ABCMdb

PMID: 21209084

Ohnsorg PM, Rohrer L, Perisa D, Kateifides A, Chroni A, Kardassis D, Zannis VI, von Eckardstein A
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.
J Biol Chem. 2011 Mar 11;286(10):7744-54. Epub 2011 Jan 5., [PubMed]

Sentences

No. Mutations Sentence Comment

45 ABCA1 p.Leu222Ala 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(èc;BglII) plasmid as described (30). Login to comment 46 ABCA1 p.Leu222Ala ABCA1 p.Leu222Ala 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(⌬BglII) plasmid as described (30). Login to comment 47 ABCA1 p.Leu222Ala 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. Login to comment 55 ABCA1 p.Leu222Ala 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). Login to comment 56 ABCA1 p.Leu222Ala 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). Login to comment 92 ABCA1 p.Leu222Ala The two mutants with a defective carboxyl-terminal sequence, apoA-I(èc;185-243) and apoA-I(L218A/L219A/V221A/L222A), showed 50 and 80% decreases in total and specific endothelial binding, respectively (Fig. 1). Login to comment 94 ABCA1 p.Leu222Ala The two mutants with a defective carboxyl-terminal sequence, apoA-I(⌬185-243) and apoA-I(L218A/L219A/V221A/L222A), showed 50 and 80% decreases in total and specific endothelial binding, respectively (Fig. 1). Login to comment 101 ABCA1 p.Leu222Ala The results are represented as means afe; S.D. (error bars) of at least three individual experiments. Transendothelial ApoA-I Transport Is a Two-step Process 7746 binant WT apoA-I, and apoA-I(èc;144-165) did not differ from each other whereas the apoA-I(èc;185-243) and apoA-I(L218A/ L219A/V221A/L222A) mutants showed strongly reduced total and specific cell association, and the apoA-I(èc;1-59) and apoA-I(èc;1-59/èc;185-243) mutants showed massively increased total cell association which could not be competed by WT apoA-I (data not shown). Login to comment 103 ABCA1 p.Leu222Ala The results are represented as means Ϯ S.D. (error bars) of at least three individual experiments. Transendothelial ApoA-I Transport Is a Two-step Process 7746 binant WT apoA-I, and apoA-I(⌬144-165) did not differ from each other whereas the apoA-I(⌬185-243) and apoA-I(L218A/ L219A/V221A/L222A) mutants showed strongly reduced total and specific cell association, and the apoA-I(⌬1-59) and apoA-I(⌬1-59/⌬185-243) mutants showed massively increased total cell association which could not be competed by WT apoA-I (data not shown). Login to comment 107 ABCA1 p.Leu222Ala In contrast, the specific transports of apoA-I(èc;185-243) and apoA-I(L218A/L219A/V221A/L222A) were decreased by 90%. Login to comment 109 ABCA1 p.Leu222Ala In contrast, the specific transports of apoA-I(⌬185-243) and apoA-I(L218A/L219A/V221A/L222A) were decreased by 90%. Login to comment 110 ABCA1 p.Leu222Ala 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). Login to comment 112 ABCA1 p.Leu222Ala ABCA1 p.Leu222Ala 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). Login to comment 114 ABCA1 p.Leu222Ala ABCA1 p.Leu222Ala 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). Login to comment 115 ABCA1 p.Leu222Ala We therefore assume that this fraction represents lipid-free aggregates of apoA-I(L218A/ L219A/V221A/L222A). Login to comment 116 ABCA1 p.Leu222Ala 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. Login to comment 117 ABCA1 p.Leu222Ala We therefore assume that this fraction represents lipid-free aggregates of apoA-I(L218A/ L219A/V221A/L222A). Login to comment 136 ABCA1 p.Leu222Ala ABCA1 p.Leu222Ala 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. Login