Wengang Chai

Imperial College London, Londinium, England, United Kingdom

Are you Wengang Chai?

Claim your profile

Publications (105)662.89 Total impact

  • Source
    Chao Gao · Yibing Zhang · Yan Liu · Ten Feizi · Wengang Chai

    Full-text · Dataset · Nov 2015
  • Source
    Chao Gao · Yibing Zhang · Yan Liu · Ten Feizi · Wengang Chai
    [Show abstract] [Hide abstract]
    ABSTRACT: Type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) sequences are constituents of the backbones of a large family of glycans of glycoproteins and glycolipids whose branching and peripheral substitutions are developmentally-regulated. It is highly desirable to have micro-sequencing methods that can be used to precisely identify and monitor these oligosaccharide sequences with high sensitivity. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation has been used for characterization of branching points, peripheral substitutions and partial assignment of linkages in reducing oligosaccharides. We now extend this method to characterizing entire sequences of linear type 1 and type 2 chain-based glycans, focusing on the type 1 and -2 units in the internal regions including the linkages connecting type 1 and type 2 disaccharide units. We apply the principles to sequence analysis of closely related isomeric oligosaccharides and demonstrate by microarray analyses distinct binding 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 and type 2-based developmentally-regulated glycan sequences.
    Full-text · Article · Nov 2015 · Analytical Chemistry

  • No preview · Conference Paper · Nov 2015

  • No preview · Conference Paper · Sep 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Glucans 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, anti-cancer 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. The 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 signalling 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. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Full-text · Article · Feb 2015 · Molecular & Cellular Proteomics
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: →3)GalNAcβ4,6S(1→4) [FucαX(1→3)]GlcAβ(1→, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Lex blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu2+-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.
    Full-text · Article · Aug 2014 · Journal of Biological Chemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl.
    Preview · Article · Aug 2014 · Journal of Biological Chemistry
  • Source

    Full-text · Article · Aug 2014 · Current Opinion in Chemical Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: ZG16p is a soluble mammalian lectin, the first to be described with a Jacalin-related β-prism-fold. ZG16p has been reported to bind both to glycosaminoglycans and mannose. To determine the structural basis of the multiple sugar-binding properties, we conducted glycan microarray analyses of human ZG16p. We observed that ZG16p preferentially binds to α-mannose-terminating short glycans such as Ser/Thr-linked O-mannose, but not to high mannose-type N-glycans. Among sulfated glycosaminoglycan oligomers examined, chondroitin sulfate B and heparin oligosaccharides showed significant binding. Crystallographic studies of human ZG16p lectin in the presence of selected ligands revealed the mechanism of multiple sugar recognition. Manα1–3Man and Glcβ1–3Glc bound in different orientations: the nonreducing end of the former and the reducing end of the latter fitted in the canonical shallow mannose binding pocket. Solution NMR analysis using 15N-labeled ZG16p defined the heparin-binding region, which is on an adjacent flat surface of the protein. On-array competitive binding assays suggest that it is possible for ZG16p to bind simultaneously to both types of ligands. Recognition of a broad spectrum of ligands by ZG16p may account for the multiple functions of this lectin in the formation of zymogen granules via glycosaminoglycan binding, and in the recognition of pathogens in the digestive system through α-mannose-related recognition.
    Preview · Article · Apr 2014 · Journal of Biological Chemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Monoclonal antibody F77 was previously raised against human prostate cancer cells and has been shown to recognize a carbohydrate antigen, but the carbohydrate sequence of the antigen was elusive. Here, we make multifaceted approaches to characterize F77 antigen, including binding analyses with the glycolipid extract of the prostate cancer cell line PC3, microarrays with sequence-defined glycan probes, and designer arrays from the O-glycome of an antigen-positive mucin, in conjunction with mass spectrometry. Our results reveal F77 antigen to be expressed on blood group H on a 6-linked branch of a poly-N-acetyllactosamine backbone. We show that mAb F77 can also bind to blood group A and B analogs but with lower intensities. We propose that the close association of F77 antigen with prostate cancers is a consequence of increased blood group H expression together with up-regulated branching enzymes. This is in contrast to other epithelial cancers that have up-regulated branching enzymes but diminished expression of H antigen. With knowledge of the structure and prevalence of F77 antigen in prostate cancer, the way is open to explore rationally its application as a biomarker to detect F77-positive circulating prostate cancer-derived glycoproteins and tumor cells.
    Full-text · Article · Apr 2014 · Journal of Biological Chemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The neoglycolipid (NGL) technology is the basis of a state-of-the-art oligosaccharide microarray system. The NGL-based microarray system in the Glycosciences Laboratory Imperial College London (http://www3.imperial.ac.uk/glycosciences) is one of the two leading platforms for glycan microarrays, being offered for screening analyses to the broad biomedical community. Highlighted in this review are the sensitivity of the analysis system and, coupled with mass spectrometry, the provision for generating 'designer' microarrays from glycomes to identify novel ligands of biological relevance. Among recent applications are assignments of ligands for apicomplexan parasites, pandemic 2009 influenza virus, polyoma and reoviruses, an innate immune receptor against fungal pathogens, Dectin-1, and a novel protein of the endoplasmic reticulum, malectin; also the characterization of an elusive cancer-associated antigen. Some other contemporary advances in glycolipid-containing arrays and microarrays are also discussed.
    Full-text · Article · Feb 2014 · Current opinion in chemical biology

