21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

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• 作者：G茅rald Gaibelet ; Fran莽ois Terc茅 ; Justine Bertr ; -Michel ; Sophie Allart ; Vincent Azalbert ; Marie-France Lecompte ; Xavier Collet ; St茅phane Orlowski
• 关键词：HDL ; high-density lipoproteins ; (V)LDL ; (very) low-density lipoproteins ; apo ; apolipoprotein ; NBD ; nitrobenzoxadiazole ; Pyr-met ; methylpyrenyl ; Chol ; cholesterol ; SM ; sphingomyelin ; LCAT ; lecithin ; cholesterol acyltransferase
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：1 November, 2013
• 年：2013
• 卷：440
• 期：4
• 页码：533-538
• 全文大小：927 K

文摘

Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described 鈥渓ipid rafts鈥?in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD-Chol non-specifically delivered to the cells.