Lipidomic strategies to study structural and functional defects of ABC-transporters in cellular lipid trafficking

FEBS Letters 580 (2006) 5597–5610

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Lipidomic strategies to study structural and functional defects of ABC-transporters in cellular lipid trafficking Gerd Schmitz*, Gerhard Liebisch, Thomas Langmann Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Franz-Josef-Strauss Allee 11, D-93053, Germany Received 3 July 2006; revised 28 July 2006; accepted 8 August 2006 Available online 17 August 2006 Edited by Bernd Helms

Abstract The majority of the human ATP-binding cassette (ABC)-transporters function in cellular lipid trafficking and in the regulation of membrane lipid composition associating their dysfunction with human disease phenotypes related to sterol, phospholipid and fatty acid homeostasis. Based on findings from monogenetic disorders, animal models, and in vitro systems, major clues on the expression, function and cellular localization of human ABC-transporters have been gained. Here we review novel lipidomic technologies including quantitative mRNA expression monitoring by realtime RT-PCR and DNA-microarrays, lipid mass spectrometry, cellular fluorescence imaging and flow cytometry as promising tools to further define regulatory networks, lipid species patterns and subcellular domains important for ABC-transporter-mediated lipid trafficking. Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Lipidomics; ABC-transporters; Lipid trafficking disorders

1. Introduction Members of the ATP-binding cassette (ABC)-transporter superfamily are found throughout most species and represent one of the biggest protein families described so far. ABC proteins are usually multispan membrane proteins which mediate the active uptake or efflux of specific substrates, mainly lipophilic molecules, across biological membrane systems including the plasma membrane, the endoplasmic reticulum, the Golgi compartment, mitochondria and peroxisomes [1]. The group of membrane spanning ABC-transporters can be split into two different sections depending on their mode of action. The active transporters or pumps, such as members of the ABCB (MDR/TAP) subfamily, couple the hydrolsis of ATP and the resulting free energy to movement of molecules across membranes against a chemical concentration gradient [2]. In contrast, recent work has identified several ABC proteins, which show nucleotide binding and a subsequent conformational change but very low ATP hydrolysis including ABCA1, the cystic fibrosis membrane conductance regulator (CFTR/ ABCC7) and the sulfonylurea receptors (SUR1/2, ABCC8/

* Corresponding author. Fax: +49 941 944 6202. E-mail address: [email protected] (G. Schmitz).

ABCC9) [3]. To date, 48 human ABC transporters, which are subdivided into seven subfamilies, have been identified and many of these proteins exert their biological activities in lipid metabolism and are themselves regulated by lipids including phospholipids, sterols and fatty acids [4] (Table 1). In the following chapters, we review genetic diseases in human ABC-transporters related to lipid metabolism, provide insights into the regulation and functional characteristics of these lipid transporters and present novel lipidomic technologies for their in vitro and in vivo analysis.

2. Diseases and phenotypes caused by ABC transporters Twenty-two out of 48 currently known human ABC proteins have been linked to human monogenic disorders or cause special disease phenotypes (Table 2). Since ABC transporters represent a combination of enzymes and structural proteins, homozygous mutations cause severe human diseases, which are inherited in a recessive manner. As shown in Table 2, these genetic diseases are found in five of the seven ABC subfamilies. In addition, heterozygous mutations and single nucleotide polymorphisms (SNPs) in ABC transporter genes have been connected with susceptibility to complex, multigenic disorders, such as inflammatory bowel disease [5] or Alzheimer’s disease [6]. In the following paragraphs, a selection of important ABC transporter related inherited disorders with a special focus on lipid metabolism are presented, including ABCA1, ABCA3, ABCA7, ABCA12, ABCB4, ABCB11, ABCD1, ABCG5, and ABCG8. 2.1. ABCA1 and familial HDL deficiency The ABCA1 gene contains 50 exons and is located on chromosome 9q31. Its open reading frame consists of 6783 bp, encoding a 2261 amino acid protein. Mutations in the human ABCA1 gene cause familial HDL deficiency syndromes such as classical Tangier disease (Table 2) [7–9]. The most striking feature of these patients is the almost complete absence of plasma HDL, low serum cholesterol levels and a markedly reduced efflux of cholesterol and phospholipids from cells. The low HDL levels seen in Tangier disease are mainly due to an enhanced catabolism of these HDL precursors [10]. Interestingly, obligate heterozygotes for TD mutations have approx. 50% of plasma HDL, but normal LDL levels [11]. Studying 13 different mutations in 77 heterozygous individuals, Clee et al. described a more than threefold risk to develop coronary

0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.08.014

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Table 1 Overview of human ABC gene subfamilies Gene

Alternat. name

Domain structure

NH2-term. –LL–

PDZ-bind.

Tissue expression and cellular location

Lipid reg.

Known or putative transported molecule

ABCA1 ABCA2 ABCA3 ABCA4 ABCA5 ABCA6 ABCA7

ABC1 ABC2 ABC3 ABCR

(TMD-ABC)2 (TMD-ABC)2 (TMD-ABC)2 (TMD-ABC)2 (TMD-ABC)2 (TMD-ABC)2 (TMD-ABC)2

+ + + + + + +

+ +

+ + +

Choline-phospholipids and cholesterol Estramustine, steroids Surfactant phospholipids N-retinylidene-PE Sterols Phospholipids Sphingolipids (e.g. ceramide) and phospholipids

ABCA8 ABCA9

(TMD-ABC)2 (TMD-ABC)2

+ +

ABCA10

(TMD-ABC)2

ABCA12

(TMD-ABC)2

ABCA13

(TMD-ABC)2

Macrophages, liver, brain Brain Lung, brain, heart, pancreas Photoreceptors Muscle, heart, testes Liver, lung, heart, brain, ovary Spleen, thymus, bone marrow, peripheral leukocytes, brain, adrenal glands, uterus Ovary, brain Heart, lung, brain, liver, prostate, ovary uterus Muscle, heart, gastrointestinal tract, spleen, lung Keratinocytes, placenta, testis, lung, brain, stomach, prostate, lymphocytes Trachea, testis, bone marrow Excretory organs, apical membrane

