Glycosyltransferases are enzymes that catalyze the transfer of a monosaccharide moiety from a glycosyl donor to an acceptor substrate. The majority are classified as Leloir enzymes that utilize nucleotide sugars as donors and produce nucleotide phosphates as part of the reaction. Assaying glycosyltransferase activity can be challenging. The most common method has been to monitor the transfer of radiolabeled sugars from donor to acceptor molecules. Various non-radioactive assays have also been developed; however, most of these have been customized for specific glycosyltransferases.
R&D Systems has developed a versatile, non-radioactive assay for measuring glycosyltransferase activity (Figure 1).1 The assay involves the use of a specific phosphatase that liberates phosphate from nucleotides generated during the glycosyltransferase reaction. The phosphate levels are then assessed using malachite green phosphate detection reagents. Because the concentration of released phosphate is directly proportional to the number of sugar molecules transferred, kinetic parameters of glycosyltransferases can be measured. In addition, this colorimetric assay is conducted in a 96-well plate, making it amenable to high-throughput studies.
To demonstrate the utility of this technique, we assessed the activities of the enzymes Clostridium difficile toxin B (TcdB; Figure 2A), human O-Glucosyltransferase I (KTELC1; Figure 2B), and human ST6GAL1 (Figure 2C). TcdB and KTELC1 are glucosyltransferases with UDP-Glucose hydrolase activity that produce UDP as a byproduct of the reaction. The phosphatase CD39L3 hydrolyzes nucleotide beta-phosphates and was used to liberate free phosphate from UDP. In contrast, ST6GAL1 is a sialyltransferase that catalyzes the transfer of sialic acid in alpha 2,6 linkage to Gal beta 1-4GlcNAc structures on N-glycans. For this reaction, CMP-NeuAc was used as a donor substrate and N-acetyllactosamine (LN) was used as the acceptor. The 5' nucleotidase CD73 was used to liberate free phosphate from CMP produced in the reaction. For all three glycosyltransferase reactions, accurate measurements of the specific activity (pmol/min/µg) versus donor substrate concentration were obtained by measuring free phosphate levels using malachite green detection reagents (Figure 2).
R&D Systems has used this technique to examine the activities of a wide range of glycosyltransferases. For more details, see our recent publication1 or visit our website at www.RnDSystems.com/go/Glycosyltransferase to request a copy of our scientific poster, Universal Phosphatase-Coupled Glycosyltransferase Assay. R&D Systems Glycosyltransferase Activity Kit (Catalog # EA001) is also now available. This kit contains the specific phosphatase CD39L3, buffers, and phosphate detection reagents to measure the activity of glycosyltransferases that use diphosphonucleotide sugars as donor substrates.
Phosphatase-coupled Glycosyltransferase Reactions
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Figure 1. The Phosphatase-coupled Glycosyltransferase Assay. This non-radioactive assay (Catalog # EA001)couples glycosyltransferase reactions with specific phosphatases to generate free phosphate groups, which are then measured. The specific phosphatase is chosen based on the phosphonucleotide produced during the reaction. Liberated free phosphate is then measured using malachite green detection reagents, and the levels are used as a measure of enzyme activity. |
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Figure 2. Measuring Enzyme Activity Using the Phosphatase-coupled Glycosyltransferase Assay. (A) The activities of Recombinant TcdB (Catalog # 6246-GT) and (B) Recombinant Human KTELC1 (Catalog # 6437-GT) were measured using the donor substrate UDP-Glucose (acceptor H2O). Both glycosyltransferase reactions were carried out in the presence of Recombinant Human CD39L3 phosphatase (Catalog # 4400-EN). Free phosphate was detected using the Malachite Green Phosphate Detection Kit (Catalog # DY996). (C) The activity of Recombinant Human ST6GAL1 (Catalog # 5924-GT) was measured using the donor substrate CMP-NeuAC (acceptor LN). The glycosyltransferase reaction was carried out in the presence of Recombinant Human CD73 phosphatase (Catalog # 4488-EN). Free phosphate was detected using the Malachite Green Phosphate Detection Kit (Catalog # DY996). |
References
- Wu, Z.L. et al. (2010) Glycobiology [Epub ahead of print].
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