Lipidomic Strategies to Study Structural and Functional Defects of ABC-Transporters in Cellular Lipid Traﬃcking

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FEBS Letters 580 (2006) 5597–5610

Minireview 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

ABCC9) [3]. To date, 48 human ABC transporters, which Abstract The majority of the human ATP-binding cassette (ABC)-transporters function in cellular lipid trafficking and in are subdivided into seven subfamilies, have been identified the regulation of membrane lipid composition associating their and many of these proteins exert their biological activities in dysfunction with human disease phenotypes related to sterol, lipid metabolism and are themselves regulated by lipids includ- phospholipid and fatty acid homeostasis. Based on findings from ing phospholipids, sterols and fatty acids [4] (Table 1). In the monogenetic disorders, animal models, and in vitro systems, following chapters, we review genetic diseases in human major clues on the expression, function and cellular localization ABC-transporters related to lipid metabolism, provide insights of human ABC-transporters have been gained. Here we review into the regulation and functional characteristics of these lipid novel lipidomic technologies including quantitative mRNA transporters and present novel lipidomic technologies for their expression monitoring by realtime RT-PCR and DNA-micro- in vitro and in vivo analysis. arrays, 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. 2. Diseases and phenotypes caused by ABC transporters Ó 2006 Federation of European Biochemical Societies. Pub- lished by Elsevier B.V. All rights reserved. Twenty-two out of 48 currently known human ABC proteins Keywords: Lipidomics; ABC-transporters; Lipid trafficking have been linked to human monogenic disorders or cause spe- disorders cial 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. 1. Introduction In addition, heterozygous mutations and single nucleotide polymorphisms (SNPs) in ABC transporter genes have been Members of the ATP-binding cassette (ABC)-transporter connected with susceptibility to complex, multigenic disorders, superfamily are found throughout most species and represent such as inflammatory bowel disease [5] or Alzheimer’s disease one of the biggest protein families described so far. ABC pro- [6]. In the following paragraphs, a selection of important ABC teins are usually multispan membrane proteins which mediate transporter related inherited disorders with a special focus on the active uptake or efflux of specific substrates, mainly lipo- lipid metabolism are presented, including ABCA1, ABCA3, philic molecules, across biological membrane systems includ- ABCA7, ABCA12, ABCB4, ABCB11, ABCD1, ABCG5, ing the plasma membrane, the endoplasmic reticulum, the and ABCG8. Golgi compartment, mitochondria and peroxisomes [1]. The group of membrane spanning ABC-transporters can be split 2.1. ABCA1 and familial HDL deficiency into two different sections depending on their mode of action. The ABCA1 gene contains 50 exons and is located on chro- The active transporters or pumps, such as members of the mosome 9q31. Its open reading frame consists of 6783 bp, ABCB (MDR/TAP) subfamily, couple the hydrolsis of ATP encoding a 2261 amino acid protein. Mutations in the human and the resulting free energy to movement of molecules across ABCA1 gene cause familial HDL deficiency syndromes such as membranes against a chemical concentration gradient [2].In classical Tangier disease (Table 2) [7–9]. The most striking fea- contrast, recent work has identified several ABC proteins, ture of these patients is the almost complete absence of plasma which show nucleotide binding and a subsequent conforma- HDL, low serum cholesterol levels and a markedly reduced ef- tional change but very low ATP hydrolysis including ABCA1, flux of cholesterol and phospholipids from cells. The low HDL the cystic fibrosis membrane conductance regulator (CFTR/ levels seen in Tangier disease are mainly due to an enhanced ABCC7) and the sulfonylurea receptors (SUR1/2, ABCC8/ catabolism of these HDL precursors [10]. Interestingly, obli- gate heterozygotes for TD mutations have approx. 50% of plasma HDL, but normal LDL levels [11]. Studying 13 differ- *Corresponding author. Fax: +49 941 944 6202. ent mutations in 77 heterozygous individuals, Clee et al. de- E-mail address: [email protected] (G. Schmitz). scribed a more than threefold risk to develop coronary

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 ABC1 (TMD-ABC)2 + + Macrophages, liver, brain + Choline-phospholipids and cholesterol ABCA2 ABC2 (TMD-ABC)2 + + Brain + Estramustine, steroids ABCA3 ABC3 (TMD-ABC)2 + À Lung, brain, heart, pancreas + Surfactant phospholipids ABCA4 ABCR (TMD-ABC)2 + À Photoreceptors À N-retinylidene-PE ABCA5 (TMD-ABC)2 + À Muscle, heart, testes + Sterols ABCA6 (TMD-ABC)2 + À Liver, lung, heart, brain, ovary + Phospholipids ABCA7 (TMD-ABC)2 + + Spleen, thymus, bone marrow, peripheral + Sphingolipids (e.g. ceramide) and leukocytes, brain, adrenal glands, uterus phospholipids ABCA8 (TMD-ABC)2 + À Ovary, brain À ABCA9 (TMD-ABC)2 + À Heart, lung, brain, liver, prostate, + ovary uterus ABCA10 (TMD-ABC)2 À + Muscle, heart, gastrointestinal tract, + spleen, lung ABCA12 (TMD-ABC)2 ÀÀKeratinocytes, placenta, testis, lung, brain, À Lamellar granule lipids and enzymes stomach, prostate, lymphocytes ABCA13 (TMD-ABC)2 + À Trachea, testis, bone marrow À

