Publications by authors named "Angelina S Palma"

44 Publications

Non-Covalent Microarrays from Synthetic Amino-Terminating Glycans-Implications in Expanding Glycan Microarray Diversity and Platform Comparison.

Glycobiology 2021 May 8. Epub 2021 May 8.

Glycosciences Laboratory, Imperial College London, London W12 0NN, United Kingdom.

Glycan microarrays have played important roles in detection and specificity assignment of glycan-recognition by proteins. However, the size and diversity of glycan libraries in current microarray systems are small compared to estimated glycomes, and these may lead to missed detection or incomplete assignment. For microarray construction, covalent and non-covalent immobilization are the two types of methods used, but a direct comparison of results from the two platforms is required. Here we develop a chemical strategy to prepare lipid-linked probes from both naturally-derived aldehyde-terminating and synthetic amino-terminating glycans that addresses the two aspects: expansion of sequence-defined glycan libraries and comparison of the two platforms. We demonstrate the specific recognition by plant and mammalian lectins, carbohydrate-binding modules and antibodies, and the overall similarities from the two platforms. Our results provide new knowledge on unique glycan-binding specificities for the immune-receptor Dectin-1 towards β-glucans and the interaction of rotavirus P[19] adhesive protein with mucin O-glycan cores.
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http://dx.doi.org/10.1093/glycob/cwab037DOI Listing
May 2021

Cracking the Breast Cancer Glyco-Code through Glycan-Lectin Interactions: Targeting Immunosuppressive Macrophages.

Int J Mol Sci 2021 Feb 17;22(4). Epub 2021 Feb 17.

iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.

The immune microenvironment of breast cancer (BC) is composed by high macrophage infiltrates, correlated with the most aggressive subtypes. Tumour-associated macrophages (TAM) within the BC microenvironment are key regulators of immune suppression and BC progression. Nevertheless, several key questions regarding TAM polarisation by BC are still not fully understood. Recently, the modulation of the immune microenvironment has been described via the recognition of abnormal glycosylation patterns at BC cell surface. These patterns rise as a resource to identify potential targets on TAM in the BC context, leading to the development of novel immunotherapies. Herein, we will summarize recent studies describing advances in identifying altered glycan structures in BC cells. We will focus on BC-specific glycosylation patterns known to modulate the phenotype and function of macrophages recruited to the tumour site, such as structures with sialylated or N-acetylgalactosamine epitopes. Moreover, the lectins present at the surface of macrophages reported to bind to such antigens, inducing tumour-prone TAM phenotypes, will also be highlighted. Finally, we will discuss and give our view on the potential and current challenges of targeting these glycan-lectin interactions to reshape the immunosuppressive landscape of BC.
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http://dx.doi.org/10.3390/ijms22041972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922062PMC
February 2021

Combined in silico and in vitro studies to identify novel antidiabetic flavonoids targeting glycogen phosphorylase.

Bioorg Chem 2021 Mar 15;108:104552. Epub 2020 Dec 15.

LAQV-REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal.

Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4'-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines.
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http://dx.doi.org/10.1016/j.bioorg.2020.104552DOI Listing
March 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

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

Molecular basis for the preferential recognition of β1,3-1,4-glucans by the family 11 carbohydrate-binding module from Clostridium thermocellum.

FEBS J 2020 07 19;287(13):2723-2743. Epub 2019 Dec 19.

UCIBIO, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Caparica, Portugal.

Understanding the specific molecular interactions between proteins and β1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3-1,4-mixed-linked over β1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the β1,3-linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3-1,4-mixed-linked glucans. This is mediated by a conformation-selection mechanism of the ligand in the binding cleft through CH-π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3-linkage at the reducing end and at specific positions along the β1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked β-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. DATABASE: Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.
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http://dx.doi.org/10.1111/febs.15162DOI Listing
July 2020

Chemoenzymatic Synthesis of -Mannose Glycans Containing Sulfated or Nonsulfated HNK-1 Epitope.

J Am Chem Soc 2019 12 2;141(49):19351-19359. Epub 2019 Dec 2.

National Glycoengineering Research Center, State Key Laboratory of Microbial Technology, Shandong Key Laboratory of Carbohydrate Chemistry and Glycobiology , Shandong University , Qingdao 266237 , China.

