ATP BINDING CASSETTE GENES AND PROTEINS FOR DIAGNOSIS AND TREATMENT OF LIPID DISORDERS AND INFLAMMATORY DISEASES

Abstract: Modulation of the activity of transmembrane proteins belonging to the ATP binding cassette (ABC) transporter protein family which are etiologically involved in cholesterol driven atherogenic processes and inflammatory diseases like psoriasis, lupus erythematodes and others provides therapeutic means to treat such diseases. Furthermore, detection of herein identified ABC transporter proteins of their respective biochemical activities involved in such atherogenic and inflammatory processes provides diagnostic means for clinical application of diagnosis and monitoring of dyslipidemias, atherosclerosis or inflammatory diseases like psoriasis and lupus erythematodes.

Description: ATP binding cassette genes and proteins for diagnosis and treatment of lipid disorders and inflammatory diseases Background of the invention Reverse cholesterol transport mediated by HDL provides a " protective" mechanism for cell membrane integrity and foam cell formation and cellular cholesterol is taken up by circulating HDL or its precursor molecules. The precise mechanism of reverse cholesterol transport however is currently not fully understood and the mechanism of cellular cholesterol efflux and transfer from the cell surface to an acceptor-particle, such as HDL, is yet unclear. Certain candidate gene products have been postulated playing a role in the process of reverse cholesterol transport [1 ]. Apolipoproteins (e.g. ApoA-I, ApoA-IV), lipid transfer proteins (e.g. CETP, PLTP) and enzymes (e.g. LCAT, LPL, HL) are essential to exchange cholesterol and phospholipids in lipoprotein-lipoprotein and lipoprotein-cell interactions. Different plasma membrane receptors, such as SR-BI [2; 3], HB1/2 [4], and GPI-linked proteins (e.g. 120 kDa and 80 kDa) [5] as well as the sphingolipid rich microdomains (Caveolae, Rafts) of the plasma membrane have been implicated being involved in the process of reverse cholesterol transport and the exchange of phospholipids. How these membrane - microdomains are organized is in the current focus of interest for the identification of therapeutic targets. In recent studies SR-BI function as receptor for uptake of HDL into the liver and steroidogenic tissues could be demonstrated and the effectivity of this process is highly dependent on the phospholipid environment [2]. Cholesterol and phospholipid homeostasis in monocytes/macrophages and other cells involved in the atherosclerotic process is a critical determinant in atherosclerotic vessel disease. The phagocytic function of macrophages in host defense, tissue remodelling, uptake and lysosomal degradation of atherogenic lipoproteins and membrane fragments or other lipid containing particles has to be balanced by effective release mechanisms to avoid foam cell formation. HDL mediated reverse cholesterol transport, supported by endogenous ApoE and CETP synthesis and secretion provides an effective mechanism to release excessive cholesterol from macrophages and other vascular cells. Alternatively, reduced cholesterol and triglyceride/fatty acid absorption by intestinal mucosa cells as well as increased lipid secretion from hepatocytes into the bile will lower plasma lipids and the concentration of atherosclerotic lipoproteins. Summary of the invention New cholesterol responsive genes were identified with differential display method in human monocytes from peripheral blood that were subjected to macrophage differentiation and cholesterol loading with acetylated LDL and subsequent deloading with HDL In an initial screen ABCGl (ABC8). a member of the rapidly growing family of ABC (ATP-Binding Cassette) transport systems, that couple the energy of ATP hydrolysis to the translocation of solutes across biological membranes, was identified as a cholesterol sensitive switch. ABCGl is upregulated by M-CSF dependent phagocytic differentiation but expression is massively induced by cholesterol loading and almost completely set back to differentiation dependent levels by HDL In a more detailed analysis 37 already characterised ABC members and 8 Fragment - sequences (Table 2) were analysed in monocyte/macrophage cells by RT-PCR (linear range) for differentiation dependent changes and cholesterol sensitivity. Among the 45 tested ABC-transporter genes 18 of the characterized ABC transporters and 2 of the Fragment -sequence based ABC-transporters are cholesterol sensitive (Example 4). The cholesterol sensitive ABC-transporter are named according to the new ABC- nomenclature and listed in Table 3 with the new and the old designations, respectively. The most sensitive gene was ABCGl . ABCGl is the human homologue of the drosophila white gene. Sequencing of the promoter of ABCGl (Example 7) shows important transcription factor binding sites relevant for phagocytic differentiation and lipid sensitivity. Antisense treatment of macrophages during cholesterol loading and HDL mediated deloading clearly identified ABCGl as a cholesterol transporter and the efflux of choline-containing phospholipids (phosphatidylcholine. sphingomyelin) was also modulated. Northern- and Western-blot analysis provided further support that inhibition of cholesterol transport is associated with lower ABCGl mRNA expression and ABCGl protein levels (Example 5). Considerable evidence was derived from energy transfer experiments (Example 3) that ABCGl in the cell membrane is in a regulated functional cooperation (e.g. cell differentiation, activation, cholesterol loading and deloading) with other membrane receptors that have either transport- (e.g. LRP-LDL receptor related protein) or signalling- and adhesion-function (e.g. integrins, integrin associated proteins) which is also supported by sequence homology of extracellular domains as well as other parts of the ABCGl sequence. For example the protein sequence of the region of the third extracellular loop of ABCGl, i.e. aminoacid residues 580 through 644, shares homology with fibronectin (aa 317-327), integrinβ5 (aa 538-547), RAP (aa 1 19-127), LRP (aa 2874-2894), apoB-100 precursor (aa 4328-4369), glutathion-S-tranferase (aa 54-78) and glucose transporter (aa 371 -380). Sequence comparison of all cholesterol sensitive transporters indicates this as a general principle of ABC transporter function and regulation. Among the other cholesterol sensitive genes ABCAl (ABCl) was further characterized. ABCAl was identified in the mouse as an IL-lbeta transporter involved also in apoptotic cell processing. We show here, by RT-PCR (Table 2) and confirmation by Northern analysis, based on the newly detected human ABCAl cDNA sequence (Example 6), that ABCAl follows the same regulation as ABCGl. Moreover, the ABCAl -knockout mice (ABCAl-/-) show massively reduced levels of serum lipids and lipoproteins. The expression of ABCAl in mucosa cells of the small intestine and the altered lipoprotein metabolism in ABCAl-/- mice allows the conclusion that ABCAl plays a major role in intestinal absorption and translocation of lipids into the lymph-system Analysis of genetic defects that affect macrophage cholesterol homeostasis identified dysregulated ABCAl