Assaying sialyltransferase activity with surface plasmon resonance

Area of Science:

• Biochemistry
• Enzymology
• Glycobiology

Background:

• Sialyltransferases are crucial enzymes involved in synthesizing sialylated glycans, which play vital roles in numerous biological processes.
• Accurate and efficient assays are needed to study sialyltransferase function and screen for inhibitors or activators.
• Existing methods for assessing sialyltransferase activity can be time-consuming or require large amounts of substrate.

Purpose of the Study:

• To develop and validate a novel surface plasmon resonance (SPR) based activity assay for sialyltransferases.
• To demonstrate the utility of the SPR assay for ST3Gal-III (EC 2.4.99.6) using various acceptor substrates.
• To establish a method for rapid screening of sialyltransferase enzymatic activities, including those in complex biological samples.

Main Methods:

• Immobilization or synthesis of natural and synthetic oligosaccharides as acceptor substrates on SPR chips.
• Incubation of immobilized substrates with sialyltransferase (ST3Gal-III) and CMP-Neu5Ac.
• Detection of enzymatic turnover using lectins specific for either the acceptor or the sialylated product.
• Correlation of SPR data with a classical radio assay for quantification of enzymatic activity.
• Regeneration of the SPR chip using sialidase for repeated experiments.

Main Results:

• Successful development of an SPR-based assay for determining sialyltransferase activity.
• Demonstrated ability to measure ST3Gal-III activity with high sensitivity, requiring only nanomolar quantities of acceptor substrates.
• Validation of the SPR assay by correlating results with a traditional radio assay.
• The SPR chip is regenerable, allowing for multiple experimental runs.

Conclusions:

• The developed SPR assay provides a rapid, sensitive, and reusable method for quantifying sialyltransferase activity.
• This technique is particularly valuable for high-throughput screening of enzyme activities in diverse samples, such as cell lysates.
• The assay facilitates the study of sialyltransferases and the discovery of modulators of their activity.