The human natural killer-1 (HNK-1) epitope is a unique sulfated trisaccharide sequence presented on - and -glycans of various glycoproteins and on glycolipids. It is overexpressed in the nervous system and plays crucial roles in nerve regeneration, synaptic plasticity, and neuronal diseases. However, the investigation of functional roles of HNK-1 in a more complex glycan context at the molecular level remains a big challenge due to lack of access to related structurally well-defined complex glycans. Herein, we describe a highly efficient chemoenzymatic approach for the first collective synthesis of HNK-1-bearing -mannose glycans with different branching patterns, and for their nonsulfated counterparts. The successful strategy relies on both chemical glycosylation of a trisaccharide lactone donor for the introduction of sulfated HNK-1 branch and substrate promiscuities of bacterial glycosyltransferases that can tolerate sulfated substrates for enzymatic diversification. Glycan microarray analysis with the resulting complex synthetic glycans demonstrated their recognition by two HNK-1-specific antibodies including anti-HNK-1/N-CAM (CD57) and Cat-315, which provided further evidence for the recognition epitopes of these antibodies and the essential roles of the sulfate group for HNK-1 glycan-antibody recognition.
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http://dx.doi.org/10.1021/jacs.9b08964DOI Listing
December 2019

Structural analysis and potential immunostimulatory activity of Nannochloropsis oculata polysaccharides.

Carbohydr Polym 2019 Oct 4;222:114962. Epub 2019 Jun 4.

QOPNA & LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; CICECO, Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal. Electronic address:

The relevance of microalgae biotechnology for producing high-value compounds with biomedical application, such as polysaccharides, has been increasing. Despite this, the knowledge about the composition and structure of microalgae polysaccharides is still scarce. In this work, water-soluble polysaccharides from Nannochloropsis oculata were extracted, fractionated, structurally analysed, and subsequently tested in terms of immunostimulatory activity. A combination of sugar and methylation analysis with interaction data of carbohydrate-binding proteins using carbohydrate microarrays disclosed the complex structural features of the different polysaccharides. These analyses showed that the water-soluble polysaccharides fractions from N. oculata were rich in (β1→3, β1→4)-glucans, (α1→3)-, (α1→4)-mannans, and anionic sulphated heterorhamnans. The immunostimulatory assay highlighted that these fractions could also stimulate murine B-lymphocytes. Thus, the N. oculata water-soluble polysaccharides show potential to be further explored for immune-mediated biomedical applications.
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http://dx.doi.org/10.1016/j.carbpol.2019.06.001DOI Listing
October 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

Novel monoclonal antibody L2A5 specifically targeting sialyl-Tn and short glycans terminated by alpha-2-6 sialic acids.

Sci Rep 2018 08 15;8(1):12196. Epub 2018 Aug 15.

UCIBIO-REQUIMTE, Department of Life Sciences, Faculty of Science and Technology, NOVA University of Lisbon, Lisbon, 2829, Portugal.

Incomplete O-glycosylation is a feature associated with malignancy resulting in the expression of truncated glycans such as the sialyl-Tn (STn) antigen. Despite all the progress in the development of potential anti-cancer antibodies, their application is frequently hindered by low specificities and cross-reactivity. In this study, a novel anti-STn monoclonal antibody named L2A5 was developed by hybridoma technology. Flow cytometry analysis showed that L2A5 specifically binds to sialylated structures on the cell surface of STn-expressing breast and bladder cancer cell lines. Moreover, immunoblotting assays demonstrated reactivity to tumour-associated O-glycosylated proteins, such as MUC1. Tumour recognition was further observed using immunohistochemistry assays, which demonstrated a high sensitivity and specificity of L2A5 mAb towards cancer tissue, using bladder and colorectal cancer tissues. L2A5 staining was exclusively tumoural, with a remarkable reactivity in invasive and metastasis sites, not detectable by other anti-STn mAbs. Additionally, it stained 20% of cases of triple-negative breast cancers, suggesting application in diseases with unmet clinical needs. Finally, the fine specificity was assessed using glycan microarrays, demonstrating a highly specific binding of L2A5 to core STn antigens and additional ability to bind 2-6-linked sialyl core-1 probes. In conclusion, this study describes a novel anti-STn antibody with a unique binding specificity that can be applied for cancer diagnostic and future development of new antibody-based therapeutic applications.
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http://dx.doi.org/10.1038/s41598-018-30421-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093877PMC
August 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

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

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

Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.

