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Quantifying lysosomal glycosidase activity within cells using bis-acetal substrates.

Samy Cecioni1,2, Roger A Ashmus1, Pierre-André Gilormini1

  • 1Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

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Researchers developed novel bis-acetal-based (BAB) fluorescent substrates for quantitative monitoring of glycoside hydrolase activity in live cells. This tool aids in developing diagnostics and therapeutics for enzyme-related diseases.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Enzymology

Background:

  • Enzyme activity within cells is crucial for physiological processes.
  • Existing tools for monitoring enzyme activity in live cells have limitations.
  • Accurate measurement of enzyme function in vivo is essential for understanding disease and developing treatments.

Purpose of the Study:

  • To develop a novel strategy for quantitative monitoring of glycoside hydrolase activity in live cells.
  • To design and synthesize bright, modular bis-acetal-based (BAB) fluorescence-quenched substrates.
  • To apply these substrates for sensitive quantitation of specific human glycosidase activities relevant to disease.

Main Methods:

  • Design and synthesis of bis-acetal-based (BAB) fluorescence-quenched substrates.
  • Application of BAB substrates for live-cell imaging and quantitation.
  • Utilizing chemical perturbagens to investigate cellular trafficking and lysosomal homeostasis.
  • Measuring inhibitor engagement and pharmacological chaperone efficiency.

Main Results:

  • Demonstrated successful design and synthesis of BAB substrates.
  • Enabled sensitive and quantitative monitoring of human α-galactosidase and α-N-acetylgalactosaminidase activities in live cells.
  • Showed utility in patient-derived cells to assess enzyme inhibitor and chaperone efficacy.
  • Highlighted the importance of in-cell activity measurements.

Conclusions:

  • BAB substrates provide a powerful tool for quantitative analysis of glycosidases in live cells.
  • This strategy overcomes limitations of previous methods for enzyme activity monitoring.
  • The substrates facilitate the development of therapeutics and diagnostics for glycosidase-related disorders.
  • Enables deeper interrogation of enzyme regulation and cellular processes.