+

+

+

+

+ + Lamellar granule lipids and enzymes

MDR1

(TMD-ABC)2

ABCB2 ABCB3 ABCB4 ABCB5 ABCB6 ABCB7 ABCB8 ABCB9 ABCB10 ABCB11

TAP1 TAP2 MDR3

MTABC2 BSEP

TMD-ABC TMD-ABC (TMD-ABC)2 (TMA-ABC)2 TMD-ABC TMD-ABC TMD-ABC TMD-ABC TMD-ABC (TMD-ABC)2

Ubiquitous, ER Ubiquitous, ER Liver, apical membrane Ubiquitous Mitochondria Mitochondria Mitochondria Heart, brain, lysosomes Mitochondria Liver, apical membrane

ABCC1

MRP1

TMD0-(TMD-ABC)2

Lung, testes, PBMC

+

ABCC2

MRP2

TMD0-(TMD-ABC)2

Liver

+

ABCC3

MRP3

TMD0-(TMD-ABC)2

Lung, intestine, liver, basolateral membrane

ABCC4

MRP4

(TMD-ABC)2

Prostate

+

ABCC5

MRP5

(TMD-ABC)2

Ubiquitous

+

ABCC6

MRP6

TMD0-(TMD-ABC)2

Kidney, liver

ABCC7

CFTR

(TMD-ABC)2

+

+

Exocrine tissue

+ +

Phospholipids, PAF, aldosterone, cholesterol, amphiphiles, b-amyloid peptide Peptides Peptides Phosphatidylcholine Fe/S clusters Fe/S clusters Fe/S clusters

+ +

Fe/S clusters Monovalent bile salts (e.g. TC) GSH-, glucuronate-, sulfate-conjugates, GSSG, sphingolipids, LTC4, PGA1, PGA2, 17b-glucuronosyl estradiol, sulfatides, GM1 GSH-, glucuronate-, sulfate-conjugates, bilirubin glucuronide, LTC4, 17b-glucuronosyl estradiol, taurocholate, taurolithocholate 3-sulfate, anionic drugs glucuronate-, sulfate-conjugates, 17b-glucuronosyl estradiol, taurolithocholate 3-sulfate Xenobiotics, nucleosides (ATP/ADP/AMP/Adenosin, GTP/GDP) Xenobiotics, nucleosides (ATP/ADP/AMP/Adenosin, GTP/GDP) Anionic cyclopentapeptides (e.g. BQ123), LTC4, GSH-conjugates Chloride ions, ATP

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ABCB1

MTABC3 ABC7 MABC1

+

+ + +

translation elongation initiation factor 2 Ubiquitous Ubiquitous Ubiquitous

Ubiquitous Placenta,, liver, kidney, intestine, stem cells Liver Liver, instestine Liver, instestine

(ABC)2 (ABC)2 (ABC)2

ABC-TMD ABC-TMD ABC-TMD ABC-TMD ABC-TMD

+ + +

+

Phospholipids, cholesterol Xenobiotics Cholesterol Plant- and shellfish sterols Plant- and shellfish sterols

Oligoadenylate Ovary, testes, spleen

+ Peroxisomes

ABC50

White MXR White2 White3 White 4

ABCF1 ABCF2 ABCF3

ABCG1 ABCG2 ABCG4 ABCG5 ABCG8

(ABC)2 OABP ABCE1

TM-ABC PMP69 ABCD4

+ ALD ALDR PMP70 ABCD1 ABCD2 ABCD3

TM-ABC TM-ABC TM-ABC

+ + + + +

+

Peroxisomes Peroxisomes Peroxisomes

+ +

Very-long-chain fatty acids Very-long-chain fatty acids long-chain fatty acid-CoA, 2-methylacyl-CoA Very-long-chain fatty acids

Sulfonylureas Sulfonylureas Pancreas Heart, muscle Low in all tissues Low in all tissues Low in all tissues + + + + TMD0-(TMD-ABC)2 TMD0-(TMD-ABC)2 TMD0-(TMD-ABC)2 (TMD-ABC)2 (TMD-ABC)2 SUR1 SUR2 MRP7 MRP8 MRP9 ABCC8 ABCC9 ABCC10 ABCC11 ABCC12

NH2-term. –LL– Alternat. name Gene

Domain structure

PDZ-bind.

Tissue expression and cellular location

Lipid reg.