to comment 137 ABCA1 p.Leu222Ala We first lipidated WT apoA-I or apoA-I(L218A/L219A/V221A/L222A). Login to comment 138 ABCA1 p.Leu222Ala ABCA1 p.Leu222Ala 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. Login to comment 139 ABCA1 p.Leu222Ala ABCA1 p.Leu222Ala We first lipidated WT apoA-I or apoA-I(L218A/L219A/V221A/L222A). Login to comment 140 ABCA1 p.Leu222Ala rHDL containing apoA-I(L218A/L219A/ V221A/L222A) were slightly less negatively charged than rHDL containing WT apoA-I (Fig. 6A). Login to comment 141 ABCA1 p.Leu222Ala ABCA1 p.Leu222Ala 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. Login to comment 142 ABCA1 p.Leu222Ala rHDL containing apoA-I(L218A/L219A/ V221A/L222A) were slightly less negatively charged than rHDL containing WT apoA-I (Fig. 6A). Login to comment 143 ABCA1 p.Leu222Ala ABCA1 p.Leu222Ala 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). Login to comment 145 ABCA1 p.Leu222Ala ABCA1 p.Leu222Ala Electron microscopy revealed that both WT apoA-I and apoA-I(L218A/L219A/ V221A/L222A) formed discoidal particles (Fig. 6C). Login to comment 146 ABCA1 p.Leu222Ala Thus prelipidation can overcome the binding and transport defects of the dysfunctional apoA-I(L218A/L219A/V221A/L222A) mutant. Login to comment 147 ABCA1 p.Leu222Ala 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). Login to comment 148 ABCA1 p.Leu222Ala Thus prelipidation can overcome the binding and transport defects of the dysfunctional apoA-I(L218A/L219A/V221A/L222A) mutant. Login to comment 152 ABCA1 p.Leu222Ala Particle size of WT apoA-I and apoA-I(L218A/L219A/V221A/L222A) before and after transport through ECs. Login to comment 154 ABCA1 p.Leu222Ala Particle size of WT apoA-I and apoA-I(L218A/L219A/V221A/L222A) before and after transport through ECs. Login to comment 156 ABCA1 p.Leu222Ala B, 125 I-apoA-I(L218A/L219A/V221A/L222A). Login to comment 158 ABCA1 p.Leu222Ala B, 125 I-apoA-I(L218A/L219A/V221A/L222A). Login to comment 164 ABCA1 p.Leu222Ala In contrast to apoA-I(L218A/L219A/V221A/L222A), prelipidation of either apoA-I(èc;1-59) or apoA-I(èc;1-59/èc;185-243) did not rescue their FIGURE 4. Login to comment 166 ABCA1 p.Leu222Ala In contrast to apoA-I(L218A/L219A/V221A/L222A), prelipidation of either apoA-I(⌬1-59) or apoA-I(⌬1-59/⌬185-243) did not rescue their FIGURE 4. Login to comment 176 ABCA1 p.Leu222Ala Binding and transport of rHDL containing WT apoA-I or apoA-I(L218A/L219A/V221A/L222A). Login to comment 178 ABCA1 p.Leu222Ala Binding and transport of rHDL containing WT apoA-I or apoA-I(L218A/L219A/V221A/L222A). Login to comment 179 ABCA1 p.Leu222Ala D, specific binding at 4 &#b0;C of 10 òe;g/ml 125 I-rHDL particles containing either WT apoA-I or apoA-I(L218A/L219A/V221A/L222A) was determined. Login to comment 180 ABCA1 p.Leu222Ala E, specific transport of 10 òe;g/ml 125 I-rHDL particles containing either WT apoA-I or apoA-I(L218A/L219A/V221A/L222A) through a monolayer of ECs was determined. Login to comment 181 ABCA1 p.Leu222Ala D, specific binding at 4 °C of 10 ␮g/ml 125 I-rHDL particles containing either WT apoA-I or apoA-I(L218A/L219A/V221A/L222A) was determined. Login to comment 182 ABCA1 p.Leu222Ala E, specific transport of 10 ␮g/ml 125 I-rHDL particles containing either WT apoA-I or apoA-I(L218A/L219A/V221A/L222A) through a monolayer of ECs was determined. Login to comment 185 ABCA1 p.Leu222Ala Transendothelial ApoA-I Transport Is a Two-step Process MARCH 11, 2011ߦVOLUME 286ߦNUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY down 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). Login to