Expression and Functional Analyses of Novel Mutations of ATP-Binding Cassette Transporter-1 in Japanese Patients with High-Density Lipoprotein Deficiency

Biochemical and Biophysical Research Communications 290, 713–721 (2002) doi:10.1006/bbrc.2001.6219, available online at http://www.idealibrary.com on

Expression and Functional Analyses of Novel Mutations of ATP-Binding Cassette Transporter-1 in Japanese Patients with High-Density Lipoprotein Deficiency Yoshiharu Nishida,* Ken-ichi Hirano,* ,1 Kosuke Tsukamoto,* Makoto Nagano,* ,† Chiaki Ikegami,* Kirsten Roomp,‡ Mitsuaki Ishihara,† Naoki Sakane,§ Zhongyan Zhang,* Ken-ichi Tsujii,* Akifumi Matsuyama,* Tohru Ohama,* Fumihiko Matsuura,* Masato Ishigami,* Naohiko Sakai,* Hisatoyo Hiraoka,* Hiroaki Hattori,† Cheryl Wellington,‡ Yoshihide Yoshida,§ Susumu Misugi, ¶ Michael R. Hayden,‡ Toru Egashira,† Shizuya Yamashita,* and Yuji Matsuzawa* *Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Osaka, Japan; †Research Department, R&D Center, BML, Saitama, Japan; §First Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan; ‡Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada; and ¶Misugi Clinic, Osaka, Japan

Received December 7, 2001

ATP-binding cassette transporter-1 (ABCA1) gene is mutated in patients with familial high-density lipoprotein deficiency (FHD). In order to know the molecular basis for FHD, we characterized three different ABCA1 mutations associated with FHD (G1158A/ A255T, C5946T/R1851X, and A5226G/N1611D) with respect to their expression in the passaged fibroblasts from the patients and in the cells transfected with the mutated cDNAs. Fibroblasts from the all patients showed markedly decreased cholesterol efflux to apolipoprotein (apo)-Al. In the fibroblasts homozygous for G1158A/A255T, the immunoreactive mass of ABCA1 could not be detected, even when stimulated by 9-cisretinoic acid and 22-R-hydroxycholesterol. In the fibroblasts homozygous for C5946T/R1851X, ABCA1 mRNA was comparable. Because the mutant ABCA1 protein (R1851X) was predicted to lack the epitope for the antibody used, we transfected FLAG-tagged truncated mutant (R1851X/ABCA1-FLAG) cDNA into Cos-7 cells, showing that the mutant protein expression was markedly reduced. The expression of N1611D ABCA1 protein was comparable in both fibroblasts and overexpressing cells, although cholesterol efflux from the cells was markedly reduced. These data indicated Abbreviations used: ABCA1, ATP-binding cassette transporter-1; DMEM, Dulbecco’s modified Eagle medium; FHD, familial HDL deficiency; HDL, high-density lipoprotein; NBD, nucleotide binding domain; RCT, reverse cholesterol transport; TD, Tangier disease; TM, transmembrane. 1 To whom correspondence and reprint requests should be addressed at Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suita 565-0871, Osaka, Japan. Fax: ⫹81-6-6879-3739. E-mail: [email protected].

that, in the three patients investigated, the abnormalities and dysfunction of ABCA1 occurred at the different levels, providing important information about the expression, regulation, and function of ABCA1. © 2002 Elsevier Science

Key Words: atherosclerosis; ATP-binding cassette transporter-1 (ABCA1); familial HDL deficiency; Tangier disease; reverse cholesterol transport.

Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which small high density lipoprotein (HDL) or free apolipoprotein A-l (apo A-l) removes cholesterol from the peripheral cells and delivers it to the liver (1, 2). We have investigated the molecular mechanism for RCT by analyzing the pathophysiology of patients with abnormal HDL metabolism and clarified the significance of cholesteryl ester transfer protein and hepatic triglyceride lipase in human RCT (3, 4). Tangier disease (TD) is a familial HDL deficiency, which is a model for impairment of cholesterol efflux, the initial step of RCT (5), and is frequently associated with cardiovascular disease (6, 7). The positional cloning approaches revealed that TD is associated with mutations in the ATP-binding cassette transporter-1 (ABCA1) genes (8 – 10). Growing evidence showed that ABCA1 was mutated in many patients with familial HDL deficiency (FHD) (8, 11), which did not have typical TD phenotype. Genetic engineered mice technology indicated that the ABCA1 null mice had similar phenotypes to those observed in patients with TD (12) and that overexpres-

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FIG. 1. Identification of three novel substitutions in the patients with familial HDL deficiency. (A) Substitution in Case 1. Case 1 was homozygous for a G to A substitution at nt 1158 of the cDNA; the control is homozygous for guanine at this position. This mutation corresponds to an alanine (A) to threonine (T) substitution in ABCA1. The alanine was conserved between human ABCA1 and murine ABCA1. (B) Substitution in Case 2. Case 2 was homozygous for a C to T substitution at nt 5946 of the cDNA; the control is homozygous for cytosine at this position. This substitution corresponds to an arginine (R) to nonsense change in ABCA1, creating a truncated ABCA1 protein, which lacks the second nucleotide binding domain. This truncation lost the epitope for the polyclonal antibody against ABCA1. (C) Substitution in Case 3. Case 3 was homozygous for a A to G substitution at nt 5226 of the cDNA; the control is homozygous for adenine at this position. This substitution corresponds to an asparagine (N) to aspartic acid (D) in ABCA1. The asparagine was conserved between human ABCA1 and murine ABCA1. 714

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Clinical Profiles of Patients with Familial HDL Deficiency

ABCA1 substitutions found (nt/aa) Age (years)/sex (M, F) Total cholesterol (mmol/L) HDL-cholesterol (mmol/L) Triglyceride (mmol/L) Apo-Al (mg/dL) Atherosclerosis Typical TD phenotype Cholesterol efflux (% of control)

Case 1

Case 2

Case 3

G1158A/A255T 56/M 0.72 0.16 2.6 3.9 ⫹ ⫹ 5.0

C5946T/R1851X 71/F 1.47 0.05 3.27 5.0 ⫹ ⫹ 2.0

A5226G/N1611D 53/F 2.7 0.11 1.75 11.0 ⫹ ⫺ 7.0

Note. Abbreviations used: ABCA1, ATP-binding cassette transporter-1; TD, Tangier disease. Atherosclerosis: Case 1 suffered from severe coronary atherosclerosis, as reported previously (Reference 7). Case 2 died of acute myocardial infarction at the age of 71. Case 3 had marked atherosclerosis with calcification in her abdominal aorta and carotid and splenic arteries. Typical TD phenotype: Cases 1 and 2 had hepatosplenomegaly and orange tonsils. Cholesterol efflux from patients’ fibroblasts: The values were expressed as % of cholesterol efflux from normal fibroblasts (N ⫽ 3) (see Materials and Methods).

sion of ABCA1 increased HDL-cholesterol levels (13– 15), supporting that ABCA1 is one of the requisite regulators for production of HDL particles. From these data, the up-regulation of ABCA1 is thought to be one of the major strategies for the development of novel treatments for atherosclerosis. On the other hand, recent experimental data indicated that ABCA1 has other possible functions such as regulation of apoptosis (16), actin cytoskeletons (17), and cholesterol absorption (18), other than mediating cholesterol efflux. It is a fact that the clinical profiles and characteristics of ABCA1 mutants are heterogeneous. Therefore, the investigation of expression and function of diseaseassociated mutations and genotype/phenotype correlation would be very important, since these data could provide a number of information not only for understanding the structure and function of ABCA1 in relation to the above diverse functions but also for considering the therapeutic strategies for overexpressing this molecule. In the present study, we have found three novel mutations in the ABCA1 gene in Japanese patients with HDL deficiency and analyzed endogenous expression of ABCA1 in their fibroblasts as well as function of the mutant ABCA1 cDNA in the overexpressing cells.

