Activity-based probes for functional interrogation of retaining β-glucuronidases

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Disease Mechanisms

Background:

• Humans possess distinct β-glucuronidase enzymes, exo-acting GUSB and endo-acting HPSE, implicated in diseases like mucopolysaccharidosis type VII, inflammation, and cancer.
• Accurate methods for assaying GUSB and HPSE activity in biological samples are crucial for understanding their roles in health and disease.

Purpose of the Study:

• To develop and validate novel β-glucuronidase-specific activity-based probes (ABPs) for the rapid and quantitative visualization of GUSB and HPSE.
• To utilize these ABPs to investigate the enzymatic activities of GUSB and HPSE in various biological contexts.
• To explore potential novel functions or structural insights related to HPSE and its proenzyme form.

Main Methods:

• Design and synthesis of a suite of β-glucuronidase-specific activity-based probes (ABPs).
• Application of ABPs for the visualization and activity profiling of GUSB and HPSE in biological samples.
• Biochemical and structural analysis to characterize enzyme-probe interactions and enzyme functionality.

Main Results:

• The developed ABPs enable rapid and quantitative visualization of both GUSB and HPSE activities in biological samples.
• Unexpectedly, the proenzyme form of heparanase, proHPSE, was also labeled by the ABPs, indicating previously unrecognized biochemical activity or binding.
• These findings provide new insights into the structural relationships between proHPSE, mature HPSE, and their bacterial counterparts.

Conclusions:

• β-glucuronidase ABPs are powerful tools for tracking pathologically relevant enzymes like GUSB and HPSE in normal and disease states.
• The study highlights the utility of ABP-driven approaches for uncovering unanticipated structural and biochemical functionalities, as demonstrated by the proHPSE findings.
• This work advances the understanding of β-glucuronidase enzymes and offers a new methodology for enzymatic activity assessment in biomedical research.