Publications by authors named "Ten Feizi"

102 Publications

A Major Subset of Mutated CLL Expresses Affinity-selected and Functionally Proficient Rheumatoid Factors.

Hemasphere 2021 Apr 23;5(4):e550. Epub 2021 Mar 23.

Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands.

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http://dx.doi.org/10.1097/HS9.0000000000000550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990012PMC
April 2021

Helicobacter pylori lipopolysaccharide structural domains and their recognition by immune proteins revealed with carbohydrate microarrays.

Carbohydr Polym 2021 Feb 2;253:117350. Epub 2020 Nov 2.

Glycosciences Laboratory, Department of Metabolism, Digestion and Reproduction, Imperial College London, W12 0NN, UK; UCIBIO, Department of Chemistry, School of Science and Technology, NOVA University of Lisbon, 2829-516 Lisbon, Portugal.

The structural diversity of the lipopolysaccharides (LPSs) from Helicobacter pylori poses a challenge to establish accurate and strain-specific structure-function relationships in interactions with the host. Here, LPS structural domains from five clinical isolates were obtained and compared with the reference strain 26695. This was achieved combining information from structural analysis (GC-MS and ESI-MS) with binding data after interrogation of a LPS-derived carbohydrate microarray with sequence-specific proteins. All LPSs expressed Lewis and N-acetyllactosamine determinants. Ribans were also detected in LPSs from all clinical isolates, allowing their distinction from the 26695 LPS. There was evidence for 1,3-d-galactans and blood group H-type 2 sequences in two of the clinical isolates, the latter not yet described for H. pylori LPS. Furthermore, carbohydrate microarray analyses showed a strain-associated LPS recognition by the immune lectins DC-SIGN and galectin-3 and revealed distinctive LPS binding patterns by IgG antibodies in the serum from H. pylori-infected patients.
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http://dx.doi.org/10.1016/j.carbpol.2020.117350DOI Listing
February 2021

Chikungunya Virus Strains from Each Genetic Clade Bind Sulfated Glycosaminoglycans as Attachment Factors.

J Virol 2020 11 23;94(24). Epub 2020 Nov 23.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

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 are 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 enzyme-linked immunosorbent assays (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. 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 to severe and debilitating arthritis. Despite the severity of CHIKV disease, there are no licensed therapeutics. Since attachment factors and receptors are determinants of viral tropism and pathogenesis, understanding these virus-host interactions can enhance our knowledge of CHIKV infection. We analyzed over 670 glycans and identified GAGs as the main glycan bound by CHIKV. We defined specific GAG components required for CHIKV binding and assessed strain-specific differences in GAG binding capacity. These studies provide insight about cell surface molecules that CHIKV binds, which could facilitate the development of antiviral therapeutics targeting the CHIKV attachment step.
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http://dx.doi.org/10.1128/JVI.01500-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925169PMC
November 2020

Siglec-15 recognition of sialoglycans on tumor cell lines can occur independently of sialyl Tn antigen expression.

Glycobiology 2021 Jan;31(1):44-54

Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin expressed on osteoclast progenitors, which plays an important role in osteoclast development and function. It is also expressed by tumor-associated macrophages and by some tumors, where it is thought to contribute to the immunosuppressive microenvironment. It was shown previously that engagement of macrophage-expressed Siglec-15 with tumor cells expressing its ligand, sialyl Tn (sTn), triggered production of TGF-β. In the present study, we have further investigated the interaction between Siglec-15 and sTn on tumor cells and its functional consequences. Based on binding assays with lung and breast cancer cell lines and glycan-modified cells, we failed to see evidence for recognition of sTn by Siglec-15. However, using a microarray of diverse, structurally defined glycans, we show that Siglec-15 binds with higher avidity to sialylated glycans other than sTn or related antigen sequences. In addition, we were unable to demonstrate enhanced TGF-β secretion following co-culture of Siglec-15-expressing monocytic cell lines with tumor cells expressing sTn or following Siglec-15 cross-linking with monoclonal antibodies. However, we did observe activation of the SYK/MAPK signaling pathway following antibody cross-linking of Siglec-15 that may modulate the functional activity of macrophages.
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http://dx.doi.org/10.1093/glycob/cwaa048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799145PMC
January 2021

Mannan detecting C-type lectin receptor probes recognise immune epitopes with diverse chemical, spatial and phylogenetic heterogeneity in fungal cell walls.