Known or putative transported molecule

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artery disease in affected family members and earlier onset compared to unaffected members [12,13]. In addition to the absence of plasma HDL, patients with genetic HDL deficiency syndromes display accumulation of cholesteryl esters either in the cells of the reticulo-endothelial system (RES) leading to splenomegaly and enlargement of tonsils or lymph nodes, or in the vascular wall, leading to premature atherosclerosis [14,15]. To date, more than 50 mutations and several single nucleotide polymorphisms in the ABCA1 gene have been reported [16]. 2.2. ABCA3 and neonatal surfactant deficiency ABCA3 is a full-size transporter and encodes a 1704 amino acid protein with the gene located on chromosome 16p13.3. ABCA3 shows highest expression in the lung but is also expressed in brain, heart, pancreas and the mammary gland [17]. The ABCA3 promoter has been partially characterized and binding of the estrogen receptor has been observed in human breast cancer cells [18], but no alternatively spliced mRNA isoforms have been detected so far. Electron microscopy studies revealed that in the lung ABCA3 is expressed exclusively on the limiting membranes of lamellar bodies of alveolar type II pneumocytes [19] (Fig. 1A). Pulmonary surfactant is a complex mixture of lipids, primarily di-palmitoylphosphatidylcholine and di-palmitoyl-phosphatidylglycerol with surfactant proteins (SP) that are synthesized by type-IIpneumocytes. The hydrophobic SP-B and SP-C as well as surfactant lipids are assembled and stored in lamellar bodies. ABCA3 selectively facilitates the transfer of phosphatidylcholine, sphingomyelin, and cholesterol to lamellar bodies [20]. ABCA3 mutations have been identified in full-term newborns with unexplained respiratory distress syndrome (URDS), associated with a defective assembly of lamellar bodies and fatal surfactant deficiency [21,22]. Recently, Brasch et al. identified a strong expression of precursors of surfactant protein B, but only low levels and aggregates of mature surfactant protein B within electron-dense bodies in type-II-pneumocytes of URDS patients [23]. Surfactant protein A (SP-A) was localized in small intracellular vesicles, but not in electron-dense bodies (Fig. 1A). SP-A and proSP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and surfactant protein C (SP-C) were reduced or absent, respectively. These data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. 2.3. ABCA7 and Sjo¨gren syndrome The ABCA7 gene consists of 46 exons and overexpression experiments in human HeLa and HEK293 cells suggest that ABCA7 is implicated in the efflux of ceramide and phospholipids from the cells [24,25], and novel data also demonstrate enhanced export of both cholesterol and phospholipids in ABCA7 transfected HEK293 cells [26]. ABCA7 knockout mice display a gender dependent reduction in visceral fat and serum cholesterol levels representing novel insights into the function of ABCA7 in human lipid metabolism [27]. The ABCA7 gene is in close proximity to the gene for the minor histocompatibility antigen HA-1 on chromosome 19p13.3 in a head-to-tail array with only 1.7 kb distance between the terminal exon of

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Table 2 Human ABC transporter genes and corresponding diseases or phenotypes Gene

Synonyms

OMIM

Disorder or phenotype

ABCA1

ABC1

600046

Familial HDL deficiency Tangier disease

ABCA2 ABCA3

ABC2 ABC3

600047 601615

Alzheimer’s disease Neonatal surfactant deficiency

ABCA4

ABCR

601691

Retinopathies Macula degeneration Cone-rod dystrophy

ABCA7

ABC7

605414

Sjo¨gren syndrome

ABCA12

ABC12

607800

Lamellar ichthyosis type II Harlequin ichthyosis

ABCB1 ABCB2 ABCB3

MDR1 TAP1 TAP2

171050 170260 170261

Ulcerative colitis Immune deficiency Wegener-like granulomatosis

ABCB4

MDR3

171060

Progressive familial intrahepatic cholestasis type 3 (PFIC-3) Intrahepatic cholestasis of pregnancy (ICP)

ABCB7 ABCB11

ABC7 BSEP

300135 603201

X-linked sideroblastosis and anemia (XLSA/A) Progressive familial intrahepatic cholestasis type 2 (PFIC-2)

ABCC2 ABCC6 ABCC7 ABCC8 ABCC9

MRP2 MRP6 CFTR SUR1 SUR2

601107 603234 602421 600509 601439

Dubin–Johnson syndrome (DJS) Pseudoxanthoma elasticum (PXE) Cystic fibrosis (CF) Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) Dilated cardiomyopathy with ventricular tachycardia

ABCD1 ABCD3

ALD PXMP1

300371 170995

Adrenoleukodystrophy (ALD) Zellweger syndrome 2 (ZWS2)

ABCG2 ABCG5 ABCG8

MXR, BCRP White3 White 4

603756 605459 605460

Protoporphyria IX b-Sitosterolemia b-Sitosterolemia

Fig. 1. ABCA3 and ABCA12 in lamellar body lipid metabolism. (A) Type II pneumocyte cells. (B) Keratinocytes. PUFA, poly unsaturated fatty acids; PC, phosphatidylcholine; DPPC, dipalmitoyl-PC; MVE, multivesicular endosome; SR-BI, scavenger receptor-BI; TJ, tight junction; AJ, adherens junction; D, desmosomes; CER, ceramide; CS, cholesterol sulfate.

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ABCA7 and the first exon of the HA-1 gene [28]. In addition to the major transcript form, a second main mRNA isoform resulting from alternative splicing (termed type II mRNA) of human ABCA7 has been detected [29]. A novel exon is located between former exons 5 and 6, designated as exon 5B, and contains two termination codons and another translation initiation codon. Thus, translation of type II mRNA produces an ABCA7 protein containing 28 novel N-terminal amino acids instead of the 1–166 amino acid sequence in full length (type I) ABCA7. Interestingly, most of type II ABCA7 is localized intracellularly when expressed in HEK 293 cells, indicating that the N-terminal domain is important for trafficking of ABCA7 to the plasma membrane. Tissue-specific RT-PCR analysis has shown that the expression of type II mRNA is higher than type I mRNA in spleen, thymus, lymph node, and trachea, while type I mRNA was equal to or more than type II mRNA in bone marrow and brain [29,30]. The detailed molecular function of the 127-kDa HA-1 minor histocompatibility antigen is unknown, but it potentially transports minor histocompatibility peptides together with MHC class I molecules via the Golgi complex to the cell surface, where they are presented to T cells. In the case of HA-1, which has properties of small G-protein family interacting proteins, a defined N-terminal nonapeptide bearing the His168Arg dimorphism has been identified as the target immune epitope recognized by T cells [31]. Between ABCA7 and HA-1 genes a functional and regulatory interlink might exist, which is especially interesting since ABCA7 has been shown to be regulated by sterols via sterol-regulatory-element binding protein 2 (SREBP2) [32]. ABCA7/HA-1 may be involved in autoimmune disorders since both transcripts are expressed in macrophages and lymphocytes, major effector cells of chronic inflammation. Furthermore, a hallmark of autoimmune related diseases is disturbed autophagy of cell organelles and thereby persistence of protein degradation products that undergo antigen presentation. A polypeptide consisting of amino acids 186–320 within the first extracellular domain of ABCA7 encodes the autoantigen SS-N suggesting that ABCA7 is associated with the pathogenesis of Sjo¨gren’s syndrome [33]. Furthermore, recent studies revealed that the 168H variant of the minor histocompatibility antigen HA-1 is associated with reduced risk of primary Sjo¨gren’s syndrome [34] and that ABCA7 is highly expressed in salivary glands of patients with Sjo¨gren’s syndrome [35]. Recent data also indicate that ABCA7 might be important for hematopoietic development, differentiation, and immunological functions related to phagocytosis [32].

