Carboxyl Terminus of Apolipoprotein A-I (ApoA-I) Is Necessary for the Transport of Lipid-free ApoA-I but Not Prelipidated ApoA-I Particles through Aortic Endothelial Cells^*

Cell Culture

ECs were isolated from bovine aortas as described previously (23) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 5% fetal calf serum (FCS) at 37 °C in a humidified 5% CO₂, 95% air incubator.

Small Interfering (si)RNA Transfection

ECs were transfected with 100 nm Stealth siRNA against ABCA1, ABCG1, ABCA1/ABCG1, or SR-BI and 34 nm BLOCK-iT fluorescent oligonucleotide (Invitrogen) as described previously (15). Binding and transport assays were conducted between 65 and 72 h after transfection. The efficiency of the silencing was evaluated by quantitative RT-PCR and Western blotting, as shown previously (15, 17).

Isolation of HDL and ApoA-I from Plasma

Human HDL (1.063<d<1.21 kg/liter) was isolated from fresh normolipidemic plasmas of blood donors by sequential ultracentrifugation (24). The purity of the lipoprotein preparation was verified by SDS-PAGE to ensure no contamination with LDL or albumin. Lipid-free human plasma WT apoA-I was further purified from delipidated HDL as described previously (25).

Production and Isolation of Recombinant ApoA-I

Production of the recombinant WT apoA-I and of the apoA-I mutants Δ(185–243), Δ(144–165), Δ(1–59), and Δ(1–59)/Δ(185–243) was described previously (22, 26–29).

Generation of Adenoviruses Expressing ApoA-I(L218A/L219A/V221A/L222A)

The apoA-I gene lacking the BglII restriction site (that is present at nucleotide positions 181 of the genomic sequence relative to the ATG codon of the gene) was cloned into the pcDNA3.1 vector to generate the pcDNA3.1-apoA-I(ΔBglII) plasmid as described (30). This plasmid was used as a template to introduce the apoA-I(L218A/L219A/V221A/L222A) mutations in apoA-I using the mutagenesis kit QuikChange® XL (Stratagene) and the mutagenic primers. The forward and reverse primers used are: forward, 5′-GGACCTCCGCCAAGGCGCGGCGCCCGCGGCGGAGAGCTTCAAGGTC-3′ and reverse, 5′-GACCTTGAAGCTCTCCGCCGCGGGCGCCGCGCCTTGGCGGAGGTCC-3′ (sites of the mutagenesis are underlined). Following 18 cycles of PCR amplification of the template DNA, the PCR product was treated with DpnI to digest plasmids containing methylated DNA in one or both of their strands. The reaction product consisting of plasmids containing newly synthesized DNA carrying the mutations of interest were used to transform competent XL-10 blue bacteria cells (Stratagene). Ampicillin-resistant clones were selected, and plasmid DNA was isolated from these clones and subjected to sequencing to confirm the presence of the point mutations. The 2.2-kb apoA-I inserts containing the apoA-I mutant were cloned into the pAdTrack CMV vector that was used to generate the adenoviral constructs by recombination with the Ad-Easy-1 helper virus in the bacteria cells BJ-5183-pAD1(Stratagene), which contain the Ad-Easy-1 helper virus. Correct clones were propagated in DH5a bacteria cells. The recombinant adenoviral constructs were linearized after incubation with PacI and used to transfect 911 cells. Following large scale infection of human embryonic kidney 293 cell cultures, the recombinant adenoviruses were purified by two consecutive CsCl ultracentrifugation steps, dialyzed, and titrated. The mutant protein was isolate from the culture medium of HTB-13 cells infected with the apoA-I(L218A/L219A/V221A/L222A)-expressing adenovirus as described (22, 26).

Lyophilized apoA-I mutants were dissolved in 5 m guanidinium hydrochloride and dialyzed against 0.01 m Tris-HCl, pH 8, 0.15 m NaCl. The iodination was carried out by the same procedure as described before for WT apoA-I adjusted to pH 8, and the extensive dialysis was against 0.01 m Tris-HCl, pH 8, 0.15 m NaCl.

