GlycoChip: multiarray for the study of carbohydrate-binding proteins (original) (raw)
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Analytical Biochemistry, 2010
Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidincoated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins.
Journal of Cereal Science, 2009
New technologies based on microarray methods have begun to feature widely in carbohydrate chemistry and biology. These 'glycoarray' techniques, which in a number of cases emulate what has been achieved with DNA microarrays, allow for high throughput, quantitative analysis of protein-carbohydrate interactions. Lectin, antibody and enzyme specificity have been evaluated with these new techniques, which also extend to the detection of viruses and bacteria, and serodiagnosis of infection. In the plant field, high throughput mapping of cell wall carbohydrate structures has been reported, giving information not only on the localisation of given glycans within a plant, but also allowing systematic comparison between mutants and species. This review outlines some of the basic principles of 'glycoarrays' and illustrates recent reports of their development and application.
Nature Biotechnology, 2002
We describe microarrays of oligosaccharides as neoglycolipids and their robust display on nitrocellulose. The arrays are sourced from glycoproteins, glycolipids, proteoglycans, polysaccharides, whole organs, or from chemically synthesized oligosaccharides. We show that carbohydrate-recognizing proteins single out their ligands not only in arrays of homogeneous oligosaccharides but also in arrays of heterogeneous oligosaccharides. Initial applications have revealed new findings, including: (i) among O-glycans in brain, a relative abundance of the Lewis x sequence based on N-acetyllactosamine recognized by anti-L5, and a paucity of the Lewis x sequence based on poly-N-acetyllactosamine recognized by anti-SSEA-1; (ii) insights into chondroitin sulfate oligosaccharides recognized by an antiserum and an antibody (CS-56) to chondroitin sulfates; and (iii) binding of the cytokine interferon-γ (IFN-γ) and the chemokine RANTES to sulfated sequences such as HNK-1, sulfo-Lewis x , and sulfo-Lewis a , in addition to glycosaminoglycans. The approach opens the way for discovering new carbohydrate-recognizing proteins in the proteome and for mapping the repertoire of carbohydrate recognition structures in the glycome.
Glycoarrays—tools for determining protein–carbohydrate interactions and glycoenzyme specificity
Chemical Communications, 2008
Carbohydrate arrays (glycoarrays) have recently emerged as a high-throughput tool for studying carbohydrate-binding proteins and carbohydrate-processing enzymes. A number of sophisticated array platforms that allow for qualitative and quantitative analysis of carbohydrate binding and modification on the array surface have been developed, including analysis by fluorescence spectroscopy, mass spectrometry and surface plasmon resonance spectroscopy. These platforms, together with examples of biologically-relevant applications are reviewed in this Feature Article.
Carbohydrate microarrays — a new set of technologies at the frontiers of glycomics
Current Opinion in Structural Biology, 2003
Carbohydrate microarray technologies are new developments at the frontiers of glycomics. Results of 'proof of concept' experiments with carbohydrate-binding proteins of the immune system-antibodies, selectins, a cytokine and a chemokineand several plant lectins indicate that microarrays of carbohydrates (glycoconjugates, oligosaccharides and monosaccharides) will greatly facilitate not only surveys of proteins for carbohydrate-binding activities but also elucidation of their ligands. It is predicted that both naturally occurring and synthetic carbohydrates will be required for the fabrication of microarrays that are sufficiently comprehensive and representative of entire glycomes. New leads to biological pathways that involve carbohydrate-protein interactions and new therapeutic targets are among biomedically important outcomes anticipated from applications of carbohydrate microarrays.
Carbohydrate microarrays: key developments in glycobiology
bchm, 2009
Carbohydrate chains of glycoproteins, glycolipids, proteoglycans, and polysaccharides mediate processes of biological and medical importance through their interactions with complementary proteins. The unraveling of these interactions is therefore a priority in biomedical sciences. Carbohydrate microarray technology is a new development at the frontier of glycomics that is revolutionizing the study of carbohydrate-protein interactions and the elucidation of their specificities in endogenous biological processes, microbe-host interactions, and immune defense mechanisms. In this review, we briefly refer to the principles of numerous platforms since the introduction of carbohydrate microarrays in 2002, and we highlight platforms that are beyond proof-of-concept and have provided new biological information.
PloS one, 2013
Improved detection of anti-carbohydrate antibodies is a need in clinical identification of biomarkers for cancer cells or pathogens. Here, we report a new ELISA approach for the detection of specific immunoglobulins (IgGs) against carbohydrates. Two nanometer gold glyconanoparticles bearing oligosaccharide epitopes of HIV or Streptococcus pneumoniae were used as antigens to coat ELISA-plates. A ~3,000-fold improved detection of specific IgGs in mice immunized against S. pneumoniae respect to the well known BSA-glycoconjugate ELISA was achieved. Moreover, these multivalent glyconanoparticles have been employed in solid phase assays to detect the carbohydratedependent binding of human dendritic cells and the lectin DC-SIGN. Multivalent glyconanoparticles in ELISA provide a versatile, easy and highly sensitive method to detect and quantify the binding of glycan to proteins and to facilitate the identification of biomarkers.
Biosensors & Bioelectronics, 2006
Carbohydrate-protein interactions play important biological roles in biological processes. But there is a lack of high-throughput methods to elucidate recognition events between carbohydrates and proteins. This paper reported a convenient and efficient method for preparing oligosaccharide microarrays, wherein the underivatized oligosaccharide probes were efficiently immobilized on aminooxyacetyl functionalized glass surface by formation of oxime bonding with the carbonyl group at the reducing end of the suitable carbohydrates via irreversible condensation. Prototypes of carbohydrate microarrays containing 10 oligosaccharides were fabricated on aminooxyacetyl functionalized glass by robotic arrayer. Utilization of the prepared carbohydrate microarrays for the characterization of carbohydrate-protein interaction reveals that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. The limit of detection (LOD) for lectin ConA on the fabricated carbohydrate microarrays was determined to be ∼0.008 g/mL. Inhibition experiment with soluble carbohydrates also demonstrated that the binding affinities of lectins to different carbohydrates could be analyzed quantitatively by determining IC 50 values of the soluble carbohydrates with the carbohydrate microarrays. This work provides a simple procedure to prepare carbohydrate microarray for high-throughput parallel characterization of carbohydrate-protein interaction.