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Journal articleParker 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: 1465-2099Influenza 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.
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Journal articleLiu 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: 2352-3409This 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.
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Journal articleGao 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: 1520-6882Type 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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Conference paperSilva L, Childs RA, Palma AS, et al., 2015,
Influence of carrier lipid composition on glycan recognition in NGL-based microarrays
, Annual Meeting of the Society-for-Glycobiology on Glycobiology - Accelerating Impact across the Biomedical Sciences, Publisher: OXFORD UNIV PRESS INC, Pages: 1260-1260, ISSN: 0959-6658 -
Journal articleCecilio NT, Carvalho FC, Liu Y, et al., 2015,
Yeast expressed ArtinM shares structure, carbohydrate recognition, and biological effects with native ArtinM
, International Journal of Biological Macromolecules, Vol: 82, Pages: 22-30, ISSN: 0141-8130Recent advances in glycobiology have revealed the essential role of lectins in deciphering the glycocodes at the cell surface to generate important biological signaling responses. ArtinM, a d-mannose-binding lectin isolated from the seeds of jackfruit (Artocarpus heterophyllus), is composed of 16 kDa subunits that are associated to form a homotetramer. Native ArtinM (n-ArtinM) exerts immunomodulatory and regenerative effects, but the potential pharmaceutical applicability of the lectin is highly limited by the fact that its production is expensive, laborious, and impossible to be scaled up. This led us to characterize a recombinant form of the lectin obtained by expression in Saccharomyces cerevisiae (y-ArtinM). In the present study, we demonstrated that y-ArtinM is similar to n-ArtinM in subunit arrangement, oligomerization and carbohydrate binding specificity. We showed that y-ArtinM can exert n-ArtinM biological activities such as erythrocyte agglutination, stimulation of neutrophil migration and degranulation, mast cell degranulation, and induction of interleukin-12 and interleukin-10 production by macrophages. In summary, the expression of ArtinM in yeast resulted in successful production of an active, recombinant form of ArtinM that is potentially useful for pharmaceutical application.
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Journal articleFeizi TEN, Haltiwanger RS, 2015,
Editorial overview: Carbohydrate-protein interactions and glycosylation: Glycan synthesis and recognition: finding the perfect partner in a sugar-coated life.
, Curr Opin Struct Biol, Vol: 34, Pages: vii-ixOligosaccharides expressed on the surface of cells and in biological fluids as glycoproteins, glycolipids, proteoglycans and polysaccharides can be recognized by partner proteins, and these interactions have been shown to mediate fundamental biological events such as occur in the immune system, signal transduction, development and cancer metastasis. The specificities of these partner proteins (lectins) for their glycan ligands are determined by factors such as glycan composition, shape and density of expression and the involvement of the aglycone moiety as part of the recognition motif. There is increasing knowledge on the mechanisms of these interactions as new secondary binding sites continue to be elucidated adding to the functional awareness of sugar-binding proteins. This issue focuses on recent advances in understanding how C-type lectins in the immune system work, how novel motifs involving asymmetric glycan branch recognition and protein-protein interactions influence critical biological functions including signal transduction and bactericidal pore formation, recent studies on novel glycan-binding proteins produced by bacteriophage, analysis of the interactions between heparin/heparan sulphate and their binding proteins, and recent findings on the molecular interactions between chondroitin-dermatan sulphate and various bioactive protein components. We conclude with a review on a recent fascinating class of processive enzymes responsible for synthesis of high-molecular weight extracellular polysaccharides such as hyaluronic acid, chitin and alginate.
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Journal articleHirose H, Tamai H, Gao C, et al., 2015,
Total syntheses of disulphated glycosphingolipid SB1a and the related monosulphated SM1a
, Organic and Biomolecular Chemistry, Vol: 13, Pages: 11105-11117, ISSN: 1472-7781Total syntheses of two natural sulphoglycolipids, disulphated glycosphingolipid SB1a and the structurally related monosulphated SM1a, are described. They have common glycan sequences and ceramide moieties and are associated with human epithelial carcinomas. The syntheses featured efficient glycan assembly and the glucosyl ceramide cassette as a versatile building block. The binding of the synthetic sulphoglycolipids by the carcinoma-specific monoclonal antibody AE3 was investigated using carbohydrate microarray technology.
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Journal articleHanashima S, Goetze S, Liu Y, et al., 2015,
Defining the Interaction of Human Soluble Lectin ZG16p and Mycobacterial Phosphatidylinositol Mannosides
, CHEMBIOCHEM, Vol: 16, Pages: 1502-1511, ISSN: 1439-4227- Author Web Link
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Journal articleKakugawa S, Langton PF, Zebisch M, et al., 2015,
Notum deacylates Wnt proteins to suppress signalling activity
, Nature, Vol: 519, Pages: 187-192, ISSN: 0028-0836Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.
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Journal articlePalma AS, Liu Y, Zhang H, et al., 2015,
Unravelling Glucan Recognition Systems by Glycome Microarrays Using the Designer Approach and Mass Spectrometry
, Molecular & Cellular Proteomics, Vol: 14, Pages: 974-988, ISSN: 1535-9484Glucans are polymers of D-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.
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Carbohydrate microarray analyses
Professor Ten Feizi
t.feizi@imperial.ac.uk
+44 (0) 20 7594 7207
Dr Yan Liu
yan.liu2@imperial.ac.uk
+44 (0) 20 7594 2598
Dr Antonio Di Maio
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Carbohydrate structural analyses
Dr Wengang Chai
w.chai@imperial.ac.uk
+44 (0) 20 7594 2596