Materials Human apo A-l was purchased from Sigma. Anti-FLAG M2 antibody (Cat. No. F3165) was purchased from Sigma. Polyclonal antibody against human ABCA1, which was raised against the carboxyterminus (KNQTVVDVAVLTSFLQDEKVKES), was kindly provided by Dr. Reijiro Arakawa and Professor Shinji Yokoyama (Nagoya City University Medical School, Nagoya, Japan) (20). ACAT inhibitor, F-1394, was kindly provided from Fujirebio Inc. (Tokyo, Japan).

Sequence of ABCA1 Gene All exons were amplified from genomic DNA by using specific primers. Polymerase chain reaction (PCR) products were directly sequenced on ABI Prism Sequencers (Applied Biosystems), as reported previously (8).

Cloning of Human ABCA1 cDNA and Plasmid Construction Full-length cDNA of human ABCA1 was cloned into pcDNA3.1 (Invitrogen, CA), as reported previously (17). FLAG-tagged hABCA1 cDNA with the FLAG epitope (DYKDDDDK) incorporated at its carboxyl terminus (ABCA1-FLAG) was generated by PCR, and was cloned into mammalian expression vector pcDNA3.1 (Invitrogen, CA) using the full-length ABCA1 cDNA as a parental clone. In order to make ABCA1-FLAG constructs carrying the substitutions we found, mutagenesis was performed using Quik Change Site-Directed Mutagenesis Kit (Stratagene). The introduction of mutations was confirmed by the sequencing analysis.

Stimulation of ABCA1 by 9-cis-Retinoic Acid (9-cis-RA) and 22-R-Hydroxycholesterol (22-OH-Chol)

MATERIALS AND METHODS Patients Three patients with HDL deficiency were subjected to this study (Table 1). For the DNA analyses we obtained the informed consents from all patients. All patients had apparent atherosclerotic changes. Case 1 suffered from severe coronary heart disease, as reported previously (7, 19). Case 2 died of acute myocardial infarction at the age of 71. Both of Cases 1 and 2 had typical TD phenotype such as hepatosplenomegaly and orange tonsils. Case 3, who did not have the TD phenotype, had marked calcifications in her abdominal aorta and carotid and splenic arteries. Apo Al-mediated cholesterol efflux was markedly reduced in fibroblasts from all patients investigated.

ABCA1 was stimulated by the replacement of the growth medium with media containing 2 mg/mL of lipoprotein-deficient serum (LPDS) with either ethanol (control) or 10 ␮M 9-cis retinoic acid (9-cis-RA) (10 mg/mL stock solution in ethanol, Sigma) and 4 ␮g/mL of 22-R-hydroxycholesterol (22-R-OH-Chol) (4 mg/mL stock solution in ethanol, Sigma). After 24 h incubation, the cells were subjected to the preparation of mRNA and membrane fractions (21).

Reverse Transcription-based Polymerase Chain Reaction (RT-PCR) In order to know the expression levels of endogenous ABCA1 mRNA, we performed the following two different types of RT-PCR.

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FIG. 2. A model for ABCA1 structure and possible locations of the substitutions found. Recently, the existence of extracellular loop at the amino-terminus, where the A255T may be located, was proposed (Refs. 24, 25). R1851X appeared to lack the second nucleotide binding domain. N1611D appeared to be located in the middle of extracellular loop from codons 1369 to 1654 (Ref. 24).