Radiolabeling of ApoA-I

ApoA-I was labeled with ¹²⁵I using Iodination Beads (Pierce) and Na¹²⁵I (Hartmann Analytic) according to the manufacturers' instructions. In a typical reaction, we used 0.5 mCi of Na¹²⁵I, 0.7 mg of apoA-I, and two beads. Protein was separated from unincorporated ¹²⁵I with a Sephadex G-25 column (Amersham Biosciences) followed by extensive dialysis (against 0.15 m NaCl, 0.3 mm EDTA, pH 7.4) to remove residual free iodine. The specific activity expressed as cpm/ng protein was calculated based on the protein concentration measured by the DC protein assay (Bio-Rad) and the activity measured using a γ-counter (PerkinElmer Life Sciences). Specific activities of 600–1200 cpm/ng protein were obtained.

Preparation of Reconstituted HDL (rHDL)

Discoidal rHDL particles were produced by the cholate dialysis method (18) and contained WT or mutant apoA-I, POPC (Sigma), and sodium cholate (Sigma) in a molar ratio of 1:40:100 ((mutant) ApoA-I 1:40). Reconstituted HDL was iodinated as described above for apoA-I. Electron microscopy analysis of the particles was performed as described (22).

Binding and Cell Association Assays

Binding and cell association assays with ¹²⁵I-apoA-I or ¹²⁵I-rHDL were performed as described previously (16). ECs were incubated with the indicated concentrations of ¹²⁵I-apoA-I or ¹²⁵I-rHDL without (total) or with (nonspecific) a 40-fold excess of the indicated competitor for 2 h at 4 °C (binding) or 1 h at 37 °C (cell association). Specific binding/cell association was calculated by subtracting the values of nonspecific binding/cell association from those of total binding/cell association. All experiments were performed at least in triplicate.

Transport Assays

Transport assays were performed as previously described (16). In brief, ECs were seeded 2 days in advance on inserts (0.4 μm; BD Biosciences) precoated with collagen type I (BD Biosciences). Medium containing either ¹²⁵I-apoA-I or ¹²⁵I-rHDL at the indicated concentrations was added to the apical compartment together with (nonspecific transport) or in the absence (total transport) of a 40-fold excess of unlabeled apoA-I and rHDL, respectively. After incubation for 60 min at 37 °C the media of the basolateral compartment were collected to measure radioactivity. Specific transport was calculated by subtracting the values of nonspecific transport from those of total transport.

Gel Filtration Chromatography

The size of apoA-I and mutant apoA-I before and after transendothelial transport was analyzed by gel filtration chromatography as described (31). In brief, transport assays were performed in four transwell cell culture wells as described above. The media (2 ml) were isolated from the basolateral compartments, combined, and concentrated to 50 μl by centrifugal concentrators (Vivaspin 500; Sartorius Stedim Biotech). The concentrate was loaded onto a Superdex^TM 200 preparation grade HiLoad^TM 16/60 column (GE Healthcare) of an Akta fast protein liquid chromatography (FPLC) system and eluted with Tris saline (0.01 m Tris, 0.15 m NaCl, 0.1 mm EDTA, pH 7.5) at a flow rate of 1.5 ml/min. Fractions (0.5 ml) were collected, and the amounts of transported radioactivity were determined using a PerkinElmer Life Sciences γ-counter.

Native Agarose Gel Electrophoresis

Equal amounts of proteins were used for mobility analysis and were loaded on a 1% native agarose gel (0.05 m barbital buffer, pH 8.6). Electrophoresis was performed at 4 °C. After that, the gel was stained with Coomassie Blue (protein staining) and Sudan Black (lipid staining) using standard protocols.

Statistical Analyses

The data for all experiments were analyzed using the GraphPad Prism 5 software program. Comparisons between groups were performed using t test methods. Experiments were routinely performed in triplicate or quadruplicate. Each experiment shown is a representative of at least three similar experiments. If not indicated otherwise, the data are graphically represented as means ± S.D.

