bind Sulfated Glycosaminoglycans as Attachment Factors (vol 94, e01500-20, 2020), JOURNAL OF VIROLOGY, Vol: 95, ISSN: 0022-538X
Garnett JA, Palma AS, Liu B, et al., 2021, Editorial: glycans as players in host-microbe interactions: a structural perspective, Frontiers in Molecular Biosciences, Vol: 8, Pages: 1-2, ISSN: 2296-889X
Gyapon-Quast F, Goicoechea de Jorge E, Malik T, et al., 2021, Defining the glycosaminoglycan interactions of complement factor H-related protein 5, Journal of Immunology, Vol: 207, Pages: 534-541, ISSN: 0022-1767
Complement activation is an important mediator of kidney injury in glomerulonephritis. Complement factor H (FH) and FH-related protein 5 (FHR-5) influence complement activation in C3 glomerulopathy and IgA nephropathy by differentially regulating glomerular complement. FH is a negative regulator of complement C3 activation. Conversely, FHR-5 in vitro promotes C3 activation either directly or by competing with FH for binding to complement C3b. The FH-C3b interaction is enhanced by surface glycosaminoglycans (GAGs) and the FH-GAG interaction is well-characterized. In contrast, the contributions of carbohydrates to the interaction of FHR-5 and C3b are unknown. Using plate-based and microarray technologies we demonstrate that FHR-5 interacts with sulfated GAGs and that this interaction is influenced by the pattern and degree of GAG sulfation. The FHR-5-GAG interaction that we identified has functional relevance as we could show that the ability of FHR-5 to prevent binding of FH to surface C3b is enhanced by surface kidney heparan sulfate. Our findings are important in understanding the molecular basis of the binding of FHR-5 to glomerular complement and the role of FHR-5 in complement-mediated glomerular disease.
Bonnardel F, Haslam SM, Dell A, et al., 2021, Proteome-wide prediction of bacterial carbohydrate-binding proteins as a tool for understanding commensal and pathogen colonisation of the vaginal microbiome, npj Biofilms and Microbiomes, Vol: 7, Pages: 1-10, ISSN: 2055-5008
Bacteria use carbohydrate-binding proteins (CBPs), such as lectins and carbohydrate-binding modules (CBMs), to anchor to specific sugars on host surfaces. CBPs in the gut microbiome are well studied, but their roles in the vagina microbiome and involvement in sexually transmitted infections, cervical cancer and preterm birth are largely unknown. We established a classification system for lectins and designed Hidden Markov Model (HMM) profiles for data mining of bacterial genomes, resulting in identification of >100,000 predicted bacterial lectins available at unilectin.eu/bacteria. Genome screening of 90 isolates from 21 vaginal bacterial species shows that those associated with infection and inflammation produce a larger CBPs repertoire, thus enabling them to potentially bind a wider array of glycans in the vagina. Both the number of predicted bacterial CBPs and their specificities correlated with pathogenicity. This study provides new insights into potential mechanisms of colonisation by commensals and potential pathogens of the reproductive tract that underpin health and disease states.