(1) Conventional RT-PCR. Two ␮g of total RNA was subjected to the RT reaction using downstream priming primers, P1 for ABCA1 and P2 for GAPDH, in the presence of 50 units Superscript Reverse Transcriptase (GibcoBRL) at 42°C for 60 min. Two hundred fiftynine bp and 244 bp fragments from ABCA1 and GAPDH, respectively, were amplified from cDNA samples in the different tubes. Sample sizes and the number of amplification cycles were optimized to produce measurements within a linear range. After heating the RT reaction at 75°C for 5 min, one-tenth volume was used for PCR with Advantage cDNA Polymerase Mix (Clontech). After heating the cDNA mixture at 95°C for 5 min, PCR was conducted: denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 68°C for 60 s, respectively. The cycle numbers were 24 for ABCA1 and 20 for GAPDH. (2) Real time quantitative RT-PCR (22, 23). After the optimization of conditions, we have performed the fluorescent quantitative RT-PCR. RT-PCR was performed with 500 ng of total RNA and downstream priming with oligo dT in the presence of 50 units Superscript Reverse Transcriptase (GibcoBRL) at 42°C for one hour. After heating the reaction mixture at 75°C for 5 min, one-fiftieth volume was used for PCR. PCR amplification and detection were achieved using SYBR Green PCR and RT-PCR Reagens kit (Applied Biosystems) and ABI Prism 7700 Sequence Detection System (Applied Biosystems) according to the manufacturer’s protocol. After incubation of the cDNA mixture at 95°C for 10 min to activate AmpliTaq Gold DNA Polymerase (Applied Biosystems), PCR was conducted; 45 cycles of denaturation at 95°C for 15 s and annealing/ extension at 60°C for 1 min. Fluorescence data were acquired at the end of the extension step. Product identity was confirmed by ethidium bromide-stained agarose gel electrophoresis.

solved with 0.1 N NaOH and residual radioactivity in the cells was determined.

Preparation of Membrane Fraction and Western Blot Analysis For preparing the crude membrane fractions, cells were scraped into scraping buffer containing 150 mM NaCl, 10 mM Tris–HCl (pH 7.4), 1 mM EDTA, and proteinase inhibitors. The scraped samples were sonicated, and then centrifuged at 800g for 20 min to remove the nuclear fraction. The supernatant was ultracentrifuged at 10,000g for 1 h and the pellet was dissolved with lysis buffer, which consisted of scraping buffer plus detergent. The samples were run on SDS–PAGE, blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore). Detection was performed using enhanced chemiluminescence (ECL) plus kit (Amersham) according to manufacturer’s protocol.

Primers Used in the Present Study P1. 5⬘-GGT GTT TTG CTT TGC TGA CCC-3⬘, ABCA1 cDNA nucleotide 5349 to 5329 (GenBank Accession No. AF285167). P2. 5⬘-GGC AGT GAT GGC ATG GAC TGT G-3⬘, GAPDH cDNA nucleotide 609 to 588 (GenBank Accession No. M33197). P3. 5⬘-GCA ATC AGC TCT TTC CTG AAT-3⬘, ABCA1 cDNA nucleotide 5091 to 5111 (GenBank Accession No. AF285167). P4. 5⬘-CAC CAC CAT GGA GAA GGC TGG-3⬘, GAPDH cDNA nucleotide 366 to 386 (GenBank Accession No. M33197).

RESULTS

Cell Culture and Lipid Efflux Assay Cos-7 cells or HEK293 cells were cultured in DMEM supplemented with 10% fetal calf serum and antibiotics (DMEM/FCS). One day before transfection, cells were plated with the density of 1.6 ⫻ 10 5/well or 2.5 ⫻ 10 5/well onto 12-well plates coated with type I collagen, respectively. The next day, cells with about 95% confluence were transfected using Lipofectamine 2000 reagent (Life Technologies) and corresponding plasmid constructs. For lipid efflux assays, cells were labeled with 1.0 ␮Ci/mL [ 3H]cholesterol on the next day of transfection. After labeling for 18 h, the cells were washed three times with PBS, incubated at 37°C for 2 h with DMEM plus 0.2% essential fatty acid-free bovine serum albumin (DMEM/BSA). The media was then replaced with fresh DMEM/BSA with and without presence of 15 ␮g/ml apoA-l and incubated for 4 h at 37°C. The media was collected and counted for radioactivity, and the cells were dis-

We sequenced all exons and exon/intron boundaries of the ABCA1 gene in the patients investigated. Each of them was found to be a homozygote (Ho) for the following novel substitutions in the coding regions. We demonstrated the sequencing patterns (Fig. 1) as well as possible localizations of substitutions found in a topological model of ABCA1 protein (Fig. 2) (24, 25). Case 1 was homozygous for a G to A substitution at nucleotide position 1158, possibly causing a change of amino acid from alanine (A) to threonine (T) at codon 255 (Fig. 1A). His daughter, whose plasma HDLcholesterol was lower than controls, was heterozygous