comment 186 ABCA1 p.Leu222Ala The reduction of the transport capacity of lipidated apoA-I(L218A/L219A/V221A/L222A) by knockdown of ABCG1 or SR-BI was smaller (afa;44% afe;7%) compared with lipidated WT apoA-I (afa;60% afe; 15%). Login to comment 187 ABCA1 p.Leu222Ala down 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). Login to comment 188 ABCA1 p.Leu222Ala The reduction of the transport capacity of lipidated apoA-I(L218A/L219A/V221A/L222A) by knockdown of ABCG1 or SR-BI was smaller (-44% Ϯ7%) compared with lipidated WT apoA-I (-60% Ϯ 15%). Login to comment 192 ABCA1 p.Leu222Ala Also, in our endothelial transwell cell culture model, both apoA-I(èc;185-243) and apoA-I(L218A/L219A/V221A/L222A) failed to form HDL-like particles although only the specific but not the nonspecific fraction of transendothelial transport was abolished. Login to comment 194 ABCA1 p.Leu222Ala Also, in our endothelial transwell cell culture model, both apoA-I(⌬185-243) and apoA-I(L218A/L219A/V221A/L222A) failed to form HDL-like particles although only the specific but not the nonspecific fraction of transendothelial transport was abolished. Login to comment 205 ABCA1 p.Leu222Ala The formation of a high affinity complex of apoA-I with ABCA1 is thought to play FIGURE8.RoleofABCA1,ABCG1,andSR-BIinthetransportofrHDLcontainingWTapoA-IorapoA-I(L218A/L219A/V221A/L222A).ECsweretransfected with siRNA coding for ABCA1, ABCG1, SR-BI and not coding siRNA. Login to comment 207 ABCA1 p.Leu222Ala The formation of a high affinity complex of apoA-I with ABCA1 is thought to play FIGURE8.RoleofABCA1,ABCG1,andSR-BIinthetransportofrHDLcontainingWTapoA-IorapoA-I(L218A/L219A/V221A/L222A).ECsweretransfected with siRNA coding for ABCA1, ABCG1, SR-BI and not coding siRNA. Login to comment 209 ABCA1 p.Leu222Ala B, specific transport of rHDL containing apoA-I(L218A/ L219A/V221A/L222A). Login to comment 211 ABCA1 p.Leu222Ala B, specific transport of rHDL containing apoA-I(L218A/ L219A/V221A/L222A). Login to comment 216 ABCA1 p.Leu222Ala The apoA-I(L218A/L219A/V221A/L222A) mutant, which shows strongly reduced specific but normal nonspecific transendothelial transport (data not shown), was not recovered as a lipidated particle (Fig. 3). Login to comment 218 ABCA1 p.Leu222Ala The apoA-I(L218A/L219A/V221A/L222A) mutant, which shows strongly reduced specific but normal nonspecific transendothelial transport (data not shown), was not recovered as a lipidated particle (Fig. 3). Login to comment 219 ABCA1 p.Leu222Ala These downstream pathways may be shared with the transport of HDL because knockdown of ABCG1 or SR-BI inhibited the specific transport of native HDL (15) as well as rHDL containing either WT apoA-I or apoA-I(L218A/L219A/ V221A/L222A) (Fig. 8). Login to comment 220 ABCA1 p.Leu222Ala In agreement with normal binding of rHDL containing apoA-I(L218A/L219A/V221A/L222A), ldlA-7 cells expressing SR-BI were previously found to bind rHDL containing apoA-I(èc;185-243) with affinity similar to that of rHDL with WT apoA-I (46). Login to comment 221 ABCA1 p.Leu222Ala These downstream pathways may be shared with the transport of HDL because knockdown of ABCG1 or SR-BI inhibited the specific transport of native HDL (15) as well as rHDL containing either WT apoA-I or apoA-I(L218A/L219A/ V221A/L222A) (Fig. 8). Login to comment 222 ABCA1 p.Leu222Ala In agreement with normal binding of rHDL containing apoA-I(L218A/L219A/V221A/L222A), ldlA-7 cells expressing SR-BI were previously found to bind rHDL containing apoA-I(⌬185-243) with affinity similar to that of rHDL with WT apoA-I (46). Login to comment