Cholesterol, L-tyrosine, horseradish peroxidase, hydrogen peroxide (30%, ACS grade), dietheylenetriaminepentaacetic acid (DTPA; free acid form), essentially fatty acid-free bovine serum albumin (BSA), N-acetyl-Leu-Leu-norleucinal (ALLN), LXR agonist T0901317 and fetal bovine serum were purchased from Sigma. [14C]Oleate (55 mCi/mmol) was from GE Healthcare. Dulbecco's modified Eagle's medium (DMEM) was purchased from Hyclone. PE-SIL G plastic backed flexible plates used for thin-layer chromatography analysis were from Whatman. Nitrocellulose membranes, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) reagents, pre-stained protein molecular mass markers and Chelex 100 resin were from Bio-Rad. μ-Calpain was from Calbiochem, and Trizol from Invitrogen. Cross-linking agent dithiobis(succinimidyl) propionate (DSP) was from Pierce.
Preparation of HDL, apolipoprotein A-I, and tyrosylated HDL
was isolated from pooled plasma from healthy fasting donors by density gradient ultracentrifugation [10
]. Apo A-I was purified from human plasma using Q-Sepharose Fast Flow chromatography as previously described [11
]. Tyrosylation of HDL was carried out at 37°C for 24 h in 66 mM potassium phosphate buffer, pH 8.0, which had been passed over Chelex 100 resin to remove transition metal ions. The reaction mixture contained a final concentration of 1 mg/mL HDL protein, 100 μ
M diethylenetriaminepentaacetic acid (to inhibit metal ion-catalyzed oxidation), 100 nM horseradish peroxidase (250 units/mg), 100 μ
M H2O2, and 100 μM L-tyrosine. The reaction mixture was subjected to size exclusion chromatography as previously described [7
] prior to use in cell culture experiments.
Human skin fibroblasts were cultured in DMEM supplemented with 10% FBS containing 50 units/ml penicillin-streptomycin solution (Invitrogen) and grown in humidified 95% air and 5% CO2 at 37°C. Confluent cells were rinsed twice with phosphate-buffered saline (PBS) containing 1 mg/ml BSA (PBS-BSA) and incubated for 24 h in DMEM containing 2 mg/ml BSA with 30 μg/ml non-lipoprotein cholesterol. To allow equilibration of added cholesterol, cells were rinsed twice with PBS-BSA and incubated for an additional 24 h in DMEM containing 1 mg/ml BSA (DMEM-BSA).
Cholesterol esterification assay
Cholesterol-loaded cells were incubated for 16 h in DMEM-BSA and the indicated additions, washed once with PBS, and incubated for 1 h at 37°C with DMEM containing 9 mM [14
C] oleate bound to 3 mM BSA [12
]. Cells were chilled on ice and rinsed twice with ice-cold PBS-BSA, twice with PBS, and stored at -20°C until extraction. Cellular lipids were extracted with hexane:isopropanol (3:2, v/v). Sterol species were separated by thin layer chromatography (TLC) on PE SIL G plastic-backed plates (Whatman) developed in hexane/diethyl ether/acetic acid (130:40:1.5 v/v/v). Lipid spots were identified by staining with I2
vapor and co-migration with standard. After allowing I2
stain to evaporate, cholesteryl ester spots were taken for determination of radioactivity by liquid scintillation counting. Cell protein was extracted by incubation of cells with 0.5 mL of 0.1 N NaOH for 1 h on a rotary shaker. Total cell protein was determined by the Lowry assay [13
] using albumin as a standard.
Reverse transcriptase-PCR analysis of ABCA1 mRNA
Total RNA was isolated from cells using Trizol, following manufacturer's protocols. Single-strand cDNA was synthesized by a SuperScript pre-amplification system (Invitrogen) from 2 μg of the total RNA. ABCA1 DNA amplification was performed by initial denaturation at 95°C for 3 mins. Thereafter, denaturing was at 95°C for 20 seconds, annealing at 58°C for 20 seconds, and extension at 72°C for 40 seconds for at total of 40 cycles (10). SYBR Green (Quanta Biosciences) was used to detect PCR products in real-time using a Realplex2 Mastercycler thermocycler (Eppendorf). The human housekeeping gene cyclophilin cDNA was amplified using the same conditions. ABCA1 mRNA levels were calculated using the comparative CT method relative to cyclophilin. The following primers were used: human ABCA1, 5'-GAC ATC CTG AAG CCA ATC CTG (forward), 5'-CCT TGT GGC TGG AGT GTC AGG T (reverse); human cyclophilin, 5'- ACC CAA AGG GAA CTG CAG CGA GAG C (forward), 5'-CCG CGT CTC CTT TGA GCT GTT TGC AG (reverse).
