Carbohydrate affinity PAGE for the study of carbohydrate-binding proteins (original) (raw)
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GlycoChip: multiarray for the study of carbohydrate-binding proteins
Lab on a Chip, 2003
Biotinylated glycoconjugates which were designed as oligosaccharides attached to 30 kDa polyacrylamide were coated on a microarray platform XNAonGOLD™, which was developed earlier for an oligonucleotide assay. The specificity of antibodies to carbohydrate antigens was analyzed using the glyco-microarray. Comparison of the obtained results with those of common 96-well plate ELISA completely coincided with the found antibody specificities. However, parameters such as the analytical sensitivity of the method and the amount of biotinylated material coated on the microarray platform proved to be worse than expected.
Studies on the specificity of rabbit hepatic carbohydrate-binding protein using neoglycoproteins
Biochemistry, 1980
The binding of amidinoneoglycoproteins of bovine serum albumin to rabbit liver membranes was measured. Derivatives of bovine serum albumin to which equivalent amounts of P-D-Gal, 6-O-Me-@-~-Gal, P-D-Fuc, a-L-Ara, P-D-G~c, @-D-Xyl, and @-DGalNAc had been attached bound to the membranes equally well. The attachment of aD Man , p-L-FUC, P-D-G~cNAc, p-D-allose, 3-O-Me-@-~-Glc, and 2deoxy-P-DGlc did not promote strong binding. The specificity of binding to the membranes was confirmed by measuring the binding of neoglycoproteins to the purified rabbit hepatic
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.
Enzyme and Microbial Technology, 2000
Affinity electrophoresis was used to identify and quantify the interaction of carbohydrate-binding modules (CBMs) with soluble polysaccharides. Association constants determined by AE were in excellent agreement with values obtained by isothermal titration calorimetry and fluorescence titration. The method was adapted to the identification, study and characterization of mutant carbohydratebinding modules with altered affinities and specificities. Competition affinity electrophoresis was used to monitor binding of small, soluble mono-and disaccharides to one of the modules.
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.
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.
Gene, 1987
antibodies directed against carbohydrate-binding protein 35 (CBP35), a galactose-specific lectin, were used to screen a 2gt 11 expression library derived from mRNA of 3T3 fibroblasts. This screening yielded several putative clones containing cDNA for CBP35, one of which was characterized in terms of its expression of a fusion protein containing /I-galactosidase and CBP35 sequences. Limited proteolysis of lysates containing the fusion protein, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-CBP35, yielded a peptide mapping pattern comparable to that obtained from parallel treatment of authentic CBP35. Such a limited proteolysis followed by afftnity chromatography on a Sepharose column coupled with galactose also yielded a 30-kDa polypeptide that exhibited carbohydrate-binding activity. This polypeptide can be immunoblotted with anti-CBP35, but not with antibodies directed against /I-galactosidase.
Carbohydrate-binding domains: multiplicity of biological roles
Applied Microbiology and Biotechnology, 2010
Insoluble polysaccharides can be degraded by a set of hydrolytic enzymes formed by catalytic modules appended to one or more non-catalytic carbohydrate-binding modules (CBM). The most recognized function of these auxiliary domains is to bind polysaccharides, bringing the biocatalyst into close and prolonged vicinity with its substrate, allowing carbohydrate hydrolysis. Examples of insoluble polysaccharides recognized by these enzymes include cellulose, chitin, β-glucans, starch, glycogen, inulin, pullulan, and xylan. Based on their amino acid similarity, CBMs are grouped into 55 families that show notable variation in substrate specificity; as a result, their biological functions are miscellaneous. Carbohydrate or polysaccharide recognition by CBMs is an important event for processes related to metabolism, pathogen defense, polysaccharide biosynthesis, virulence, plant development, etc. Understanding of the CBMs properties and mechanisms in ligand binding is of vital significance for the development of new carbohydrate-recognition technologies and provide the basis for fine manipulation of the carbohydrate–CBM interactions.