  • No preview · Conference Paper · Nov 2013
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.
    Full-text · Article · Sep 2013 · Virology
  • Source

    Full-text · Article · Jul 2013 · Nature
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: ι-Carrageenases belong to family 82 of glycoside hydrolases that degrade sulfated galactans in the red algae known as ι-carrageenans. The catalytic mechanism and some substrate-binding residues of family GH82 have been studied but the substrate recognition and binding mechanism of this family have not been fully elucidated. We report here the purification, cloning and characterization of a new ι-carrageenase CgiA_Ce from the marine bacterium Cellulophaga sp. QY3. CgiA_Ce was the most thermostable carrageenase described so far. It was most active at 50°C and pH 7.0 and retained more than 70% of the original activity after incubation at 50°C for 1 h at pH 7.0 or at pH 5.0-10.6 for 24 h. CgiA_Ce was an endo-type ι-carrageenase; it cleaved ι-carrageenan yielding neo-ι-carrabiose and neo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was the minimum substrate. Sequence analysis and structure modeling showed that CgiA_Ce is indeed a new member of family GH82. Moreover, sequence analysis of ι-carrageenases revealed that the amino acid residues at subsites -1 and +1 were more conserved than those at other subsites. Site-directed mutagenesis followed by kinetic analysis identified three strictly conserved residues at subsites -1 and +1 of ι-carrageenases, G228, Y229 and R254 in CgiA_Ce, which played important roles for substrate binding. Furthermore, our results suggested that Y229 and R254 in CgiA_Ce interacted specifically with the sulfate groups of the sugar moieties located at subsites -1 and +1, shedding light on the mechanism of ι-carrageenan recognition in the family GH82.
    Full-text · Article · May 2013 · PLoS ONE
  • [Show abstract] [Hide abstract]
    ABSTRACT: Blood group antigens, such as those containing fucose and bearing the ABO(H)- and Lewis-type determinants expressed on the carbohydrate chains of glycoproteins and glycolipids, and also on unconjugated free oligosaccharides in human milk and other secretions, are associated with various biological functions. We have previously shown the utility of negative-ion electrospay ionization tandem mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) for typing of Lewis (Le) determinants, e.g. Lea, Lex, Leb, and Ley on neutral and sialylated oligosaccharide chains. In the present report we extended the strategy to characterization of blood-group A-, B- and H-determinants on type 1 and type 2, and also on type 4 globoside chains to provide a high sensitivity method for typing of all the major blood-group antigens, including the A, B, H, Lea, Lex, Leb, and Ley determinants, present in oligosaccharides. Using the principles established we identified two minor unknown oligosaccharide components present in the products of enzymatic synthesis by bacterial fermentation. We also demonstrated that the unique fragmentations derived from the D- and 0,2A-type cleavages observed in ESI-CID-MS/MS, which are important for assigning blood-group and chain types, only occur under the negative-ion conditions for reducing sugars but not for reduced alditols or under positive-ion conditions.
    No preview · Article · May 2013 · Analytical Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV-16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell-binding. Here, we analyze the phenomenon that preincubation of HPV-16 with increasing concentrations of heparin results in partial restoration rather than more efficient inhibition of infection. While corroborating that the HSPGs are cell-binding receptors for HPV-16, heparin-preincubated virus bound to the extracellular matrix (ECM) via laminin-332. Furthermore, the interaction of virions with heparin, a representative of the highly sulfated S-domains of heparan sulfate (HS) chains of HSPGs, allowed HPV-16 infection in the absence of cell surface HSPGs. Therefore, we concluded that specific glycan moieties but not specific HSPG protein backbones are required for infection. The increased binding of an epitope-specific antibody to the viral capsid after heparin-binding suggested that initial conformational changes in the HPV-16 virion occur during infection by interaction with 'heparin-like' domains of cellular HSPGs. We propose that HS sequences with specific sulfation patterns are required to facilitate HPV-16 infection.
    No preview · Article · Apr 2013 · Cellular Microbiology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background: The aim is to compare the structures, anticoagulant and antithrombotic activities of two fucosylated chondroitin sulfates isolated from sea cucumbers Isostichopus badionotus (fCS-Ib) and Pearsonothuria graeffei (fCS-Pg), which were known to have different sulfation patterns on the fucose branches. Methods: The structures of fCSs were identified using 2D NMR. Anticoagulant activities were measured by activated partial thromboplastin time (APTT) and thrombin time (TT), and inhibition of factors IIa, Xa and XIIa was assessed in vitro. Antithrombotic activity was determined ex vivo by measuring the length and weight of the thrombus generated. Results: The two fCSs had identical chondroitin sulfate E backbones and similar fucose branches, but different sulfation patterns of the fucose branches. The fucose branch in fCS-Ib was mainly 2,4-O-sulfated whereas that in fCS-Pg was mainly 3,4-O-sulfated. In vitro assay indicated that fCS-Pg possessed much lower potency than fCS-Ib in prolonging APTT/TT and in inhibition of thrombin. However, they both exhibited similar inhibitory effects on factor X activation by intrinsic tenase complex, and on thrombus generation. Furthermore, both fCSs significantly activated factor XII, which has been proved to be associated with adverse clinical events associated with heparin contaminated by oversulfated chondroitin sulfate. Conclusion: The 2,4-O-sulfated fucose branch is the key structural factor of fCSs for prolonged APTT/TT and inhibition of thrombin, whereas the inhibitory effect of fCSs on factor X, XII activation and thrombus generation was attributed to the overall structure of fCS polysaccharide. GENERAL IMPORTANCE: Both fCSs have well defined structures and can be readily quality-controlled, and therefore may be potential alternatives for heparin as anticoagulant and antithrombotic drugs.
    No preview · Article · Jan 2013 · Biochimica et Biophysica Acta
  • Source