as a gene locus involved in the HDL-deficiency syndrome (Tangier-Disease). This disease is associated with hypertriglyceridemia and splenomegaly. Another as yet not described HDL-deficiency syndrome associated with early onset of coronary heart disease and psoriasis showed a dysregulation of the chromosome 17 associated ABC-sequences (ABCC4 (MRP3); ABCC3 (MRP3); ABCA5 (Fragment 90625); ABCA6 (Fragment 155051) : 17q21-24). This points to an association with the predicted gene locus for psoriasis at chromosome 17. A recently sequenced human ABC-transporter (ABCA8, Example 9) shows high homology to ABCAl and also belongs to the group of cholesterol sensitive ABC- transporter. ABCC5 (MRP5, sMRP) is a member of the MRP-subfamily among which ABCC2 (MRP2, cMOAT) was characterized as the hepatocyte canalicular membrane transporter that is involved in bilirubin glucoronide secretion [9] and identified as the gene locus for Dubin-Johnson Syndrome [10] a disorder associated with mild chronic conjugated hyperbilirubinemia. Furthermore, the identification of ABCAl as a transporter for IL-1 β identifies this gene as a candidate gene for treatment of inflammatory diseases including rheumatoid arthritis and septic shock. The cytokine IL-1 β is a broadly acting proinflammatory mediator that has been implicated in the pathogenesis of these diseases. Moreover, we could demonstrate, that glyburide as an inhibitor of IL-1 β secretion inhibits not only Caspase I mediated processing of pro-IL-1 β and release of mature IL-1 β but simultaneously inhibits ceramide formation from sphingomyelin mediated by neutral sphingomyelinase and thereby releases human fibroblasts from G 2 -phase cell cycle arrest. These data provide a further mechanism indicative for a function of ABCAl in signalling and cellular lipid metabolism. Autoimmune disorders that are associated with the antiphospholipid syndrome (e.g. lupus erythematodes) can be related to dysregulation of B-cell and T-cell function, aberrant antigen processing, or aberrations in the asymmetric distribution of membrane phospholipids. ABC-transporters are, besides their transport function, candidate genes for phospholipid translocases, floppases and scramblases that regulate phospholipid asymmetry (outer leaflet: PC+SPM; inner leaflet: PS+PE) of biological membranes [1 1]. There is considerable evidence for a dysregulation of the analysed ABC-transporters in patient cells. We conclude that these ABC-cassettes are also candidate genes for a genetic basis of antiphospholipid syndromes such as in Lupus erythematodes. In summary, the ABC genes ABCGl, ABCAl and the other cholesterol-sensitive ABC genes as specified herein, can be used for diagnostic and therapeutic applications as well as for biochemical or cell-based assays to screen for pharmacologically active compounds which can be used for treatment of lipid disorders, atherosclerosis or other inflammatory diseases. Thus it is an objective of the present invention to provide assays to screen for pharmacologically active compounds which can be used for treatment of lipid disorders, atherosclerosis or other inflammatory diseases. Further the invention provides tools to identify modulators of these genes and gene products. These modulators can be used for the treatment of lipid disorders, atherosclerosis or other inflammatory diseases or for the the preparation of medicaments for treatment of lipid disorders, atherosclerosis or other inflammatory diseases. The medicaments comprise besides the modulator acceptable and usefull pharmaceutical carriers. Abbreviations aa Amino acid ABC ATP-binding cassette ABCA# ATP-binding cassette, sub-family A (ABC 1 ), member # ABCB# ATP-binding cassette, sub-family B (MDR/TAP), member # ABCC# ATP-binding cassette, sub-family C (CFTR/MRP), member # ABCD# ATP-binding cassette, sub-family D (ALD), member # ABCE# ATP-binding cassette, sub-family E (OABP). member # ABCF# ATP-binding cassette, sub-family F (GCN20). member # ABCG# ATP-binding cassette, sub-family G (WHITE), member # ABCR Homo sapiens rim ABC transporter AcLDL Acetylated LDL ADP1 ATP-dependent permease ALDP Adrenoleukodystrophy protein ALDR Adrenoleukodystrophy related protein ApoA Apolipoprotein A ApoE Apolipoprotein E ARA Anthracycline resistance associated protein AS Antisense ATP Adenosine triphosphate CETP Cholesteryl ester transfer protein CFTR Cystic fibrosis transmembrane conductance regulator CGT ceramide glucoxyl transferase CH Cholesterol cMOAT Canalicular multispecific organic anion transporter dsRNA Double stranded RNA Fragment Gen Fragment FABP plasma membrane fatty acid binding protein FACS Fluorescence activated cell sorter FATP intracellular fatty acid binding protein FCS foetal calve serum FFA free fatty acids GAPDH Glyceraldehyde-3 -phosphate dehydrogenase GCN20 protein kinase that phosphorylates the alpha-subunit of translation initiation factor 2 GPI Glycosylphosphatidylinositol HaCaT keratinocytic cell line HDL High density lipoprotein HL Hepatic lipase HlyB haemolysin translocator protein B HMT1 yeast heavy metal tolerance protein HPTLC High performance thin layer chromatography IL Interleukin LCAT Lecithin holesterol acyltransferase LDL Low density lipoprotein LPL Lipoprotein lipase LRP LDL receptor related protein MDR Multidrug resistance MRP Multidrug resistance-associated protein PC Phosphatidylcholine PE Phosphatidylethanolamin PL Phospholipid PLTP Phospholipid transferprotein PMP peroxisomal membrane protein PS Phosphatidylserine RNA Ribonucleic acid RT-PCR Reverse transcription - polymerase chain reaction SDS Sodium dodecyl sulfate SL Sphingolpid sMRP Small form of MRP SPM Sphingomyelin SR-BI Scavenger receptor Bl SUR Sulfonylurea receptor TAP Antigen peptide transporter TG Triglycerides TSAP TNF-alpha stimulated ABC protein UTR untranslated region References cited 1. 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SCHMITZ G: Identification and characterization of a novel monocyte/macrophage differentiation-dependent gene that is responsive to lipopolysaccharide, ceramide. and lysophos- phatidylcholine. Biochem Biophys Res Commun 1997, 235:4-9. 22. LOHMANN J, SCHICKLE H, BOSCH TC: REN display, a rapid and efficient method for nonradioactive differential display and mRNA isolation. Biotechniques 1995, 18:200-202. 23. VIRCA GD, NORTHEMANN W, SHIELS BR. WIDERA G, BROOME S: Simplified northern blot hybridization using 5% sodium dodecyl sulfate. Biotechniques 1990, 8:370-371. 24. CHIRGWIN JM, PRZYBYLA AE, MACDONALD RJ, RUTTER WJ: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979, 18:5294-5299. 25. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein meaABCC8 (SUR)ement with the folin phenol reagent. J Biol Chem 193, 265-275. 1951. (GENERIC) Ref Type: Generic 26. BLIGH EG, DYER WJ: A rapid method of total lipid extraction and purification. Can J Biochem Phys 1959, 37:91 1-917. 