McAllister N, Liu Y, Silva LM, et al., 2020, Chikungunya virus strains from each genetic clade bind sulfated glycosaminoglycans as attachment factors, Journal of Virology, Vol: 94, ISSN: 0022-538X
Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes debilitating musculoskeletal disease. CHIKV displays broad cell, tissue, and species tropism, which may correlate with the attachment factors and entry receptors used by the virus. Cell-surface glycosaminoglycans (GAGs) have been identified as CHIKV attachment factors. However, the specific types of GAGs and potentially other glycans to which CHIKV binds and whether there are strain-specific differences in GAG binding is not fully understood. To identify the types of glycans bound by CHIKV, we conducted glycan microarray analyses and discovered that CHIKV preferentially binds GAGs. Microarray results also indicate that sulfate groups on GAGs are essential for CHIKV binding and that CHIKV binds most strongly to longer GAG chains of heparin and heparan sulfate. To determine whether GAG-binding capacity varies among CHIKV strains, a representative strain from each genetic clade was tested. While all strains directly bound to heparin and chondroitin sulfate in ELISAs and depended on heparan sulfate for efficient cell-binding and infection, we observed some variation by strain. Enzymatic removal of cell-surface GAGs and genetic ablation that diminishes GAG expression reduced CHIKV binding and infectivity of all strains. Collectively, these data demonstrate that GAGs are the preferred glycan bound by CHIKV, enhance our understanding of the specific GAG moieties required for CHIKV binding, define strain differences in GAG engagement, and provide further evidence for a critical function of GAGs in CHIKV cell attachment and infection.IMPORTANCE Alphavirus infections are a global health threat, contributing to outbreaks of disease in many parts of the world. Recent epidemics caused by CHIKV, an arthritogenic alphavirus, resulted in more than 8.5 million cases as the virus has spread into new geographic regions, including the Western Hemisphere. CHIKV causes disease in the majority of people infected, leading
Vendele I, Willment JA, Silva LM, et al., 2020, Mannan detecting C-type lectin receptor probes recognise immune epitopes with diverse chemical, spatial and phylogenetic heterogeneity in fungal cell walls, PLoS Pathogens, Vol: 16, Pages: 1-29, ISSN: 1553-7366
During the course of fungal infection, pathogen recognition by the innate immune system is critical to initiate efficient protective immune responses. The primary event that triggers immune responses is the binding of Pattern Recognition Receptors (PRRs), which are expressed at the surface of host immune cells, to Pathogen-Associated Molecular Patterns (PAMPs) located predominantly in the fungal cell wall. Most fungi have mannosylated PAMPs in their cell walls and these are recognized by a range of C-type lectin receptors (CTLs). However, the precise spatial distribution of the ligands that induce immune responses within the cell walls of fungi are not well defined. We used recombinant IgG Fc-CTLs fusions of three murine mannan detecting CTLs, including dectin-2, the mannose receptor (MR) carbohydrate recognition domains (CRDs) 4–7 (CRD4-7), and human DC-SIGN (hDC-SIGN) and of the β-1,3 glucan-binding lectin dectin-1 to map PRR ligands in the fungal cell wall of fungi grown in vitro in rich and minimal media. We show that epitopes of mannan-specific CTL receptors can be clustered or diffuse, superficial or buried in the inner cell wall. We demonstrate that PRR ligands do not correlate well with phylogenetic relationships between fungi, and that Fc-lectin binding discriminated between mannosides expressed on different cell morphologies of the same fungus. We also demonstrate CTL epitope differentiation during different phases of the growth cycle of Candida albicans and that MR and DC-SIGN labelled outer chain N-mannans whilst dectin-2 labelled core N-mannans displayed deeper in the cell wall. These immune receptor maps of fungal walls of in vitro grown cells therefore reveal remarkable spatial, temporal and chemical diversity, indicating that the triggering of immune recognition events originates from multiple physical origins at the fungal cell surface.
Wu N, Silva LM, Liu Y, et al., 2019, Glycan Markers of Human Stem Cells Assigned with Beam Search Arrays., Mol Cell Proteomics, Vol: 18, Pages: 1981-2002, ISSN: 1535-9476
Glycan antigens recognized by monoclonal antibodies have served as stem cell markers. To understand regulation of their biosynthesis and their roles in stem cell behavior precise assignments are required. We have applied state-of-the-art glycan array technologies to compare the glycans bound by five antibodies that recognize carbohydrates on human stem cells. These are: FC10.2, TRA-1-60, TRA-1-81, anti-i and R-10G. Microarray analyses with a panel of sequence-defined glycans corroborate that FC10.2, TRA-1-60, TRA-1-81 recognize the type 1-(Galβ-3GlcNAc)-terminating backbone sequence, Galβ-3GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and anti-i, the type 2-(Galβ-4GlcNAc) analog, Galβ-4GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and we determine substituents they can accommodate. They differ from R-10G, which requires sulfate. By Beam Search approach, starting with an antigen-positive keratan sulfate polysaccharide, followed by targeted iterative microarray analyses of glycan populations released with keratanases and mass spectrometric monitoring, R-10G is assigned as a mono-sulfated type 2 chain with 6-sulfation at the penultimate N-acetylglucosamine, Galβ-4GlcNAc(6S)β-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc. Microarray analyses using newly synthesized glycans corroborate the assignment of this unique determinant raising questions regarding involvement as a ligand in the stem cell niche.