    Full-text · Article · Sep 2012
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A charge and size uniform polysaccharide GW2M was extracted with cold water from red alga Gloiopeltis furcata and purified by strong anion ion-exchange and gel permeation chromatography. Its chemical structure was identified by methylation, 1H–1H COSY, 1H–13C HMQC and 1H–13C HMBC techniques. The experimental data showed that GW2M was composed of galactose (40.3%), 3,6-anhydro-galactose (34.1%) and sulfate (24.8%) with an average molecular mass of 20.6 kDa. The results proved GW2M was a linear repeating sequence of alternating (1 → 3)-linked 6-O-sulfated-β-d-galactose (G6S) and (1 → 4)-linked 3,6-anhydro-α-l-galactose (A) which made it to be an ideal 6-O-sulfated-agarose. The sequences of serial oligosaccharides prepared by mild acid and reductive acid hydrolysis from GW2M were confirmed using electrospray collision induced dissociation tandem mass spectrometry (ES-CID-MS/MS) technique.
    Full-text · Article · Jul 2012 · Carbohydrate Polymers

Publication Stats

5k Citations
662.89 Total Impact Points

Institutions

  • 2003-2015
    • Imperial College London
      • • Department of Medicine
      • • Faculty of Medicine
      Londinium, England, United Kingdom
  • 2012
    • Ocean University of China
      • Key Laboratory of Marine Drugs, Ministry of Education
      Tsingtao, Shandong Sheng, China
  • 2005
    • The Scripps Research Institute
      La Jolla, California, United States
  • 2002
    • Royal Melbourne Hospital
      Melbourne, Victoria, Australia
  • 1990-1995
    • MRC Clinical Sciences Centre
      London Borough of Harrow, England, United Kingdom