7. ROGLER G, TRUMBACH B, KLIMA B, LACKNER KJ, SCHMITZ G: HDL-mediated efflux of intracellular cholesterol is impaired in fibroblasts from Tangier disease patients. Arterioscler Thromb Vase Biol 1995, 15:683- 690. Description of the Figures Figures 1 to 5 are showing nucleotide and protein sequences described in this application. The sequences are repeated in the sequence listing. Description of Tabels: Table 1: Levels of RNA transcripts of ABCGl (ABC8), ABCAl (ABCl ) and ABCA8 in human tissues were determined by Northern blot analysis of a multiple tissue dot-blot (Human RNA MasterBlot, Clontech Laboratories, Inc.. CA, USA ). The relative amount of expression is indicated by different numbers of filled circles. Table 2: The expression pattern of ABC-transporters in monocytes, monocyte derived macrophages (3 days cultivated monocytes in serum free Macrophage-SFM medium containing 50 ng/ml M-CSF), AcLDL incubated monocytes (3 days with 100 μg/ml) followed by HDL 3 (100 μg/ml) incubated monocytes is shown. Expressed genes are tested for cholesterol sensitivity by semiquantitative PCR. For known ABC-Transporter the chromosomal location and the transported molecules are also presented. Table 3: Disorders, that are associated with ABC-transporters are shown. The chromosomal location is indicated and the relevant accession number in OMIN (Online Mendelian Inheritance in Man). Table 4: Expression of ABC-Transporters in HaCaT keratinocytic cells during differentiation Table 1 Table 2: Cholesterol dependent gene regulation of human ABC transporters + = expressed 0 = not expressed nr=not regulated fl = upregulated l)= downregulated half (hs) or full size (fs) transporter as deduced from the mRNA size Table 3 Table 4: Expression of ABC-Transporters in HaCaT keratinocytic cells during differentiation + relative expression n d not determined φ upregulated Φ downregulated 7! Si Diphasic expression Description of specific embodiments Candidate gene identification during cholesterol loading and deloading of human monocyte derived macrophages In order to discover genes that are involved in the cholesterol loading and/or deloading in vitro assays were set up. Particularly, gene expression in human blood derived monocytes and macrophages elicited by cholesterol and its physiological transport formulation, i.e. various low density lipoprotein (LDL) particle species like AcLDL, was studied. Elutriated human monocytes were cultivated in M-CSF containing but serum free macrophage medium supplemented with AcLDL (100 μg protein/ml medium) for three days, followed by cholesterol depletion replacing AcLDL by HDL, (100 μg protein/ml medium) for twelve hours. Differential display screening for new candidate genes, regulated by cholesterol loading/deloading, was performed (Example 1). Identification of a new cholesterol sensitive gene ABCGl (ABC8) was discoverd as a novel cholesterol sensitive gene. ABCGl belongs to the ATP binding cassette (ABC) transporter gene family. ABCGl was recently published as the human analogue of the drosophila white gene [6-8]. The gene is strongly upregulated by AcLDL-mediated cholesterol loading, and almost completely downregulated by HDL, mediated-cholesterol deloading. as confirmed by Northern blot (Example 2). Nothern blot analysis oh mRNA from human monocyte-derived macrophages obtained from the peripherical blood probands clearly show upregulation of ABCGl mRNA formation upon AcLDL incubation. In sharp contrast, ABCGl mRNA expression was decreased in such macrophages upon incubation with HDL, containing medium. ABCG1 expression in cholesterol loaded and deloaded cells after four days pre- differentiation For effective cholesterol loading monocytes must be differentiated to phagocytic- macrophage like cells. During this period scavenger receptors are upregulated and promote AcLDL uptake leading to cholesteryl ester accumulation. After four days preincubation period we have incubated the cells for one, two and three days with AcLDL (100 μg/ml) to show cholesteryl ester accumulation. After two days of loading we deloaded the cells with HDL 3 for 12 hours, 24 hours and 48 hours, respectively. ABCGl is time dependently upregulated during the AcLDL loading period and downregulated by HDL, deloading (Examples 2 and 3) In order to confirm time dependent increase of ABCGl mRNA expression after AcLDL challenge in human monocyte derived macrophages, Nothern blot analyses for ABCGl mRNA quantification were made, RNA samples from the macrophages were harvested at day zero and day four as controls and mRNA samples were taken one, two, and three days after AcLDL treatment of macrophages. which started at day four. A dramatic increase of ABCGl mRNA content of the macrophages could be detected from day five through day seven by Nothern blot analyses. This regulation shows the same pattern as changes of cellular cholesteryl ester content (Example3). Cholesterol ester accumulation starts in monocyte-derived macrophages upon AcLDL stimulation from a base level below 5 nmol/mg cell protein at day four up to 120 nmol/mg cell protein at day seven (i.e. three days after AcLDL application). Tissue expression Besides cholesterol loaded macrophages ABCGl is prominently expressed in brain, spleen, lung, placenta, adrenal gland, thymus and fetal tissues (Table 1 ). Chromosomal location and associated genes and diseases The ABCGl gene maps to human chromosome 21q 22.3. Also localized in this region 21q 22.3 are the following genes: integrin β 2 (CD 18), brain specific polypeptide 19, down syndrome cell adhesion molecule, dsRNA specific adenosine deaminase, cystathionine β synthase, collagen VI alpha-2. collagen XVIII alpha-1, autosomal recessive deafness, and amyloid beta precursor. This chromosomal region is in close proximity to other regions involved in Down syndrome, autosomal dominant bipolar affective disorder, and autosomal recessive non-syndromic deafness. Extracellular loop of ABCGl (ABC8) for antibody generation The putative structure of the hydrophobic transmembrane region of ABCGl shows 6 transmembrane spanning domains, and 3 extracellular loops, two of them are 9- and 8-amino acids-long, respectively, while the third one is 66-amino acids-long. The larger one of the two intracellular loops consists of 30 amino acids. Similarity- survey in protein databases for homologies the 3rd extracellular loop (Illex) with other genes resulted in the identification of fibronectin, integrinβ5, RAP, LRP (LDL receptor related protein) apo-lipoprotein B 100 precursor protein, glutathion S- transferase and glucose transporter. A polyclonal antiserum was generated against the 3rd extracellular loop (Illex) of ABCGl in order to perform flow cytometric analysis, energy transfer experiments and Western-blotting (see Example 3). In the amino acid sequence of ABCGl the 3rd extracellular loop (Illex) comprises 66 amino acids comprises 66 amino acids from amino acid 580 through 644. The peptide fragment for antibody generation comprises the amino acid residues 613 through 628 of ABCGl polypeptide. ABCGl obviously interacts with endogenous sequence motivs with other membrane receptors involved in transport (e.g. LRP, RAP), signalling and adhesion (e.g. integrins, integrin associated proteins) as a basis of ABCGl -function and regulation. Moreover sequence comparisons of all ABC-transporters listed in Table 3 indicates functional cooperation with other membrane receptors as a general principle of the whole gene family. Subfamily-Analysis Evolutionary relationship studies with the