| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Departments of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706
Department of Biochemistry, Centre d'Immunologie de Marseille Luminy, 13288 CEDEX 09, France
Xenon Genetics Inc., Vancouver, BC, V5G 4W8, Canada
** Department of Animal Sciences, University of Wisconsin-Madison, Madison, WI 53706
Department of Vascular Medicine, Academic Medical Centre, Amsterdam, The Netherlands
Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics and Department of Medical Genetics, Children's and Women's Hospital, University of British Columbia, Vancouver, BC V5Z 4H5, Canada
DOI 10.1194/jlr.M200223-JLR200
1 To whom correspondence should be addressed. e-mail: attie{at}biochem.wisc.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The WHAM chicken is a naturally occurring animal model for Tangier Disease.
Abbreviations: ABCA1, ATP-binding cassette protein-1; FHA, familial hypoalphalipoproteinemia; PC, phosphatidylcholine; SM, sphingomyelin; WHAM, Wisconsin hypoalpha mutant
Supplementary key words HDL • hypoalphalipoproteinemia • Tangier Disease
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A decade later, Poernama et al. discovered that the WHAM chickens have a severe deficiency of HDL (2). Unlike conditions leading to defective VLDL production, however, this syndrome involves normal synthesis and secretion of apolipoproteins. Most notably, the rate of synthesis of apoA-I, the principal protein of HDL, is normal (3). Moreover, the apoA-I gene locus is excluded as a candidate gene for this mutant phenotype because the WHAM mutation is sex-linked in the chicken (1), whereas apoA-I is autosomal in chickens (3) as it is in mammals (4, 5). Since HDL production is drastically reduced while apoA-I synthesis is normal, this animal model exposed a post-secretory step that is rate-limiting for HDL production (3).
Metabolic studies in WHAM chickens provided the key to understanding their defect in HDL metabolism. When 125I-labeled HDL particles were injected into WHAM chickens, their disappearance from the circulation was only moderately increased relative to normal chickens. However, when lipid-free 125I-apoA-I was injected, it was removed by the kidneys from the circulation 4-fold more rapidly in WHAM than in normal chickens (3). Because apoA-I synthesis and secretion are normal in WHAM chickens, we reasoned that another factor affecting the stability of apoA-I was limiting. Further analysis of serum lipids revealed a 70% reduction in phospholipids, implying that the primary defect is in phospholipid efflux (3). The dissociation between the metabolism of 125I-labeled HDL compared with the rapid catabolism of 125I-apoA-I, together with the defect in efflux, suggested that the primary defect in the WHAM chicken relates to lipidation of the lipid-depleted apoA-I particle.
Tangier Disease and familial hypoalphalipoproteinemia (FHA) are HDL deficiency disorders that are also characterized by hypercatabolism of apoA-I (6). Studies in fibroblasts from Tangier Disease and FHA patients reveal defects in phospholipid and cholesterol efflux (7–9). Consequently, Tangier Disease manifests as a cholesterol ester storage disorder (10, 11). Mutations in the ATP-binding cassette protein-1 (ABCA1) gene are responsible for both Tangier Disease and FHA (12–15), implying that this protein functions as a phospholipid and/or cholesterol transporter.
The phenotypes of WHAM chickens and of Tangier Disease patients share key common features. First, in both instances apoA-I was ruled out as a candidate gene. Second, apoA-I synthesis is normal. Finally, the in vitro studies in Tangier fibroblasts suggested a defect in lipid efflux (12–16), analogous to the in vivo findings in the WHAM chicken (3). Mapping of the Tangier Disease mutation to human chromosome 9 and its synteny with the region of the chicken Z-chromosome harboring the WHAM mutation provided genetic evidence that individuals with Tangier Disease and WHAM chickens may have mutations in the same gene. We have cloned and sequenced the chicken ABCA1 gene and show that a single missense mutation (E89K) in the amino terminus of ABCA1 results in altered trafficking of ABCA1 with its retention in the endoplasmic reticulum and loss of function at the plasma membrane. The WHAM chicken thus represents a naturally occurring animal model for Tangier disease.