Vendele I, Willment JA, Silva LM, et al., 2019, Mannan detecting C-type lectin receptor probes recognise immune epitopes with diverse chemical, spatial and phylogenetic heterogeneity in fungal cell walls
<jats:title>Abstract</jats:title><jats:p>During the course of fungal infection, pathogen recognition by the innate immune system is critical to initiate efficient protective immune responses. The primary event that triggers immune responses is the binding of Pattern Recognition Receptors (PRRs), which are expressed at the surface of host immune cells, to Pathogen-Associated Molecular Patterns (PAMPs) located predominantly in the fungal cell wall. Most fungi have mannosylated PAMPs in their cell walls and these are recognized by a range of C-type lectin receptors (CTLs). However, the precise spatial distribution of the ligands that induce immune responses within the cell walls of fungi are not well defined. We used recombinant IgG Fc-CTLs fusions of three murine mannan detecting CTLs, including dectin-2, the mannose receptor (MR) carbohydrate recognition domains (CRDs) 4-7 (CRD4-7), and human DC-SIGN (hDC-SIGN) and the β-1,3 glucan-binding lectin dectin-1 to map PRR ligands in the fungal cell wall. We show that epitopes of mannan-specific CTL receptors can be clustered or diffuse, superficial or buried in the inner cell wall. We demonstrate that PRR ligands do not correlate well with phylogenetic relationships between fungi, and that Fc-lectin binding discriminated between mannosides expressed on different cell morphologies of the same fungus. We also demonstrate CTL epitope differentiation during different phases of the growth cycle of<jats:italic>Candida albicans</jats:italic>and that MR and DC-SIGN labelled outer chain<jats:italic>N</jats:italic>-mannans whilst dectin-2 labelled core<jats:italic>N</jats:italic>-mannans displayed deeper in the cell wall. These immune receptor maps of fungal walls therefore reveal remarkable spatial, temporal and chemical diversity, indicating that the triggering of immune recognition events originates from multiple physical origins at the fungal cell surface.</jats:p>
Chandra N, Liu Y, Liu J-X, et al., 2019, Sulfated glycosaminoglycans as viral decoy receptors for human adenovirus type 37, Viruses, Vol: 11, ISSN: 1999-4915
Glycans on plasma membranes and in secretions play important roles in infection by many viruses. Species D human adenovirus type 37 (HAdV-D37) is a major cause of epidemic keratoconjunctivitis (EKC) and infects target cells by interacting with sialic acid (SA)-containing glycans via the fiber knob domain of the viral fiber protein. HAdV-D37 also interacts with sulfated glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, we investigated the molecular requirements of HAdV-D37 fiber knob:GAG interactions using a GAG microarray and demonstrated that fiber knob interacts with a broad range of sulfated GAGs. These interactions were corroborated in cell-based assays and by surface plasmon resonance analysis. Removal of heparan sulfate (HS) and sulfate groups from human corneal epithelial (HCE) cells by heparinase III and sodium chlorate treatments, respectively, reduced HAdV-D37 binding to cells. Remarkably, removal of HS by heparinase III enhanced the virus infection. Our results suggest that interaction of HAdV-D37 with sulfated GAGs in secretions and on plasma membranes prevents/delays the virus binding to SA-containing receptors and inhibits subsequent infection. We also found abundant HS in the basement membrane of the human corneal epithelium, which may act as a barrier to sub-epithelial infection. Collectively, our findings provide novel insights into the role of GAGs as viral decoy receptors and highlight the therapeutic potential of GAGs and/or GAG-mimetics in HAdV-D37 infection.