ABCB1 MDR1 (TMD-ABC)2 À + Excretory organs, apical membrane + Phospholipids, PAF, aldosterone, cholesterol, amphiphiles, b-amyloid peptide ABCB2 TAP1 TMD-ABC ÀÀUbiquitous, ER À Peptides ABCB3 TAP2 TMD-ABC ÀÀUbiquitous, ER À Peptides ABCB4 MDR3 (TMD-ABC)2 ÀÀLiver, apical membrane + Phosphatidylcholine ABCB5 (TMA-ABC)2 ÀÀUbiquitous À ABCB6 MTABC3 TMD-ABC ÀÀMitochondria + Fe/S clusters ABCB7 ABC7 TMD-ABC ÀÀMitochondria À Fe/S clusters ABCB8 MABC1 TMD-ABC ÀÀMitochondria À Fe/S clusters

ABCB9 TMD-ABC ÀÀHeart, brain, lysosomes + 5597–5610 (2006) 580 Letters FEBS / al. et Schmitz G. ABCB10 MTABC2 TMD-ABC ÀÀMitochondria À Fe/S clusters ABCB11 BSEP (TMD-ABC)2 ÀÀLiver, apical membrane + Monovalent bile salts (e.g. TC)

ABCC1 MRP1 TMD0-(TMD-ABC)2 ÀÀLung, testes, PBMC + GSH-, glucuronate-, sulfate-conjugates, GSSG, sphingolipids, LTC4, PGA1, PGA2,17b-glucuronosyl estradiol, sulfatides, GM1 ABCC2 MRP2 TMD0-(TMD-ABC)2 À + Liver + GSH-, glucuronate-, sulfate-conjugates, bilirubin glucuronide, LTC4, 17b-glucuronosyl estradiol, taurocholate, taurolithocholate 3-sulfate, anionic drugs ABCC3 MRP3 TMD0-(TMD-ABC)2 ÀÀLung, intestine, liver, basolateral membrane À glucuronate-, sulfate-conjugates, 17b-glucuronosyl estradiol, taurolithocholate 3-sulfate ABCC4 MRP4 (TMD-ABC)2 ÀÀProstate + Xenobiotics, nucleosides (ATP/ADP/AMP/Adenosin, GTP/GDP) ABCC5 MRP5 (TMD-ABC)2 ÀÀUbiquitous + Xenobiotics, nucleosides (ATP/ADP/AMP/Adenosin, GTP/GDP) ABCC6 MRP6 TMD0-(TMD-ABC)2 Kidney, liver À Anionic cyclopentapeptides (e.g. BQ123), LTC4, GSH-conjugates ABCC7 CFTR (TMD-ABC)2 À + Exocrine tissue À Chloride ions, ATP G. Schmitz et al. / FEBS Letters 580 (2006) 5597–5610 5599

artery disease in affected family members and earlier onset compared to unaffected members [12,13]. In addition to the ab- ecule sence 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 Oligoadenylate translation elongation initiation factor 2 Sulfonylureas Sulfonylureas long-chain fatty acid-CoA, 2-methylacyl-CoA Xenobiotics 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 À À À À À À À À À À À 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-palmitoyl- phosphatidylcholine and di-palmitoyl-phosphatidylglycerol with surfactant proteins (SP) that are synthesized by type-II- pneumocytes. 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 Ovary, testes, spleen Ubiquitous Ubiquitous Ubiquitous Ubiquitous + Phospholipids, cholesterol Peroxisomes Placenta,, liver, kidney, intestine, stem cells Heart, muscle Low in all tissues Low in all tissues Peroxisomes + Very-long-chain fatty acids Low in all tissues Liver + Cholesterol Liver, instestine + Plant- and shellfish sterols Liver, instestine + Plant- and shellfish sterols Peroxisomes + Very-long-chain fatty acids 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 À + PeroxisomesÀ + Very-long-chain fatty acids À À À À À À 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 ÀÀ ÀÀ ÀÀ ÀÀ ÀÀ À ÀÀ ÀÀ ÀÀ 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 ABCF1ABCF2 ABC50ABCF3ABCG1 White (ABC)2 (ABC)2 ABC-TMD (ABC)2 + ABCE1 OABP (ABC)2 ABCG2 MXR ABC-TMD + Gene Alternat. name Domain structure NH2-term. –LL– PDZ-bind. Tissue expression and cellular location Lipid reg. Known or putative transported mol ABCC8ABCC9 SUR1 SUR2 TMD0-(TMD-ABC)2 TMD0-(TMD-ABC)2 + + + Pancreas ABCC10 MRP7ABCD3 TMD0-(TMD-ABC)2 + PMP70ABCD4 PMP69 TM-ABC TM-ABC ABCG4 White2 ABC-TMD + ABCG5 White3 ABC-TMD + ABCG8 White 4 ABC-TMD + ABCC11ABCC12 MRP8 MRP9ABCD1 ALD (TMD-ABC)2 (TMD-ABC)2 TM-ABC + ABCD2 ALDR TM-ABC array with only 1.7 kb distance between the terminal exon of 5600 G. Schmitz et al. / FEBS Letters 580 (2006) 5597–5610

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 ABC2 600047 Alzheimer’s disease ABCA3 ABC3 601615 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 MDR1 171050 Ulcerative colitis ABCB2 TAP1 170260 Immune deﬁciency ABCB3 TAP2 170261 Wegener-like granulomatosis