|
MATERIAL AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Phospholipid analysis
Plasma lipoproteins were fractionated on a Superose 6HR 10/30 FPLC column (Pharmacia). The equivalent of 100 µl of plasma was injected onto the column. Five-hundred microliter fractions were collected and used for total cholesterol measurements (Sigma kit #352-50). Values represent total cholesterol mass per fraction. The identities of the lipoproteins have been confirmed by utilizing anti-apoB immunoreactivity for LDL and anti-apoA-I immunoreactivity for HDL (not shown). Triglyceride profiles were used to identify VLDL. Lipids were extracted from a 200 µl aliquot of whole plasma. Lipids were extracted (17), dried down under nitrogen, resuspended in 35 µl of CHCl3 and spotted onto an activated TLC plate (Silica Gel 60, Aldrich No. Z29297-4), developed in the first dimension consisting of CHCl3-MeOH-28% NH3 (65:25:5, v/v/v) allowed to dry overnight and then developed in the second dimension consisting of CHCl3-acetone-MeOH-glacial acetate-H2O (6:8:2:2:1, v/v/v/v/v). To visualize lipids, plates were sprayed with 5% sulfuric acid, 5% glacial acetate and 0.5 mg/ml FeCl3, followed by baking at 100°C for 30 min.
DNA sequencing, RT-PCR amplification, and sequence analysis
Total RNA was isolated from control and WHAM chicken liver and reverse transcribed with oligo-(dT)18 primer using SuperScript II reverse transcriptase (Life Technologies). cDNA was amplified using Taq DNA polymerase and primers derived from the published human and mouse ABCA1 cDNA sequences (15, 18), and primers derived from initial chicken sequence. Fifteen sets of primer pairs were used to amplify WHAM and control chicken cDNA samples, generating 15 overlapping DNA fragments covering 6,773 bp. To determine the 5' untranslated portion of the mRNA, we performed 5' RACE using the Marathon cDNA amplification kit (Clontech). DNA sequencing was performed directly on PCR products using an ABI 373 automated DNA sequencer (Applied Biosystems).
Detection of the WHAM mutation
The G265A mutation was detected by comparison of the cDNA sequence of normal and WHAM male chickens. Genotyping of the variant in normal and WHAM White Leghorn and normal Rhode Island Red chicken genomic DNA was performed by PCR amplification with primers in exon 4 (Forward: 5'-GTCACTTCCCAAACAAAGCTA-3', Reverse: 5'-ATGGACGCATTGAAGTTTCC-3'). PCR product (15 µl) was incubated with Hinf I (10 units) in total volume (25 µl) for 1 h at 37°C, and products separated on 2% agarose gels. The presence of the G265A mutation destroys a Hinf I site in the PCR product.
Sequence alignment
Clustal W 1.8 with modifications, accessed through the Baylor College of Medicine search launcher (http://dot.imgen.bcm.tmc.edu:9331/multi-align/Options/clustalw.html), was used for multiple sequence alignments, with Boxshade for graphical enhancement (http://www.ch.embnet.org/software/BOX_form.html).
Generation and analysis of ABCA1 harbouring the WHAM mutation
The E to K mutation at position 89, corresponding to an A to G shift of nucleotide 348 in GB X75926, was introduced on the mouse ABCA1 backbone by fusion PCR with the following oligonucleotides: a-TATAAGCAGAGAGCTCGTTTA corresponding to the sequence at bp 94–118 in pBI vector, d- GATGCTTGATCTGCCGTA-bp 478–495 of GB X75926); b- and c-TCCCGGCAAGGCTCCC and GGGAGCCTTGCCGGGA, complementary oligonucleotides spanning the mutated nucleotide. The amplified fragment was reinserted into the ABCA1/(enhanced green fluorescent protein) backbone in pBI vector (19) by restriction digestion with Not I/Bsr GI. Introduction of the point mutation was confirmed by sequencing with the Dynamic ET terminator Cycle sequencing kit (Amersham Pharmacia Biotech, Uppsala Sweden).
HeLa cells were transiently transfected for 16 h with EXGEN 500 (Euromedex, Mundolsheim, France) accordingly to manufacturer's instruction and immediately seeded for immunofluorescence, biochemical and functional analysis.
Transfection efficiency, assessed by flow cytometric evaluation of (green flourescent protein) RFI (relative fluorescence intensity) in the whole cell population, was consistently higher than 30%. Intracellular trafficking was monitored by both immunofluorescence analysis and surface biotinilation at 60 h after transfection.
Immunofluorescence was carried out by standard protocols on slides seeded with 3–5 x 104 cells and analyzed in X-Y dimensions by a Leica TS100 confocal microscope.