Akune Y, Arpinar S, Silva LM, et al., 2018, CarbArrayART: Carbohydrate Array Analysis and Reporting Tool New software for glycan array for data processing, storage and presentation, Annual Meeting of the Society-for-Glycobiology (SFG), Publisher: OXFORD UNIV PRESS INC, Pages: 1034-1035, ISSN: 0959-6658
Lenman A, Liaci AM, Liu Y, et al., 2018, Polysialic acid is a cellular receptor for human adenovirus 52, Proceedings of the National Academy of Sciences of the United States of America, Vol: 115, Pages: E4264-E4273, ISSN: 0027-8424
Human adenovirus 52 (HAdV-52) is one of only three known HAdVs equipped with both a long and a short fiber protein. While the long fiber binds to the coxsackie and adenovirus receptor, the function of the short fiber in the virus life cycle is poorly understood. Here, we show, by glycan microarray analysis and cellular studies, that the short fiber knob (SFK) of HAdV-52 recognizes long chains of α-2,8-linked polysialic acid (polySia), a large posttranslational modification of selected carrier proteins, and that HAdV-52 can use polySia as a receptor on target cells. X-ray crystallography, NMR, molecular dynamics simulation, and structure-guided mutagenesis of the SFK reveal that the nonreducing, terminal sialic acid of polySia engages the protein with direct contacts, and that specificity for polySia is achieved through subtle, transient electrostatic interactions with additional sialic acid residues. In this study, we present a previously unrecognized role for polySia as a cellular receptor for a human viral pathogen. Our detailed analysis of the determinants of specificity for this interaction has general implications for protein–carbohydrate interactions, particularly concerning highly charged glycan structures, and provides interesting dimensions on the biology and evolution of members of Human mastadenovirus G.
Stappers MHT, Clark AE, Aimanianda V, et al., 2018, Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus, NATURE, Vol: 555, Pages: 382-386, ISSN: 0028-0836
Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity1. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin2,3, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.
Liu Y, Palma AS, Ten F, et al., 2018, Insights Into Glucan Polysaccharide Recognition Using Glucooligosaccharide Microarrays With Oxime-Linked Neoglycolipid Probes, CHEMICAL GLYCOBIOLOGY, PT B: MONITORING GLYCANS AND THEIR INTERACTIONS, Editors: Imperiali, Publisher: ELSEVIER ACADEMIC PRESS INC, Pages: 139-167
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Akune Y, Arpinar S, Stoll M, et al., 2017, New software for glycan array for data processing, storage and presentation, Annual Meeting of the Society-for-Glycobiology, Publisher: OXFORD UNIV PRESS INC, Pages: 1204-1204, ISSN: 0959-6658
Li Z, Gao C, Zhang Y, et al., 2017, O-Glycome beam search arrays for carbohydrate ligand discovery, Molecular and Cellular Proteomics, Vol: 17, Pages: 121-133, ISSN: 1535-9476
O-glycosylation is a post-translational modification of proteins crucial to molecular mechanisms in health and disease. O-glycans are typically highly heterogeneous. The involvement of specific O-glycan sequences in many bio-recognition systems is yet to be determined due to a lack of efficient methodologies. We describe here a targeted microarray approach: O-glycome beam search that is both robust and efficient for O-glycan ligand-discovery. Substantial simplification of the complex O-glycome profile and facile chromatographic resolution is achieved by arraying O-glycans as branches, monitoring by mass spectrometry, focusing on promising fractions, and on-array immuno-sequencing. This is orders of magnitude more sensitive than traditional methods. We have applied beam search approach to porcine stomach mucin and identified extremely minor components previously undetected within the O-glycome of this mucin that are ligands for the adhesive proteins of two rotaviruses. The approach is applicable to O-glycome recognition studies in a wide range of biological settings to give insights into glycan recognition structures in natural microenvironments.