ABCB4 MDR3 171060 Progressive familial intrahepatic cholestasis type 3 (PFIC-3) Intrahepatic cholestasis of pregnancy (ICP)

ABCB7 ABC7 300135 X-linked sideroblastosis and anemia (XLSA/A) ABCB11 BSEP 603201 Progressive familial intrahepatic cholestasis type 2 (PFIC-2)

ABCC2 MRP2 601107 Dubin–Johnson syndrome (DJS) ABCC6 MRP6 603234 Pseudoxanthoma elasticum (PXE) ABCC7 CFTR 602421 Cystic ﬁbrosis (CF) ABCC8 SUR1 600509 Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) ABCC9 SUR2 601439 Dilated cardiomyopathy with ventricular tachycardia

ABCD1 ALD 300371 Adrenoleukodystrophy (ALD) ABCD3 PXMP1 170995 Zellweger syndrome 2 (ZWS2)

ABCG2 MXR, BCRP 603756 Protoporphyria IX ABCG5 White3 605459 b-Sitosterolemia ABCG8 White 4 605460 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. G. Schmitz et al. / FEBS Letters 580 (2006) 5597–5610 5601

ABCA7 and the first exon of the HA-1 gene [28]. In addition to the milder form manifesting as lamellar ichthyosis type 2. Dur- the major transcript form, a second main mRNA isoform ing keratinocyte differentiation, lipids accumulate in lamellar resulting from alternative splicing (termed type II mRNA) of granules which are extruded in the intercellular space for enzy- human ABCA7 has been detected [29]. A novel exon is located matic modification to produce a water permeability barrier between former exons 5 and 6, designated as exon 5B, and consisting of ceramides, cholesterol and saturated long-chain contains two termination codons and another translation initi- fatty acids [41]. ABCA12 is present in the upper epidermal ation codon. Thus, translation of type II mRNA produces an layer in lamellar granules of normal epidermal keratinocytes ABCA7 protein containing 28 novel N-terminal amino acids and likely functions in the secretion of lipids [39] (Fig. 1B). instead of the 1–166 amino acid sequence in full length Keratinocytes from patients with harlequin ichthyosis display (type I) ABCA7. Interestingly, most of type II ABCA7 is local- an accumulation of glucosylceramide around the nuclei and ized intracellularly when expressed in HEK 293 cells, indicat- ABCA12 gene transfer into deficient cells normalizes glucosyl- ing that the N-terminal domain is important for trafficking ceramide staining present in healthy keratinocytes, assuming of ABCA7 to the plasma membrane. Tissue-specific RT-PCR that ABCA12 is involved in the intracellular translocation of analysis has shown that the expression of type II mRNA is lamellar granule sphingolipids [39]. higher than type I mRNA in spleen, thymus, lymph node, and trachea, while type I mRNA was equal to or more than 2.5. ABCB4 (MDR3) and ABCB11 (BSEP) in progressive type II mRNA in bone marrow and brain [29,30]. familial intrahepatic cholestasis (PFIC) The detailed molecular function of the 127-kDa HA-1 minor Mutations in three hepatic canalicular membrane proteins histocompatibility antigen is unknown, but it potentially trans- have been identified that result in cholestasis and later chronic ports minor histocompatibility peptides together with MHC liver disease. These three diseases are progressive familial class I molecules via the Golgi complex to the cell surface, intrahepatic cholestasis (PFIC) types 1, 2 and 3 and are caused where they are presented to T cells. In the case of HA-1, which by mutations in the genes coding for the familial intrahepatic has properties of small G-protein family interacting proteins, a cholestasis 1 protein (FIC1), and the ABC-transporters bile defined N-terminal nonapeptide bearing the His168Arg dimor- salt export pump (BSEP, ABCB11) and the phospholipid phism has been identified as the target immune epitope recog- translocase MDR3 (ABCB4), respectively [42]. Ten autosomal nized by T cells [31]. Between ABCA7 and HA-1 genes a recessive mutations in the coding region of the ABCB11 functional and regulatory interlink might exist, which is espe- (BSEP) gene have been reported so far in different PFIC2 pa- cially interesting since ABCA7 has been shown to be regulated tients. ABCB11 is the major bile salt carrier in the canalicular by sterols via sterol-regulatory-element binding protein 2 membrane and mutations very often cause low expression or (SREBP2) [32]. ABCA7/HA-1 may be involved in autoim- aberrant membrane localization [43]. PFIC3 is also an auto- mune disorders since both transcripts are expressed in macro- somal recessive disorder and the ABCB4 (MDR3) gene local- phages and lymphocytes, major effector cells of chronic izes to chromosome 7q21. In most cases where mutations inflammation. Furthermore, a hallmark of autoimmune re- causing truncations occur in the gene, a complete loss of lated diseases is disturbed autophagy of cell organelles and ABCB4 protein at the canalicular membrane has been ob- thereby persistence of protein degradation products that un- served, whereas low expression levels correlate with missense dergo antigen presentation. A polypeptide consisting of amino mutations [44]. The function of ABCB4 protein was analyzed acids 186–320 within the first extracellular domain of ABCA7 in a knock-out model with deletion of the mouse homolog encodes the autoantigen SS-N suggesting that ABCA7 is asso- (mdr2). These knock-out mice had a lack of phospholipid ciated with the pathogenesis of Sjo¨gren’s syndrome [33]. Fur- translocation into bile, normal bile acid secretion, progressive thermore, recent studies revealed that the 168H variant of liver disease, and histology consistent with PFIC3 patients the minor histocompatibility antigen HA-1 is associated with leading to the assumption that ABCB4 is a phosphatidylcholine reduced risk of primary Sjo¨gren’s syndrome [34] and that translocase [45]. The purpose of phospholipids in bile is to form ABCA7 is highly expressed in salivary glands of patients with micelles with bile acid and to prevent crystallization of choles- Sjo¨gren’s syndrome [35]. Recent data also indicate that terol. Without the formation of micelles with bile acids, the del- ABCA7 might be important for hematopoietic development, eterious detergent effect of bile acids on the biliary epithelium differentiation, and immunological functions related to phago- and cholangiocytes is not prevented and causes PFIC. cytosis [32].