PLoS Pathog 2020 01 30;16(1):e1007927. Epub 2020 Jan 30.

MRC Centre for Medical Mycology, Aberdeen Fungal Group, College of Life Sciences and Medicine, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, United Kingdom.

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.
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http://dx.doi.org/10.1371/journal.ppat.1007927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012452PMC
January 2020

Nanolithography of biointerfaces.

Authors:
Ten Feizi

Faraday Discuss 2019 10;219(0):262-275

Faculty of Medicine, Imperial College London, London, UK.

This article is based on the Concluding remarks made at the Faraday Discussion meeting on Nanolithography of Biointerfaces, held in London, UK, 3-5th July 2019.
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http://dx.doi.org/10.1039/c9fd00082hDOI Listing
October 2019

Glycan Markers of Human Stem Cells Assigned with Beam Search Arrays.

Mol Cell Proteomics 2019 10 15;18(10):1981-2002. Epub 2019 Jul 15.

Glycosciences Laboratory, Department of Medicine, Imperial College London, London W12 0NN, United Kingdom. Electronic address:

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, -3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and anti-i, the type 2-(Galβ-4GlcNAc) analog, -3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and we determine substituents they can accommodate. They differ from R-10G, which requires sulfate. By 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 -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.
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http://dx.doi.org/10.1074/mcp.RA119.001309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773554PMC
October 2019

Editorial overview: Carbohydrates: O-glycosylation.

Curr Opin Struct Biol 2019 06 12;56:iii-v. Epub 2019 Jun 12.

Glycosciences Laboratory, Department of Surgery and Cancer, Imperial College London, London, UK. Electronic address:

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http://dx.doi.org/10.1016/j.sbi.2019.05.010DOI Listing
June 2019

Sulfated Glycosaminoglycans as Viral Decoy Receptors for Human Adenovirus Type 37.

Viruses 2019 03 12;11(3). Epub 2019 Mar 12.

Section of Virology, Department of Clinical Microbiology, Umeå University, SE-90185 Umeå, Sweden.

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.
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http://dx.doi.org/10.3390/v11030247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466042PMC
March 2019

Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis.

Nat Commun 2018 12 11;9(1):5288. Epub 2018 Dec 11.

Medical Research Council Centre for Medical Mycology at the University of Aberdeen, Aberdeen, AB25 2ZD, UK.

The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis.
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http://dx.doi.org/10.1038/s41467-018-07738-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290022PMC
December 2018

The neoglycolipid (NGL) technology-based microarrays and future prospects.

Authors:
Zhen Li Ten Feizi

FEBS Lett 2018 12 29;592(23):3976-3991. Epub 2018 Aug 29.

Glycosciences Laboratory, Imperial College London, UK.

The neoglycolipid (NGL) technology is the basis of a state-of-the-art oligosaccharide microarray system, which we offer for screening analyses to the broad scientific community. We review here the sequential development of the technology and its power in pinpointing and isolating naturally occurring ligands for glycan-binding proteins (GBPs) within glycan populations. We highlight our Designer Array approach and Beam Search Array approach for generating natural glycome arrays to identify novel ligands of biological relevance. These two microarray approaches have been applied for assignments of ligands or antigens on glucan polysaccharides for effector proteins of the immune system (Dectin-1, DC-SIGN and DC-SIGNR) and carbohydrate-binding modules (CBMs) on bacterial hydrolases. We also discuss here the more recent applications to elucidate the structure of a prostate cancer- associated antigen F77 and identify ligands for adhesins of two rotaviruses, P[10] and P[19], expressed on an epithelial mucin glycoprotein.
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http://dx.doi.org/10.1002/1873-3468.13217DOI Listing
December 2018

Assignment by Negative-Ion Electrospray Tandem Mass Spectrometry of the Tetrasaccharide Backbones of Monosialylated Glycans Released from Bovine Brain Gangliosides.