Mol Cell Proteomics 2015 Apr 10;14(4):974-88. Epub 2015 Feb 10.

From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom;

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, 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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http://dx.doi.org/10.1074/mcp.M115.048272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390274PMC
April 2015

Conformational analysis of the Streptococcus pneumoniae hyaluronate lyase and characterization of its hyaluronan-specific carbohydrate-binding module.

J Biol Chem 2014 Sep 6;289(39):27264-27277. Epub 2014 Aug 6.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada,. Electronic address:

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.
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http://dx.doi.org/10.1074/jbc.M114.578435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175358PMC
September 2014

Carbohydrate sequence of the prostate cancer-associated antigen F77 assigned by a mucin O-glycome designer array.

J Biol Chem 2014 Jun 21;289(23):16462-77. Epub 2014 Apr 21.

From the Glycosciences Laboratory, Department of Medicine, Imperial College London, W12 0NN London, United Kingdom,

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.
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http://dx.doi.org/10.1074/jbc.M114.558932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047413PMC
June 2014

The neoglycolipid (NGL)-based oligosaccharide microarray system poised to decipher the meta-glycome.

Curr Opin Chem Biol 2014 Feb 7;18:87-94. Epub 2014 Feb 7.

The Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cbpa.2014.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105633PMC
February 2014

Structures of B-lymphotropic polyomavirus VP1 in complex with oligosaccharide ligands.

PLoS Pathog 2013 Oct 31;9(10):e1003714. Epub 2013 Oct 31.

Interfaculty Institute of Biochemistry, University of Tuebingen, Tuebingen, Germany.

B-Lymphotropic Polyomavirus (LPyV) serves as a paradigm of virus receptor binding and tropism, and is the closest relative of the recently discovered Human Polyomavirus 9 (HPyV9). LPyV infection depends on sialic acid on host cells, but the molecular interactions underlying LPyV-receptor binding were unknown. We find by glycan array screening that LPyV specifically recognizes a linear carbohydrate motif that contains α2,3-linked sialic acid. High-resolution crystal structures of the LPyV capsid protein VP1 alone and in complex with the trisaccharide ligands 3'-sialyllactose and 3'-sialyl-N-acetyl-lactosamine (3SL and 3SLN, respectively) show essentially identical interactions. Most contacts are contributed by the sialic acid moiety, which is almost entirely buried in a narrow, preformed cleft at the outer surface of the capsid. The recessed nature of the binding site on VP1 and the nature of the observed glycan interactions differ from those of related polyomaviruses and most other sialic acid-binding viruses, which bind sialic acid in shallow, more exposed grooves. Despite their different modes for recognition, the sialic acid binding sites of LPyV and SV40 are half-conserved, hinting at an evolutionary strategy for diversification of binding sites. Our analysis provides a structural basis for the observed specificity of LPyV for linear glycan motifs terminating in α2,3-linked sialic acid, and links the different tropisms of known LPyV strains to the receptor binding site. It also serves as a useful template for understanding the ligand-binding properties and serological crossreactivity of HPyV9.
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http://dx.doi.org/10.1371/journal.ppat.1003714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814675PMC
October 2013

A structure-guided mutation in the major capsid protein retargets BK polyomavirus.

PLoS Pathog 2013 10;9(10):e1003688. Epub 2013 Oct 10.

Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.

Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.
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http://dx.doi.org/10.1371/journal.ppat.1003688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795024PMC
May 2014

Changes in the hemagglutinin of H5N1 viruses during human infection--influence on receptor binding.

Virology 2013 Dec 17;447(1-2):326-37. Epub 2013 Sep 17.

Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam; Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1016/j.virol.2013.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820038PMC
December 2013

The C-type lectin receptor CLECSF8 (CLEC4D) is expressed by myeloid cells and triggers cellular activation through Syk kinase.

J Biol Chem 2012 Jul 11;287(31):25964-74. Epub 2012 Jun 11.

Institute of Infectious Disease and Molecular Medicine, Central Laboratory Services, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town 7925, South Africa.