2.6. ABCD1 in adrenoleukodystrophy 2.4. ABCA12 and genetic keratinization disorders The X-linked adrenoleukodystrophy (ALD) is an inherited The ABCA12 gene, which comprises 53 exons spanning peroxisomal disorder caused by mutations in ABCD1 (Table 2) 206 kb, has been mapped to chromosome 2q34, the region resulting in progressive neurological dysfunction, occasionally for the keratinization disorder lamellar ichthyosis, an auto- associated with adrenal insufficiency [46]. ALD is character- somal recessive form of ichthyosis characterized by whole ized by the accumulation of unbranched saturated fatty acids body skin desquamation [36,37]. Thereafter, missense muta- with a chain length of 24–30 carbons, particularly hexacosano- tions in the ABCA12 gene have been identified [38] and two re- ate (C26), in cholesteryl esters of brain white matter and in cent independent studies demonstrated that mutations in the adrenal cortex and in certain brain sphingolipids. Adreno- ABCA12 gene also underlie the very severe form of another leukodystrophy belongs to a group of defects in peroxisomal keratinization disorder-harlequin ichthyosis [39,40]. Based on b-oxidation [47]. The first step in the oxidation of very-long- the identified mutations, it can be assumed that the two clinical chain fatty acids (VLCFA) involves the activation by conver- phenotypes arise from different alterations of the ABCA12 sion into CoA esters and the transport into peroxisomes [48]. protein causing either the severe form harlequin ichthyosis or The ABCD1 protein mediates this transport process as 5602 G. Schmitz et al. / FEBS Letters 580 (2006) 5597–5610 evidenced by overexpression of human cDNAs encoding the the liver, intestinal ABCA1 expression is a rate-limiting factor ABCD1 protein and its closest relative, the ABCD2 (ALDR) for plasma HDL production [62]. Although ABCA1 in macro- protein in fibroblasts of ALD patients [49]. The accumulation phages has little influence on HDL, it is an important factor in of very-long-chain fatty acids could also be prevented by over- the prevention of foam cell formation of macrophages in the expression of the ABCD2 protein in immortalized ALD cells arterial wall [63]. To control lipid efflux, ABCA1 is tightly reg- and the peroxisomal b-oxidation defect in the liver of ulated at the level of transcription and translation. The major ABCD1-deficient mice could be restored by stimulation of human ABCA1 gene promoter was first reported upstream of ABCD2 and ABCD4 gene expression through treatment with exon 1 (221 bp) followed by a 24-kb intron 1 [64,65]. Cavelier fenofibrate [49]. These results implicate that a therapy of adre- et al. [66] have identified an alternative exon 1 (exon 1a), noleukodystrophy might be possible by drug-induced overex- 136 bp in length and located 2210 bp upstream of exon 2. This pression or ectopic expression of ABCD genes. alternative transcript contains the entire coding sequence of human ABCA1 and is expressed in testis and liver but not 2.7. ABCG5 and ABCG8 in b-Sitosterolemia macrophages. Upstream of exon 1a, an alternative promoter ABCG5 and ABCG8 have been recently implicated in the ef- that contains a TATA box and CAAT sites as well as other po- flux of dietary sterols from intestinal epithelial cells back into tential binding sites for sterol-sensing nuclear receptors was the gut lumen and from the liver to the bile duct. These mech- identified. The importance of this alternative promoter in reg- anisms are disrupted in b-Sitosterolemia or phytosterolemia or ulating ABCA1 gene expression, however, has to be deter- shellfishsterolemia, a rare autosomal recessive disorder. The mined in human tissues [67]. ABCA1 gene expression is disease is characterized by enhanced trapping of cholesterol stimulated by liver-X-receptors (LXRs) and the retinoid-X- and other sterols, including plant and shellfish sterols, within receptor (RXR) which form heterodimers and are activated the intestinal cells and the inability to concentrate these sterols by oxysterols and retinoids, respectively. In macrophages, in the bile. As a consequence affected individuals have strongly the physiological ligand for LXRa and LXRb is 27-hydroxy- increased plasma levels of