RRC ID 36034
Author Shimada Y, Maruya M, Iwashita S, Ohno-Iwashita Y.
Title The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains.
Journal Eur. J. Biochem.
Abstract There is much evidence to indicate that cholesterol forms lateral membrane microdomains (rafts), and to suggest their important role in cellular signaling. However, no probe has been produced to analyze cholesterol behavior, especially cholesterol movement in rafts, in real time. To obtain a potent tool for analyzing cholesterol dynamics in rafts, we prepared and characterized several truncated fragments of theta-toxin (perfringolysin O), a cholesterol-binding cytolysin, whose chemically modified form has been recently shown to bind selectively to rafts. BIAcore and structural analyses demonstrate that the C-terminal domain (domain 4) of the toxin is the smallest functional unit that has the same cholesterol-binding activity as the full-size toxin with structural stability. Cell membrane-bound recombinant domain 4 was detected in the floating low-density fractions and was found to be cofractionated with the raft-associated protein Lck, indicating that recombinant domain 4 also binds selectively to cholesterol-rich rafts. Furthermore, an enhanced green fluorescent protein-domain 4 fusion protein stains membrane surfaces in a cholesterol-dependent manner in living cells. Therefore, domain 4 of theta-toxin is an essential cholesterol-binding unit targeting to cholesterol in membrane rafts, providing a very useful tool for further studies on lipid rafts on cell surfaces and inside cells.
Volume 269(24)
Pages 6195-203
Published 2002-12
PII 3338
PMID 12473115
MeSH Amino Acid Sequence Bacterial Toxins / chemistry* Centrifugation, Density Gradient Cholesterol / metabolism* Chromatography, Thin Layer Circular Dichroism Clostridium perfringens / metabolism Detergents / pharmacology Endopeptidases / metabolism Escherichia coli / metabolism Hemolysin Proteins Humans Kinetics Lipid Metabolism Liposomes / metabolism Membrane Microdomains / metabolism* Molecular Sequence Data Octoxynol / pharmacology Plasmids / metabolism Protein Binding Protein Structure, Tertiary Recombinant Proteins / metabolism Spectrometry, Fluorescence Subcellular Fractions Surface Plasmon Resonance Tryptophan / metabolism Tumor Cells, Cultured
Times Cited 84
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material pET28/His6-EGFP-D4 (RDB13961)