whole ABC transporter family have shown that ABCGl (ABC8) forms a subfamily together ABCG2 (estl 57481) and this subfamily is closely related to the full-size transporters ABCAl (ABC l ). ABCA2 (ABC2), ABCA3 (ABC3), ABCA4 (ABCR) and the half-size transporter ABCF1 (TSAP) . Recent studies by Allikmets et al. have identified 21 new genes as ABC transporters by expressed sequence tags database search [13]. General description of the ABC transporter family The ATP-binding cassette (ABC) transporter superfamily contains some of the most functionally diverse proteins known. Most of the members of the ABC family (also called traffic ATP-ases) function as ATP-dependent active transporters (Table 3). The typical functional unit consists of a pair of ATP-binding domains and a set of transmembrane (TM) domains. The TM-domains determine the specificity for the type of molecule transported, and the ATP-binding domains provide the energy to move the molecule through the membrane [14; 15]. The variety of substrates handled by different ABC-transporters is enormous and ranges from ions to peptides. Specific transporters are found for nutrients, endogenous toxins, xenobiotics. peptides, aminoacids, sugars, organic/inorganic ions, vitamins, steroid hormones and drugs [16; 17]. ABC-transporter associated diseases The search for human disease genes (Table 3) provided a number of previously undiscovered ABC proteins [16]. The best characterized disease caused by a mutation in an ABC transporter is cystic fibrosis (ABCC7 (CFTR)). Inherited disorders of peroxisomal metabolism as Adrenoleukodystrophy and Zellweger's syndrome also show alterations in ABC transporters. They are involved in peroxisomal beta-oxidation, necessary for very long chain fatty acid metabolism [18]. Antisense against ABCGl inhibits cholesterol efflux to HDL 3 Since ABCGl is a cholesterol sensitive gene and other ABC transporters are known to be involved in certain lipid transport processes, the question arises whether ABCGl plays a role in transport of cholesterol, phospholipids, fatty acids or glycerols. Therefore antisense experiments were performed to test the influence of ABCGl on lipid loading and deloading. The inhibition of ABCGl with specific antisense oligonucleotides decreased the efflux of cholesterol and phosphatidyl- choline to HDL,. (Example 5) Other cholesterol sensitive ABC transporter Cloning and sequencing of the human ABCAl (ABCl) provided the information to characterize ABCAl for cholesterol sensitivity, and tissue distribution (Example 6). Another cholesterol sensitive human ABC transporter (ABCA8 ) has been cloned and sequenced (Example 8) Characterization of the ABCGl promoter region The ABCGl promoter has the characteristic binding sites for transcription factors that are involved in the differentiation of monocytes into phagocytic macrophages. The cholesterol sensitivity of the expression of ABCGl is represented by the transcription factor pattern that is relevant for phagocytic differentiation (Example 7). Examples Example 1 Identification of cholesterol loading and deloading candidate genes Monocyte isolation and cell culture Monocytes were obtained from peripheral blood of healthy normolipidemic volunteers by leukapheresis and purified by counterflow elutriation. Purity of isolated monocytes was >95% as revealed by FACS analysis. l OxlO 6 monocytes were seeded into 100 mm 2 diameters cell culture dishes under serum free conditions in macrophage medium for 12 hours in a humidified 37°C incubator maintained with a 5% CO2, 95% air atmosphere. After 12 hours medium containing unattached cells was replaced by fresh macrophage medium supplemented with 50 ng/ml human recombinant M-CSF (this medium is the standard medium for any further incubations). Isolation of lipoproteins and preparation of AcLDL Lipoproteins were prepared from human plasma from healthy volunteer donors by standard sequential ultracentrifugation methods in a Beckman L-70 ultracentrifuge equipped with a 70 Ti rotor at 4°C to obtain LDL (d= 1 ,006 to 1,063 g/ml) and HDL, (d=l,125 to 1,21 g/ml). All densities were adjusted with solid KBr. Lipoprotein fractions are extensively dialyzed with phosphate-buffered saline (PBS) containing 5 mM EDTA. The final dialysis step was in 0,15 mol/L NaCl in the absence of EDTA. Lipoproteins were made sterile by filtration through a 0.45 μm (pore-size) sterile filter (Sartorius). LDL was acetylated by repeated addition of acetic anhydride followed by dialysis against PBS [19]. Modified LDL showed enhanced mobility on agarose gel electrophoresis. Incubation of monocyte-macrophages with AcLDL and HDL 3 After 12 hours of preincubation cells were grown in the presence or absence (control) of 100 μg protein /ml AcLDL for further 3 day in medium. Then, the incubation medium was replaced with fresh medium and incubated with or without the addition of HDL 3 (100 μg/ml) for another 12 hours. Differential display Differential display screening was performed for new candidate genes that are regulated by cholesterol loading/deloading as described [20; 21]. In brief, 0,2 μg of total RNA isolated from monocytes at various incubations was reverse transcribed with specific anchored oligo-dT primers, using a commercially available kit (GeneAmp RNA PCR Core Kit, Perkin Elmer, Germany). The oligo-dT primers used had two additional nucleotides at their 3' end consisting of an invariable A at the second last position (3'-end) and A, C, G or T at the last position to allow a subset of mRNAs to be reverse transcribed. Here, a 13-mer oligo-dT (TI 01 : 5'Tl lAG-2' ) was used in a 20-μl reaction at 2,5 μM concentration. One tenth of the cDNA was amplified in a 20-μl PCR reaction using the same oligo-dT and an arbitrary 10-mer upstream primer (D20 5'-GATCAATCGC-3'), 2,5 μM each, using 2,5 units of TAQ DNA Polymerase and 1.25 mM MgC12. Amplification was for 40 cycles with denaturation at 94°C for 30 sec, annealing at 41 °C for 1 min and elongation at 72°C for 30 sec with a 5 min extension at 72°C following the last cycle. All PCR reactions were carried out in a Perkin Elmer 9600 thermocycler (Perkin Elmer, Germany). PCR-products were separated on ready to use 10% polyacrylamide gels with a 5% stacking gel (CleanGel Large- 10/40 ETC, Germany) under non-denaturating conditions using the Multiphor II electrophoresis apparatus (Pharmacia, Germany). The DNA fragments were visualized by silverstaining of the gel as previously described [22]. Cloning and sequencing of differentially expressed cDNAs cDNA bands of interest were cut out of the gel and DNA was isolated by boiling the gel slice for 10 min in 20 μl of water. A 4 μl aliquot was used for the following PCR- reaction in a 20μl volume. The cDNA was reamplified using the same primer set and PCR conditions as above, except, that the final dNTP concentration was ImM each. Reamplified cDNAs were cloned in the pUC18-vector using ABCC8 (SUR)eClone- Kit (Pharmacia), sequenced on an automated fluorescence DNA sequencer using the AutoRead Sequencing Kit (Pharmacia, Germany) and used as probes for Northern blot analysis [23]. Example 2 Northern Blot analyses of monocytes and macrophages after 3 days AcLDL incubation followed