plant sterols e.g. b-sitosterol, cam- cholesterol, which is produced by the mitochondrial 27-choles- pesterol, stigmasterol, avenosterol, and 5a-saturated stanols, terol hydroxylase (CYP27). In addition to the induction of whereas total sterol levels remain normal or are just moder- ABCA1 expression, cAMP also stimulates ABCA1 phosphor- ately elevated [50]. Using a combination of positional cloning, ylation [68,69]. Interestingly, apoA-I activates cAMP signal- genome database survey as well as DNA-microarray analysis, ling and thereby the phosphorylation of ABCA1. Janus Lee et al. [51] and Berge et al. [52] identified ABCG5 and kinase 2 (JAK2) also phosphorylates ABCA1 [70], whereas ABCG8 mutations in b-Sitosterolemia patients. ABCG5 and in the absence of apolipoproteins, phosphorylation of the ABCG8 have a bi-directional promoter and share common ABCA1 PEST motif is a signal for calpain-mediated degrada- regulatory elements [53]. The highest expression level of both tion of ABCA1 [71] (Fig. 2). Furthermore, ABCA1 protein transporters is found in liver and intestine and high-cholesterol destabilization is performed by unsaturated fatty acids via a diet feeding in mice induces the expression of both genes. phospholipase D2-dependent pathway [72,73]. These findings together with the observed clinical and bio- ABCA1, which has also a conserved N-terminal dileucine chemical features of b-Sitosterolemia patients assume that motif, assembles functional complexes with heteromeric pro- ABCG5 and ABCG8 play an important role in reducing intes- teins and the intracellular targeting of ABCA1 is regulated tinal absorption and promote biliary excretion of sterols. Until by diverse trafficking proteins (Fig. 2). The Rho family today several mutations and a number of polymorphisms have GTPase cdc42 directly interacts with ABCA1 to control been identified in ABCG5 and ABCG8 [54]. A striking finding filopodia formation and intracellular lipid transport [74,75]. is that mutations in b-Sitosterolemia patients occur exclusively Furthermore, the PDZ-domain proteins b2-syntrophin, a1- either in ABCG5 or ABCG8, but never in both genes together. syntrophin, and Lin7 bind to the KESYV-motif at the C-ter- ABCG5 and ABCG8 proteins form a functional heterodimer minus of ABCA1 and regulate its targeting via vesicular to transport plant sterols directly back to the gut lumen as a trafficking, turnover, and intracellular localization in polarized kick-back mechanism, which may also efflux cholesterol, there- cells [76,77]. The ADP-ribosylation factor (ARF)-like 7 by regulating total sterol absorption. In the liver, expression of (ARL7) is involved in vesicular budding from the trans-Gol- ABCG5 and ABCG8 functions in regulating biliary cholesterol gi-network and has been implicated in the intracellular translo- secretion [55–57], which also requires biliary phospholipid cation of lipids via ABCA1 and the secretory lysosome transport by ABCB4 [58]. 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 3. Regulation, trafficking, and function of ABCA1 and ABCG1 interacting protein pallidin are major constituents of the adap- in lipid metabolism ter protein 3 (AP3) pathway and the AP3/secretory lysosome pathway seems to play a major role in ABCA1-dependent lipid ABCA1 is crucial for the efflux of phospholipids and sterols traffic. to apoA-I containing HDL precursors. Like Tangier disease The cell-type expression and lipid-sensitivity of the half-size patients, ABCA1 knockout mice exhibit HDL deficiency and transporter ABCG1 is very similar to ABCA1 although the have strongly reduced cellular lipid efflux activity [59]. Total transcriptional regulation of ABCG1 is very complex and mul- body and selective hepatic overexpression of ABCA1 in mice tiple alternative promoters and mRNA isoforms exist [80–83]. results in an increase of HDL [60] and conversely, HDL levels A comparison of human and murine orthologue promoter se- are strongly reduced in mice with a liver-selective deletion of quences and cDNAs suggests that one major transcript ABCA1 [61]. There is increasing evidence that in addition to (termed ABCG1a) is expressed in most tissues, whereas the G. Schmitz et al. / FEBS Letters 580 (2006) 5597–5610 5603