2.4. ABCA12 and genetic keratinization disorders The ABCA12 gene, which comprises 53 exons spanning 206 kb, has been mapped to chromosome 2q34, the region for the keratinization disorder lamellar ichthyosis, an autosomal recessive form of ichthyosis characterized by whole body skin desquamation [36,37]. Thereafter, missense mutations in the ABCA12 gene have been identified [38] and two recent independent studies demonstrated that mutations in the ABCA12 gene also underlie the very severe form of another keratinization disorder-harlequin ichthyosis [39,40]. Based on the identified mutations, it can be assumed that the two clinical phenotypes arise from different alterations of the ABCA12 protein causing either the severe form harlequin ichthyosis or

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the milder form manifesting as lamellar ichthyosis type 2. During keratinocyte differentiation, lipids accumulate in lamellar granules which are extruded in the intercellular space for enzymatic modification to produce a water permeability barrier consisting of ceramides, cholesterol and saturated long-chain fatty acids [41]. ABCA12 is present in the upper epidermal layer in lamellar granules of normal epidermal keratinocytes and likely functions in the secretion of lipids [39] (Fig. 1B). Keratinocytes from patients with harlequin ichthyosis display an accumulation of glucosylceramide around the nuclei and ABCA12 gene transfer into deficient cells normalizes glucosylceramide staining present in healthy keratinocytes, assuming that ABCA12 is involved in the intracellular translocation of lamellar granule sphingolipids [39]. 2.5. ABCB4 (MDR3) and ABCB11 (BSEP) in progressive familial intrahepatic cholestasis (PFIC) Mutations in three hepatic canalicular membrane proteins have been identified that result in cholestasis and later chronic liver disease. These three diseases are progressive familial intrahepatic cholestasis (PFIC) types 1, 2 and 3 and are caused by mutations in the genes coding for the familial intrahepatic cholestasis 1 protein (FIC1), and the ABC-transporters bile salt export pump (BSEP, ABCB11) and the phospholipid translocase MDR3 (ABCB4), respectively [42]. Ten autosomal recessive mutations in the coding region of the ABCB11 (BSEP) gene have been reported so far in different PFIC2 patients. ABCB11 is the major bile salt carrier in the canalicular membrane and mutations very often cause low expression or aberrant membrane localization [43]. PFIC3 is also an autosomal recessive disorder and the ABCB4 (MDR3) gene localizes to chromosome 7q21. In most cases where mutations causing truncations occur in the gene, a complete loss of ABCB4 protein at the canalicular membrane has been observed, whereas low expression levels correlate with missense mutations [44]. The function of ABCB4 protein was analyzed in a knock-out model with deletion of the mouse homolog (mdr2). These knock-out mice had a lack of phospholipid translocation into bile, normal bile acid secretion, progressive liver disease, and histology consistent with PFIC3 patients leading to the assumption that ABCB4 is a phosphatidylcholine translocase [45]. The purpose of phospholipids in bile is to form micelles with bile acid and to prevent crystallization of cholesterol. Without the formation of micelles with bile acids, the deleterious detergent effect of bile acids on the biliary epithelium and cholangiocytes is not prevented and causes PFIC. 2.6. ABCD1 in adrenoleukodystrophy The X-linked adrenoleukodystrophy (ALD) is an inherited peroxisomal disorder caused by mutations in ABCD1 (Table 2) resulting in progressive neurological dysfunction, occasionally associated with adrenal insufficiency [46]. ALD is characterized by the accumulation of unbranched saturated fatty acids with a chain length of 24–30 carbons, particularly hexacosanoate (C26), in cholesteryl esters of brain white matter and in adrenal cortex and in certain brain sphingolipids. Adrenoleukodystrophy belongs to a group of defects in peroxisomal b-oxidation [47]. The first step in the oxidation of very-longchain fatty acids (VLCFA) involves the activation by conversion into CoA esters and the transport into peroxisomes [48]. The ABCD1 protein mediates this transport process as