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for the substitution (data not shown). We could not find the G1158A/A255T substitution in 48 unrelated Americans or 176 Japanese control subjects (data not shown). Case 2 was homozygous for a C to T substitution at nucleotide position 5946. This mutation caused a premature stop at the codon 1851 (R1851X), resulting that the predicted ABCA1 protein was truncated from lacking the second nucleotide binding domain (NBD) (Figs. 1B and 2). Case 3 was homozygous for a A to G substitution at nucleotide position 5226, causing a change of amino acid from asparagine (N) to aspartic acid (D) at codon 1611 (N1611D), as shown in Fig. 1C. The codon 1611 appeared to be located around the proximal part of the second transmembrane domain (Fig. 2). Although it was reported that ABCA1 gene was mutated in many patients with HDL deficiency, the endogenous expression of ABCA1 has not fully been reported. We analyzed the endogenous expression levels of ABCA1 mRNA (Figs. 3A and 3B) and protein (Fig. 3C) in their fibroblasts with and without the stimulation by 9-cis-RA and 22-R-OH-Chol, which is the known method to stimulate the transcription of ABCA1. Fibroblasts from Case 1 (Ho/A255T) had markedly low levels of ABCA1 mRNA in the condition without the stimulation, whereas these levels became comparable after the stimulation. In Cases 2 (Ho/ R1851X) and 3 (Ho/N1611D), the expression of mRNA did not appear to be altered with and without the stimulation (Figs. 3A and 3B). Figure 3C shows the results of Western blot analyses for ABCA1 protein. The immunoreactive mass of ABCA1 could not be detected in fibroblasts from Case 1 even when the cells were stimulated by 9-cis-RA and 22-R-OH-Chol, which markedly up-regulated ABCA1 mRNA levels in the same patient (Figs. 3A and 3B). It was likely that no-trace amount of ABCA1 protein contributed to the FHD phenotype in Case 1 (Ho/A255T). As it was noted that the predicted mutant ABCA1 (R1851X) lacked the epitope for the antibody we used, we could not see any immunoreactive mass in fibroblasts from Case 2 (Ho/ R1851X). We could observe the comparable expression of ABCA1 protein in fibroblasts from Case 3 (Ho/ N1611D). To further analyze the underlying molecular mechanism, we made epitope (FLAG)-tagged ABCA1 cDNA constructs carrying one of the above substitutions found. As shown in Figs. 4A and 4B, the expression of each ABCA1 cDNA construct was successful in Cos-7 cells and HEK293 cells (data not shown). It was noted that we obtained the comparable protein expression of A255T/ABCA1-FLAG cDNA construct, though we could observe no ABCA1 protein in the fibroblasts from Case 1 (Ho/A255T). It was striking that the protein expression of cDNA construct carrying the truncated mutation (R1851X) was extremely low, though we could see the expected band with smaller size.