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 iso- ABCG1 and ABCG4 expressed in Sf9 cells both display ATP forms, which differ at the N-terminus of ABCG1 remains to binding and hydrolysis with a significant higher activity of be determined. A striking similarity between human ABCG1 ABCG1 compared to ABCG4. Importantly, co-expression of and Drosophila ABCG transporters (white, brown, scarlet a catalytic site mutant of ABCG4 showed a dominant negative and E23) is their transcriptional regulation by nuclear hor- effect and selectively abolished the ATPase activity of ABCG1 mone receptors and their potential cellular localization. Dro- [88]. Therefore, it can be concluded that ABCG1 may either sophila white and scarlet proteins are involved in the act as a homodimer in the absence of ABCG4 or function as formation of eye color pigments by the transport of 3-hydr- a heterodimer in combination with ABCG4 in coexpressed tis- oxykynurenine from the cytoplasm into pigment granules. The sues to increase lipid efflux. Informations about the in vivo garnet gene product, which also modulates eye color biogene- function of ABCG1 came from a recent study with ABCG1 sis of Drosophila, shows strong homology with the delta sub- knockout mice, showing that cholesterol, triglyceride, and unit of the human AP-3 complex, which is mainly associated phospholipid concentrations were significantly increased in with the trans-Golgi network and other peripheral membranes the liver and lung of Abcg1-null mice fed the high-fat/high- [84]. This adaptor complex is involved in intracellular targeting cholesterol diet, as compared to their wild-type littermates of proteins to specialized vesicular compartments and these [89]. Despite the lack of changes in plasma lipid levels, macro- proteins often harbor dileucine motifs, which are recognized phage foam cells highly enriched in lipid droplets and choles- by the AP-3 complex. A sequence alignment of white, brown, terol crystals were observed in the lung of Abcg1-null mice. and scarlet from Drosophila and human ABCG proteins re- These findings are in contrast to earlier reports showing that veals a conserved region near the first membrane domain con- plasma HDL and cholesteryl ester levels were reduced by infu- taining a putative dileucine motif [85]. These findings raise the sion of mice with adenovirus expressing ABCG1 [90], which possibility that ABCG1 and other family members may inter- might result from artifically high overexpression of ABCG1 act with AP-3 in order to reach their subcellular compartment in hepatocytes [91]. and that the different ABCG1 protein isoforms might be tar- There is little doubt that ABCA1 interacts directly with geted to different intracellular compartments. apoA-I. Moreover, there is evidence that ABCA1 modulates ABCG1-specific antisense oligonucleotides significantly im- the internalization of apoA-I for lipidation and resecretion paired cholesterol and phospholipid efflux to HDL3 [86].In (retroendocytosis). For the first time the HDL retroendocyto- HEK293-cells, transient overexpression of murine ABCG1 or sis pathway was suggested by Schmitz et al. in 1985 [92] using ABCG4 caused a strong increase in HDL-dependent choles- electron microscopy and gold-conjugated HDL particles. terol efflux and RNA interference-directed against macro- Beside a saturable HDL binding to macrophages loaded with phage ABCG1 mRNA also resulted in diminished lipid efflux cholesterol, gold-labeled HDL were endocytosed via coated [87]. Interestingly, in ABCG1 overexpressing cells, lipid-poor pits and resecreted into the culture medium without degrada- apoA-I was ineffective in lipid deloading of cells, which indi- tion. Furthermore, apoA-I and ABCA1 colocalise in endocytic cates that, in contrast to ABCA1, acceptor-particles for compartments [93,94]. Additionally, there is evidence that ABCG1-derived lipids are more mature a-HDL subclasses apoA-I binding to ABCA1 occurs at cholesterol/sphingomye- (HDL3 and HDL2) than preb-HDL [87]. Human recombinant lin rich membrane domains. Although, ABCA1 is excluded 5604 G. Schmitz et al. / FEBS Letters 580 (2006) 5597–5610 from lipid rafts isolated by Triton X-100 or detergent-free DNA microarrays are now frequently applied for the fast methods, some ABCA1 was detected in Lubrol-resistant mem- detection of expression levels of a large number of genes simul- branes [95,96]. Moreover, apoA-I interaction with plasma taneously and the generation of molecular finger prints [102]. membrane lipid rafts controls cholesterol export from macro- A specific microarray dedicated to ABC transporters has been phages [97] and sphingomyelinase treatment, which also dis- recently published by Annereau and coworkers [103]. This rupts rafts, inhibited cholesterol efflux to apoA-I [98]. Thus, ABC-ToxChip contains cDNA probes and 70mer oligonucleo- an early apoA-I interaction with lipid rafts seems to be essen- tides targeting 36 human ABC transporters and specific over- tial for efflux initiation, although the majority of exported cho- expression of ABCB1 and ABCC2 could be detected in lesterol and phospholipids may not originate from raft tumor cells [103]. Huang et al. developed a 70mer oligonucleo- domains [97] (Liebisch et al. unpublished observation). tide microarray covering 40 ABC transporters and ABCB1, Together with recent data from Gelissen