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evidenced by overexpression of human cDNAs encoding the ABCD1 protein and its closest relative, the ABCD2 (ALDR) protein in fibroblasts of ALD patients [49]. The accumulation of very-long-chain fatty acids could also be prevented by overexpression of the ABCD2 protein in immortalized ALD cells and the peroxisomal b-oxidation defect in the liver of ABCD1-deficient mice could be restored by stimulation of ABCD2 and ABCD4 gene expression through treatment with fenofibrate [49]. These results implicate that a therapy of adrenoleukodystrophy might be possible by drug-induced overexpression or ectopic expression of ABCD genes. 2.7. ABCG5 and ABCG8 in b-Sitosterolemia ABCG5 and ABCG8 have been recently implicated in the efflux of dietary sterols from intestinal epithelial cells back into the gut lumen and from the liver to the bile duct. These mechanisms are disrupted in b-Sitosterolemia or phytosterolemia or shellfishsterolemia, a rare autosomal recessive disorder. The disease is characterized by enhanced trapping of cholesterol and other sterols, including plant and shellfish sterols, within the intestinal cells and the inability to concentrate these sterols in the bile. As a consequence affected individuals have strongly increased plasma levels of plant sterols e.g. b-sitosterol, campesterol, stigmasterol, avenosterol, and 5a-saturated stanols, whereas total sterol levels remain normal or are just moderately elevated [50]. Using a combination of positional cloning, genome database survey as well as DNA-microarray analysis, Lee et al. [51] and Berge et al. [52] identified ABCG5 and ABCG8 mutations in b-Sitosterolemia patients. ABCG5 and ABCG8 have a bi-directional promoter and share common regulatory elements [53]. The highest expression level of both transporters is found in liver and intestine and high-cholesterol diet feeding in mice induces the expression of both genes. These findings together with the observed clinical and biochemical features of b-Sitosterolemia patients assume that ABCG5 and ABCG8 play an important role in reducing intestinal absorption and promote biliary excretion of sterols. Until today several mutations and a number of polymorphisms have been identified in ABCG5 and ABCG8 [54]. A striking finding is that mutations in b-Sitosterolemia patients occur exclusively either in ABCG5 or ABCG8, but never in both genes together. ABCG5 and ABCG8 proteins form a functional heterodimer to transport plant sterols directly back to the gut lumen as a kick-back mechanism, which may also efflux cholesterol, thereby regulating total sterol absorption. In the liver, expression of ABCG5 and ABCG8 functions in regulating biliary cholesterol secretion [55–57], which also requires biliary phospholipid transport by ABCB4 [58].

3. Regulation, trafficking, and function of ABCA1 and ABCG1 in lipid metabolism ABCA1 is crucial for the efflux of phospholipids and sterols to apoA-I containing HDL precursors. Like Tangier disease patients, ABCA1 knockout mice exhibit HDL deficiency and have strongly reduced cellular lipid efflux activity [59]. Total body and selective hepatic overexpression of ABCA1 in mice results in an increase of HDL [60] and conversely, HDL levels are strongly reduced in mice with a liver-selective deletion of ABCA1 [61]. There is increasing evidence that in addition to

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the liver, intestinal ABCA1 expression is a rate-limiting factor for plasma HDL production [62]. Although ABCA1 in macrophages has little influence on HDL, it is an important factor in the prevention of foam cell formation of macrophages in the arterial wall [63]. To control lipid efflux, ABCA1 is tightly regulated at the level of transcription and translation. The major human ABCA1 gene promoter was first reported upstream of exon 1 (221 bp) followed by a 24-kb intron 1 [64,65]. Cavelier et al. [66] have identified an alternative exon 1 (exon 1a), 136 bp in length and located 2210 bp upstream of exon 2. This alternative transcript contains the entire coding sequence of human ABCA1 and is expressed in testis and liver but not macrophages. Upstream of exon 1a, an alternative promoter that contains a TATA box and CAAT sites as well as other potential binding sites for sterol-sensing nuclear receptors was identified. The importance of this alternative promoter in regulating ABCA1 gene expression, however, has to be determined in human tissues [67]. ABCA1 gene expression is stimulated by liver-X-receptors (LXRs) and the retinoid-Xreceptor (RXR) which form heterodimers and are activated by oxysterols and retinoids, respectively. In macrophages, the physiological ligand for LXRa and LXRb is 27-hydroxycholesterol, which is produced by the mitochondrial 27-cholesterol hydroxylase (CYP27). In addition to the induction of ABCA1 expression, cAMP also stimulates ABCA1 phosphorylation [68,69]. Interestingly, apoA-I activates cAMP signalling and thereby the phosphorylation of ABCA1. Janus kinase 2 (JAK2) also phosphorylates ABCA1 [70], whereas in the absence of apolipoproteins, phosphorylation of the ABCA1 PEST motif is a signal for calpain-mediated degradation of ABCA1 [71] (Fig. 2). Furthermore, ABCA1 protein destabilization is performed by unsaturated fatty acids via a phospholipase D2-dependent pathway [72,73]. ABCA1, which has also a conserved N-terminal dileucine motif, assembles functional complexes with heteromeric proteins and the intracellular targeting of ABCA1 is regulated by diverse trafficking proteins (Fig. 2). The Rho family GTPase cdc42 directly interacts with ABCA1 to control filopodia formation and intracellular lipid transport [74,75]. Furthermore, the PDZ-domain proteins b2-syntrophin, a1syntrophin, and Lin7 bind to the KESYV-motif at the C-terminus of ABCA1 and regulate its targeting via vesicular trafficking, turnover, and intracellular localization in polarized cells [76,77]. The ADP-ribosylation factor (ARF)-like 7 (ARL7) is involved in vesicular budding from the trans-Golgi-network and has been implicated in the intracellular translocation of lipids via ABCA1 and the secretory lysosome pathway [78]. Along with these findings, the phagosomal proteins syntaxin 13 and flotillin 1 form a complex with ABCA1 in plasma membrane raft microdomains and thereby modulate phospholipid and cholesterol efflux [79]. Syntaxin 13 and its interacting protein pallidin are major constituents of the adapter protein 3 (AP3) pathway and the AP3/secretory lysosome pathway seems to play a major role in ABCA1-dependent lipid traffic. The cell-type expression and lipid-sensitivity of the half-size transporter ABCG1 is very similar to ABCA1 although the transcriptional regulation of ABCG1 is very complex and multiple alternative promoters and mRNA isoforms exist [80–83]. A comparison of human and murine orthologue promoter sequences and cDNAs suggests that one major transcript (termed ABCG1a) is expressed in most tissues, whereas the

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Fig. 2. Domain structure of ABCA1 and sites regulating turnover, trafficking and function. The amino acid numbers denominate transmembrane domains. NBD, nucleotide binding domains; A, B, Walker A/B motifs; S, signature motif.