Binding and Association of Lipid-free ApoA-I Mutants to ECs

Initially we compared the endothelial binding properties of WT apoA-I either isolated from plasma or produced as a recombinant protein. ECs were incubated with radiolabeled lipid-free WT apoA-I at 4 °C in the absence (total) or presence of a 40-fold excess of unlabeled plasma WT apoA-I (nonspecific). The difference between the total and the nonspecific binding corresponds to the specific binding. The recombinant and plasma-derived WT isoproteins of apoA-I showed the same total and specific binding to ECs. Therefore, in further experiments, plasma WT apoA-I was used as a control to compare the behavior of the different apoA-I mutants.

The apoA-I(Δ144–165) mutant showed total and specific binding to ECs similar to those of WT apoA-I. The two mutants with a defective carboxyl-terminal sequence, apoA-I(Δ185–243) and apoA-I(L218A/L219A/V221A/L222A), showed 50 and 80% decreases in total and specific endothelial binding, respectively (Fig. 1). In contrast, apoA-I(Δ1–59) and apoA-I(Δ1–59/Δ185–243) showed a dramatic 25-fold increase in total binding. The binding of apoA-I(Δ1–59) could not be competed by unlabeled WT apoA-I so that no specific binding could be recorded (Fig. 1). The calculated specific binding of apoA-I(Δ1–59/Δ185–243) did not differ from that of WT apoA-I.

We repeated these experiments at 37 °C where ligands are not only bound but also internalized by ECs (16, 32), and therefore the absolute amount of cell-associated radioactivity is higher. In principle, we made the same observations as described for binding at 4 °C: WT apoA-I from plasma, recombinant WT apoA-I, and apoA-I(Δ144–165) did not differ from each other whereas the apoA-I(Δ185–243) and apoA-I(L218A/L219A/V221A/L222A) mutants showed strongly reduced total and specific cell association, and the apoA-I(Δ1–59) and apoA-I(Δ1–59/Δ185–243) mutants showed massively increased total cell association which could not be competed by WT apoA-I (data not shown).

Transport of Lipid-free ApoA-I Mutants through ECs

Next, we analyzed the transport of the different apoA-I mutants though ECs cultivated in a transwell system. Radiolabeled lipid-free WT or mutant apoA-I was added to the apical side with or without 40-fold excess of unlabeled competitor (WT apoA-I). After a 1-h incubation at 37 °C, the medium of the basolateral compartment was collected to measure radioactivity. The specific transport was calculated as the difference between total transport (radioactivity after incubation without competitor) and nonspecific transport (radioactivity after incubation with competitor). As shown in Fig. 2, specific transports of plasma WT apoA-I, recombinant WT apoA-I, and apoA-I(Δ144–165) were similar. In contrast, the specific transports of apoA-I(Δ185–243) and apoA-I(L218A/L219A/V221A/L222A) were decreased by 90%. No specific transports could be calculated for apoA-I(Δ1–59) and apoA-I(Δ1–59/Δ185–243) mutants because both in the presence or absence of the competitor the same amounts of radioactivity were recovered in the basolateral compartment of the transwell cell culture dish.

ApoA-I with Mutations in the Carboxyl Terminus Is Not Lipidated after Transendothelial Transport