Gel electrophoresis and immunoblotting
Cells were scraped into N-dodecyl-β-D-maltoside--containing lysis buffer (20 mM tris, 5 mM EDTA, 5 mM EGTA, 0.5% N-dodecyl-β-D-maltoside, pH 7.5) with complete protease inhibitor (Roche Molecular Biochemicals) and stored at -80°C. Cells were homogenized by sonication, centrifuged to remove the cellular debris, and the lysate collected for ABCA1 protein detection. The cell lysate was run on a 5-15% gradient SDS-PAGE gel under reducing conditions and transferred to nitrocellulose membrane for 16 h at 4°C. Immunoblotting was performed using a polyclonal rabbit anti-human ABCA1 antibody from Novus Biologicals (#NB400-105, 1:1000 dilution) and a goat anti-rabbit IgG horseradish perodixase-conjugated secondary antibody from Sigma (#A-0545, 1:10,000 dilution). Immunoblots were re-probed with rabbit polyclonal anti-actin (Abcam, #AB8227-50, 1:2000) as loading control. SDS-PAGE was also performed for the detection of apoA-I and apoA-II using 12% polyacrylamide gels under reducing conditions followed by staining with 0.25% Coomassie Brilliant Blue. Immunoblot analysis of apoA-I, HDL and tyrHDL were performed using rabbit anti-human apoA-I polyclonal antibody (Calbiochem #178422, 1:10,000) and goat anti-human apoA-II monoclonal antibody (Calbiochem, #178464, 1:20,000).
Calpain and ALLN studies
Sensitivity of ABCA1 to digestion by exogenous calpain following incubation with apoA-I, HDL, or tyrHDL was performed using digitonin permeabilization and treatment with 0.5 μM μcalpain for 20 min as previously described [4
]. To assess cellular levels of ABCA1 following inhibition of endogenous calpain-dependent degradation by ALLN, cells were incubated for 16 h with the indicated conditions plus 50 μM ALLN as previously described [3
]. Cell lysates were then analyzed by ABCA1 immunoblotting as described above.
Biotinylation of cell-surface proteins
Proteins on the surface of cells treated with the indicated additions were biotinylated, harvested, and purified on streptavidin resin using a Pierce Cell Surface Protein Isolation Kit (Pierce) following manufacturer's protocols [14
]. Briefly, after surface biotinylation and quenching, cells were lysed and debris was removed by centrifugation at 10,000 × g for 10 min. Biotinylated proteins in the supernatant (500 μl) were bound to a streptavidin column and eluted in 250 μl of SDS sample buffer. The total and cell surface fraction were treated with 50 mM dithiothreitol to remove biotin, resolved by SDS-PAGE, and immunoblotted using antibodies against ABCA1, β-actin (Abcam), and ERK (New England Biolabs).
Crosslinking of apoA-I, HDL or tyrHDL to ABCA1
Chemical cross-linking was performed as described by Wang et al. [15
] with minor modifications as follows. Cells were grown to confluence in 100 mm culture dishes, cholesterol loaded with non-lipoprotein cholesterol, equilibrated, and then treated with 5 μM LXR Agonist (T0901317) in DMEM-BSA for 24 h to up-regulate ABCA1 expression. The cells were washed twice with PBS and then treated with DMEM/BSA or the same media containing 10 μg/mL apoA-I, HDL, or tyrHDL in DMEM-BSA for 2 h at 37°C. Cells were then placed on ice for 15 min and washed three times with PBS. Cross linking agent DSP was dissolved immediately before use in dimethyl sulfoxide (Sigma) and diluted to 250 μM with PBS. 10 ml of DSP solution was added to the cells for 1 h at room temperature, the medium removed, and dishes were washed twice with ice-cold PBS. Cells were lysed at 4°C in buffer containing 20 mM Tris, 0.5 mM EDTA, 0.5 mM EGTA, 1% Triton X-100 (BDH Chemicals, pH 7.5), with complete protease inhibitor (Roche). by aspiration with a fine-tipped needle, and the mixture was left to rotate at 4°C for 30 min. Cell lysates were centrifuged at 1,500 rpm for 10 min at 4°C to remove cellular debris The supernatant was collected and incubated with loading buffer in the absence or presence of β-mercaptoethanol before loading onto 4-20% gradient SDS-PAGE gels for electrophoresis followed by electrotransfer to a nitrocellulose sheet for immunoblot analysis. ApoA-I cross-linked to ABCA1 was detected by blotting the membranes with rabbit polyclonal anti-human apoA-I antibody and goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody as noted above. To detect ABCA1, the membrane was stripped and reprobed with rabbit polyclonal antibody to ABCA1 and goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody as noted above.
Lipid-free apoA-I, HDL and tyrHDL were incubated with 0.13 U enteropeptidase (Calbiochem) per g of protein for 6 h at 37°C as previously described for apoA-I [16
]. Cleaved products were run on 12% SDS-PAGE under reducing conditions and stained with 0.25% Coomassie brilliant blue. Immunoblot analysis of apoA-I and apoA-II were performed using rabbit anti-human apoA-I polyclonal antibodies and goat anti-human apoA-II monoclonal antibodies.
Results for Figures and were analyzed using GraphPad Prism version 5.0 and are presented as the mean ± S.D. Significant differences between experimental groups were determined using the Student's t test.
Figure 1 tyrHDL requires ABCA1 to enhance depletion of ACAT-accessible cholesterol. Human skin fibroblasts from a normal donor and 2 unrelated Tangier Disease (TD) subjects were grown to confluence, loaded with non-lipoprotein cholesterol for 24 h, equilibrated (more ...)
Figure 2 tyrHDL increases ABCA1 protein but not mRNA levels. Fibroblasts grown to confluence, cholesterol loaded, and equilibrated as in Figure 1 were treated with medium containing 1 mg/ml BSA alone or plus 10 μg/ml apoA-I, HDL or tyrHDL for 2 or 16 h. (more ...)