function or specific expression of two alternative mRNA isoforms, which differ at the N-terminus of ABCG1 remains to be determined. A striking similarity between human ABCG1 and Drosophila ABCG transporters (white, brown, scarlet and E23) is their transcriptional regulation by nuclear hormone receptors and their potential cellular localization. Drosophila white and scarlet proteins are involved in the formation of eye color pigments by the transport of 3-hydroxykynurenine from the cytoplasm into pigment granules. The garnet gene product, which also modulates eye color biogenesis of Drosophila, shows strong homology with the delta subunit of the human AP-3 complex, which is mainly associated with the trans-Golgi network and other peripheral membranes [84]. This adaptor complex is involved in intracellular targeting of proteins to specialized vesicular compartments and these proteins often harbor dileucine motifs, which are recognized by the AP-3 complex. A sequence alignment of white, brown, and scarlet from Drosophila and human ABCG proteins reveals a conserved region near the first membrane domain containing a putative dileucine motif [85]. These findings raise the possibility that ABCG1 and other family members may interact with AP-3 in order to reach their subcellular compartment and that the different ABCG1 protein isoforms might be targeted to different intracellular compartments. ABCG1-specific antisense oligonucleotides significantly impaired cholesterol and phospholipid efflux to HDL3 [86]. In HEK293-cells, transient overexpression of murine ABCG1 or ABCG4 caused a strong increase in HDL-dependent cholesterol efflux and RNA interference-directed against macrophage ABCG1 mRNA also resulted in diminished lipid efflux [87]. Interestingly, in ABCG1 overexpressing cells, lipid-poor apoA-I was ineffective in lipid deloading of cells, which indicates that, in contrast to ABCA1, acceptor-particles for ABCG1-derived lipids are more mature a-HDL subclasses (HDL3 and HDL2) than preb-HDL [87]. Human recombinant

ABCG1 and ABCG4 expressed in Sf9 cells both display ATP binding and hydrolysis with a significant higher activity of ABCG1 compared to ABCG4. Importantly, co-expression of a catalytic site mutant of ABCG4 showed a dominant negative effect and selectively abolished the ATPase activity of ABCG1 [88]. Therefore, it can be concluded that ABCG1 may either act as a homodimer in the absence of ABCG4 or function as a heterodimer in combination with ABCG4 in coexpressed tissues to increase lipid efflux. Informations about the in vivo function of ABCG1 came from a recent study with ABCG1 knockout mice, showing that cholesterol, triglyceride, and phospholipid concentrations were significantly increased in the liver and lung of Abcg1-null mice fed the high-fat/highcholesterol diet, as compared to their wild-type littermates [89]. Despite the lack of changes in plasma lipid levels, macrophage foam cells highly enriched in lipid droplets and cholesterol crystals were observed in the lung of Abcg1-null mice. These findings are in contrast to earlier reports showing that plasma HDL and cholesteryl ester levels were reduced by infusion of mice with adenovirus expressing ABCG1 [90], which might result from artifically high overexpression of ABCG1 in hepatocytes [91]. There is little doubt that ABCA1 interacts directly with apoA-I. Moreover, there is evidence that ABCA1 modulates the internalization of apoA-I for lipidation and resecretion (retroendocytosis). For the first time the HDL retroendocytosis pathway was suggested by Schmitz et al. in 1985 [92] using electron microscopy and gold-conjugated HDL particles. Beside a saturable HDL binding to macrophages loaded with cholesterol, gold-labeled HDL were endocytosed via coated pits and resecreted into the culture medium without degradation. Furthermore, apoA-I and ABCA1 colocalise in endocytic compartments [93,94]. Additionally, there is evidence that apoA-I binding to ABCA1 occurs at cholesterol/sphingomyelin rich membrane domains. Although, ABCA1 is excluded

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from lipid rafts isolated by Triton X-100 or detergent-free methods, some ABCA1 was detected in Lubrol-resistant membranes [95,96]. Moreover, apoA-I interaction with plasma membrane lipid rafts controls cholesterol export from macrophages [97] and sphingomyelinase treatment, which also disrupts rafts, inhibited cholesterol efflux to apoA-I [98]. Thus, an early apoA-I interaction with lipid rafts seems to be essential for efflux initiation, although the majority of exported cholesterol and phospholipids may not originate from raft domains [97] (Liebisch et al. unpublished observation). Together with recent data from Gelissen et al. [99] showing that overexpression of ABCG1 promotes cholesterol efflux not only to HDL but also to other lipid acceptors like phosphatidylcholine vesicles, a two-step model for ABCA1 and ABCG1 mediated cellular lipid efflux could be proposed: Initially ABCA1 may promote cholesterol and phospholipid efflux involving membrane microdomains to lipid-poor apoA-I and ABCG1 subsequently promotes the efflux of additional cellular cholesterol to preformed lipid–protein complexes.

4. Novel lipidomic technologies 4.1. mRNA detection by realtime RT-PCR and DNA microarrays Quantitative realtime reverse transcription PCR (RT-PCR) is a fast and sensitive detection method which allows reproducible quantification of minute amounts of RNA. Total RNA from tissue specimens or single cells is reverse transcribed to generate cDNA as a template for quantitative PCR. Two different approaches have been reported to quantify all known human ABC transporters in tissue samples with highly active lipid metabolism and tumor cell lines [17,100]. We have developed a rapid, specific and sensitive realtime RT-PCR method to monitor the mRNA levels of 47 human ABC transporters based on the TaqManÒ technology using specific primers and probes and 50 ng of total RNA [17]. We have recently extended, improved and simplified the quantification of all 47 ABC transporters by creating a Human ABC Transporter TaqMan Low Density Array based on an Applied Biosystems 7900HT Micro Fluidic Card [101]. The method allows simultaneous analysis of all 47 human ABC transporters and the reference gene 18S rRNA in two replicates of four different samples per run in a 2 ll small-volume format. Transcript profiling in macrophages has revealed several novel LXR/RXR regulated ABC transporters including ABCB1 (MDR1), ABCB9, ABCB11 (BSEP), ABCC2 (MRP2), ABCC5 (MRP5), ABCD1 (ALD), ABCD4, and ABCG2. This assay will be a very useful tool to monitor dysregulated ABC transporter mRNA profiles in human lipid disorders and cancer-related multidrug resistance. Furthermore, the pharmacological and metabolic regulation of ABC transporter expression important for drug development could be analyzed in large screening approaches using this method. In a recent report by Szakacs et al. the expression levels of the same gene panel was measured by realtime RT-PCR using SYBR Green and the LightCyclerä instrument with 150 ng RNA template. Although less specific and more laborious than our TaqManÒ approach, the LightCyclerä assay was successfully used to correlate the mRNA expression pattern of ABC transporters with anticancer drug response in 60 diverse cancer cell lines [100].

