The development of a broad-spectrum retaining β-exo-galactosidase activity-based probe

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Acid β-galactosidase (GLB1) and galactocerebrosidase (GALC) are crucial retaining exo-β-galactosidases in lysosomal glycoconjugate metabolism.
• Deficiencies in GLB1 and GALC are linked to severe lysosomal storage disorders like GM1 gangliosidosis, Morquio B syndrome, galactosialidosis, and Krabbe disease.
• Activity-based protein profiling (ABPP) is an effective method for assessing enzyme activity in biological contexts.

Purpose of the Study:

• To develop and utilize novel fluorescent and biotin-carrying activity-based probes (ABPs) for assessing GLB1 and GALC activity.
• To demonstrate the utility of these ABPs in various biological samples including cell lysates, culture media, and tissue extracts.
• To provide tools that support fundamental and clinical research on β-galactosidases and associated inherited metabolic disorders.

Main Methods:

• Synthesis and application of novel fluorescent and biotin-carrying activity-based probes (ABPs).
• Activity-based protein profiling (ABPP) applied to cell lysates, culture media, and tissue extracts.
• Utilizing cyclophellitol-based configurational isomer scaffolds for probe design.

Main Results:

• Successful development of ABPs capable of detecting the activity of both GLB1 and GALC.
• Demonstrated efficacy of the probes across diverse biological sample types.
• The new ABPs complement existing tools for studying retaining glycosidases.

Conclusions:

• The developed ABPs are valuable tools for assessing GLB1 and GALC activity.
• These probes facilitate research into the mechanisms and consequences of lysosomal storage disorders.
• The findings contribute to the advancement of diagnostic and therapeutic strategies for β-galactosidase deficiencies.