Abstract
The phospholipids of lipoproteins can be transferred to cells by an endocytosis-independent uptake pathway. We analyzed the role of scavenger receptor BI (SR-BI) for the selective cellular phospholipid import. Human monocytes rapidly acquired the pyrene (py)-labeled phospholipids sphingomyelin (SM), phosphatidylcholine, and phosphatidylethanolamine from different donors (low and high density lipoproteins (LDL, HDL), lipid vesicles). The anti-SR-BI antibody directed against the extracellular loop of the membrane protein lowered the cellular import of the phospholipids by 40-80%. The phospholipid transfer from the lipid vesicles into the monocytes was suppressed by LDL, HDL, and apoprotein AI. Transfection of BHK cells with the cDNA for human SR-BI enhanced the cellular import of the vesicle-derived py-phospholipids by 5-6-fold. In the case of the LDL donors, transfer of py-SM to the transfected cells was stimulated to a greater extent than the uptake of the other py-phospholipids. Similar differences were not observed when the vesicles and HDL were used as phospholipid donors. The concentration of LDL required for the half-maximal phospholipid import was close to the previously reported apparent dissociation constant for LDL binding to SR-BI. The low activation energy of the SR-BI-mediated py-phospholipid import indicated that the transfer occurs entirely in a hydrophobic environment. Disruption of cell membrane caveolae by cyclodextrin treatment reduced the SR-BI-catalyzed incorporation of py-SM, suggesting that intact caveolae are necessary for the phospholipid uptake. In conclusion, SR-BI mediates the selective import of the major lipoprotein-associated phospholipids into the cells, the transfer efficiency being dependent on the structure of the donor lipoprotein.
Highlights
Phospholipids are known to play essential roles in a multitude of cellular processes such as, for example, in intracellular signal transduction [1] and as cofactors of enzyme complexes mediating the proteolytic activation of coagulation proteins [2]
low density lipoproteins (LDL) and high density lipoproteins (HDL) particles enriched with the indicated py-phospholipids were incubated for 10 min at 37 °C with the monocytes [106] in the absence or presence of the anti-scavenger receptor BI (SR-BI) antibody (0.1 mg/ml)
The lipoproteins were added at 20 g of protein/ml corresponding to 23 nmol (LDL) and 26 nmol (HDL) of total phospholipids
Summary
Materials—1-Palmitoyl-2-pyrenedecanoyl-sn-3-glycerophosphorylcholine (py-PC), 1-palmitoyl-2-pyrenedecanoyl-sn-3-glycerophosphorylethanolamine (py-PE), and (N-pyrenedecanoyl)-sphingomyelin (pySM) were from Sigma and from Molecular Probes. [14C-choline]SM and [14C-choline]PC were purchased from PerkinElmer Life Sciences. The cell pellet obtained after the last centrifugation step containing the peripheral blood mononuclear cells was suspended in phosphate-buffered saline supplemented with 0.15% EDTA and 0.5% BSA (pH 7.4, antibody buffer). The peripheral blood mononuclear cell suspension was incubated at 8 °C for 15 min with microbeads conjugated with anti-human CD14 antibodies and thereafter passaged over a positive selection column. Incubation of Cells and Phospholipid Donors—Pyrene-labeled donors (either lipoproteins or vesicles) were suspended with monocytes and BHK cells in resuspension buffer (without calcium in the case of BHK cells) in a thermostated cuvette at 37 °C. The incorporation of the pyrene-labeled phospholipids into the monocytes was monitored by the increase of the monomer intensity and the concomitant decrease of the excimer/monomer ratio This method exclusively monitors selective phospholipid import since uptake of whole donor particles into the cells will not lower the excimer/monomer ratio of the suspensions. Fluorescence measurements were carried out using a Shimadzu RF-5001-PC spectrofluorimeter
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