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DNA microarrays are now frequently applied for the fast detection of expression levels of a large number of genes simultaneously and the generation of molecular finger prints [102]. A specific microarray dedicated to ABC transporters has been recently published by Annereau and coworkers [103]. This ABC-ToxChip contains cDNA probes and 70mer oligonucleotides targeting 36 human ABC transporters and specific overexpression of ABCB1 and ABCC2 could be detected in tumor cells [103]. Huang et al. developed a 70mer oligonucleotide microarray covering 40 ABC transporters and ABCB1, ABCC3, and ABCB5 expression was negatively correlated with drug resistance, implicating ABCB5 as a novel chemoresistant factor [104]. In another approach, a low-density cDNA microarray for 38 human ABC transporters, termed DualChip human ABC, was produced and validated in multidrug-resistant sublines previously shown to overexpress ABCB1, ABCC1, or ABCG2 [105]. These results demonstrate the proof of principle for successful microarray expression analysis of ABC transporters, which provide abundant amount of RNA. However, the limited sensitivity, e.g. the requirement of 25 lg total RNA for one ABC-ToxChip assay or of 1 lg mRNA for the DualChip human ABC, specificity problems with highly homologous DNA sequences, and the lack of standardization are major drawbacks limiting the application of this methodology in lipidomics research. In conclusion, among the published ABC-transporter mRNA profiling assays, the Taqman microfluidic card assay optimally fulfills good laboratory practice (GLP) criteria concerning specificity, sensitivity and accuracy and thereby can be regarded as a reference assay for mRNA expression analysis of this gene group.

4.2. Lipid species analysis by mass spectrometry The introduction of soft ionisation techniques like electrospray ionization (ESI) revolutionized the field of lipid analysis making polar lipids accessible to mass spectrometric analysis. Now numerous methodologies have been developed for both the structural analysis of complex lipids as well as quantitative profiling of complex lipid mixtures (for reviews see phospholipids [106,107], sphingolipids [108], glycosphingolipids [109], prostaglandins [110]). In the late 90s it became clear that nano electrospray ionization tandem mass spectrometry (ESI-MS/MS) permits a direct quantification of the major phospholipid classes from a minute amount of material without separation of crude lipid extracts [111]. These methods could be used in a high sample throughput either by direct flow injection analysis [112–114] or using an automated nanospray chip ion source [115–117]. To perform high throughput quantification of lipid species an automated data analysis is a prerequisite including the correction of isotope overlap [113], species assignment [118] as well as quantification. Analysis of unseparated lipid extracts by mass spectrometry may be hampered by signal suppression effects due to other lipid components as well as lack of specificity in the presence of isobaric molecules or uncharacteristic molecule fragmentation. Therefore, sample separation prior analysis either by direct mass spectrometer coupling or offline separation are used. Beside classical approaches like liquid and gas chromatography coupling to mass spectrometry, capillary electrophoresis is increasingly applied especially in the field of glycosphingolipid analysis [109].

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We applied ESI-MS/MS for a detailed analysis of cellular lipid efflux to apoA-I and HDL, which is related to ABCA1 and ABCG1 function (Schifferer et al. submitted JBC). Both free cholesterol (FC) loaded human primary fibroblasts and human monocyte-derived macrophages loaded with enzymatically modified LDL (E-LDL) revealed a higher apoA-I specific efflux for monounsaturated phosphatidylcholine (PC) species together with a decreased contribution of polyunsaturated PC species. Moreover, medium chain sphingomyelin (SM) species SM 14:0 and SM 16:1 were translocated preferentially to apoA-I in both cell types. In contrast to fibroblasts, monocyte-derived macrophages displayed a considerable proportion of cholesteryl esters (CE) in basal and apoA-I specific efflux media, most likely due to secretion of CE associated to apoE. Analysis of HDL3 mediated lipid efflux from monocytederived macrophages using D9-choline and 13C3-FC stable isotope labeling revealed significantly different D9-PC and D9-SM species pattern for apoA-I and HDL3 specific media, which indicates a contribution of distinct cellular phospholipid pools to apoA-I and HDL3 mediated efflux. This example shows that a detailed lipid species analysis by ESI-MS/MS may provide an even more powerful tool to understand cellular lipid transport mechanism also related to ABC transporter function. Several ABC transporters are involved in the formation of bile (ABCB4 [58], ABCB11 [42], ABCG5, ABCG8 [55–57]). Mass spectrometric techniques could serve to further analyze the detailed composition of lipid species secreted into the bile, which may together with biophysical models help to further understand the formation of toxic bile. Lipid species analysis may include bile acids and their conjugates, which may be analyzed by LC-MS/MS [119]. Additionally, phytosterol analysis, which may be useful also in plasma, could be achieved by LCMS/MS using atmospheric pressure photoionization (APPI) [120]. Another important aspect related to bile formation is the biosynthesis of PC the main substrate of ABCB4 in the liver. PC can be derived from either choline and can be converted to PC via the CDP-choline pathway or be generated via methylation of phosphatidylethanolamine (PE) by the enzyme PE N-methyltransferase (PEMT)[121] . Moreover it has been shown that the PC/PE ratio is a key regulator of cell membrane integrity and plays a role in the progression of steatosis into steatohepatitis [122]. In this respect a detailed investigation of phospholipid metabolism using stable isotope labeling and ESI-MS/MS analysis may shed further light on mechanisms [123,124]. One major problem of these novel mass spectrometry based lipidomic techniques is the vast amount of data generated. Because commercial software for lipidomic analysis is so far not available, most groups use self-made data analysis programs. These tools are used for raw data processing as well as species assignment and quantification. Software assistance are required even more to analyze metabolic data of single lipid species, e.g. by stable isotope labelling. Finally, classical approaches like gas chromatography [125] or novel approaches based on LC-MS/MS [126] may be used to detect very long-chain fatty acids (VLCFA) in plasma and tissues indicating X-linked adrenoleukodystrophy (ABCD1) [48]. In general lipid mass spectrometric techniques may be used in the diagnosis of ABC transporter related disorders as well as to discover lipid species transported by ABC or to unravel ABC transporter function.

