Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry

Biochim Biophys Acta. 2016 Nov;1858(11):2882-2893. doi: 10.1016/j.bbamem.2016.08.015. Epub 2016 Aug 31.


Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1μm, and larger vesicles of around 10-μm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

Keywords: Aegerolysin proteins; Extracellular vesicles; Ostreolysin A; Vesiculation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Carrier Proteins / pharmacology*
  • Cell Membrane / chemistry
  • Cell Membrane / drug effects*
  • Cell Survival / drug effects
  • Cell-Derived Microparticles / chemistry*
  • Cell-Derived Microparticles / drug effects
  • Cholesterol / chemistry
  • Cholesterol / isolation & purification
  • Dogs
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Fungal Proteins / pharmacology
  • Hemolysin Proteins / genetics
  • Hemolysin Proteins / metabolism
  • Hemolysin Proteins / pharmacology*
  • Ion Transport
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Luminescent Proteins / pharmacology*
  • Lysophosphatidylcholines / chemistry
  • Lysophosphatidylcholines / isolation & purification
  • Madin Darby Canine Kidney Cells
  • Metabolomics
  • Pancreatic Elastase / genetics
  • Pancreatic Elastase / metabolism
  • Pancreatic Elastase / pharmacology*
  • Protein Binding
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Fusion Proteins / pharmacology*
  • Sphingomyelins / chemistry
  • Sphingomyelins / isolation & purification


  • Carrier Proteins
  • Fungal Proteins
  • Hemolysin Proteins
  • Luminescent Proteins
  • Lysophosphatidylcholines
  • Recombinant Fusion Proteins
  • Sphingomyelins
  • ostreolysin
  • red fluorescent protein
  • Cholesterol
  • Pancreatic Elastase
  • cholesterol-binding protein
  • Calcium