et al. [99] showing ABCC3, and ABCB5 expression was negatively correlated that overexpression of ABCG1 promotes cholesterol efflux not with drug resistance, implicating ABCB5 as a novel chemore- only to HDL but also to other lipid acceptors like phosphati- sistant factor [104]. In another approach, a low-density cDNA dylcholine vesicles, a two-step model for ABCA1 and ABCG1 microarray for 38 human ABC transporters, termed DualChip mediated cellular lipid efflux could be proposed: Initially human ABC, was produced and validated in multidrug-resis- ABCA1 may promote cholesterol and phospholipid efflux tant sublines previously shown to overexpress ABCB1, involving membrane microdomains to lipid-poor apoA-I and ABCC1, or ABCG2 [105]. These results demonstrate the proof ABCG1 subsequently promotes the efflux of additional cellular of principle for successful microarray expression analysis of cholesterol to preformed lipid–protein complexes. 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 4. Novel lipidomic technologies mRNA for the DualChip human ABC, specificity problems with highly homologous DNA sequences, and the lack of stan- 4.1. mRNA detection by realtime RT-PCR and DNA dardization are major drawbacks limiting the application of microarrays this methodology in lipidomics research. In conclusion, among Quantitative realtime reverse transcription PCR (RT-PCR) the published ABC-transporter mRNA profiling assays, the is a fast and sensitive detection method which allows reproduc- Taqman microfluidic card assay optimally fulfills good labora- ible quantification of minute amounts of RNA. Total RNA tory practice (GLP) criteria concerning specificity, sensitivity from tissue specimens or single cells is reverse transcribed to and accuracy and thereby can be regarded as a reference assay generate cDNA as a template for quantitative PCR. Two dif- for mRNA expression analysis of this gene group. ferent 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 devel- 4.2. Lipid species analysis by mass spectrometry oped a rapid, specific and sensitive realtime RT-PCR method The introduction of soft ionisation techniques like electro- to monitor the mRNA levels of 47 human ABC transporters spray ionization (ESI) revolutionized the field of lipid analysis based on the TaqManÒ technology using specific primers making polar lipids accessible to mass spectrometric analysis. and probes and 50 ng of total RNA [17]. We have recently ex- Now numerous methodologies have been developed for both tended, improved and simplified the quantification of all 47 the structural analysis of complex lipids as well as quantitative ABC transporters by creating a Human ABC Transporter profiling of complex lipid mixtures (for reviews see phospho- TaqMan Low Density Array based on an Applied Biosystems lipids [106,107], sphingolipids [108], glycosphingolipids [109], 7900HT Micro Fluidic Card [101]. The method allows simulta- prostaglandins [110]). neous analysis of all 47 human ABC transporters and the ref- In the late 90s it became clear that nano electrospray ioniza- erence gene 18S rRNA in two replicates of four different tion tandem mass spectrometry (ESI-MS/MS) permits a direct samples per run in a 2 ll small-volume format. Transcript pro- quantification of the major phospholipid classes from a minute filing in macrophages has revealed several novel LXR/RXR amount of material without separation of crude lipid extracts regulated ABC transporters including ABCB1 (MDR1), [111]. These methods could be used in a high sample through- ABCB9, ABCB11 (BSEP), ABCC2 (MRP2), ABCC5 put either by direct flow injection analysis [112–114] or using (MRP5), ABCD1 (ALD), ABCD4, and ABCG2. This assay an automated nanospray chip ion source [115–117]. To per- will be a very useful tool to monitor dysregulated ABC trans- form high throughput quantification of lipid species an auto- porter mRNA profiles in human lipid disorders and cancer-re- mated data analysis is a prerequisite including the correction lated multidrug resistance. Furthermore, the pharmacological of isotope overlap [113], species assignment [118] as well as and metabolic regulation of ABC transporter expression quantification. important for drug development could be analyzed in large Analysis of unseparated lipid extracts by mass spectrometry screening approaches using this method. may be hampered by signal suppression effects due to other In a recent report by Szakacs et al. the expression levels of lipid components as well as lack of specificity in the presence the same gene panel was measured by realtime RT-PCR using of isobaric molecules or uncharacteristic molecule fragmenta- SYBR Green and the LightCyclerä instrument with 150 ng tion. Therefore, sample separation prior analysis either by di- RNA template. Although less specific and more laborious than rect mass spectrometer coupling or offline separation are used. our TaqManÒ approach, the LightCyclerä assay was success- Beside classical approaches like liquid and gas chromatogra- fully used to correlate the mRNA expression pattern of ABC phy coupling to mass spectrometry, capillary electrophoresis transporters with anticancer drug response in 60 diverse cancer is increasingly applied especially in the field of glycosphingo- cell lines [100]. lipid analysis [109]. G. Schmitz et al. / FEBS Letters 580 (2006) 5597–5610 5605

