Biomedical subjects

Gerd Schmitz

Publications and source records attributed to Gerd Schmitz.

At least 73 records · Page 4Linked to original sources

PubMed2003

Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry.

Sphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. The currently available methods for the quantification of SPH are, in part, complicated, time-consuming, insensitive, or unselective. Therefore, a fast and convenient methodology for the quantification of SPH and the biosynthetic intermediate sphinganine (SPA) was developed. The method is based on an HPLC separation coupled to electrospray ionization tandem mass spectrometry (MS/MS). Quantitation is achieved by the use of a constant concentration of a non-naturally occurring internal standard, 17-carbon chain SPH (C17-SPH), together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA coeluted with C17-SPH, which allows an accurate correction of the analyte response. Interference of the SPH+2 isotope with SPA quantification was corrected by an experimentally determined factor. The limits of detection were 9 fmol for SPH and 21 fmol for SPA. The overall coefficients of variation were 8% and 13% for SPH and SPA, respectively. The developed HPLC-tandem mass spectrometry methodology, with an analysis time of 3.5 min, simple sample preparation, and automated data analysis, allows high-throughput quantification of sphingoid bases from crude lipid extracts and is a valuable tool for studies of cellular sphingolipid metabolism and signaling.

Animals↗

PubMed2003

ABCA10, a novel cholesterol-regulated ABCA6-like ABC transporter.

We recently identified several novel members of the A subclass of ABC transporters. In this study, we report the cloning of an additional ABC A subfamily transporter, denoted ABCA10, from macrophages. The coding sequence of ABCA10 is of 4.6 kb size and codes for a 1543-amino acid protein that bears the structural features of a full-size ABC transporter. Intriguingly, ABCA10 contains a PEST sequence downstream of the N-terminal transmembrane domain which may be potentially involved in the control of its turnover rate by proteasomal degradation. Several distinct ABCA10 transcripts are expressed in human macrophages that predict the existence of various truncated forms of the novel transporter. Moreover, we identified seven single nucleotide polymorphisms in ABCA10 transcripts. ABCA10 displays high amino acid sequence homology with ABCA6 (63%), ABCA8 (62%), and ABCA9 (63%), respectively, known members of the subgroup of ABCA6-like transporters. Like other transporters of this subfamily, ABCA10 mRNA is ubiquitously expressed and highest gene expression levels are detectable in heart, brain, and the gastrointestinal tract. Analysis of the gene structure demonstrated that the ABCA10 gene consists of 40 exons that extend across a genomic region of approximately 97kb size (Chr. 17q24.3). ABCA10 mRNA is expressed in similar quantities in monocytes and M-CSF differentiated macrophages. Importantly, ABCA10 expression is suppressed by cholesterol import into macrophages, indicating that it is a cholesterol-responsive gene. Our results identify ABCA10 as a novel member of the group of ABCA6-like transporters and suggest its involvement in macrophage lipid homeostasis.

ATP-Binding Cassette Transporters↗

PubMed2003

Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.

The fatty acid transport protein family is a group of evolutionarily conserved proteins that are involved in the cellular uptake and metabolism of long and very long chain fatty acids. However, little is known about their respective physiological roles. To analyze the functional significance of fatty acid transport protein 4 (Fatp4, Slc27a4), we generated mice with a targeted disruption of the Fatp4 gene. Fatp4-null mice displayed features of a neonatally lethal restrictive dermopathy. Their skin was characterized by hyperproliferative hyperkeratosis with a disturbed epidermal barrier, a flat dermal-epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis. The rigid skin consistency resulted in an altered body shape with facial dysmorphia, generalized joint flexion contractures, and impaired movement including suckling and breathing deficiencies. Lipid analysis demonstrated a disturbed fatty acid composition of epidermal ceramides, in particular a decrease in the C26:0 and C26:0-OH fatty acid substitutes. These findings reveal a previously unknown, essential function of Fatp4 in the formation of the epidermal barrier.

Animals↗

PubMed2003

ZNF202 is inversely regulated with its target genes ABCA1 and apoE during macrophage differentiation and foam cell formation.

The zinc finger protein ZNF202 is a transcriptional repressor that binds to promoter elements predominantly found in genes involved in lipid metabolism. Here we demonstrate that ZNF202 mRNA expression is inversely correlated with ATP binding cassette A1 (ABCA1), ABCG1, and apolipoprotein E (apoE) in human monocytes. Upregulation of ABCA1, ABCG1, and apoE expression during monocyte differentiation and foam cell formation was accompanied by a simultaneous downregulation of both ZNF202 mRNA isoforms m1 and m3. Conversely, deloading of macrophage foam cells with HDL3 caused upregulation of ZNF202 mRNA. To further characterize the transcriptional regulation of the ZNF202 gene, comparative genomic sequence analysis and reporter gene assays were performed. The ZNF202 core promoter region resides within 247 bp upstream of the transcription initiation site and is highly active in THP-1 monocytes, yet downregulated upon macrophage differentiation. Using site-directed mutagenesis, we show that two highly conserved transcription factor binding sites, a GC-box and an Ets-binding motif, are required for ZNF202 gene expression. Furthermore, electrophoretic mobility shift assays demonstrate in vitro binding of PU.1 and GC-box binding proteins to the ZNF202 proximal promoter. We conclude that the inversely correlated transcriptional activity of ZNF202 and its target genes during macrophage differentiation may reflect a direct regulatory interdependence and thus provide further evidence for ZNF202 as an important gatekeeper of lipid efflux.