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4.3. Fluorescence imaging and flow cytometry To characterize ABC transporter function fluorescent imaging methods are important to define tissue and subcellular localization including lipid domains, colocalization with other proteins as well as trafficking of ABC transporters. Frequently fusion proteins with green fluorescent protein (GFP) were used to detect the subcellular localization of ABC transporters, e.g. localization of ABCA1 on basolateral surface in hepatocytes [127] and Caco-2 cells [128]. Neufeld et al. performed an elegant study to investigate the complex intracellular trafficking of ABCA1 using a ABCA1-GFP fusion protein and confocal microscopy also in time-lapse image acquisition mode [93]. The importance of the ABCA1-PEST domain for posttranslational modification and intracellular shuttling of ABCA1 could be shown by immunofluorescence confocal microscopy. While wild type ABCA1 showed both plasma membrane localization and substantial colocalization with LAMP2 in late endosomes, PEST domain deleted ABCA1 revealed more prominent plasma membrane localization but little colocalization with LAMP2 [129]. Experiments evaluating the binding of Cy5-apoA-I and Cy5-annexin V argue against a role of phosphatidylserine translocation in the apoA-I mediated lipid efflux [130]. Moreover, the abundance and localization of protein isoforms can be evaluated by immunofluorescence confocal microscopy as for example the cellular localization of type I (cell surface and intracellularly) compared to type II (intracellularly) ABCA7 [29]. An important goal to analyze lipid transport as well as metabolism is to visualize lipids in their subcellular compartments. A frequently used approach uses fluorescent labeled lipid probes. For example sphingolipid analogs labeled with NBD or BODIPY containing fatty acids are internalized by endocytosis and subsequently sorted and transported to various intracellular compartments [131]. However, the bulky fluorescent groups may change the biophysical behaviour of the lipid probes showing a different behaviour compared to their natural analogues. An excellent example of lipid probes mimicking the conformation of their natural counterparts are polyene-fatty acids described by Ku¨rschner et al. [132]. Another important issue of lipid imaging is the detection of lipids rafts or plasma membrane microdomains. Several lipid probes have been described to monitor lipid components of rafts, e.g. lysenin, a sphingomyelin-binding protein obtained from an earthworm as well as a fluorescein ester of poly(ethyleneglycol) cholesteryl ether (fPEG-Chol) were described to partition into cholesterol-rich membranes [133]. Other frequently used probes to monitor ‘‘raftophilic’’ lipid species are cholera toxin for ganglioside GM1 [134] and cholesterol binding protein perfringolysin O (theta-toxin) [135]. Moreover, the single-molecule tracking of labeled lipid molecules like dioleoylphosphatidylethanolamine [95,136] or other membrane molecules may give novel insights into membrane compartmentalization and the dynamic assembly of large molecular complexes potentially clustered in rafts [137]. A convenient technique to quantify surface expression of lipids and lipid-modified proteins is flow cytometry. For instance phosphatidylserine translocation to the outer plasma membrane leaflet by annexin V staining is frequently used assay to investigate apoptosis. Furthermore, cell surface expression of membrane ceramide, lactosylceramide and more complex glycosphingolipids was analyzed by flow cytometry [138]. In a combination of quantitative flow cytometry and lipid

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imaging Grandl et al. [138] could show that loading of human primary macrophages with enzymatically degraded low density lipoprotein (E-LDL) or oxidized LDL (Ox-LDL) induces different membrane microdomains. A commonly used technique to study interactions between molecules is fluorescence resonance energy transfer (FRET). This method can also be combined with flow cytometry, e.g. for the investigation of lipopolysaccharide and ceramide induced clustering of the LPS receptor CD14 with other signaling receptors [139]. Finally, flow cytometry may be used to investigate the detergent insolubility of membrane markers, which has been used for a rapid screening of raft-association of blood monocyte surface antigens [140].

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[9]

[10]

[11] [12]

5. Conclusions Human ABC-transporters are associated with several monogenic and polygenic disorders. Disease phenotypes have greatly expanded the knowledge about their physiological roles in lipid metabolism as well as their precise mechanisms of action. Novel lipidomic high-throughput technologies are valuable tools for researchers to study and define important transcriptional regulators, intracellular routing steps and transported lipid molecule species. These findings will help to develop pathophysiological concepts and moreover will allow to improve diagnosis and therapy of ABC-transporter related lipid disorders.

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[14] [15]

Acknowledgement: This paper has been prepared in the context of the European Lipidomics Initiative, EC specific support action LSSG-CT2004-013032.

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