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First published online 14 November 2002
doi: 10.1242/jcs.00126

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Oxidative tyrosylation of high density lipoproteins impairs cholesterol efflux from mouse J774 macrophages: role of scavenger receptors, classes A and B

Centre de Recherche, Hôpital Sainte-Justine, Université de Montréal, Montréal, Québec, Canada
¹ Department of Nutrition, Université de Montréal, Montréal, Québec, Canada
² Department of Pediatrics, Université de Montréal, Montréal, Québec, Canada

View larger version (35K): [in a new window]   Fig. 1. Fluorescence excitation-emission spectra (A) and gradient gel electrophoresis (B) of native and tyrosylated HDL apolipoproteins. (A) Native and tyrosylated HDL3 were subjected to fluorescence spectroscopy with ex at 328 nm and em at 300 nm to 500 nm. (B) SDS-PAGE was performed using 5-20% polyacrylamide gradient gels under non-reducing conditions as described in Materials and Methods. Lanes: 1, apo A-I; 2, apoA-II; 3, native HDL3; 4, tyrosylated HDL3; 5, native total HDL; 6, tyrosylated total HDL; 7, molecular weight standards.

View larger version (11K): [in a new window]   Fig. 2. Cholesterol efflux from J774-A1 macrophages in the presence of native and tyrosylated HDL3 (A) or total HDL (B). J774-A1 cells were [3H]cholesterol-loaded with [3H]cholesteryl oleate-labeled AcLDL (50 µg apo B/ml, 7.6x105 DPM/ml) as described in Materials and Methods. After an 18 hour equilibration period, the cells were incubated for 24 hours at 37°C with the indicated final concentrations of native (•) or tyrosylated () HDL3 (A) or total HDL (B). After extensive washing with PBS, cellular and medium lipids were extracted, separated by TLC and counted to determine the [3H]cholesteryl ester and the [3H]free cholesterol fractions. Cholesterol efflux was calculated as the amount of [3H]cholesterol (free and esterified) released to the medium relative to total label in each well. Each value is the mean±s.e.m. of three determinations. *P<0.03, **P<0.02, ***P<0.01.

View larger version (88K): [in a new window]   Fig. 3. Time (A) and concentration (B) dependence of native and tyrosylated Oregon-HDL3 cell association and fluorescent staining of cells illustrating Oregon HDL3 cell association (C). J774-A1 were cholesterol-loaded with AcLDL (50 µg apoB/ml) as described in Materials and Methods. After an 18 hour equilibration period, cells were incubated (37°C) for different times with 25 µg apo A-I/ml of native (•) or tyrosylated () Oregon-HDL3 (A), or with different concentrations of native (•) or tyrosylated () Oregon-HDL3 for 3 hours (B). These assays were performed in the absence or presence of a 50-fold excess of the corresponding unlabeled HDL3. Cells were then extensively washed with DMEM-5% LPDS and PBS, and cell-associated fluorescence was measured by flow cytometry. Specific Oregon-HDL3 binding was determined by subtracting the measurements made in the presence of 50-fold excess from the corresponding unlabeled HDL3. The values are expressed as the means±s.e.m. of four independent experiments using different lipoprotein preparations. C shows the fluorescence microscopy of J774-A1 cells at 3 and 6 hours following Oregon-HDL3 uptake (100 µg apo A-I/ml). *P<0.003.

View larger version (15K): [in a new window]   Fig. 4. Time (A) and concentration (B) dependence of native and tyrosylated specific cholesterol ether uptake by J774-A1 cells. J774-A1 cells were cholesterol loaded with AcLDL (50 µg apo B/ml) as described in Materials and Methods. After an 18 hour equilibration period, cells were incubated for different times with 25 µg apo A-I/ml of native (•) or tyrosylated () [3H]cholesteryl hexadecyl ether-HDL3 (A) or for 3 hours at 37°C with the indicated final concentrations of native (•) or tyrosylated () [3H]cholesteryl hexadecyl ether-HDL3 (B), in the absence or presence of a 50-fold excess of the corresponding unlabeled HDL3. After cell washing, the specific [3H]cholesteryl hexadecyl ether cell uptake was determined by radioactivity measurement. Each value represents the mean±s.e.m. of three independent experiments using different lipoprotein preparations. *P<0.05, **P<0.02.

