ABCMdb

PMID: 23221702

Tomioka M, Toda Y, Kurisu J, Kimura Y, Kengaku M, Ueda K
The effects of neurological disorder-related codon variations of ABCA13 on the function of the ABC protein.
Biosci Biotechnol Biochem. 2012;76(12):2289-93. Epub 2012 Dec 7., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCA13 p.Arg4843Cys ABCA13 p.Thr4031Ala ABCA1 p.Thr1088Ala Here, we examined the effects of neurological disorder-related SNPs ABCA13, T4031A and R4843C in the context of ABCA1, and found that the former SNP (T1088A in ABCA1) severely impaired the ABCA1 functions of apolipoprotein A-I (apoA-I) binding and cholesterol efflux. Login to comment 3 ABCA13 p.Thr4031Ala These results suggest that the T4031A replacement affects the function of ABCA13 in the brain. Login to comment 16 ABCA13 p.Arg4843Cys ABCA13 p.Thr4031Ala ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys Two SNPs of ABCA13, T4031A and R4843C, were inserted into the corresponding amino acid residues of ABCA1, T1088A and K2031C. Login to comment 56 ABCA1 p.Trp590Ser ApoA-I-dependent free cholesterol efflux from HEK293 cells stably expressing ABCA1, non-functional mutant (MM), a Tangier mutant (W590S), and SNP mutants are shown. Login to comment 57 ABCA1 p.Trp590Ser ApoA-I-dependent free cholesterol efflux from HEK293 cells stably expressing ABCA1, non-functional mutant (MM), a Tangier mutant (W590S), and SNP mutants are shown. Login to comment 66 ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys ABCA1 p.Lys2031Arg Hence we established HEK293 cells that stably expressed ABCA1 containing ABCA13 SNPs, ABCA1-T1088A, ABCA1- K2031C, and ABCA1-K2031R (as control). Login to comment 67 ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys ABCA1 p.Lys2031Arg Hence we established HEK293 cells that stably expressed ABCA1 containing ABCA13 SNPs, ABCA1-T1088A, ABCA1- K2031C, and ABCA1-K2031R (as control). Login to comment 68 ABCA1 p.Trp590Ser We also analyzed cholesterol efflux from cells expressing non-functional ABCA1-MM, in which two critical lysine residues in the NBDs were replaced with methionine residues and a Tangier disease mutant, ABCA1-W590S. Login to comment 69 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ABCA1 p.Thr1088Ala When the cells were incubated with a physiological concentration of apoA-I (10 mg/mL) for 24 h, a significant amount of cholesterol was transported from them to the medium by ABCA1 (Fig. 2A), but we found that ABCA1-MM did not mediate apoA-I-dependent cholesterol efflux, and that W590S Tangier mutation reduced cholesterol efflux by about 55%, as reported previously.12) Furthermore, the T1088A SNP drastically reduced cholesterol efflux, by about 90%. Login to comment 70 ABCA1 p.Trp590Ser ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys ABCA1 p.Lys2031Arg However, the K2031C SNP did not significantly affect cholesterol efflux as compared to the wild type or K2031R (Fig. 2A). Login to comment 71 ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys ABCA1 p.Lys2031Arg ApoA-I binding of HEK/ABCA1-SNP mutants at the plasma membrane We have reported that the first step in high-density lipoprotein (HDL) formation is apoA-I binding to the extracellular domains of ABCA1.13) Hence we examined to determine whether the loss in the cholesterol efflux ability of T1088A was due to a change in apoA-I binding. Login to comment 72 ABCA1 p.Thr1088Ala ApoA-I binding of HEK/ABCA1-SNP mutants at the plasma membrane We have reported that the first step in high-density lipoprotein (HDL) formation is apoA-I binding to the extracellular domains of ABCA1.13) Hence we examined to determine whether the loss in the cholesterol efflux ability of T1088A was due to a change in apoA-I binding. Login to comment 73 ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys W509S retained the ability to interact with apoA-I, while MM abolished that ability, as found previously.10) Furthermore, apoA-I binding was severely reduced (by 76%) by the T1088A SNP, while K2031C showed only a mild reduction (44%) (Fig. 2B). Login to comment 74 ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys These results suggest that HEK/ABCA1-T1088A has low apoA-I-binding activity. Login to comment 75 ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala Total and surface expression of the ABCA1-SNP mutants The fluorescence intensity of the GFP that fused to the C-terminus of ABCA1 suggests that T1088A did not severely impair total expression as compared to the wild-type ABCA1 (Fig. 2B), but it was difficult to compare surface expression because ABCA1 also located to the intracellular vesicles.14) Hence we examined the relative surface expression of ABCA1 by biotinylating cell-surface proteins. Login to comment 76 ABCA1 p.Trp590Ser ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala The total expression of T1088A was similar to that of the wild-type and the other mutants, with the exception of W590S (Fig. 3A). Login to comment 77 ABCA1 p.Trp590Ser ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala However, the amount of biotinylated ABCA1-T1088A was low as compared with the other mutants, and the fraction of surface localization was reduced by about 70% as compared to ABCA1-MM (Fig. 3B). Login to comment 78 ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala To confirm this change in the subcellular localization of ABCA1-T1088A, we examined glycosylation via EndoH, which cleaves within the chitobiose core of high mannose-type N-linked oligosaccharides, and PNGaseF, which cleaves all types of N-linked oligosaccharides. Login to comment 79 ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys ABCA1 p.Lys2031Arg For K2031C and wild-type K2031R, most of the protein was resistant to EndoH, but 50% or more of ABCA1-T1088 was sensitive to EndoH (Fig. 4), suggesting that the T1088A mutation caused improper folding of the protein. Login to comment 80 ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys ABCA1 p.Lys2031Arg For K2031C and wild-type K2031R, most of the protein was resistant to EndoH, but 50% or more of ABCA1-T1088 was sensitive to EndoH (Fig. 4), suggesting that the T1088A mutation caused improper folding of the protein. Login to comment 81 ABCA13 p.Thr4031Ala Detection of ABCA13 in the mouse brain The above results suggest that SNP T4031A, reported to be linked with risk of neurological disorder, can Fig. 4. Login to comment 82 ABCA13 p.Thr4031Ala Detection of ABCA13 in the mouse brain The above results suggest that SNP T4031A, reported to be linked with risk of neurological disorder, can Fig. 4. Login to comment 87 ABCA1 p.Trp590Ser A B W590S Fig. 3. Login to comment 88 ABCA1 p.Trp590Ser A B W590S Fig. 3. Login to comment 97 ABCA13 p.Arg4843Cys ABCA13 p.Thr4031Ala Discussion In this study, we examined the effects of neurological disorder-related SNPs of ABCA13, T4031A, and R4843C, in the context of ABCA1, and found that the former SNP severely impaired ABCA1 function. Login to comment 98 ABCA13 p.Arg4843Cys ABCA13 p.Thr4031Ala ABCA13 p.Thr4031Ala Because the antibody against ABCA13 reacted with neuronal cells in the cerebral cortex, hippocampus, and cerebellum, T4031A replacement in ABCA13 could affect its function in neurons. Login to comment 99 ABCA13 p.Thr4031Ala Because the antibody against ABCA13 reacted with neuronal cells in the cerebral cortex, hippocampus, and cerebellum, T4031A replacement in ABCA13 could affect its function in neurons. Login to comment 100 ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys ABCA1 p.Lys2031Cys No SNPs have been reported at these positions in ABCA1, although more than 100 coding variants have been identified in ABCA1.15) Using PolyPhen-2, T1088A and K2031C were both predicted to impair the function of ABCA1, with scores of 1.000 and 0.999, respectively,16,17) but the effects of T1088A and K2031C replacement were quite different. Login to comment 101 ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys ABCA1 p.Lys2031Cys ABCA1 p.Lys2031Cys T1088A severely impaired the subcellular localization and the functions of ABCA1, specifically apoA-I binding and cholesterol efflux, but K2031C showed only a mild effect on apoA-I binding, and no effect on cholesterol efflux. Login to comment 102 ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala ABCA1 p.Lys2031Cys Based on the structure of mouse mdr1a,18) T1088A can be mapped at the interface between two NBDs and K2031 at the domain protruding to the outside (Fig. 6). Login to comment 103 ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala The T1088A replacement of a hydrophilic by a hydrophobic amino acid might change the environment around its side chain and affect protein folding. Login to comment 104 ABCA1 p.Thr1088Ala ABCA1 p.Thr1088Ala Biotinylation of surface proteins and glycosylation analysis suggested that some fraction of ABCA1-T1088A reached the plasma membrane. Login to comment 105 ABCA1 p.Thr1088Ala Biotinylation of surface proteins and glycosylation analysis suggested that some fraction of ABCA1-T1088A reached the plasma membrane. Login to comment 106 ABCA1 p.Lys2031Cys On the other hand, K2031C did not affect the subcellular localization or cholesterol efflux of ABCA1. Login to comment 107 ABCA13 p.Arg4843Cys ABCA1 p.Lys2031Cys The R4843C replacement in the protruded domain of NBD2 in ABCA13 might have an effect Fig. 5. Login to comment 108 ABCA13 p.Arg4843Cys The R4843C replacement in the protruded domain of NBD2 in ABCA13 might have an effect Fig. 5. Login to comment