Sticholysin II Identifies Intracellular Lipid Deposits in Niemann Pick C Fibroblasts

Gabriele Chieppa ,Fiorella Piemonte ,Roberta Taurisano ,Giovanna D’arcangelo ,A Zaratti ,C Dionisi Vici ,Claudio Frank ,Federica Deodato ,Rosalba Carrozzo ,Stefano Rufini

doi:10.13188/2328-1723.1000011

Abstract

The cells of patients of Niemann-Pick C disease are characterized by the presence of sphingolipids and cholesterol atypical intracellular storages. The existence of a cause-effect relation between the composition, nature and kinetics of the lipid storages and the pathogenesis of the disease still not clear, mainly due to the scarcity and low reliability of the tools that are able to unambiguously recognize the different lipids. In this study we report the use of sticholysine II, a sea-anemone protein able to bind to sphingomyelin, along with its antibody in the analysis of cell lipids. Results demonstrate that the protein recognizes a well-defined lipid-storage vesicles class in the NPC patients fibroblasts. A confocal fluorescence microscopy analysis of the cells labelled with sticholysin together with the cholesterol- dye filipin and cholera toxin that binds specifically GM1, shows that cholesterol and sphingolipids accumulate in different classes of vesicles. In conclusion, we propose Sticholysin as a novel and valuable tool for the study of the nature and the dynamic of lipid storage in so called lysosomal-diseases.

Highlights

Sphingolipids are ubiquitous and critical components of eukaryotic cell biological membranes typically interacting with cholesterol to drive the formation of plasma membrane rafts [1]
StnII belongs to a wider family of protein such as Equinotoxin and Lysenin that bind selectively SM [16] and using a monoclonal antibody anti-StnII (A10) we demonstrated that Sticholysin II (Stn II) is an efficient tool to localise the SM in the plasma membrane of cultured cells [17]
Images obtained by fluorescence microscopy show that in H-DF only a diffuse red staining is detectable, this is associated to the plasma membranes (Figure 1a) while in Niemann-Pick disease type C (NPC)-DF, StnII dyes brightly discrete granules (Figure 1b)

Summary

Introduction

Sphingolipids are ubiquitous and critical components of eukaryotic cell biological membranes typically interacting with cholesterol to drive the formation of plasma membrane rafts [1]. NPC1 is an integral membrane protein on the limiting membrane of late endosome/lysosome (LE/LY) that mediates cholesterol transport from LE/LY to endoplasmic reticulum (ER) and plasma membrane in a vesicle - or oxysterol-binding protein (OSBP) - related protein 5 (ORP5) - dependent manner [9]. Mutations in the NPC1 gene cause a defective mechanism of lipid fate [10] or of the authophagic process [11] with a massive accumulation of intracellular stored granule lipids. The lipid deposit is always composed of non-esterified cholesterol while the presence of different sphingolipides classes is related to the cell type [7]. We compared the cytoplasmic non-esterified cholesterol localization with the sphingomyelin (SM) and GM1 deposits, in both healthy and NPC disease patient fibroblast cultures using the probes Filipin, Cholera toxin (CtxB) and Sticholysin II (Stn II). StnII belongs to a wider family of protein such as Equinotoxin and Lysenin that bind selectively SM [16] and using a monoclonal antibody anti-StnII (A10) we demonstrated that Stn II is an efficient tool to localise the SM in the plasma membrane of cultured cells [17]

Methods

Results

Conclusion