In previous studies we observed a change in particle size and electrophoretic mobility of lipid-free WT apoA-I after the transport through ECs which we interpreted as a result of lipidation (16, 17). Therefore, we compared the particle sizes of WT apoA-I and the dysfunctional apoA-I(L218A/L219A/V221A/L222A) mutant before and after transport through ECs (Fig. 3). After transport and recovery from the basolateral compartment, WT apoA-I was fractionated by gel filtration into two peaks, one peak with identical elution volume (87.6 ml ± 0.5 ml) and hence size of the starting material and one new peak with lower elution volume (72.6 ml ± 0.5 ml) and hence larger particle size (Fig. 3A). By contrast, the comparison of the gel filtration profiles of the binding- and transport-defective apoA-I(L218A/L219A/V221A/L222A) mutant before and after incubation with ECs did not reveal the occurrence of any new peak (Fig. 3B). Already before incubation with cells and hence in the lipid-free state, this mutant was eluted in two peaks, one with the size of WT apoA-I (87.6 ml ± 0.5 ml) and one corresponding to larger particle size (77.1 ml ± 0.5 ml). The larger sized fraction of apoA-I(L218A/L219A/V221A/L222A) has a higher elution volume than the fraction formed after transport of WT apoA-I and did not change after transport. We therefore assume that this fraction represents lipid-free aggregates of apoA-I(L218A/L219A/V221A/L222A).

Role of ABCA1, ABCG1, and SR-BI for Binding, Cell Association, and Transport of Lipid-free WT ApoA-I

Using specific siRNAs (15), we investigated the effects of ABCA1, ABCG1, and SR-BI knockdown alone and the silencing of both ABCA1 and ABCG1 together on endothelial binding, cell association, and transport of lipid-free WT apoA-I (Fig. 4). ABCA1, ABCG1, and SR-BI transcription were reduced by approximately 80–90% in cells transfected with specific siRNA. The remaining protein expression of ABCA1, ABCG1, and SR-BI after silencing was ∼50% as assessed by Western blotting and already shown previously (15, 17). Also as reported previously, knockdown of ABCA1, but not ABCG1 or SR-BI, reduced the binding of lipid-free WT apoA-I at 4 °C (Fig. 4A). The knockdown of ABCA1 and ABCG1 together revealed the same reduction of apoA-I binding as the single knock-down of ABCA1 by approximately 60%. At 37 °C, suppression of ABCA1 and ABCG1 either individually or both together but not the knockdown of SR-BI diminished the cell association (Fig. 4B) and transendothelial transport of initially lipid-free apoA-I (Fig. 4C) by approximately 40%.

These at first sight discrepant observations at 4 °C and 37 °C could be explained by a previously proposed two-step model in which ABCA1-mediated lipid efflux generates a lipidated particle that secondarily interacts with ABCG1 (18–20). To test this hypothesis we analyzed the binding, association, and transport of HDL reconstituted artificially with WT or mutant apoA-I.

Interactions of rHDL with ECs

We first compared the binding (at 4 °C) and association properties (at 37 °C) of WT apoA-I in either the lipid-free or prelipidiated forms. The binding of lipid-free apoA-I was competed by rHDL to a similar degree as by lipid-free apoA-I itself or native HDL (Fig. 5A). The binding of rHDL was not competed by lipid-free apoA-I but by both reconstituted and native HDL (Fig. 5A). Cell association experiments yielded similar findings. EC association of lipid-free apoA-I was competed with 40-fold excesses of unlabeled apoA-I, lipidated apoA-I, or HDL to a similar degree (Fig. 5B). Cell association of rHDL was competed with an excess of either rHDL or native HDL (Fig. 5B). By contrast with the binding experiment, we however observed that also lipid-free apoA-I competed the cell association of rHDL, although to less extent (approximately 35%) than rHDL or native HDL (approximately 60%). These observations provide further evidence that at 37 °C the lipidation of apoA-I by ABCA1 generates a lipidated particle that can then compete with the cellular interaction of prelipidated apoA-I.

Binding and Transport of Reconstituted HDL Containing WT ApoA-I or ApoA-I(L218A/L219A/V221A/L222A)

