Uptake and protein targeting of fluorescent oxidized phospholipids in cultured RAW 264.7 macrophages

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• 作者：U. Stemmer^a ; C. Ramprecht^a ; E. Zenzmaier^a ; B. Stoj膷i膰^a ; G. Rechberger^b ; M. Kollroser^c ; A. Hermetter^a ; ^{albin.hermetter@tugraz.at}
• 关键词：aSMase ; acid sphingomyelinase ; BSA ; bovine serum albumin ; BY ; BODIPY&trade ; DMEM ; Dulbecco's modified Eagle's medium ; HAEC ; human aortic endothelial cells ; IL ; interleukin ; LDL ; low density lipoprotein ; MAPK ; mitogen-activated protein kinase ; mmLDL ; mini
• 刊名：Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids
• 出版年：2012
• 期刊代码：18_13881981
• 类别：bio
• 出版时间：April, 2012
• 卷：1821
• 期：4
• 页码：706-718
• 文件大小：1720 K

摘要

The truncated phospholipids 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are oxidation products of 1-palmitoyl-2-arachidonoyl phosphatidylcholine. Depending on concentration and the extent of modification, these compounds induce growth and death, differentiation and inflammation of vascular cells thus playing a role in the development of atherosclerosis. Here we describe the import of fluorescent POVPC and PGPC analogs into cultured RAW 264.7 macrophages and the identification of their primary protein targets. We found that the fluorescent oxidized phospholipids were rapidly taken up by the cells. The cellular target sites depended on the chemical reactivity of these compounds but not on the donor (aqueous lipid suspension, albumin or LDL). The great differences in cellular uptake of PGPC and POVPC are a direct consequence of the subtle structural differences between both molecules. The former compound (carboxyl lipid) can only physically interact with the molecules in its immediate vicinity. In contrast, the aldehydo-lipid covalently reacts with free amino groups of proteins by forming covalent Schiff bases, and thus becomes trapped in the cell surface. Despite covalent binding, POVPC is exchangeable between (lipo)proteins and cells, since imines are subject to proton-catalyzed base exchange. Protein targeting by POVPC is a selective process since only a limited subfraction of the total proteome was labeled by the fluorescent aldehydo-phospholipid. Chemically stabilized lipid-protein conjugates were identified by MS/MS. The respective proteins are involved in apoptosis, stress response, lipid metabolism and transport. The identified target proteins may be considered primary signaling platforms of the oxidized phospholipid.