by 12 hours HDL 3 incubation Elutriated monocytes were incubated with AcLDL (100 μg/ml medium) for 2.5 days or differentiated for the same time without the addition of AcLDL as control. ABCGl (ABC8) expression is 4 times stronger upregulated with AcLDL incubation than in differentiated monocytes .After the AcLDL incubation period cells were washed and incubated with HDL, for the next 12 hours or with medium alone as control. ABCGl expression is almost completely downregulated by HDL3 incubation and only moderatly decreased in control incubation as confirmed by Northern blot. For effective cholesterol loading monocytes must be differentiated to macrophage like cells. During this period scavenger receptors are upregulated and promote AcLDL uptake leading to cholesteryl ester accumulation. To differentiated the cells prior to AcLDL-dependent cholesterol loading, we cultured the cells for four days in standard medium. At day four, cells were washed and incubated with AcLDL (lOOμg/ml medium) or in the absence of AcLDL as control for further one. two and three days to load the cells with cholesterol. At each timepoint cells were lysed with 0.1 % SDS and lipid was extracted as described in materials and methods and cellular cholesteryl ester was determined by HPTLC-separation. Cells were loaded time dependently up to 120 nmol/mg cell protein after 3 days AcLDL loading, whereas in unloaded cells no cholesteryl ester accumulation could be observed. To distinguish HDL, dependent and independent cholesterol efflux cells were pulsed with AcLDL (100 μg/ml) for three days with the coincubation of M C-cholesterol (1,5 μCi/ml medium). Cells were washed and deloaded with HDL, ( 100 μg/ml) for 12 hours, 24 hours and 48 hours, respectively. Cells were incubated without the addition of exogenous lipid-acceptors as a control. After chase period the content of l C- cholesterol was determined in the medium and in the cells by liquid scintillation as described in material and methods. The efflux of cholesterol is expressed in percent of cellular DPMs of total DPMs (counts in the cells plus medium) With HDL, the efflux is faster and more intense, than the efflux without the addition of HDL, as an endogenous lipid acceptor. After 12 hours cellular cholesterol content was reduced to 68 % with HDL,-dependent deloading, and 86 % in HDL,-independent deloading. After 48 hours only 35 % of loaded 14C-cholesterol was observed in the cells treated with HDL, . In contrast, 70 % of loaded l4 C-cholesterol was found in untreated cells In AcLDL pulsed cells the RNA-expression of ABCGl is upregulated whereas no upregulation appears in the cells that were not loaded with AcLDL. Cells that were loaded for two days with AcLDL were deloaded with HDL, for 12. 24 and 48 hours (12h; 24h; 48h), and in the absence of exogenous lipid acceptors. The RNA- expression is downregulated again, in HDL 3 treated cells more intense than in cells treatet without any exogenous lipid acceptor. Materials: Macrophage medium (Macrophage-SFM) was obtained from Gibco Life Technologies, Germany. Human recombinant M-CSF was obtained from Genzyme Diagnostics, Germany, and antisense phosphorothioate oligonucleotides were supplied by Biognostics, Germany. All other chemicals were purchased from Sigma. Nylon membranes and a32P-dCTP were obtained from Amersham, Germany, 14C- cholesterol and 3H-choline chloride from NEN, Germany, and cell culture dishes are Becton Dickinson, Germany Isolation of total RNA and northern blotting Total RNA was isolated at each time-point, before and after AcLDL incubation, and after HDL 3 incubation, respectivly, Washed cells were solubilized in guanidine isothiocyanate followed by sedimentation of the extract through cesium chloride [24]. For Northern analysis. 10 μg/lane of total RNA samples were fractionated by electrophoresis in 1 ,2% agarose agarose gel containing 6% formaldehyde and blotted onto nylon membranes (Schleicher & Schϋll, Germany). After crosslinking with UV- irradiation (Stratalinker model 1800, Stratagene, USA), the membranes were hybridized with a cDNA probe for ABCGl (ABC8). Hybridization and washing conditions were performed as recommended by the manufacturer of the membrane. Example 3 Westernblot analysis of monocytes and macrophages after cholesterol loading and deloading Protein expression of ABCGl (ABC8) is upregulated in AcLDL-loaded and dovvn- regulated in HDL 3 -deloaded monocyte-derived macrophages. Western blotting with a peptide antibody against ABCGl as described in materials and methods is performed with 40 μg of total protein for each lane of SDS-PAGE. ABCGl -protein expression is shown in freshly isolated monocytes (day zero) and in differentiated monocytes (day four). From day four to day seven (5d; 6d; 7d) monocyte-derived macrophages were loaded with AcLDL or without AcLDL as control. AcLDL loaded cells from day 6 (6d) were deloaded with HDL, for 12, 24, and 48 hours and without exogenous added HDL lipid-acceptor. AcLDL increases the protein-expression, whereas HDL, decreases the expression to normal levels again. Protein isolation and determination At each timepoint cells were lysed with 0.1% SDS and the protein content was determined by the method of Lowry et al. [25]. Generation of ABCGl specific antibodies ABCGl specific peptide antibodies were generated by immunization of chickens and rabbits with a synthetic peptide (Fa. Pineda, Berlin). The peptide sequence was chosen from the extracellular domain exIII amino acid residues 613-628 of ABCGl comprising the amino acids REDLHCDIDETCHFQ (see sequence listing ID No. 53). After 58 days of immunization western blotting was performed with 1 : 1000 diluted serum and 1 : 10000 secondary peroxidase labelled antibody. Electrophoresis and immunoblotting SDS-polyacrylamide gelelectrophoresis was performed with 40μg total cellular protein per lane. Proteins were transferred to Immobilon as reported. Transfer was confirmed by Coomassie Blue staining of the gel after the electroblot. After blocking for at least 2 hours in 5% nonfat dry milk the blot was washed 3 times for 15 minutes in PBS. Antiserum generated as described was used at 1 :1000 dilution in 5% nonfat dry milk in PBS. The blot was incubated for 1 hour. After 4 times washing with PBS at room-temperature a secondary peroxidase-labelled rabbit anti chicken IgG- antibody (1 :10000 diluted, Sigma) was incubated in 5% nonfat dry milk in PBS for 1 hour. After 2 times washing with PBS, detection of the immune complexes was carried out with the ECL Western blot detection system (Amersham International PLC, UK). Fluorescence resonance energy transfer: Monocytes were labelled with the specific antibodies for 15 minutes on ice, one antibody is labelled by biotin, the other one is labelled by phycoerythrin. After washing the cells were incubated with a Cy5-conjugated streptavidin for another 15 minutes. Distances between antibody labelled proteins on the cell surface is measured by energy transfer with a FACScan (Becton Dickinson). Following single laser excitation at 488 nm the Cy5 specific emmission represents an indirect excitation of Cy5 dependent on the proximity of the PE-conjugated antibody. The relative transfer