Surface biotinylation was carried out on 3–5 x 106 cells with 1 mg/ml NHS-LC-biotin (Pierce) in ice cold PBS for 30 min, followed by lysis in RIPA buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA and 1% Triton X-100) for 30 min at 4°C. Similar amounts of ABCA1, normalised with respect to both protein concentration in the samples and transfection efficiency, were immunoprecipitated with an anti-GFP antibody (clone7.1/13.1, Boehringer Indianapolis, IN), accordingly to standard protocols. The immunoprecipitated samples were fractionated by SDS-PAGE and blotted for 20 h onto nitrocellulose paper (Schleicher & Schuell, Dassel, Germany). The biotinylated protein was then revealed by ECL (Amersham Pharmacia Biotech, Uppsala, Sweden) after hybridization to streptavidin HRP (Amersham Pharmacia Biotech, Uppsala Sweden).
For functional analysis, fluorescence based assays for surface binding of cyanilated apoA-I or annexin V were carried out as described (20) at 60 h after transfection on 0.5–1 x 106 cells. Results, averaged from a minimum of four individual experiments, are expressed as percent of the binding elicited by wild-type ABCA1/EGFP chimera transfected in parallel.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
|
The sequences of the normal and mutant chickens ABCA1 CDNAs were identical with the exception of a G to A transition in WHAM DNA at nucleotide 265, corresponding to a glutamic acid to lysine substitution at amino acid 89 (E89K) (Fig. 3B). The G265A mutation eliminates a Hinf I restriction site present in the normal chicken sequence, and facilitated development of a PCR-based Hinf I RFLP assay to confirm the mutation in chicken genomic DNA (Fig. 3C). This alteration is a non-conservative amino acid substitution at a residue that is conserved in the ABCA1 gene between human, mouse, chicken, and Takifugu rubripes ("fugu" (Fig. 3D). The mutation segregates with the phenotype of HDL deficiency in the WHAM chickens and is not seen in wild-type White Leghorn chickens or in another strain of chicken that was investigated, New Hampshire (not shown).
Functional analysis of the E89K mutation in the ABCA1 gene
In order to establish the impact of the WHAM mutation on the function of the ABCA1 transporter, we engineered a construct harbouring the E89K mutation on a murine ABCA1/EGFP chimeric backbone. The effects of this mutation on the intracellular trafficking and function of the transporter were then analyzed in transiently transfected HeLa cells. Morphological analysis highlighted a dramatic retention of the protein in the endoplasmic reticulum at 48 h and 60 h after transfection (Fig. 4A, B)
. At these time points, the wild-type product is predominantly located at the plasma membrane (Fig. 4A). This mistargeting was further supported by the virtual absence of transporter accessible to cell surface biotinylation (Fig. 4C). A minor amount of WHAM ABCA1, however, does reach the plasma membrane, but does not exceed 10–20% of the wild-type ABCA1 detected in similar conditions.
|
In both assays, flow cytometric evaluation of the GFP expressing transfected cell failed to detect any significant binding (Fig. 4D) (values expressed as percent of the binding elicited by wild-type ABCA1/EGFP are 13.8% ± 5; n = 6 for annexin V and 20% ± 11 for apoA-I; n = 4.). The WHAM mutant therefore acts as an essentially complete-loss-of-function mutation.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Comparative gene sequencing is an effective tool for the study of the functional importance of specific amino acid residues. The functional conservation of glutamic acid-89 over 400 million years in the ABCA1 gene of the fugu, chicken, mouse, and human genomes provides evidence for this glutamic acid to lysine change having significant effects on ABCA1 function in the chicken. The dramatically reduced HDL levels in the WHAM chicken provides further compelling evidence for the rate-limiting role of the ABCA1 gene in HDL synthesis and conclusively demonstrates that ABCA1 is absolutely required for maintenance of HDL levels in different species.
The non-conservative E89K mutation described here makes a strong case that this is indeed the WHAM mutation. The mutation is in an N-terminal segment of ABCA1 whose topology has been controversial. The original descriptions of ABCA1 proposed that the first 640 amino acids are cytoplasmic and precede the first transmembrane domain. However, recent studies of Fitzgerald et al. (26) have shown that several of the N-glycosylation sites within this segment are in fact glycosylated, indicating that this segment had to be translocated across the ER and must therefore have an exofacial orientation. Thus, it is proposed that the first transmembrane domain is in a type 2 orientation, followed by amino acids 44–640. The WHAM mutation would therefore be exposed to the endoplasmic reticulum lumen and be in a position to disrupt the folding of the protein. It is also important to note that this segment also includes a 60-amino acid N-terminal region that was initially excluded from the presumed open reading frame of ABCA1 and was subsequently shown to be essential to its function (26).
