Flow Cytometry-Based Assay to Detect Alpha Galactosidase Enzymatic Activity at the Cellular Level
Nóra Fekete1,2, Luca Kamilla Li3, Gergely Tibor Kozma4,5
1Department of Genetics, Cell and Immunobiology, Semmelweis University, 1085 Budapest, Hungary.
Cells
|April 26, 2024
Summary
A new flow cytometry assay quantifies alpha-galactosidase A (AGAL) activity in individual cells. This method aids in diagnosing Fabry disease, particularly in heterozygous women where traditional tests fall short.
Area of Science:
- Biochemistry
- Genetics
- Cell Biology
Background:
- Fabry disease is an X-linked lysosomal disorder causing multi-organ dysfunction due to deficient alpha-galactosidase A (AGAL) activity.
- Accumulation of glycosphingolipids like globotriaosyl-ceramide (Gb3) occurs in cells.
- Current assays measuring mean AGAL activity often fail to detect dysfunction in heterozygous women due to variable symptomatology.
Purpose of the Study:
- To develop a flow cytometry assay for measuring AGAL activity at the single-cell level.
- To improve the diagnosis of Fabry disease, especially in cases with heterogeneous presentation.
Main Methods:
- Utilized conventional and multispectral imaging flow cytometry to detect AGAL activity.
- Validated assay specificity using a GLA knockout Jurkat cell line and an AGAL inhibitor.
- Generated and validated the GLA KO cell line using CRISPR-Cas9, exome sequencing, and gene expression analysis.
Main Results:
- Demonstrated the feasibility of detecting specific AGAL activity using fluorescently labeled Gb3 via flow cytometry.
- Determined optimal substrate concentration (2.83 nmol/mL) and incubation time (6 hours) for Jurkat cells.
- Established the necessity of quenching non-specific Gb3 binding with trypan blue for accurate detection of lysosomal substrate accumulation.
Conclusions:
- Developed a quantitative, single-cell flow cytometry assay for AGAL activity.
- This assay holds potential for improved diagnosis of Fabry disease patients.