Finally, we tested apo Al-mediated cholesterol efflux from the Cos-7 cells and HEK293 cells transfected with various ABCA1 constructs (Figs. 5A and 5B). Significant cholesterol efflux was observed in cells expressing wild type of ABCA1 (WT/ABCA1-FLAG). The degree of cholesterol efflux from cells expressing the mutants was variable. We could detect a significant amount of cholesterol efflux from cells expressing A255T/ABCA1FLAG. As expected, no significant cholesterol efflux could be detected from cells transfected with R1851X/ ABCA1-FLAG cDNA. From cells expressing N1611D/ ABCA1-FLAG, no significant cholesterol efflux could be observed, although we obtained the comparable expression of the mutant protein (Figs. 4A and 4B). DISCUSSION In the present study, we have found the novel substitutions associated with patients with FHD and analyzed the endogenous expression of ABCA1 in fibroblasts from the patients as well as the functional ability of ABCA1 carrying the substitutions we found. The results were interesting but complicated. As summarized in Table 2, the molecular basis was heterogeneous and the abnormalities and dysfunction of ABCA1 occurred at the different levels in each patient as discussed below. In Case 1, we found a novel substitution (G1158A/ A255T). The codon 255 is located at the putative extracellular loop of ABCA1 protein in some topological models (24, 25). Because the recent clinical mutational analyses in patients with TD appeared to show that many potential loss-of-function mutations are located around this lesion (8 –11), we had initially speculated that this predicted mutant ABCA1 protein (A255T) could have any dysfunction of this loop. However, it did not appear to be the case. No ABCA1 protein could be detected in the fibroblasts from Ho/G1158A even after the stimulation, which is likely to be responsible for the phenotypic expression of FHD in Case 1. Our transfection experiment has given us the discrepancy of the expression of ABCA1 protein between the patients’ fibroblasts and overexpressing Cos-7 cells and HEK293 cells (Figs. 2C, 3A, and 3B). The ABCA1 cDNA construct carrying the G1158A/A255T substitution appeared to be functionally normal in the overexpressing cells (Figs. 5A and 5B). Considering that his daughter, whose HDL-cholesterol was low, was heterozygous for the substitution and that the substitution was not seen either in the American or Japanese control subjects, we could, at least, say that the G1158A substitution may be related to the phenotypic expression of FHD in Case 1 family. From the above data, we speculated that dysfunction of ABCA1 in Case 1 occurred most likely at mRNA level (Table 2). Detailed survey of ABCA1 mRNA is underway in our laboratory.

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FIG. 3. Analyses of endogenous expression of ABCA1 mRNA (A and B) and protein (C) in fibroblasts from cases 1–3 with and without the stimulation by 9-cis-RA and 22-OH-Chol. Total RNA and membrane fractions were prepared as described under Materials and Methods. Passaged fibroblasts were cultured with and without the stimulation by 9-cis-RA and 22-OH-Chol. (A) Agarose gel electrophoresis of conventional RT-PCT products. Two micrograms of total RNA was subjected to RT reaction with superscript Reverse Transcriptase at 42°C for 1 h. After heating the RT reaction at 75°C for 10 min, one-tenth of volume was used for PCR with Advantage cDNA Polymerase Mix (Clontech). After heating the reaction at 95°C for 5 min, the following parameters were used: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 68°C for 1 min. Cycle number was 24 for ABCA1 and 20 for GAPDH. (B) Patterns of real time quantitative RT-PCR analysis of ABCA1 mRNA. SYBR Green I ds DNA dye was used for the RT-PCR analysis of ABCA1 mRNA. Log fluorescence versus cycle number was plotted for each sample to permit simple visualization of the exponential phase of amplification. The arrows indicated patterns from samples with and without the stimulation. (C) Western blot analyses of the expression of endogenous ABCA1 protein in patients’ fibroblasts. It should be noted that the predicted ABCA1 mutant in Case 2 was truncated form which lacks the epitope for the antibody used.

In Case 2, we found another novel mutation resulting in the presence of truncated ABCA1 which lacks the second NBD (R1851X). Our transfection study demonstrated an interesting data to show that the expression level of truncated mutant (R1851X) was much lower than those of other mutants and wild type tested. Analyses for the other member of ABC transporters, cystic fibrosis transmembrane conductance regulator (CFTR), provided us the similar phenomenon (26, 27). The truncated mutant protein of CFTR had a very short half-life, which was possibly caused by the

promotion of protease-dependent degradation. Recently it has been speculated that the expression levels of ABCA1 protein may be mainly regulated by the degradation of protein rather than transcription (28). Because this mutant lacks the epitope for the polyclonal antibody used, we could not investigate the endogenous expression of truncated ABCA1 protein at this time point. Based upon the results of our transfection experiment (Fig. 3), we speculated that ABCA1 expression was severely impaired at the protein level in Case 2 (Ho/R1851X).

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FIG. 4. Expression of transfected ABCA1 cDNA constructs in Cos-7 cells (A and B). (A, B) The indicated ABCA1 plasmids were transfected into Cos-7 cells and HEK293 cells by Lipofectamine 2000 reagent. The whole cell lysates were prepared and subjected to Western blot analyses. The expressed ABCA1 was detected by anti-ABCA1 antibody (A) or anti-FLAG M2 antibody (B).