efficiency was calculated following standardisation for the intensity of PE and Cy5 labelling and nonspecific overlap of fluorescence based on dual laser excitation and comparison to separately stained control samples. Example 4 Cholesterol sensitivity of ABCGl (ABC8) and other members of the ABC- transporter family The influence of cholesterol loading and deloading on other members of the ABC- family was also investigated to find out the potential second half-size ABC transporter. Further analysis has been performed to examine the expression pattern of all human ABC transporters in monocytes and monocyte derived macrophages as well as in cholesterol loaden and deloaden mononuclear phagocytes. The experiments were performed by RT-PCR with cycle-variation to compare the expression in the quantitative part of the distinct PCR. Primer sets were generated from the published sequences of the ABC-transporters. A RT-PCR with GAPDH primers was used as control. Several ABC-transporters are also cholesterol sensitive which further supports the function of ABC-transporters in cellular lipid trafficking (Table 2). Semi-quantitative RT-PCR All known ABC-transporters are tested for AcLDL/HDL, sensitive regulation of expression using RT-PCR with cycle-variation to compare the expression in the quantitative part of the distinct PCR. 1 μg of total RNA was used in a 40 μl reverse transcription reaction, using the Reverse Transkription System (Promega, Corp. WI, USA). Aliquots of 5 μl of this RT-reaction was used in 50μl PCR reaction. After denaturing for 1,5 min at 94°C, 35 or less cycles of PCR were performed with 92,3 °C for 44s, 60,8°C for 40s (standard annealing temperature differs in certain primer-combinations), 71,5°C for 46s followed by a final 5-min extension at 72°C. The Primer sets were generated from the published sequences of the ABC- transporters. A RT-PCR with primers specific for GAPDH was performed as control. The expression pattern of ABC-transporters in monocytes, monocyte derived macrophages (3 days cultivated monocytes in serum free macrophage-SFM medium containing 50 ng/ml M-CSF), AcLDL incubated monocytes (3 days with 100 μg/ml) followed by HDL 3 (100 μg/ml) incubated monocytes is shown in Table 2. Expressed genes are tested for cholesterol sensitivity by semi-quantitative PCR. Example 5: Functional analyses of the cholesterol sensitive ABCGl (ABC8) transporter gene by antisense oligonucleotide experiments Antisense experiments were conducted in order to address the question, that beyond being regulated by cholesterol loading and deloading ABCGl is directly involved in lipid loading and deloading processes. In various experiments antisense oligonucleotides decreased the efflux of cholesterol and phosphatidylcholine to HDL,. During the loading period with AcLDL the cells were coincubated with 17 different antisense oligonucleotides. To measure the efflux of cholesterol and phospholipids the cells were pulsed in the loading period with 1,5 μCi/ml 14 C-cholesterol and 3μCi/ml 3 H-choline chloride. The medium was changed and during the chase period cells were incubated with or without HDL, for 12 hours. The 14 C-cholesterol and Η-choline content in the medium and in the cell lysate was measured and the efflux was determined in percent of total l4 C-cholesterol and 3 H- choline loading. The most effective antisense oligonucleotide (AS Nr.2) inhibited cholesterol and phospholipids efflux relative to cells that were treated with control antisense (AS control). A dose dependent decrease in cholesterol efflux of 16,79% (5nmol AS) and 32,01% (10 nmol AS) could be shown, respectively. Antisense incubation To inhibit the induction of ABCGl cells were treated with three different antisense oligonucleotides targeting ABCGl or one scrambled control-antisense oligonucleotide during the AcLDL-incubation period. Determination of cholesterol and phosphatidylcholine efflux from monocytes in dependency of antisense oligonucleotide treatment To measure the efflux of cholesterol and phospholipids the cells were pulsed in addition to AcLDL-incubation with 1,5 μCi/ml , C-cholesterol and 3μCi/ml H- choline chloride. The medium was changed and in chase period the cells were incubated with or without HDL, for 12 hours. Lipid extraction was performed according to the method of Bligh and Dyer [26]. The l4 C-cholesterol and 3 H-choline content in the medium and in the cell lysate was measured by liquid scintillation counting and the efflux was determined in percent of total l C-cholesterol and 3 H- choline loading as described [27] Computer analyses DNA and protein sequence analyses were conducted using programs provided by HUSAR, Heidelberg, Germany: http://genius.embnet.dkfz-heidelberg.de:8080. Example 6 Complete cDNA sequence of the human ATP binding cassette transporter 1 (ABCAl (ABCl)) and assessing the cholesterol sensitive regulation of ABCAl mRNA expression cDNA Cloning and Primary Protein Structure We have cloned a 6880-bp cDNA containing the complete coding region of the human ABCAl gene (Figure 8) The open reading frame of 6603 bp encodes a 2201- amino acid protein with a predicted molecular weight of 220 kDa. This protein displays a 94% identity on the amino acid level in an alignment with mouse ABCAl and can therefore be considered as the human ortholog. Tissue Distribution of ABCAl mRNA Expression In order to examine the tissue-specific expression of ABCAl a multiple tissue RNA master blot containing poly A + RNA from 50 human tissues was carried out. Northern Blot analysis demonstrates the presence of a ABCAl specific signal in all tissues. It is mostly prominent in adrenal gland, liver, lung, placenta and all fetal tissues examined so far (Table 1). The weakest signals are found in kidney, pancreas, pituitary gland, mammary gland and bone marrow. Sterol Regulation of ABCAl mRNA Expression In order to determine the regulation of ABCAl in monocytes/macrophages during cholesterol loading/depletion Northern Blot analysis was performed. The cloned 1000-bp DNA fragment derived from PCR amplification of RNA from five day differentiated monocytes with primers ABCAl 3622f (CGTCAGCACTCTGATGATGGCCTG-T) and ABCAl 4620r (TCTCTGCTATCTCCAACCTCA-V) was hybridized to Northern Blots containing RNA of differentially cultivated monocytes (figure 12) As can be seen in lanes one to five, the ABCAl mRNA is increased during in vitro differentiation of freshly isolated monocytes until day five. Longer cultivation results in a total loss of expression. When the cells were incubated in the presence of AcLDL to induce sterol loading (lanes 6-8) beginning at day four, a much stronger accumulation of mRNA can be detected in comparison to control cells (lanes 2-5). When these cells were cultured with HDL, as cholesterol acceptor for 12h, 24h and 48h (lanes 9-1 1) the ABCAl signal significantly decreases with respect to control cells incubated in the absence of HDL 3 (lanes 12-14). Taken together, these results indicate that ABCAl is a sterol-sensitive gene which is induced by cholesterol loading and downregulated by cholesterol depletion. Cell culture. Peripheral blood monocytes were isolated by leukapheresis and counterflow elutriation (19JBC). To