J Am Soc Mass Spectrom 2018 Jun 11;29(6):1308-1318. Epub 2018 May 11.

Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, W12 0NN, UK.

Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a "gangliome" microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series. Graphical abstract.
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http://dx.doi.org/10.1007/s13361-018-1944-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003998PMC
June 2018

Polysialic acid is a cellular receptor for human adenovirus 52.

Proc Natl Acad Sci U S A 2018 05 19;115(18):E4264-E4273. Epub 2018 Apr 19.

Division of Virology, Department of Clinical Microbiology, and Laboratory for Molecular Infection Medicine Sweden, Umeå University, SE-90185 Umeå, Sweden.

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 .
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http://dx.doi.org/10.1073/pnas.1716900115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939068PMC
May 2018

Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus.

Nature 2018 03 28;555(7696):382-386. Epub 2018 Feb 28.

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal.

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 immunity. 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 melanin, 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.
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http://dx.doi.org/10.1038/nature25974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5857201PMC
March 2018

Insights Into Glucan Polysaccharide Recognition Using Glucooligosaccharide Microarrays With Oxime-Linked Neoglycolipid Probes.

Methods Enzymol 2018 21;598:139-167. Epub 2017 Nov 21.

Glycosciences Laboratory, Imperial College London, London, United Kingdom.

Glucans are polysaccharides of increasing biomedical interest because of their involvement in mechanisms of pathogen recognition, modulation of the immune system and anticancer, and health-promoting activities. Most of these biological activities occur through specific interactions with glucan-recognizing proteins. However, detailed molecular studies of glucan recognition remain a challenge mainly due to the inherent sequence heterogeneity and polydispersity of glucan polysaccharides, and associated difficulties in their purification and sequence characterization. It is thus ideal to have a series of sequence-defined glucooligosaccharides to represent the sequence diversity of glucan polysaccharides and to apply these to gain insight into glucan recognition processes. In this chapter, we describe the the methods for developing of oligosaccharide microarrays derived from a collection of glucans with different linkages based on the neoglycolipid (NGL) microarray system. The microscale oxime-ligation method has provided access in microarrays to over 150 sequence-defined glucooligosaccharides with different chain lengths, linkages, and branching patterns. We focus on the essential steps in the preparation of NGL-based glucooligosaccharide microarrays, which include (1) the depolymerization and purification methods to obtain oligosaccharide fractions of defined chain lengths; (2) a mass spectrometry-based method for linkage and sequence analysis of glucooligosaccharides; (3) improved procedures for preparation of oxime-linked NGLs from glucooligosaccharides for construction of microarrays; and (4) analyses of the recognition of these oligosaccharide sequences by various glucan-recognizing proteins: monoclonal antibodies, other proteins of the immune system such as Dectin-1 and DC-SIGN, and carbohydrate-binding modules of bacterial glycoside hydrolases.
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http://dx.doi.org/10.1016/bs.mie.2017.09.001DOI Listing
January 2019

O-Glycome Beam Search Arrays for Carbohydrate Ligand Discovery.

Mol Cell Proteomics 2018 01 28;17(1):121-133. Epub 2017 Nov 28.

From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, W12 0NN, UK;

-glycosylation is a post-translational modification of proteins crucial to molecular mechanisms in health and disease. -glycans are typically highly heterogeneous. The involvement of specific -glycan sequences in many bio-recognition systems is yet to be determined because of a lack of efficient methodologies. We describe here a targeted microarray approach: -glycome that is both robust and efficient for -glycan ligand-discovery. Substantial simplification of the complex -glycome profile and facile chromatographic resolution is achieved by arraying -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 -glycome of this mucin that are ligands for the adhesive proteins of two rotaviruses. The approach is applicable to -glycome recognition studies in a wide range of biological settings to give insights into glycan recognition structures in natural microenvironments.
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http://dx.doi.org/10.1074/mcp.RA117.000285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750842PMC
January 2018

Binding of CLL subset 4 B-cell receptor immunoglobulins to viable human memory B lymphocytes requires a distinctive IGKV somatic mutation.

Mol Med 2017 Mar 12;23:1-12. Epub 2017 Jan 12.