CLECSF8 is a poorly characterized member of the "Dectin-2 cluster" of C-type lectin receptors and was originally thought to be expressed exclusively by macrophages. We show here that CLECSF8 is primarily expressed by peripheral blood neutrophils and monocytes and weakly by several subsets of peripheral blood dendritic cells. However, expression of this receptor is lost upon in vitro differentiation of monocytes into dendritic cells or macrophages. Like the other members of the Dectin-2 family, which require association of their transmembrane domains with signaling adaptors for surface expression, CLECSF8 is retained intracellularly when expressed in non-myeloid cells. However, we demonstrate that CLECSF8 does not associate with any known signaling adaptor molecule, including DAP10, DAP12, or the FcRγ chain, and we found that the C-type lectin domain of CLECSF8 was responsible for its intracellular retention. Although CLECSF8 does not contain a signaling motif in its cytoplasmic domain, we show that this receptor is capable of inducing signaling via Syk kinase in myeloid cells and that it can induce phagocytosis, proinflammatory cytokine production, and the respiratory burst. These data therefore indicate that CLECSF8 functions as an activation receptor on myeloid cells and associates with a novel adaptor molecule. Characterization of the CLECSF8-deficient mice and screening for ligands using oligosaccharide microarrays did not provide further insights into the physiological function of this receptor.
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http://dx.doi.org/10.1074/jbc.M112.384164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3406680PMC
July 2012

Neoglycolipid-based "designer" oligosaccharide microarrays to define β-glucan ligands for Dectin-1.

Methods Mol Biol 2012 ;808:337-59

The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK.

In this chapter, we describe the key steps of the "designer" oligosaccharide microarray approach we followed to prove the carbohydrate binding activity and define the oligosaccharide ligands for Dectin-1, an atypical C-type lectin-like signaling receptor of the mammalian innate immune system with a key role in anti-fungal immunity. The term "designer" microarray, which we introduced in the course of the Dectin-1 study refers to a microarray of oligosaccharide probes generated from ligand-bearing glycoconjugates to reveal the oligosaccharide ligands they harbor, so that these can be isolated and characterized. Oligosaccharide probes were generated from two polysaccharides, one that was bound by Dectin-1 and known to be rich in β1,3-glucose sequence and another that was not bound and was rich in β1,6-glucose sequence and served as a negative control. The approach involved: classic ELISA-type binding assays to select the polysaccharides; partial depolymerization of the polysaccharides by chemical hydrolysis; fractionation by size of the glucan oligosaccharides obtained and determination of their chain lengths by mass spectrometry; detection of Dectin-1 ligand-positive and ligand-negative oligosaccharides using the neoglycolipid (NGL) technology; methylation analysis of oligosaccharides to derive glucose linkage information, and incorporation of the newly generated glucan oligosaccharide probes into microarrays encompassing diverse mammalian-type and exogenous sequences for microarray analysis of Dectin-1.
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http://dx.doi.org/10.1007/978-1-61779-373-8_23DOI Listing
March 2012

Neoglycolipid-based oligosaccharide microarray system: preparation of NGLs and their noncovalent immobilization on nitrocellulose-coated glass slides for microarray analyses.

Methods Mol Biol 2012 ;808:117-36

The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK.

Carbohydrate microarrays, since their advent in 2002, are revolutionizing studies of the molecular basis of protein-carbohydrate interactions both in endogenous recognition systems and pathogen-host interactions. We have developed a unique carbohydrate microarray system based on the neoglycolipid (NGL) technology, a well-validated microscale approach for generating lipid-tagged oligosaccharide probes for use in carbohydrate recognition studies. This chapter provides an overview of the principles and key features of the NGL-based oligosaccharide microarrays, and describes in detail the basic techniques - from the preparation of NGL probes to the generation of microarrays using robotic arraying hardware, as well as a general protocol for probing the microarrays with carbohydrate-binding proteins.
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http://dx.doi.org/10.1007/978-1-61779-373-8_8DOI Listing
March 2012

The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella.

PLoS Pathog 2011 Oct 13;7(10):e1002296. Epub 2011 Oct 13.

Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, United Kingdom.

