Related Experiment Videos
The binding in vitro of modified LDL to the intermediate filament protein vimentin
A K Heidenthal1, P C Weber, F Lottspeich
1Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universität München, Pettenkoferstrasse 9, Münich, D-80336, Germany.
Abstract:
Membrane-associated proteins with specific binding properties to modified LDL were investigated in J774 macrophages and Mono Mac 6 sr cells. Ligand blotting of membrane proteins revealed a 54-kDa protein which bound oxidized and acetylated but not native LDL. The 54-kDa protein, isolated by 2D-PAGE, was identified as vimentin. (125)I-AcLDL bound to purified vimentin and desmin in a saturable manner, with an approximate K(d) of 1.7 x 10(-7) M (89 microgram/ml) and 8.0 x 10(-8) M (41 microgram/ml), respectively. Blots of vimentin mutant proteins with deletions in the positively charged N-terminal head domain showed that amino acids 26-39 are essential for the binding of AcLDL by vimentin. Taken together, our data indicate that vimentin binds modified LDL, but not native LDL, in a specific and saturable manner. Vimentin filaments extend throughout the cytoplasm as far as the inner surfaces of plasma and vesicular membranes. Vimentin may thus play a role in membrane-associated steps involved in the intracellular processing of oxidized LDL, contributing to its unregulated uptake and intracellular retention by cells of the atherogenic plaque.
Insights
Vimentin, a cellular protein, binds modified low-density lipoprotein (LDL) but not native LDL. This specific binding suggests vimentin
Area of Science:
- Cell Biology
- Biochemistry
- Atherosclerosis Research
Background:
- Modified low-density lipoprotein (LDL) accumulation is central to atherosclerosis.
- Cellular mechanisms of modified LDL uptake and retention are not fully understood.
Purpose of the Study:
- To identify membrane-associated proteins that bind modified LDL in macrophages.
- To elucidate the role of identified proteins in modified LDL processing.
Main Methods:
- Ligand blotting of macrophage membrane proteins.
- Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein isolation.
- Binding assays with purified vimentin and desmin using iodinated acetylated LDL ((125)I-AcLDL).
- Site-directed mutagenesis of vimentin to identify binding domains.
Main Results:
- A 54-kDa protein, identified as vimentin, specifically bound oxidized and acetylated LDL, but not native LDL.
- Purified vimentin and desmin exhibited saturable binding of (125)I-AcLDL.
- Amino acids 26-39 in vimentin's N-terminal head domain were essential for AcLDL binding.
- Vimentin's cytoplasmic localization suggests a role in intracellular processing of modified LDL.
Conclusions:
- Vimentin directly binds modified LDL in a specific and saturable manner.
- Vimentin may contribute to the unregulated uptake and retention of oxidized LDL in atherogenic plaque cells.
- Vimentin's interaction with modified LDL offers a potential target for understanding and treating atherosclerosis.