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MM3D-Fluorogenic Probe for Real-Time Measurement of ENGase Activity

 

M3174

MM3D is hydrolyzed by endo-β-N-acetylglucosaminidase (ENGase) to generate fluorescence. MM3D is a useful tool for real-time measurement of the enzymatic activity of ENGase.

Principle

 

MM3D-Fluorogenic Probe for Real-Time Measurement of ENGase Activity

Endo-β-N-acetylglucosaminidase (ENGase) first hydrolyzes the chitobiose moiety of N-glycan. MM3D, a N-glycan which features both a fluorescent group (N-methyl anthraniloyl, MANT) and a quencher group (dinitrophenyl, DNP) in the structure. Cleavage of MM3D by ENGase removes the self-quenching effect, leading to an increase in fluorescence intensity.1) MM3D has shown to be useful for high throughput analysis with plate readers, and in the search for new types of ENGase or inhibitor of ENGase. These inhibitors can be served as therapeutic agents for NGLY1-deficiency, a very rare genetic disease.2,3)
*Note: MM3D is not hydrolyzed by Endo-H which belongs to GH Family 18, and therefore is not suitable for measurement of Endo-H activity.

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Measurement of Endo-M [A1651] activity using MM3D1)

(Data are provided by professor Matsuo, Gunma university.)

Comparing the fluorescence intensity of MM3D with that of the hydrolyzed product, the fluorescence of MANT is decreased by 98% due to the quenching effect of the DNP group (a). However, since the cleaved product of MM3D is highly sensitive, the concentration of MM3D is sufficient in 0.5 µM to measure Endo-M [A1651] activity (b). Next, addition of DMSO to hinder the enzymatic reaction suppresses enzyme activity in a concentration-dependent manner (c). These results indicate that MM3D is suitable for screening for potential inhibitors of ENGase.

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This product was produced by collaboration with Prof. Ichiro Matsuo at Gunma University.

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