The strict conservation of this glutamate (E89) residue (from fugu to chicken) suggests a functional importance that we have confirmed by the in vitro analysis of the WHAM transporter in transfected cells. The presence of the WHAM mutation impairs physiological intracellular trafficking since most of the transporter appears to be retained in the endoplasmic reticulum. Naturally occurring mutations in the ABC transporters also have been shown to affect intracellular trafficking. Indeed, the most frequent mutation causal for cystic fibrosis, (the 
F508 mutation) leads to a temperature sensitive defect in protein folding and impaired trafficking along the secretory pathway (27).
As a result of the WHAM mutation, only limited amounts of ABCA1 reach the plasma membrane, and fail to elicit the functional effects associated with the expression of wild-type ABCA1. In particular, the complete absence of apoA-I binding upon expression of WHAM transporter is sufficient to impair cellular release of PL to lipid poor HDL particles.
The serum from WHAM chickens is colorless, a consequence of greatly reduced levels of carotenoids. Tangier serum has the same carotenoid content as normal human serum. In the WHAM chicken, carotenoids are absorbed normally (unpublished observations) and are cleared from the circulation. Unlike the human patients, the WHAM chicken is deficient in virtually all lipoproteins, thus the reduced carrying capacity for carotenoids is the likely explanation for the colorless serum.
The phenotype of Tangier Disease and of WHAM chickens establishes a critical role for ABCA1 in the supply of lipids to the lipid-poor HDL particle. A large body of literature proposes a role for HDL in transport of cholesterol from extrahepatic tissues to the liver, a process termed "reverse cholesterol transport." Implicit in this model is that the bulk of the lipids that end up in HDL particles originate in extraheptic tissues. The drastic effect that the ABCA1 mutation has on HDL levels in Tangier Disease and in the WHAM chickens supports a major role for ABCA1 in the supply of lipids for HDL. The fact that ABCA1 functions to export lipids from cells is consistent with HDL assembly as an extracellular event in which the first step is the binding of apoA-I to phospholipids. Indeed, apoA-I spontaneously forms HDL precursor particles when exposed to phospholipids (28).
The WHAM chicken supplied the first genetic evidence that vertebrates, like invertebrates, have an extracellular lipoprotein assembly pathway. The key observation was that despite normal apoA-I synthesis and secretion, the chickens are unable to produce stable HDL particles. The mutant chickens have a deficiency in plasma phospholipid which suggested that the dearth of phospholipid in the bloodstream was likely the primary defect responsible for the HDL deficiency syndrome (3). Subsequent studies in Tangier fibroblasts established that a lipid transport defect underlies this disease (7–9). It remains to be established which tissue makes the largest contribution to HDL lipids in an ABCA1-dependent fashion. Recent studies by Haghpassand et al. (29) show that macrophages, despite being highly enriched in ABCA1, do not make a significant contribution to HDL lipids. In the WHAM chicken, there is substantial cholesterol ester accumulation in the liver and intestine (unpublished observations), suggesting that ABCA1 is most active in lipid transport out of these tissues. If this is the case, then it will be important to make a distinction between the role of ABCA1 in the contribution of lipids to HDL and the role of ABCA1 in macrophages relative to atherosclerosis susceptibility. If these are indeed two separate roles, then the traditional view of reverse cholesterol transport would still be valid vis-à-vis atherosclerosis, but not necessarily be relevant to the bulk of HDL lipid transport.