obtain fractions containing >90% CD 14 positive mononuclear phagocytes, cells were pooled and cultured on plastic Petri dishes in macrophage SFM medium (Gibco BRL) containing 25 U/ml recombinant human M- CSF (Genzyme) for various times in 5% CO 2 in air at 37°C. The cells were incubated in the absence (differentiation control) or presence of AcLDL (100 μg/ml) to induce sterol loading. Following this incubation the cells were cultured in fresh medium supplemented with or without HDL 3 (100 μg/ml) for additional times in order to achieve cholesterol efflux from the cells to its acceptor HDL,. Preparation of RNA and Northern blot analysis. Total cellular RNA was isolated from the cells by guanidium isothiocyanate lysis and CsCl centrifugation (Chirgwin). The RNA isolated was quantitated spectrophotometrically and 15 μg samples were separated on a 1.2% agarose- formaldehyde gel and transferred to a nylon membrane (Schleicher & Schϋll). After crosslinking with UV-irradiation (Stratalinker model 1800. Stratagene), the membranes were hybridized with a 1000 bp DNA fragment derived from PCR amplification with primers ABCAl 3622f and ABCAl 4620r, stripped and subsequently hybridized with a human β-actin probe. In order to determine the tissue-specific expression of ABCAl a multiple tissue RNA master blot containing poly A + RNA from 50 human tissues was purchased from Clontech. The probes were radiolabeled with [γ- 32 P]dCTP (Amersham) using the Oligolabeling kit from Pharmacia. Hybridization and washing conditions were performed follwoing the method described previously (Virca). cDNA cloning of human ABCAl Based on sequence information of mouse ABCAl cDNA we designed primers for RT-PCR analysis in order to amplify the human ABCAl (ABCl ) cDNA. Approximately 1 μg of RNA from five day differentiated mononuclear phagocytes was reverse transcribed in a 20 μl reaction using the RNA PCR Core Kit from Perkin Elmer. An aliquot of the cDNA was used in a 100 μl PCR reaction performed with Amplitaq Gold (Perkin Elmer) and the following primer combinations: (primer names indicate the position in the corresponding mouse cDNA sequence): mABCl-144f(5 '-CAAACATGTCAGCTGTTACTGGA-3 ') and mABCl-643r (5 '-TAGCCTTGCAAA-AATACCTTCTG-3 ' ), mABCl-1221f(5 '-GTTGGAAAGATTCTCTATACACCTG-3 ') and mABCl-1910r (5 ' -CGTCAGCACTCTGATGATGGCCTG-3 ' ), mABCl-3622f(5 '-TCTCTGCTATCTCCAACCTCA-3 ') and mABCl-4620r (5'-ACGTCTTCACCAGGTAATCTGAA-3 ' ), mABCl-5056f(5'-CTATCTGTGTCATCTTTGCGATG-3 ') and mABCl-5857r (5 '-CGCTTCCTCCTATAGATCTTGGT-3 ' ), mABCl-6093f(5 ' -AAGAGAGCATGTGGA-GTTCTTTG-3 ' ) and mABCl-7051r (5 '-CCCTGTAATGGAATTGTGTTCTC-3 '), hABCl-540f(5'-AACCTTCTCTGGGTTCCTGTATC-3 ' ) and hABCl-1300r (5 ' -AGTTCCTGGAA-GGTCTTGTTCAC-3 '), hABCl -183 If (5 '-GCTGACCCCTTTGAGGACA TGCG-3 ') and hABCl-3701r (5 '-ATAGGTCAGCTCATGCCCTATGT-3 '), hABCl-4532f(5 '-GCTGCC-TCCTCCACAAAGAAAAC-3 ') and hABCl-5134r (5 '-GCTTTGCTGACCCGCTCC-TGGATC-3 '), hABCl-5800f(5 '-GAGGCCAGAATGACATCTTAGAA-3 ') and hABCl-6259r (5 '-CTTGACAACACTTAGGGCACAA T-3 '). All PCR products were cloned into the pUC18 plasmid vector and the nucleotide sequences were determined on a Pharmacia ALFexpress sequencer using the dideoxy chain-termination method and fluorescent dye-labeled primers. Example 7 Identification of the 5 'end of ABCGl We could partially prove the 5'-end of ABCGl published by Chen [7] that differs from the 5 '-end published by Croop [6] obtained from the mRNA of human monocytes/macrophages using a 5' RACE approach. In detail the sequence according to Chen et al. downstream of position 25 was in agreement with our own data. In contrast, our identified sequence differs from the one reported by Chen [7] and Croop [6] at a site upstream of position 25 (Chen [7]). The sequence SEQ ID NO: 32 shows the newly identified 5 '-end followed by the sequence published by Chen [7] from position 25. Molecular cloning and characterisation of the ABCGl 5 'UTR We identified several fragments by screening of a λ phage library which contained a total of app. 3 kb of the 5' UTR upstream sequence of the human ABCGl gene. The sequence that comprises the 5 'UTR and part of exon 1 (described above) are given in SEQ ID NO: 54. The promoter activity of this sequence was proven by luciferase reporter gene assays in transiently transfected CHO cells. Putative transcription factor binding sites within the promoter region with the highest likelihood ratio for the matched sequence as deduced from the TransFac database, GFB, Braunschweig, Germany. Multiple binding sites for SP-1, AP-1, AP-2 and CCAAT-binding factor (C EBP family) are present within the first 1 kb of the putative promoter region. Additionally, a transcription factor binding site involved in the regulation of apolipoprotein B was identified. Example 8 Characterization of the human ABCA8 full length cDNA The putative ABCA8 coding sequence is app. 6.5 kb in size. We successfully cloned and sequenced a lkb segment of the human ABCA8 cDNA that encodes the putative second nucleotide binding site of the mature polypeptide (the sequence is shown in the sequence listing). The nucleotide sequence exhibits a 73% homology with the known human ABCAl (ABCl) cDNA sequence. We identified an alternative transcript in the cloned 1 kb coding region which consists of a 72 bp segment (see sequence listing). Genomic analysis of this region revealed that the alternative sequence is identical with a complete intron suggesting that the alternative mRNA is generated by intron retention. The retained intron introduces a preterminal stop codon and thus may code for a truncated ABCA8 variant. ABCA8 also shows a cholesterol sensitive regulation of the mRNA expression (Table 2). Tissue expression of ABCA8 is shown in table 1. Example 9 Characterisation of the regulation of ABC transporter during differentiation of keratinocytic cells (HaCaT) Differentiation of epidermal keratinocytes is accompanied by the synthesis of specific lipids composed mainly of sphingolipids (SL), free fatty acids (FFA ), cholesterol (CH), and cholesterol sulfate, all involved in the establishment of the epidermal permeability barrier. The skin and, in particular, the proliferating layer of the epidermis is one of the most active sites of lipid synthesis in the entire organism. Cholesterol synthesis in normal human epidermis is LDL-independent, and circulating cholesterol levels do not affect the cutaneous de novo cholesterol synthesis. Fully differentiated normal human keratinocytes lack LDL receptors or its expression is very low, whereas in the normal human epidermis only basal cells express LDL receptors. During keratinocyte differentiation a shift from polar glycerophospholipids to neutral lipids (FFA, TG) and also a replacement of short chain FFA by long chain highly saturated FFA is observed. The most important lipids for the barrier function of the skin are sphingolipids that account for one third of the lipids in the cornified layer, and consist of a large ceramide fraction as a result of glucosylceramide degradation by intercellular glycosidases and de novo synthesis of ceramide . Glucosylceramide is synthesized intracellulary and stored in lamellar bodies and glucosylceramide synthase expression was found