Liu Y, McBride R, Stoll M, et al., 2016, The Minimum Information Required for a Glycomics Experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data, Glycobiology, Vol: 27, Pages: 280-284, ISSN: 1460-2423
MIRAGE (Minimum Information Required for A Glycomics Experiment) is an initiative that was created by experts in the fields of glycobiology, glycoanalytics, and glycoinformatics to produce guidelines for reporting results from the diverse types of experiments and analyses used in structural and functional studies of glycans in the scientific literature. As a sequel to the guidelines for sample preparation (Struwe et al. 2016, Glycobiology, 26, 907-910) and mass spectrometry (MS) data (Kolarich et al. 2013, Mol. Cell Proteomics. 12, 991-995), here we present the first version of guidelines intended to improve the standards for reporting data from glycan microarray analyses. For each of eight areas in the workflow of a glycan microarray experiment, we provide guidelines for the minimal information that should be provided in reporting results. We hope that the MIRAGE glycan microarray guidelines proposed here will gain broad acceptance by the community, and will facilitate interpretation and reproducibility of the glycan microarray results with implications in comparison of data from different laboratories and eventual deposition of glycan microarray data in international databases.
Bartels MF, Winterhalter PR, Yu J, et al., 2016, Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates, PLOS One, Vol: 11, ISSN: 1932-6203
Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-Man)AV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins.
Zhang H, Palma AS, Zhang Y, et al., 2016, Generation and characterization of β1,2-gluco-oligosaccharide probes from Brucella abortus cyclic β-glucan and their recognition by C-type lectins of the immune system, Glycobiology, Vol: 26, Pages: 1086-1096, ISSN: 1460-2423
The β1,2-glucans produced by bacteria are important in invasion, survival andimmunomodulation in infected hosts be they mammals or plants. However, there has been alack of information on proteins which recognize these molecules. This is partly due to theextremely limited availability of the sequence-defined oligosaccharides and derived probesfor use in the study of their interactions. Here we have used the cyclic β1,2-glucan (CβG) ofthe bacterial pathogen Brucella abortus, after removal of succinyl side chains, to preparelinearized oligosaccharides which were used to generate microarrays. We describe optimizedconditions for partial depolymerization of the cyclic glucan by acid hydrolysis and conversionof the β1,2-gluco-oligosaccharides, with degrees of polymerization 2-13, to neoglycolipids forthe purpose of generating microarrays. By microarray analyses we show that the C-type lectinreceptor DC-SIGNR, like the closely related DC-SIGN we investigated earlier, binds to theβ1,2-gluco-oligosaccharides, as does the soluble immune effector serum mannose-bindingprotein. Exploratory studies with DC-SIGN are suggestive of the recognition also of the intactCβG by this receptor. These findings open the way to unravelling mechanisms ofimmunomodulation mediated by β1,2-glucans in mammalian systems.
Struwe WB, Agravat S, Aoki-Kinoshita KF, et al., 2016, The minimum information required for a glycomics experiment (MIRAGE) project: sample preparation guidelines for reliable reporting of glycomics datasets., Glycobiology, Vol: 26, Pages: 907-910, ISSN: 0959-6658
The minimum information required for a glycomics experiment (MIRAGE) project was established in 2011 to provide guidelines to aid in data reporting from all types of experiments in glycomics research including mass spectrometry (MS), liquid chromatography, glycan arrays, data handling and sample preparation. MIRAGE is a concerted effort of the wider glycomics community that considers the adaptation of reporting guidelines as an important step towards critical evaluation and dissemination of datasets as well as broadening of experimental techniques worldwide. The MIRAGE Commission published reporting guidelines for MS data and here we outline guidelines for sample preparation. The sample preparation guidelines include all aspects of sample generation, purification and modification from biological and/or synthetic carbohydrate material. The application of MIRAGE sample preparation guidelines will lead to improved recording of experimental protocols and reporting of understandable and reproducible glycomics datasets.
