Glyco-array technology for efficient monitoring of plant cell wall glycosyltransferase activities
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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 α(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 [14C]Fuc onto xyloglucan polymer and trimer. [14C]-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.
KeywordsCell wall Glycosyltransferases Glycochip Xyloglucan Microarray
arabidopsis fucosyltransferase 1
degree of polymerization
high performance liquid chromatography
matrix-assisted laser-desorption/ionization time-of-flight
Spodoptera frugiperda 21
We would like to thank Dr. Kenneth Keegstra for providing AtFUT1 protein expressed in Spodoptera frugiperda 21 (Sf21) cells. Our thanks go also to all of the members of the Faik laboratory and cell wall group at Ohio University, in particular to Dr. Showalter and Mr Wei Zeng, for helpful discussion and technical support. This work was supported in part by grant no. II/2/2005 to center of excellence GLYCOBIOS from the Slovak Academy of Sciences and grant and no. 2/6133/06 from Grant Agency VEGA to V.F.
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