Glyco-array technology for efficient monitoring of plant cell wall glycosyltransferase activities. 2008

Matthew Shipp, and Ramya Nadella, and Hui Gao, and Vladimir Farkas, and Hans Sigrist, and Ahmed Faik
Environmental and Plant Biology department, Ohio University, Porter Hall 512, Athens, OH, 45701, USA.

The plant cell wall is a complex network of polysaccharides. The diversity in the linkage types connecting all monosaccharides within these polysaccharides would need a large set of glycosyltransferases to catalyze their formation. Development of a methodology that would allow monitoring of glycosyltransferase activities in an easy and high-throughput manner would help assign biochemical functions, and understand their roles in building this complex network. A microarray-based method was optimized for testing glycosyltransferases involved in plant wall biosynthesis using an alpha(1,2)fucosyltransferase involved in xyloglucan biosynthesis. The method is simple, sensitive, and easy to implement in any lab. Tamarind xyloglucan polymer and trimer, and a series of cello-oligosaccharides were immobilized on a thin-coated photo-activable glass slide. The slide with the attached sugars was then used to estimate the incorporation of [(14)C]Fuc onto xyloglucan polymer and trimer. [(14)C]-radiolabel incorporation is revealed with a standard phosphoimager scanner, after exposure of the glycochip to a phosphor screen and detection. The method proved to be sensitive enough to detect as low as 45 cpm/spot. Oriented anchoring of small oligosaccharides (trimer) was required for optimal transferase activities. The glycochip was also used to monitor and estimate xyloglucan fucosyltransferase activity in detergent-solubilized crude extracts from pea microsomes that are known to contain this enzyme activity. Our data indicate that the methodology can be used for efficient and rapid monitoring of glycosyltransferase activities involved in plant wall polysaccharides biosynthesis.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D002240 Carbohydrate Sequence The sequence of carbohydrates within POLYSACCHARIDES; GLYCOPROTEINS; and GLYCOLIPIDS. Carbohydrate Sequences,Sequence, Carbohydrate,Sequences, Carbohydrate
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D002473 Cell Wall The outermost layer of a cell in most PLANTS; BACTERIA; FUNGI; and ALGAE. The cell wall is usually a rigid structure that lies external to the CELL MEMBRANE, and provides a protective barrier against physical or chemical agents. Cell Walls,Wall, Cell,Walls, Cell
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D013379 Substrate Specificity A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts. Specificities, Substrate,Specificity, Substrate,Substrate Specificities
D016695 Glycosyltransferases Enzymes that catalyze the transfer of glycosyl groups to an acceptor. Most often another carbohydrate molecule acts as an acceptor, but inorganic phosphate can also act as an acceptor, such as in the case of PHOSPHORYLASES. Some of the enzymes in this group also catalyze hydrolysis, which can be regarded as transfer of a glycosyl group from the donor to water. Subclasses include the HEXOSYLTRANSFERASES; PENTOSYLTRANSFERASES; SIALYLTRANSFERASES; and those transferring other glycosyl groups. EC 2.4. Glycosyltransferase,Glycoside Transferases,Transferases, Glycoside
D017360 Arabidopsis A plant genus of the family BRASSICACEAE that contains ARABIDOPSIS PROTEINS and MADS DOMAIN PROTEINS. The species A. thaliana is used for experiments in classical plant genetics as well as molecular genetic studies in plant physiology, biochemistry, and development. Arabidopsis thaliana,Cress, Mouse-ear,A. thaliana,A. thalianas,Arabidopses,Arabidopsis thalianas,Cress, Mouse ear,Cresses, Mouse-ear,Mouse-ear Cress,Mouse-ear Cresses,thaliana, A.,thaliana, Arabidopsis,thalianas, A.
D046228 Microarray Analysis The simultaneous analysis, on a microchip, of multiple samples or targets arranged in an array format. Microarray Analytical Devices,Microarray Microchips,Nanoarray Analytical Devices,Analysis, Microarray,Analytical Device, Microarray,Analytical Device, Nanoarray,Analytical Devices, Microarray,Analytical Devices, Nanoarray,Device, Microarray Analytical,Device, Nanoarray Analytical,Devices, Microarray Analytical,Devices, Nanoarray Analytical,Microarray Analytical Device,Microarray Microchip,Microchip, Microarray,Microchips, Microarray,Nanoarray Analytical Device
D050260 Carbohydrate Metabolism Cellular processes in biosynthesis (anabolism) and degradation (catabolism) of CARBOHYDRATES. Metabolism, Carbohydrate

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