ABCMdb

PMID: 14967052

Chroni A, Liu T, Fitzgerald ML, Freeman MW, Zannis VI
Cross-linking and lipid efflux properties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1.
Biochemistry. 2004 Feb 24;43(7):2126-39., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCA1 p.Trp590Ser The WT apoA-I, amino, carboxy and double deletion mutants of apoA-I showed differences in the cross-linking to WT ABCA1 and the mutant ABCA1[W590S]. Login to comment 16 ABCA1 p.Trp590Ser To further examine the structural domains of apoA-I that affect the apoA-I/ABCA1 interactions, we tested the effect of amino-terminal, carboxy-terminal, and internal deletion mutants, as well as point mutants in the central helices of apoA-I on ABCA1-mediated lipid efflux and on the ability of apoA-I to cross-link to ABCA1. Furthermore, we tested the ability of apoA-I mutants to associate with the ABCA1[W590S] mutant, which is defective in cholesterol efflux but cross-links to apoA-I (20). Login to comment 204 ABCA1 p.Trp590Ser Effect of Amino-Terminal, Carboxy-Terminal, and Double Amino- and Carboxy-Terminal Deletions of ApoA-I on the Ability of ApoA-I to Cross-Link to the ABCA1[W590S] Mutant. Login to comment 205 ABCA1 p.Trp590Ser To further explore the interaction of apoA-I with ABCA1, we tested the ability of various apoA-I mutants to associate with the ABCA1[W590S] mutant. Login to comment 207 ABCA1 p.Trp590Ser For this purpose, we performed a direct cross-linking of iodinated WT and mutant forms of apoA-I (0.2 µM) to WT ABCA1 or the ABCA1[W590S] mutant in the presence or absence of 30-fold molar excess of the corresponding unlabeled apoA-I form as described in the Experimental Procedures. Login to comment 208 ABCA1 p.Trp590Ser As shown in Figure 5A, the WT apoA-I, the amino-terminal, and the double amino- and carboxy-terminal deletion mutants of apoA-I cross-link to ABCA1[W590S] and the cross-linking is completely inhibited by 30-fold molar excess of unlabeled apoA-I. Login to comment 209 ABCA1 p.Trp590Ser The carboxy-terminal deletion mutants apoA-I[∆(185-243)] and apoA-I[∆(220-243)] cross-link less efficiently to either WT ABCA1 or mutant ABCA1[W590S], and the cross-linking is partially competed by 30-fold molar excess of unlabeled apolipoprotein. Login to comment 210 ABCA1 p.Trp590Ser The WT apoA-I and the apoA-I[∆(1-41)∆(185-243)] mutant have higher and equal ability, respectively, to cross-link to ABCA1[W590S] as compared to WT ABCA1. Login to comment 211 ABCA1 p.Trp590Ser However, the amino-terminal deletion mutant apoA-I[∆(1-41)] and the carboxy-terminal deletion mutants apoA-I[∆(185-243)] and apoA-I[∆(220-243)] show 36-64% reduced cross-linking to ABCA1[W590S] (Figure 5B). Login to comment 212 ABCA1 p.Trp590Ser The finding suggests subtle differences in the cross-linking of WT and mutant apoA-I forms to the ABCA1[W590S] mutant as compared to the cross-linking of the same apoA-I forms to the WT ABCA1. Login to comment 241 ABCA1 p.Trp590Ser This model is supported by the observation that, with the exception of a single mutant ABCA1[W590S], all other natural mutants in the two extracellular loops of ABCA1 tested, which are defective in cholesterol efflux, cross-link inefficiently to ABCA1 (20). Login to comment 263 ABCA1 p.Trp590Ser With FIGURE 5: Cross-linking of 125I-labeled WT and mutant forms of apoA-I to WT ABCA1 and ABCA1[W590S]. Login to comment 264 ABCA1 p.Trp590Ser HEK293 cells transfected with plasmids carrying the WT ABCA1 and ABCA1[W590S] mutant were incubated for 1 h at 37 °C with 0.2 µM 125I-labeled WT or the indicated mutant forms of apoA-I alone or in the presence of a 30-fold molar excess of the respective unlabeled apolipoproteins. Login to comment 270 ABCA1 p.Trp590Ser The fmol of cross-linked WT or mutant forms of apoA-I per mg of cellular protein to WT ABCA1 and ABCA1[W590S] were calculated as described in the legend of the Figure 2D,E and are graphed to panel B. Login to comment 271 ABCA1 p.Trp590Ser Panel B shows the total cross-linking of the indicated apoA-I forms to WT ABCA1 and the ABCA1[W590S] mutant. Login to comment 274 ABCA1 p.Trp590Ser The % ratio of the cross-linking of each 125I-labeled apoA-I form to the ABCA1[W590S] mutant to the cross-linking of the same apoA-I form to the WT ABCA1 was also calculated and is shown in the lower part of the panel B. Login to comment 316 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Interactions of WT and Mutant Forms of ApoA-I with the WT ABCA1 and the ABCA1[W590S] Mutant Differ and May Account for the Inability of the ABCA1[W590S] Mutant, Which Binds Strongly to ApoA-I, To Promote Cholesterol Efflux. Login to comment 317 ABCA1 p.Trp590Ser The present, as well as a previous study (20), showed that the ABCA1[W590S] cross-links better than WT ABCA1 to apoA-I but fails to promote cholesterol efflux in vitro and HDL formation in vivo (7, 20). Login to comment 318 ABCA1 p.Trp590Ser However, as shown in this study, the amino- and carboxy-terminal deletion mutants cross-link less efficiently to the ABCA1[W590S] mutant, and the double deletion mutant cross-links with similar efficiency to the WT and the mutant ABCA1 forms. Login to comment 320 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser If the binding of apoA-I acceptors to the ABCA1 [W590S] mutant was brought about by ABCA1-dependent changes in the cell membrane (11, 12), one would expect that apoA-I mutants that bind inefficiently to the WT ABCA1 would also bind inefficiently to the ABCA1[W590S] mutant and vice versa. Login to comment 321 ABCA1 p.Trp590Ser One possible way then to interpret the inability of the ABCA1[W590S] mutant to promote cholesterol efflux, is to assume that the complex formed is a non-productive complex that prevents the lipidation of apoA-I that is bound to ABCA1. Login to comment 323 ABCA1 p.Trp590Ser In fact, recent experiments have confirmed this hypothesis by showing that following binding to the WT ABCA1, the apoA-I is released associated with cholesterol, whereas apoA-I bound to the ABCA1[W590S] mutant is released in lipid-free form (49). Login to comment 333 ABCA1 p.Trp590Ser In the case of the ABCA1[W590S] mutant, the complex is formed but the FIGURE 7: Two step model of cholesterol efflux. Login to comment 339 ABCA1 p.Trp590Ser Formation of a non-productive complex is supported by our data, which showed that WT and mutant apoA-I have differences in their cross-linking to the WT ABCA1 and the ABCA1[W590S] mutant. Login to comment