Eimeria spp. are a highly successful group of intracellular protozoan parasites that develop within intestinal epithelial cells of poultry, causing coccidiosis. As a result of resistance against anticoccidial drugs and the expense of manufacturing live vaccines, it is necessary to understand the relationship between Eimeria and its host more deeply, with a view to developing recombinant vaccines. Eimeria possesses a family of microneme lectins (MICs) that contain microneme adhesive repeat regions (MARR). We show that the major MARR protein from Eimeria tenella, EtMIC3, is deployed at the parasite-host interface during the early stages of invasion. EtMIC3 consists of seven tandem MAR1-type domains, which possess a high specificity for sialylated glycans as shown by cell-based assays and carbohydrate microarray analyses. The restricted tissue staining pattern observed for EtMIC3 in the chicken caecal epithelium indicates that EtMIC3 contributes to guiding the parasite to the site of invasion in the chicken gut. The microarray analyses also reveal a lack of recognition of glycan sequences terminating in the N-glycolyl form of sialic acid by EtMIC3. Thus the parasite is well adapted to the avian host which lacks N-glycolyl neuraminic acid. We provide new structural insight into the MAR1 family of domains and reveal the atomic resolution basis for the sialic acid-based carbohydrate recognition. Finally, a preliminary chicken immunization trial provides evidence that recombinant EtMIC3 protein and EtMIC3 DNA are effective vaccine candidates.
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http://dx.doi.org/10.1371/journal.ppat.1002296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192848PMC
October 2011

Bacterial, fungal, and algal lectins: combatants in tug of war against HIV.

Structure 2011 Aug;19(8):1035-7

The Glycosciences Laboratory, Imperial College London, Burlington Danes Building Level 5, Du Cane Road, London, W12 0NN, UK.

High-resolution X-ray crystallography and NMR studies by Koharudin and Gronenborn in this issue provide new information on the mode of N-glycan recognition by a cyanobacterial agglutinin, with anti-HIV activity pointing to the pentamannosyl core as a novel target for therapeutic intervention.
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http://dx.doi.org/10.1016/j.str.2011.07.006DOI Listing
August 2011

Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling.

J Biol Chem 2011 Jul 12;286(27):24208-18. Epub 2011 May 12.

Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.

The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1-4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.
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http://dx.doi.org/10.1074/jbc.M111.226142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3129202PMC
July 2011

The human epithelial carcinoma antigen recognized by monoclonal antibody AE3 is expressed on a sulfoglycolipid in addition to neoplastic mucins.

Biochem Biophys Res Commun 2011 May 19;408(4):548-52. Epub 2011 Apr 19.

Glycosciences Laboratory, Department of Medicine, Imperial College London, Northwick Park and St. Marks Campus, Watford Road, Harrow, Middlesex, UK.

The term human epithelial carcinoma antigen (HCA) has been applied collectively to mucin-type high molecular weight (>1000kDa) glycoproteins that are over-expressed in epithelial cancers. Since the 1990s, over 40 monoclonal antibodies have been raised that recognize HCA. There has been evidence that the antigenic determinants are mostly carbohydrates, but details have been elusive. Here we have carried out carbohydrate microarray analyses of one of the monoclonal antibodies, AE3, that has been regarded the 'most carcinoma specific' in respect to its ability to detect HCA in sera of patients with epithelial cancers. The microarrays encompassed a series of 492 sequence-defined glycan probes in the form of glycolipids and neoglycolipids. We have thus established that the antigen recognized by antibody AE3 is a carbohydrate sequence distinct from the A, B, H, Lewis(a/b), Lewis(x/y) and T antigens, but that it is strongly expressed on the monosulfated tetra-glycosyl ceramide, SM1a, Galβ1-3GalNAcβ1-4(3-O-sulfate)Galβ1-4GlcCer. This is the first report of an anti-HCA to be characterized with respect to its recognition sequence and of the occurrence of the antigen on a glycolipid as well as on glycoproteins. Knowledge of a discrete glycan sequence as target antigen now opens the way to its exploration as a serologic cancer biomarker, namely to determine if the antigen elicits an autoantibody response in early non-metastatic cancer, or if it is shed and immunochemically detectable in more advanced disease.
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http://dx.doi.org/10.1016/j.bbrc.2011.04.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100433PMC
May 2011

The interactions of calreticulin with immunoglobulin G and immunoglobulin Y.

Biochim Biophys Acta 2011 Jul 5;1814(7):889-99. Epub 2011 Apr 5.

Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark.

Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N-glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N-linked glycan, which is a ligand for calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of calreticulin. The experiments demonstrate the ability of calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY.
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http://dx.doi.org/10.1016/j.bbapap.2011.03.015DOI Listing
July 2011