Karches Center for Oncology Research, The Feinstein Institute for Medical Research, Manhasset, NY, 11030, USA.

Amino acid replacement mutations in certain CLL stereotyped B-cell receptor (BCR) immunoglobulins (IGs) at defined positions within antigen-binding sites strongly imply antigen selection. Prime examples of this are CLL subset 4 BCR IGs using IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements. Conspicuously and unlike most CLL IGs, subset 4 IGs do not bind apoptotic cells. By testing the (auto)antigenic reactivities of subset 4 IGs toward viable lymphoid-lineage cells and specific autoantigens typically bound by IGHV4-34 IGs, we found IGs from both subset 4 and non-subset 4 IGHV4-34-expressing CLL cases bind naïve B cells. However, only subset 4 IGs react with memory B cells. Furthermore, subset 4 IGs do not bind DNA nor i or I carbohydrate antigens, common targets of IGHV4-34-utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively. Notably, we found that subset 4 IG binding to memory B lymphocytes depends on an aspartic acid at position 66 of FR3 in the rearranged IGKV2-30 gene; this amino acid residue is acquired by somatic mutation. Our findings illustrate the importance of positive and negative selection criteria for structural elements in CLL IGs and suggest that autoantigens driving normal B cells to become subset 4 CLL cells differ from those driving IGHV4-34 B cells in other diseases.
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http://dx.doi.org/10.2119/molmed.2017.00003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364113PMC
March 2017

The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data.

Glycobiology 2017 04 22;27(4):280-284. Epub 2016 Nov 22.

Emory Comprehensive Glycomics Core, Emory University School of Medicine, Atlanta, GA 30322, USA.

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  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.
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http://dx.doi.org/10.1093/glycob/cww118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444268PMC
April 2017

Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates.

PLoS One 2016 3;11(11):e0166119. Epub 2016 Nov 3.

Centre for Organismal Studies, Department of Cell Chemistry, Heidelberg University, Heidelberg, Germany.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166119PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5094735PMC
June 2017

The minimum information required for a glycomics experiment (MIRAGE) project: sample preparation guidelines for reliable reporting of glycomics datasets.

Glycobiology 2016 09 21;26(9):907-910. Epub 2016 Sep 21.

Beilstein-Institut, Trakehner Str. 7-9, 60487 Frankfurt am Main, Germany

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045532PMC
http://dx.doi.org/10.1093/glycob/cww082DOI Listing
September 2016

Glycan Specificity of P[19] Rotavirus and Comparison with Those of Related P Genotypes.

J Virol 2016 Nov 14;90(21):9983-9996. Epub 2016 Oct 14.

Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). However, unlike the other P[II] RVs, which mainly infect humans, P[19] RVs commonly infect animals (pigs), making P[19] unique for the study of RV diversity and host ranges. Through in vitro binding assays and saturation transfer difference (STD) nuclear magnetic resonance (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 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 our 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 of the molecular bases for strain-specific host ranges and cross-species transmission of these human and animal RVs is important to understand RV epidemiology and disease burden, which may impact development of control and prevention strategies against RV gastroenteritis.
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http://dx.doi.org/10.1128/JVI.01494-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068545PMC
November 2016

Abnormally High Content of Free Glucosamine Residues Identified in a Preparation of Commercially Available Porcine Intestinal Heparan Sulfate.

Anal Chem 2016 07 15;88(13):6648-52. Epub 2016 Jun 15.

Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.