|
ACKNOWLEDGMENTS |
|---|
Manuscript received June 6, 2002 and in revised form July 1, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  W. E. Connor, P. B. Duell, R. Kean, and Y. Wang The Prime Role of HDL to Transport Lutein into the Retina: Evidence from HDL-Deficient WHAM Chicks Having a Mutant ABCA1 Transporter Invest. Ophthalmol. Vis. Sci., September 1, 2007; 48(9): 4226 - 4231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Dichlberger, L. A. Cogburn, J. Nimpf, and W. J. Schneider Avian apolipoprotein A-V binds to LDL receptor gene family members J. Lipid Res., July 1, 2007; 48(7): 1451 - 1456. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Wang, S. L Connor, E. J Johnson, M. L Klein, S. Hughes, and W. E Connor Effect of dietary lutein and zeaxanthin on plasma carotenoids and their transport in lipoproteins in age-related macular degeneration Am. J. Clinical Nutrition, March 1, 2007; 85(3): 762 - 769. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A Mares, T. L LaRowe, D M. Snodderly, S. M Moeller, M. J Gruber, M. L Klein, B. R Wooten, E. J Johnson, R. J Chappell, and for the CAREDS Macular Pigment Study Group and Inv Predictors of optical density of lutein and zeaxanthin in retinas of older women in the Carotenoids in Age-Related Eye Disease Study, an ancillary study of the Women's Health Initiative. Am. J. Clinical Nutrition, November 1, 2006; 84(5): 1107 - 1122. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. R. Singaraja, M. Van Eck, N. Bissada, F. Zimetti, H. L. Collins, R. B. Hildebrand, A. Hayden, L. R. Brunham, M. H. Kang, J.-C. Fruchart, et al. Both Hepatic and Extrahepatic ABCA1 Have Discrete and Essential Functions in the Maintenance of Plasma High-Density Lipoprotein Cholesterol Levels In Vivo Circulation, September 19, 2006; 114(12): 1301 - 1309. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Trompier, M. Alibert, S. Davanture, Y. Hamon, M. Pierres, and G. Chimini Transition from Dimers to Higher Oligomeric Forms Occurs during the ATPase Cycle of the ABCA1 Transporter J. Biol. Chem., July 21, 2006; 281(29): 20283 - 20290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-Y. Lee, J. M. Timmins, A. Mulya, T. L. Smith, Y. Zhu, E. M. Rubin, J. W. Chisholm, P. L. Colvin, and J. S. Parks HDLs in apoA-I transgenic Abca1 knockout mice are remodeled normally in plasma but are hypercatabolized by the kidney J. Lipid Res., October 1, 2005; 46(10): 2233 - 2245. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Agassandian, S. N. Mathur, J. Zhou, F. J. Field, and R. K. Mallampalli Oxysterols Trigger ABCA1-Mediated Basolateral Surfactant Efflux Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2): 227 - 233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-P. Duan, H. H. Wang, and D. Q-H. Wang Cholesterol absorption is mainly regulated by the jejunal and ileal ATP-binding cassette sterol efflux transporters Abcg5 and Abcg8 in mice J. Lipid Res., July 1, 2004; 45(7): 1312 - 1323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yamauchi, M. Hayashi, S. Abe-Dohmae, and S. Yokoyama Apolipoprotein A-I Activates Protein Kinase C{alpha} Signaling to Phosphorylate and Stabilize ATP Binding Cassette Transporter A1 for the High Density Lipoprotein Assembly J. Biol. Chem., November 28, 2003; 278(48): 47890 - 47897. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Bortnick, E. Favari, J.-Q. Tao, O. L. Francone, M. Reilly, Y. Zhang, G. H. Rothblat, and S. R. Bates Identification and characterization of rodent ABCA1 in isolated type II pneumocytes Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L869 - L878. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Wellington, L. R. Brunham, S. Zhou, R. R. Singaraja, H. Visscher, A. Gelfer, C. Ross, E. James, G. Liu, M. T. Huber, et al. Alterations of plasma lipids in mice via adenoviral-mediated hepatic overexpression of human ABCA1 J. Lipid Res., August 1, 2003; 44(8): 1470 - 1480. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Mulligan, M. T. Flowers, A. Tebon, J. J. Bitgood, C. Wellington, M. R. Hayden, and A. D. Attie ABCA1 Is Essential for Efficient Basolateral Cholesterol Efflux during the Absorption of Dietary Cholesterol in Chickens J. Biol. Chem., April 4, 2003; 278(15): 13356 - 13366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Kiss, D. C. McManus, V. Franklin, W. L. Tan, A. McKenzie, G. Chimini, and Y. L. Marcel The Lipidation by Hepatocytes of Human Apolipoprotein A-I Occurs by Both ABCA1-dependent and -independent Pathways J. Biol. Chem., March 14, 2003; 278(12): 10119 - 10127. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS |
