title: Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.
abstract: Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, 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.
authors: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom; §UCIBIO-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, NOVA University of Lisbon; angelina.palma@fct.unl.pt w.chai@imperial.ac.uk.
authors: Angelina S
authors: AS
authors: Palma
authors: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom;
authors: Yan
authors: Y
authors: Liu
authors: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom;
authors: Hongtao
authors: H
authors: Zhang
authors: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom;
authors: Yibing
authors: Y
authors: Zhang
authors: ¶Megazyme International, County Wicklow, Ireland;
authors: Barry V
authors: BV
authors: McCleary
authors: ‖Key Laboratory of Marine Drugs, Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, School of Medicine and Pharmacy, Ocean University of China;
authors: Guangli
authors: G
authors: Yu
authors: **Department of Chemistry, Wuhan University, Wuhan, China;
authors: Qilin
authors: Q
authors: Huang
authors: ‡‡Instituto de Investigaciones Biotecnológicas "Dr. Rodolfo A. Ugalde", Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de San Martín, Buenos Aires, Argentina;
authors: Leticia S
authors: LS
authors: Guidolin
authors: ‡‡Instituto de Investigaciones Biotecnológicas "Dr. Rodolfo A. Ugalde", Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de San Martín, Buenos Aires, Argentina;
authors: Andres E
authors: AE
authors: Ciocchini
authors: §§Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanitá, Rome, Italy;
authors: Antonella
authors: A
authors: Torosantucci
authors: ¶¶Tumor Glycomics Laboratory, SRI International Biosciences Division, Menlo Park, CA;
authors: Denong
authors: D
authors: Wang
authors: §UCIBIO-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, NOVA University of Lisbon;
authors: Ana Luísa
authors: AL
authors: Carvalho
authors: ‖‖CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Portugal.
authors: Carlos M G A
authors: CM
authors: Fontes
authors: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom;
authors: Barbara
authors: B
authors: Mulloy
authors: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom;
authors: Robert A
authors: RA
authors: Childs
authors: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom;
authors: Ten
authors: T
authors: Feizi
authors: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom; angelina.palma@fct.unl.pt w.chai@imperial.ac.uk.
authors: Wengang
authors: W
authors: Chai
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism; D005936:Glucans / Q000378:metabolism*; D007107:Immune System / Q000378:metabolism; D037181:Lectins, C-Type / Q000378:metabolism; D008024:Ligands; D051379:Mice; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D020543:Proteome / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014612:Vaccines / Q000276:immunology; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
chemical_list: D000906:Antibodies; D015815:Cell Adhesion Molecules; D005936:Glucans; D037181:Lectins, C-Type; D008024:Ligands; D009844:Oligosaccharides; D020543:Proteome; D011956:Receptors, Cell Surface; D014612:Vaccines; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin; C055213:saccharide-binding proteins