In Case 3, who did not have typical TD phenotype such as orange tonsils and hepatosplenomegaly, we found the other novel substitution (A5226G/N1611D). In some cases of FHD without TD phenotype like Case 3, various mutations or substitutions were reported to be located around two transmembrane domains, though the functional analyses have not been reported. Our analyses showed that the mutant protein expression was comparable, however cholesterol efflux was markedly reduced in both her fibroblasts and the overexpressing cells. These data suggested that asparagine at codon 1611 could be very important for mediating cholesterol efflux. Previous literatures lead to the following speculations. The asparagine at codon 1611 was located around the proximal part of the second transmembrane domain spanning from codon 1369 to codon 1654 in a model proposed by Ueda K (24). Furthermore, it is well known that ABC transporters are

highly glycosylated (29) and this asparagine at the codon 1611 is one of the potentially glycosylation sites of ABCA1. The experiments to know the detailed mechanism by which this missense mutation affects the phenotypic expression of Case 3 are underway in our laboratory. To clarify the association between ABCA1 mutations and development of atherosclerotic cardiovascular diseases awaits more progress of structure and function study of ABCA1. We have recently reported that a small G protein, Cdc42Hs, was decreased in cells from TD in association with altered actin cytoskeletons and impaired cholesterol efflux (19). We further demonstrated the possible protein–protein interaction between ABCA1 and Cdc42 (17). In addition to structure and function study of ABCA1, we believe that further analysis of the interaction between ABCA1 and such cholesterol efflux-related molecules would provide im-

FIG. 5. Apo Al-mediated cholesterol efflux from the transfected Cos-7 cells (A) and HEK293 cells (B). (A, B) The transfected cells were labeled with 1.0 ␮Ci/mL [ 3H]cholesterol for 18 h. Cells were washed three times with PBS and equilibrated with DMEM/BSA for 2 h. The medium was then replaced with fresh DMEM/BSA with and without presence of 15 ␮g/mL apoA-l and incubated at 37°C for 4 h. The radioactivity of media and cells were measured by liquid scintillation counting, and cholesterol efflux was calculated as a fraction of the cpm in media over the total (media plus cells). (* P ⬍ 0.05. P values were calculated by Mann–Whitney’s U test.) 719

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Summary of Molecular Basis of FHD Investigated Case 1 ABCA1 substitutions Passaged homozygous fibroblasts ABCA1 mRNA ABCA1 Protein Cholesterol efflux Overexpression system ABCA1-FLAG Protein Cholesterol efflux

Case 2

Case 3

G1158A/A255T

C5946T/R1851X

A5226G/N1611D

Markedly reduced Not detected Deficient

Comparable Unknown* Deficient

Comparable Comparable Deficient

Comparable Comparable

Markedly reduced Deficient

Comparable Deficient

* Note that the predicted mutant protein with C5946T/R1851X lacks the epitope for the ABCA1 antibody used in the present study.

portant information to elucidate detailed mechanism for reverse cholesterol transport, the protective system against atherosclerosis. ACKNOWLEDGMENTS 9. We thank Dr. Reijiro Arakawa and Professor Shinji Yokoyama (Nagoya City University Medical School, Nagoya, Japan) for kindly providing us the ABCA1 antibody. This work was supported by research grants from Study Group of Molecular Cardiology (Japan), from Japan Heart Foundation (Japan), from Osaka Heart Club (Japan), from Japan Heart Foundation/Pfizer Grant for Research on Hypertension and Vascular Metabolism (Japan), and from Tanabe Medical Frontier Conference (TMFC) (Japan) to K. Hirano. This work was supported by grants-in-aid to S. Yamashita (No. 11557055 and No. 10671070) and K. Hirano (No. 13671191) from the Ministry of Education, Science, Sports, and Culture of Japan and a research grant to Y. Matsuzawa from JSPS-RFTF97L00801.

10.

11.

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