A fluorogenic probe for core-fucosylated glycan-preferred ENGase

Area of Science:

• Glycobiology
• Enzymology
• Assay Development

Background:

• Endo-β-N-acetylglucosaminidases (ENGases) are crucial enzymes involved in N-glycan processing.
• Core fucosylation of N-glycans is significant in various biological processes.
• Accurate methods for assessing ENGase activity are essential for understanding glycan metabolism.

Purpose of the Study:

• To develop a novel fluorescence-quenching assay for determining ENGase hydrolytic activity.
• To evaluate the assay's performance with specific ENGases acting on core fucosylated N-glycans.
• To elucidate the substrate specificities of different ENGases.

Main Methods:

• Construction of a dual-labeled fluorescent probe with a hexasaccharide structure.
• Utilizing the probe in a fluorescence-quenching-based assay system.
• Testing the assay with purified ENGases (Endo-M W251N mutant, Endo-F3, Endo-S).

Main Results:

• The assay system successfully detected hydrolysis reactions by monitoring increased fluorescence intensity.
• Quantitative evaluation of ENGase activities was achieved for most tested enzymes.
• Substrate specificities of the examined ENGases towards core fucosylated N-glycans were clearly indicated.

Conclusions:

• A robust fluorescence-based assay for ENGase activity has been established.
• The assay facilitates the quantitative assessment of ENGase function on specific N-glycan structures.
• This tool aids in distinguishing substrate specificities among different ENGases, particularly those recognizing core fucosylated glycans.