Liu Y, Ramelot TA, Huang P, et al., 2016, Glycan specificity of P[19] rotavirus and comparison with those of other related P genotypes, Journal of Virology, Vol: 90, Pages: 9983-9996, ISSN: 1098-5514
The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). However, unlike the other P[II] RVs that mainly infects humans, P[19] RVs commonly infect animals (porcine), making P[19] unique to study RV diversity and host ranges. Through in vitro binding assays and saturation transfer difference (STD) NMR, we found that P[19] could bind mucin cores 2, 4, and 6, as well as type 1 histo-blood group antigens (HBGAs). The common sequences of these glycans serve as minimal binding units, while additional residues, such as the A, B, H, and Lewis epitopes of the type 1 HBGAs, can further define the binding outcomes and therefore, likely the host ranges for P[19] RVs. This complex binding property of P[19] is shared with those of the other three P[II] RVs (P[4], P[6] and P[8]) in that all of them recognized the type 1 HBGA precursor, although P[4] and P[8], but not P[6], also bind to mucin cores. Moreover, while essential for P[4] and P[8] binding, the addition of the Lewis epitope blocked P[6] and P[19] binding to type 1 HBGAs. Chemical shift NMR of P[19] VP8* identified a ligand binding interface that has shifted away from the known RV P-genotype binding sites but is conserved among all P[II] RVs and two P[I] RVs (P[10] and P[12]), suggesting an evolutionary connection among these human and animal RVs. Taken together, these data are important for hypotheses on potential mechanisms for RV diversity, host ranges, and cross-species transmission. IMPORTANCE: In this study, we found that this P[19] strain and other P[II] RVs recognize mucin cores and the type 1 HBGA precursors as the minimal functional units and that additional saccharides adjacent to these units can alter binding outcomes and thereby possibly host ranges. These data may help to explain why some P[II] RVs, such as P[6] and P[19], commonly infect animals but rarely humans, while others, such as the P[4] and P[8] RVs, mainly infect humans and are predominant over other P genotypes. Elucidation
Parker L, Wharton SA, Martin SR, et al., 2016, Effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza A (H3N2) vaccine viruses, Journal of General Virology, Vol: 97, Pages: 1333-1344, ISSN: 1465-2099
Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012–2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.
Liu Y, Cecilio NT, Carvalho FC, et al., 2015, Glycan microarray analysis of the carbohydrate-recognition specificity of native and recombinant forms of the lectin ArtinM, Data in Brief, Vol: 5, Pages: 1035-1047, ISSN: 2352-3409
This article contains data related to the researc.h article entitled “Yeast-derived ArtinM shares structure, carbohydrate recognition, and biological effects with native ArtinM” by Cecílio et al. (2015) [1]. ArtinM, a D-mannose-binding lectin isolated from the seeds of Artocarpus heterophyllus, exerts immunomodulatory and regenerative activities through its Carbohydrate Recognition Domain (CRD) (Souza et al., 2013; Mariano et al., 2014 [2] and [3]). The limited availability of the native lectin (n-ArtinM) led us to characterize a recombinant form of the protein, obtained by expression in Saccharomyces cerevisiae (y-ArtinM). We compared the carbohydrate-binding specificities of y-ArtinM and n-ArtinM by analyzing the binding of biotinylated preparations of the two lectin forms using a neoglycolipid (NGL)-based glycan microarray. Data showed that y-ArtinM mirrored the specificity exhibited by n-ArtinM.
Gao C, Zhang Y, Liu Y, et al., 2015, Negative-Jon Electrospray Tandem Mass Spectrometry and Microarray Analyses of Developmentally Regulated Antigens Based on Type 1 and Type 2 Backbone Sequences, Analytical Chemistry, Vol: 87, Pages: 11871-11878, ISSN: 1520-6882
Type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) sequences are constituents of the backbones of alarge family of glycans of glycoproteins and glycolipids whosebranching and peripheral substitutions are developmentallyregulated. It is highly desirable to have microsequencingmethods that can be used to precisely identify and monitorthese oligosaccharide sequences with high sensitivity. Negative-ionelectrospray tandem mass spectrometry withcollision-induced dissociation has been used for characterizationof branching points, peripheral substitutions, andpartial assignment of linkages in reducing oligosaccharides. Wenow extend this method to characterizing entire sequences oflinear type 1 and type 2 chain-based glycans, focusing on thetype 1 and type 2 units in the internal regions including the linkages connecting type 1 and type 2 disaccharide units. We applythe principles to sequence analysis of closely related isomeric oligosaccharides and demonstrate by microarray analyses distinctbinding activities of antibodies and a lectin toward various combinations of type 1 and 2 units joined by 1,3- and 1,6-linkages.These sequence-specific carbohydrate-binding proteins are in turn valuable tools for detecting and distinguishing the type 1 andtype 2-based developmentally regulated glycan sequences.
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