View larger version (16K): [in a new window]   Fig. 5. nCEH activity as a function of native and tyrosylated HDL3 concentrations. J774-A1 were cholesterol loaded with AcLDL (50 µg apoB/ml) as described in Materials and Methods. After an 18 hour equilibration period, the cells were incubated for 24 hours with the specified final concentrations of native (•) or tyrosylated () HDL3. After extensive washing with PBS, cells were homogenized by sonication in 50 mM Tris-HCL buffer, pH 7.0, containing 250 mM sucrose, 1 mM EDTA, 1 mM DTT, 20 µg/ml leupeptin and 1 µg/ml pepstatin. The mixture was then ultracentrifuged for 30 minutes at 43,000 g at 4°C. The supernatant (400 µg protein, 100 µl) was assayed for nCEH activity as described in Materials and Methods. Results were expressed as a percentage of the initial activity in the absence of HDL3. Each point represents the mean of three experiments±s.e.m. *P<0.05, **P<0.001.

View larger version (16K): [in a new window]   Fig. 6. Lipoprotein displacement of Oregon-HDL3 cell-association with J774-A1 cells. J774-A1 cells were cholesterol loaded with AcLDL (50 µg apo B/ml) as described in Materials and Methods. After an 18 hour equilibration period, the cells were incubated in duplicate with 25 µg apo A-I/ml of native (A) or tyrosylated (B) Oregon-HDL3 for 3 hours at 37°C, in the presence or absence (control) of 5, 10 or 15-fold excess concentration of the unlabeled competitors: native HDL3 (•), tyrosylated HDL3 (), AcLDL () and oxLDL (). The non-specific binding was determined in the presence of 50-fold excess of the corresponding unlabeled HDL3. After extensive cell washing, cell association of control and tyrosylated Oregon-HDL3 was determined by flow cytometry. Depicted values represent the percentages of specific binding. Data shown are the means±s.e.m. of four independent experiments.

View larger version (18K): [in a new window]   Fig. 7. Lipoprotein displacement of [3H]cholesteryl hexadecyl ether-HDL3 cell uptake. J774-A1 cells were cholesterol-loaded with AcLDL (50 µg apo B/ml) as described in Materials and Methods. After an 18 hour equilibration period, the cells were incubated in duplicate with 10 µg apo A-I/ml of native (A) or tyrosylated (B) [3H]cholesteryl hexadecyl ether-HDL3 for 3 hours, in the presence of 5-, 10- or 15-fold excess concentration of the unlabeled competitors: native HDL3 (•), tyrosylated HDL3 (), AcLDL () and oxLDL (). The non-specific binding was determined in the presence of 50-fold excess of the corresponding unlabeled HDL3. After extensive washing, [3H]cholesteryl hexadecyl ether cell uptake from native and tyrosylated HDL3 was counted and expressed in DPM per mg of cell protein. Depicted values represent the percentages of the specific cholesterol uptake with respect to the experiment carried out. Data shown were the means±s.e.m. of four independent experiments.

View larger version (10K): [in a new window]   Fig. 8. Effect of specific SR-BI/BII blocking antibody on native or tyrosylated oregon-HDL3 cell association (A) and cholesterol ether-HDL3 uptake (B). J774-A1 cells were cholesterol loaded with AcLDL (50 µg apo B/ml) as described in Materials and Methods. After an 18 hour equilibration period, the cells were incubated for 3 hours at 37°C with 10 µg/ml of native (black) or tyrosylated (grey) Oregon-HDL3 (A) or [3H]cholesteryl hexadecyl ether-HDL3 (B) with or without 0.6 mg/ml of blocking rabbit SR-BI/BII antibody (preincubated for 30 minutes with cells before the addition of HDL3, in a final volume of 0.2 ml). Control experiments were carried out with 0.6 mg/ml of rabbit IgG in the same conditions. After extensive cell washing, the specific cell-associated fluorescence and cellular [3H]cholesteryl hexadecyl ether uptake were determined by flow cytometry and radioactivity measurement, respectively. Results were expressed as a percentage of the value without antibody or rabbit IgG. Each value is the mean±s.e.m. of three independent experiments using different lipoprotein preparations.

View larger version (14K): [in a new window]   Fig. 9. Effect of Poly-I on the native and tyrosylated Oregon-HDL3 cell-association (A) and cholesterol ester-HDL3 uptake (B). J774-A1 cells were cholesterol loaded with AcLDL (50 µg apo B/ml) as described in Materials and Methods. After an 18 hour equilibration period, the cells were incubated for 3 hours at 37°C with 10 µg apoA/ml of native (•) or tyrosylated () Oregon-HDL3 (A) or [3H]cholesteryl hexadecyl ether-HDL3 (B), with or without (control) different concentrations of Poly-I. The non-specific binding was determined in the presence of a 50-fold excess of the corresponding unlabeled HDL3. After extensive cell washing, the specific cell-associated fluorescence and cellular [3H]cholesteryl hexadecyl ether uptake were determined by flow cytometry and by radioactivity measurement, respectively. Results were espressed as a percentage of the values without Poly-I. Each value is the mean±s.e.m. of three independent experiments using different lipoprotein preparations. *P<0.005.