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

Maria Angelina de Sá Palma, Yan Liu, Hongtao Zhang, Yibing Zhang, Barry V McCleary, Guangli Yu, Qilin Huang, Leticia S Guidolin, Andres E Ciocchini, Antonella Torosantucci, Denong Wang, Ana Luísa Carvalho, Carlos M G A Fontes, Barbara Mulloy, Robert A Childs, Ten Feizi, Wengang Chai

Research output: Contribution to journalArticle

34 Citations (Scopus)

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 $- or $-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.
Original languageEnglish
Pages (from-to)974-988
Number of pages15
JournalMolecular & Cellular Proteomics
Volume14
Issue number4
DOIs
Publication statusPublished - 1 Apr 2015

Keywords

  • Animals
  • Antibodies
  • Carbohydrate Sequence
  • Cell Adhesion Molecules
  • Glucans
  • Immune System
  • Lectins, C-Type
  • Ligands
  • Mice
  • Oligosaccharides
  • Protein Array Analysis
  • Protein Binding
  • Proteome
  • Receptors, Cell Surface
  • Reproducibility of Results
  • Signal Transduction
  • Spectrometry, Mass, Electrospray Ionization
  • Vaccines
  • Journal Article
  • Research Support, Non-U.S. Gov't

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