Real-time assay method of lipid extraction activity.
Intracellular lipid translocation is mediated by lipid transfer proteins and their functional impairments cause severe disorder in lipid metabolism. However, molecular mechanisms of protein-mediated lipid transfer remain unclear since conventional assay methods could not observe elementary processes in the lipid transfer reaction, such as lipid bilayer binding and lipid uptake. In this study, we found that ceramide extraction mediated by a ceramide trafficking protein (CERT) could be detected as decreasing the response of surface plasmon resonance (SPR). Based on this finding, we developed a novel real-time assay method that enables quantitative evaluation of the ceramide extraction activity of CERT, using the SPR technique. Performing this SPR-based assay using ceramide-embedded and ceramide-free lipid bilayers as ligands allows for the exclusive investigation of ceramide uptake processes, differentiating them from other CERT-membrane binding events. Furthermore, mutagenesis experiments of CERT using this SPR-based assay clearly elucidated whether an amino acid residue plays a role in the ceramide uptake process or the lipid bilayer binding process. This SPR-based assay method can separately evaluate the lipid extraction activity and lipid bilayer binding activity of the lipid transfer proteins, and provide more detailed information about lipid transfer phenomena.
Sugiki
Toshihiko
T
Japan Biological Informatics Consortium (JBiC), Aomi, Koto-ku, Tokyo 135-8073, Japan.
Takahashi
Hideo
H
Nagasu
Michiko
M
Hanada
Kentaro
K
Shimada
Ichio
I
D019943:Amino Acid Substitution; D001692:Biological Transport; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020349:Surface Plasmon Resonance / Q000379:methods*
D002518:Ceramides; D008051:Lipid Bilayers; D008055:Lipids; D011994:Recombinant Proteins; C119090:CERT1 protein, human; D017346:Protein Serine-Threonine Kinases