We applied ESI-MS/MS for a detailed analysis of cellular 4.3. Fluorescence imaging and flow cytometry lipid efflux to apoA-I and HDL, which is related to ABCA1 To characterize ABC transporter function fluorescent imag- and ABCG1 function (Schifferer et al. submitted JBC). Both ing methods are important to define tissue and subcellular free cholesterol (FC) loaded human primary fibroblasts and localization including lipid domains, colocalization with other human monocyte-derived macrophages loaded with enzymati- proteins as well as trafficking of ABC transporters. Frequently cally modified LDL (E-LDL) revealed a higher apoA-I specific fusion proteins with green fluorescent protein (GFP) were used efflux for monounsaturated phosphatidylcholine (PC) species to detect the subcellular localization of ABC transporters, e.g. together with a decreased contribution of polyunsaturated localization of ABCA1 on basolateral surface in hepatocytes PC species. Moreover, medium chain sphingomyelin (SM) spe- [127] and Caco-2 cells [128]. Neufeld et al. performed an ele- cies SM 14:0 and SM 16:1 were translocated preferentially to gant study to investigate the complex intracellular trafficking apoA-I in both cell types. In contrast to fibroblasts, mono- of ABCA1 using a ABCA1-GFP fusion protein and confocal cyte-derived macrophages displayed a considerable proportion microscopy also in time-lapse image acquisition mode [93]. of cholesteryl esters (CE) in basal and apoA-I specific efflux The importance of the ABCA1-PEST domain for posttransla- media, most likely due to secretion of CE associated to apoE. tional modification and intracellular shuttling of ABCA1 Analysis of HDL3 mediated lipid efflux from monocyte- could be shown by immunofluorescence confocal microscopy. 13 derived macrophages using D9-choline and C3-FC stable While wild type ABCA1 showed both plasma membrane local- isotope labeling revealed significantly different D9-PC and ization and substantial colocalization with LAMP2 in late D9-SM species pattern for apoA-I and HDL3 specific media, endosomes, PEST domain deleted ABCA1 revealed more which indicates a contribution of distinct cellular phospholipid prominent plasma membrane localization but little colocaliza- pools to apoA-I and HDL3 mediated efflux. This example tion with LAMP2 [129]. Experiments evaluating the binding of shows that a detailed lipid species analysis by ESI-MS/MS Cy5-apoA-I and Cy5-annexin V argue against a role of phos- may provide an even more powerful tool to understand cellu- phatidylserine translocation in the apoA-I mediated lipid efflux lar lipid transport mechanism also related to ABC transporter [130]. Moreover, the abundance and localization of protein function. isoforms can be evaluated by immunofluorescence confocal Several ABC transporters are involved in the formation of microscopy as for example the cellular localization of type I bile (ABCB4 [58], ABCB11 [42], ABCG5, ABCG8 [55–57]). (cell surface and intracellularly) compared to type II (intracel- Mass spectrometric techniques could serve to further analyze lularly) ABCA7 [29]. the detailed composition of lipid species secreted into the bile, An important goal to analyze lipid transport as well as which may together with biophysical models help to further metabolism is to visualize lipids in their subcellular compart- understand the formation of toxic bile. Lipid species analysis ments. A frequently used approach uses fluorescent labeled may include bile acids and their conjugates, which may be ana- lipid probes. For example sphingolipid analogs labeled with lyzed by LC-MS/MS [119]. Additionally, phytosterol analysis, NBD or BODIPY containing fatty acids are internalized by which may be useful also in plasma, could be achieved by LC- endocytosis and subsequently sorted and transported to vari- MS/MS using atmospheric pressure photoionization (APPI) ous intracellular compartments [131]. However, the bulky fluo- [120]. Another important aspect related to bile formation is rescent groups may change the biophysical behaviour of the the biosynthesis of PC the main substrate of ABCB4 in the lipid probes showing a different behaviour compared to their liver. PC can be derived from either choline and can be con- natural analogues. An excellent example of lipid probes mim- verted to PC via the CDP-choline pathway or be generated icking the conformation of their natural counterparts are poly- via methylation of phosphatidylethanolamine (PE) by the en- ene-fatty acids described by Ku¨rschner et al. [132]. zyme PE N-methyltransferase (PEMT)[121] . Moreover it has Another important issue of lipid imaging is the detection of been shown that the PC/PE ratio is a key regulator of cell lipids rafts or plasma membrane microdomains. Several lipid membrane integrity and plays a role in the progression of ste- probes have been described to monitor lipid components of atosis into steatohepatitis [122]. In this respect a detailed inves- rafts, e.g. lysenin, a sphingomyelin-binding protein obtained tigation of phospholipid metabolism using stable isotope from an earthworm as well as a fluorescein ester of poly(ethyl- labeling and ESI-MS/MS analysis may shed further light on eneglycol) cholesteryl ether (fPEG-Chol) were described to mechanisms [123,124]. partition into cholesterol-rich membranes [133]. Other fre- One major problem of these novel mass spectrometry based quently used probes to monitor ‘‘raftophilic’’ lipid species lipidomic techniques is the vast amount of data generated. Be- are cholera toxin for ganglioside GM1 [134] and cholesterol cause commercial software for lipidomic analysis is so far not binding protein perfringolysin O (theta-toxin) [135]. Moreover, available, most groups use self-made data analysis programs. the single-molecule tracking of labeled lipid molecules like These tools are used for raw data processing as well as species dioleoylphosphatidylethanolamine [95,136] or other mem- assignment and quantification. Software assistance are re- brane molecules may give novel insights into membrane com- quired even more to analyze metabolic data of single lipid partmentalization and the dynamic assembly of large species, e.g. by stable isotope labelling. molecular complexes potentially clustered in rafts [137]. Finally, classical approaches like gas chromatography [125] A convenient technique to quantify surface expression of lip- or novel approaches based on LC-MS/MS [126] may be used ids and lipid-modified proteins is flow cytometry. For instance to detect very long-chain fatty acids (VLCFA) in plasma and tis- phosphatidylserine translocation to the outer plasma mem- sues indicating X-linked adrenoleukodystrophy (ABCD1) [48]. brane leaflet by annexin V staining is frequently used assay In general lipid mass spectrometric techniques may be used to investigate apoptosis. Furthermore, cell surface expression in the diagnosis of ABC transporter related disorders as well of membrane ceramide, lactosylceramide and more complex as to discover lipid species transported by ABC or to unravel glycosphingolipids was analyzed by flow cytometry [138].In ABC transporter function. a combination of quantitative flow cytometry and lipid 5606 G. Schmitz et al. / FEBS Letters 580 (2006) 5597–5610 imaging Grandl et al. [138] could show that loading of human Ashbourne-Excoffon, M., Sensen, C.W., Scherer, S., Mott, S., primary macrophages with enzymatically degraded low density Denis, M., Martindale, D., Frohlich, J., Morgan, K., Koop, B., lipoprotein (E-LDL) or oxidized LDL (Ox-LDL) induces dif- Pimstone, S., Kastelein, J.J. and Hayden, M.R. (1999) Muta- tions in ABC1 in Tangier disease and familial high-density ferent membrane microdomains. lipoprotein deficiency. Nat. Genet. 22, 336–345. A commonly used technique to study interactions between [9] Rust, S., Rosier, M., Funke, H., Real, J., Amoura, Z., Piette, molecules is fluorescence resonance energy transfer (FRET). J.C., Deleuze, J.F., Brewer, H.B., Duverger, N., Denefle, P. and This method can also be combined with flow cytometry, e.g. Assmann, G. (1999) Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. 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