Next, we exploited the apoA-I(L218A/L219A/V221A/L222A) mutant with defects in ABCA1-mediated lipid efflux as well as specific endothelial binding and transport to test the hypothesis that transendothelial transport of lipid-free apoA-I occurs by a two-step mechanism in which apoA-I is first lipidated by ABCA1-dependent lipid efflux to then undergo ABCA1-independent transport through ECs. We first lipidated WT apoA-I or apoA-I(L218A/L219A/V221A/L222A). The lipidation and resulting particle formation were verified by native agarose gel electrophoresis and electron microscopy. Both WT apoA-I and apoA-I(L218A/L219A/V221A/L222A) formed particles that had a higher electrophoretic mobility than the respective lipid-free apolipoproteins. rHDL containing apoA-I(L218A/L219A/V221A/L222A) were slightly less negatively charged than rHDL containing WT apoA-I (Fig. 6A). In addition, the Sudan Black staining of the agarose gel clearly reveals the lipidation of both WT apoA-I and apoA-I(L218A/L219A/V221A/L222A). After lipid staining, the bands containing rHDL with WT or mutant apoA-I were similarly intense (Fig. 6B). Electron microscopy revealed that both WT apoA-I and apoA-I(L218A/L219A/V221A/L222A) formed discoidal particles (Fig. 6C).

We then used the prelipidated particles to perform binding and transport studies. Neither the specific binding (Fig. 6D) nor the specific transport (Fig. 6E) differed between particles containing either WT apoA-I or the mutant apoA-I(L218A/L219A/V221A/L222A). Thus prelipidation can overcome the binding and transport defects of the dysfunctional apoA-I(L218A/L219A/V221A/L222A) mutant. These findings corroborate the hypothesis that lipidation of apoA-I is necessary for specific transcytosis.

Binding and Transport of rHDL Containing ApoA-I(Δ1–59) and ApoA-I(Δ1–59/Δ185–243)

We then investigated the effects of prior cell-free lipidation on binding and transport of the two apoA-I mutants which in the lipid-free form showed excessive nonspecific endothelial binding and transport, namely apoA-I(Δ1–59) and apoA-I(Δ1–59/Δ185–243). After cholate dialysis with POPC in a molar ratio of 1:40, both mutant rHDL particles showed higher electrophoretic mobility than the respective lipid-free apolipoproteins (Fig. 7A). The efficacy of lipidation was further confirmed by lipid staining of the agarose gel (Fig. 7B). The two mutant rHDL particles, however, differed from each other and from normal rHDL by electrophoretic mobility (Fig. 7, A and B). Both mutants formed discoidal particles (Fig. 7C). With these lipidated mutants we performed binding and transport studies. We used native HDL as the competitor because reconstituted and native HDL competed equivalently (see Fig. 5). As shown in Fig. 7D, specific binding of the lipidated WT apoA-I was approximately 60% of the specific binding of lipid-free WT apoA-I. However, prelipidated apoA-I(Δ1–59) and prelipidated apoA-I(Δ1–59/Δ185–243) showed 4-fold and 8-fold higher total binding than prelipidated WT apoA-I. In contrast to lipidated WT apoA-I, it was not possible to compete this binding with excess HDL, indicating that these mutants keep their very high nonspecific endothelial binding properties also after prelipidation. In contrast to apoA-I(L218A/L219A/V221A/L222A), prelipidation of either apoA-I(Δ1–59) or apoA-I(Δ1–59/Δ185–243) did not rescue their defective specific transendothelial transport (Fig. 7E). These findings suggest that lipidation of initially lipid-free apoA-I is not sufficient for the specific transport through ECs of the apoA-I(Δ1–59) or apoA-I(Δ1–59/Δ185–243) mutants.

Role of ABCA1, ABCG1, and SR-BI for Transport of rHDL

To analyze which of the known apoA-I/HDL-binding proteins are participating in the transport of prelipidated apoA-I we used siRNAs to suppress ABCA1, ABCG1 or SR-BI. Knockdown of ABCG1 and SR-BI but not of ABCA1 decreased the specific transendothelial transport of prelipidated WT apoA-I (Fig. 8A) and prelipidated apoA-I(L218A/L219A/V221A/L222A) (Fig. 8B). The reduction of the transport capacity of lipidated apoA-I(L218A/L219A/V221A/L222A) by knockdown of ABCG1 or SR-BI was smaller (−44% ±7%) compared with lipidated WT apoA-I (−60% ± 15%).