up-regulated during the differentiation of human keratinocytes. Cholesterol sulfate is formed by the action of cholesterol sulfotransferase during keratinocyte differentiation . Cholesterol sulfate and the degrading enzyme steroid sulfatase are present in all viable epidermal layers, with the highest levels in the stratum granulosum. The gradient of cholesterol sulfate content across the stratum corneum (from inner to outer layers), and progressive desulfation of cholesterol sulfate regulate cell cohesiveness and normal stratum corneum keratinization and desquamation, respectively. Cholesterol sulfate induces transglutaminase 1 and the coordinate regulation of both factors is essential for normal keratinization . The final step in lipid barrier formation involves lamellar body secretion and the subsequent post-secretory processing of polar lipids into their nonpolar lipid products through the action of hydrolytic enzymes that are simultaneously released (β- glucocerebrosidase, phospholipases, steroid sulfatase, acid sphingomyelinase). Disruption of the permeability barrier results in an increased cholesterol, fatty acid, and ceramide synthesis in the underlying epidermis. It has been shown that mRNA levels for the key enzymes required for cholesterol, fatty acid, and ceramide synthesis increased rapidly after artificial barrier disruption . Currently the lipid transport systems in keratinocytes are poorly characterized. Several fatty acid transport related proteins have been identified in keratinocytes: plasma membrane fatty acid transport proteins (FATP) and intracellular fatty acid binding proteins (FABPs), most of them exhibiting high affinity for essential fatty acids. The expression of epidermal FABPs is up-regulated in hyperproliferative and inflammatory skin diseases, during keratinocyte differentiation and barrier disruption Based on our data on macrophages, we propose several ABC transporters as putative candidates for cellular lipid export in keratinocytes. We have examined the expres- sion of all known ABC transporters during HaCaT cells differentiation. The human HaCaT cell line has a full epidermal differentiation capacity. Keratinocytes grown in vitro as a monolayer at low calcium concentration (< 0.1 mM ) can be differentiated by increasing calcium concentration in the culture medium ( 1 -2 mM ) . We cultured HaCaT cells as a monolayer in calcium-free RMPI (Gibco) medium mixed with standard Ham's F12 medium at a ratio 3:1 supplemented with 10% chelex -treated FCS, Penicillin and Streptomycin. The final concentration of calcium in above medium was 0.06 mM. When the cells reached confluence (usually on 5 lh day of the culture), calcium concentration was enhanced up to the level of 1.2 mM. The cells were seeded at a density of 2x10 5 / cm 2 in 60 mm culture dishes. The culture medium was replaced every two day and the cells were harvested after 24 h. 48h h, 4 d, 6 da, 8 d and 10 d in culture, respectively. Total RNA from HaCaT cells was isolated using the isothiocyanate/cesium chloride-ultracentrifugation method. The expression of all known human ABC transporters was examined during HaCaT cell differentiation (24 h, 48 h, 4 d, 6 d, 8 d, lOd, respectively) using a semi- quantitative RT-PCR approach (Table 6). The primer sets were generated from the published sequences of the ABC-transporters. Primers specific for GAPDH were used as a control. As a marker of keratinocyte differentiation CGT (ceramide glucosyl transferase) gene expression was assessed. Three of the transporters examined, ABCB1 (MDR1), ABCB4 (MDR3), ABCD3 (PMP70), were not expressed. ABCC6 (MRP6), ABCAl (ABC1),ABCD2 (ALDR and ABCB9 (estl22234) were expressed at low levels (Table 6) Most of the other transporters exhibited a biphasic expression pattern or were downregulated during keratinocyte differentiation. There was, however, a high expression of ABCGl (ABC8), ABCA8 (new) and ABCC3 (MRP3) indicative for their involvement in terminal keratinocyte lipid secretion for cholesterol, FFAs and ceramide-backbone lipids.. The two peroxisomal ABC transporters, ABCD2 (ALDR) and ABCD1 (ALDP) that mediate the transport of very long chain fatty acids into peroxisomes were initially expressed at relatively low levels and subsequently downregulated during differentiation. This is in agreement with the replacement of short chain fatty acids by very long chain fatty acids during keratinocyte differentiation. Example 10: Sequencing of ABCAl cDNA and genomic structure in five families of patients with Tangier disease revealed different mutations in the ABCAl gene locus. These patients have different mutations at different positions in the ABCAl gene, that result in changes in the protein structure of ABCAl. Family members that are heterozygous for these mutations show lowered levels of serum HDL, whereas the homocygote patients have extremely reduced HDL serum levels.

Claims: Claims: 1. A polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide encoding the polypeptide as set forth in SEQ ID NO:2; (b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a polynucleotide fragment of the polynucleotide of (a) or (b). 2. The polynucleotide of claim 1 wherein the polynucleotide is DNA. 3. A vector containing one or more of the polynucleotides of claim 1 and 2. 4. A host cell containing the vector of claim 3. 5. A process for producing a polypeptide comprising: expressing from the host cell of claim 4 the polypeptide encoded by said DNA. 6. A polypeptide selected from the group consisting of (a) a polypeptide having the deduced amino acid sequence of SEQ ID NO:2 and fragments, analogs and derivatives thereof, and (b) a polypeptide comprising amino acid 1 to amino acid 2201 of SEQ ID NO:2. 7. An antibody capable to bind to the polypeptide of claim 6. 8. A diagnostic kit for the detection of the polypeptide of claim 6. . Use of a polypeptides encoded by a polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide as set forth in SEQ ID NO: 1 , 3, 4 and 6 to 31 ; (b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a polynucleotide fragment of the polynucleotide of (a) or (b) in an assay for for detecting modulators of said polypeptides. 10. Modulator of a polypeptides encoded by a polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide as set forth in SEQ ID NO: 1, 3, 4 and 6 to 31 ; (b) a polynucleotide capable of hybridizing to and which is at least 70%> identical to the polynucleotide of (a); and (d) a polynucleotide fragment of the polynucleotide of (a) or (b) 11. A pharmaceutical comprising the modulator of claim 10 12. An assay for detecting polypeptides encoded by a polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide as set forth in SEQ ID NO: 1, 3, 4 and 6 to 32 and 54; (b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a polynucleotide fragment of the polynucleotide of (a) or (b)

Inventors: KLUCKEN JOCHEN, SCHMITZ GERD, KLUCKEN, JOCHEN, SCHMITZ, GERD
Applicants: BAYER AG, KLUCKEN JOCHEN, SCHMITZ GERD, BAYER AKTIENGESELLSCHAFT, KLUCKEN, JOCHEN, SCHMITZ, GERD
Application year: 1999
Main classification: C12N15
IPC codes: A61K38, A61K45, A61K48, A61P3, A61P9, A61P29, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1, G01N33