Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

Background: Niemann-Pick disease type C (NPC) is caused by defects in Cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified Cholesterol in late endosomes/lysosomes is observed. At the level of the organism these Cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly. Until now filipin staining of Cholesterol deposits in cells has been widely used for NPC diagnostics. In this report we present an alternative method for Cholesterol visualization and estimation using a cholesterol-binding Bacterial toxin, perfringolysin O.

Methods: To detect Cholesterol deposits, a recombinant probe, perfringolysin O fused with glutathione S-transferase (GST-PFO) was prepared. GST-PFO followed by labeled Antibodies or streptavidin was applied for immunofluorescence and immunoelectron microscopy to analyze Cholesterol distribution in cells derived from NPC patients. The identity of GST-PFO-positive structures was revealed by a quantitative analysis of their colocalization with several organelle markers. Cellular ELISA using GST-PFO was developed to estimate the level of unesterified Cholesterol in NPC cells.

Results: GST-PFO recognized Cholesterol with high sensitivity and selectivity, as demonstrated by a protein/lipid overlay assay and surface plasmon resonance analysis. When applied to stain NPC cells, GST-PFO decorated abundant deposits of Cholesterol in intracellular vesicles that colocalized with filipin-positive structures. These Cholesterol deposits were resistant to 0.05%-0.2% Triton X-100 used for cells permeabilization in the staining procedure. GST-PFO-stained organelles were identified as late endosomes/lysosomes based on their colocalization with LAMP-1 and lysobisphosphatidic acid. On the other hand, GST-PFO did not colocalize with markers of the Golgi apparatus, endoplasmic reticulum, peroxisomes or with actin filaments. Only negligible GST-PFO staining was seen in fibroblasts of healthy individuals. When applied to cellular ELISA, GST-PFO followed by anti-GST-peroxidase allowed a semiquantitative analysis of Cholesterol level in cells of NPC patients. Binding of GST-PFO to NPC cells was nearly abolished after extraction of Cholesterol with methyl-β-cyclodextrin.

Conclusions: Our data indicate that a recombinant protein GST-PFO can be used to detect Cholesterol accumulated in NPC cells by immunofluorescence and cellular ELISA. GST-PFO can be a convenient and reliable probe for revealing Cholesterol deposits in cells and can be useful in diagnostics of NPC disease.