Heparan sulfate (HS) polysaccharides are ubiquitous in animal tissues as components of proteoglycans, and they participate in many important biological processes. HS carbohydrate chains are complex and can contain rare structural components such as N-unsubstituted glucosamine (GlcN). Commercially available HS preparations have been invaluable in many types of research activities. In the course of preparing microarrays to include probes derived from HS oligosaccharides, we found an unusually high content of GlcN residue in a recently purchased batch of porcine intestinal mucosal HS. Composition and sequence analysis by mass spectrometry of the oligosaccharides obtained after heparin lyase III digestion of the polysaccharide indicated two and three GlcN in the tetrasaccharide and hexasaccharide fractions, respectively. (1)H NMR of the intact polysaccharide showed that this unusual batch differed strikingly from other HS preparations obtained from bovine kidney and porcine intestine. The very high content of GlcN (30%) and low content of GlcNAc (4.2%) determined by disaccharide composition analysis indicated that N-deacetylation and/or N-desulfation may have taken place. HS is widely used by the scientific community to investigate HS structures and activities. Great care has to be taken in drawing conclusions from investigations of structural features of HS and specificities of HS interaction with proteins when commercial HS is used without further analysis. Pending the availability of a validated commercial HS reference preparation, our data may be useful to members of the scientific community who have used the present preparation in their studies.
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http://dx.doi.org/10.1021/acs.analchem.6b01662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948919PMC
July 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 2016 10 6;26(10):1086-1096. Epub 2016 Apr 6.

Glycosciences Laboratory, Department of Medicine, Imperial College London, London W12 0NN, UK

The β1,2-glucans produced by bacteria are important in invasion, survival and immunomodulation in infected hosts be they mammals or plants. However, there has been a lack of information on proteins which recognize these molecules. This is partly due to the extremely limited availability of the sequence-defined oligosaccharides and derived probes for use in the study of their interactions. Here we have used the cyclic β1,2-glucan (CβG) of the bacterial pathogen Brucella abortus, after removal of succinyl side chains, to prepare linearized oligosaccharides which were used to generate microarrays. We describe optimized conditions for partial depolymerization of the cyclic glucan by acid hydrolysis and conversion of the β1,2-gluco-oligosaccharides, with degrees of polymerization 2-13, to neoglycolipids for the purpose of generating microarrays. By microarray analyses, we show that the C-type lectin receptor DC-SIGNR, like the closely related DC-SIGN we investigated earlier, binds to the β1,2-gluco-oligosaccharides, as does the soluble immune effector serum mannose-binding protein. Exploratory studies with DC-SIGN are suggestive of the recognition also of the intact CβG by this receptor. These findings open the way to unravelling mechanisms of immunomodulation mediated by β1,2-glucans in mammalian systems.
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http://dx.doi.org/10.1093/glycob/cww041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072146PMC
October 2016

Effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza A (H3N2) vaccine viruses.

J Gen Virol 2016 06 14;97(6):1333-1344. Epub 2016 Mar 14.

Formerly Divisions of Virology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK.

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.
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http://dx.doi.org/10.1099/jgv.0.000457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394856PMC
June 2016

Editorial overview: Carbohydrate-protein interactions and glycosylation: Glycan synthesis and recognition: finding the perfect partner in a sugar-coated life.

Curr Opin Struct Biol 2015 Oct 21;34:vii-ix. Epub 2015 Nov 21.

Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602-4712, USA. Electronic address:

Oligosaccharides 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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http://dx.doi.org/10.1016/j.sbi.2015.10.005DOI Listing
October 2015

Negative-Ion Electrospray Tandem Mass Spectrometry and Microarray Analyses of Developmentally Regulated Antigens Based on Type 1 and Type 2 Backbone Sequences.

Anal Chem 2015 Dec 10;87(23):11871-8. Epub 2015 Nov 10.

Glycosciences Laboratory, Department of Medicine, Imperial College London , Hammersmith Campus, London W12 0NN, U.K.

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 microsequencing 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 type 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.
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http://dx.doi.org/10.1021/acs.analchem.5b03471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929357PMC
December 2015

Yeast expressed ArtinM shares structure, carbohydrate recognition, and biological effects with native ArtinM.

Int J Biol Macromol 2016 Jan 1;82:22-30. Epub 2015 Oct 1.

Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil. Electronic address:

Recent 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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http://dx.doi.org/10.1016/j.ijbiomac.2015.09.062DOI Listing
January 2016

Total syntheses of disulphated glycosphingolipid SB1a and the related monosulphated SM1a.

Org Biomol Chem 2015 Dec 24;13(45):11105-17. Epub 2015 Sep 24.

Department of Applied Bioorganic Chemistry, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan.

Total 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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http://dx.doi.org/10.1039/c5ob01744kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920060PMC
December 2015