ATP Binding Cassette Transporter 1↗

PubMed2003

Marked induction of sterol 27-hydroxylase activity and mRNA levels during differentiation of human cultured monocytes into macrophages.

Sterol 27-hydroxylase has been suggested to be involved in an alternative pathway for the elimination of cholesterol from macrophages and early atherosclerotic lesions. We have previously shown that human lung macrophages as well as monocyte-derived macrophages have a relatively high activity of sterol 27-hydroxylase (CYP27). This enzyme converts intracellular cholesterol into 27-hydroxycholesterol and cholestenoic acid that flux from cultured cells into the medium. It is shown here that human monocytes have very low CYP27 activity and CYP27 mRNA levels. During differentiation into macrophages, both CYP27 activity and CYP27 mRNA levels increase markedly after 4 days of culture in serum-free medium. Addition of macrophage-colony stimulating factor had no significant effect on the induction and addition of fetal calf serum had an inhibitory effect. Cholesterol synthesis was found to be a critical factor for the production of 27-oxygenated products by the macrophages cultured in serum-free medium. The increased capacity of the differentiated cells to eliminate intracellular cholesterol is of interest and supports the contention that CYP27 is an antiatherogenic factor.

Cell Differentiation↗

PubMed2003

Plasma ceramide and lysophosphatidylcholine inversely correlate with mortality in sepsis patients.

Recent data indicate that ceramide (Cer) and lysophosphatidylcholine (LPC) regulate immune cell functions. Since these bioactive lipids are generated in blood plasma by inflammatory lipases, we hypothesized that they may be involved in the process of acute systemic sepsis. In order to provide support for this hypothesis, we analyzed the plasma levels of Cer and LPC by quantitative tandem mass spectrometry in 102 sepsis patients starting with the day at which the sepsis criteria were fulfilled for the first time, as well as on day 4 and day 11. The values were compared with 56 healthy controls and correlated with sepsis-related mortality within 30 days of study entry. Most Cer species were increased in sepsis patients, while all LPC species were markedly decreased. In addition, we determined the molar ratios with their precursor molecules sphingomyelin (SPM) and phosphatidylcholine (PC), which reflect the enzymatic reactions responsible for their formation. Species-specific as well as total Cer-SPM ratios were increased, whereas LPC-PC ratios were decreased in sepsis patients. The increased Cer-SPM ratios as well as the decreased LPC-PC ratios showed a strong predictive power for sepsis-related mortality. Together with existing data from in vitro experiments and animal models, the results provide the first ex vivo indication for the role of Cer and lysophospholipids in systemic inflammation in humans.

Adolescent↗

PubMed2003

CD55/decay accelerating factor is part of the lipopolysaccharide-induced receptor complex.

Recently, we described an 80-kDa lipopolysaccharide (LPS)-binding membrane protein to be identical to CD55 [decay accelerating factor (DAF)]. Here, we demonstrate that CD55 is able to contribute to lipopolysaccharide (LPS) signaling. Transfection of Chinese hamster ovary (CHO) cells with human CD55 resulted in a translocation of NF-kappa B after stimulation with LPS as well as with free lipid A. In addition, interaction of lipid A and CD55 was shown by co-immuno-precipitation of these molecules from CHO-CD55 cells after incubation with lipid A and anti-lipid A monoclonal antibody, as well as by fluorescence resonance energy transfer (FRET) analysis in human monocytes. The comparison of LPS-induced signaling pathways in CHO-CD55 and CHO-CD14 cells revealed that p38, JNK and ERK MAP kinases are activated upon LPS stimulation in both cell lines, and that the activation by LPS can be blocked at the level of Toll-like receptor 4. Finally, through FRET analysis we could demonstrate LPS-induced clustering of CD55 and CD11/CD18 in human monocytes. Our results imply a new functional role of CD55 as a member of a multimeric LPS receptor complex.

Animals↗

PubMed2003

Adenosine triphosphate binding cassette (ABC) transporters are expressed and regulated during terminal keratinocyte differentiation: a potential role for ABCA7 in epidermal lipid reorganization.

Central aspects of the cellular lipid trafficking mechanisms that occur during keratinocyte differentiation are still not well understood. In the past years, evidence has accumulated to suggest that members of the superfamily of adenosine triphosphate binding cassette (ABC) transporters are critically involved in the transmembrane transport of cellular lipids. To test the hypothesis that ABC molecules are potentially involved in the epidermal transport of sphingolipids, glycerophospholipids, cholesterol, and fatty acids, we performed mRNA expression profiling of all currently known ABC molecules during in vitro differentiation of human keratinocytes and HaCaT cells. We identified six ABC molecules that displayed significant regulation during differentiation of these cells. The recently cloned transporter ABCA7 was highly expressed in keratinocytes and HaCaT cells and upregulated during differentiation. Overexpression of ABCA7 in HeLa cells resulted in increased expression of intracellular and cell surface ceramide and elevated intracellular phosphatidylserine levels. Given the observation that during terminal keratinocyte differentiation intracellular and surface ceramide levels are increased, our results render ABCA7 a candidate regulator of ceramide transport in this process. In addition to ABCA7, the cholesterol transporters ABCB1 and ABCG1 and the glutathione/glucuronide sulfate transporters ABCC1, ABCC3, and ABCC4, were strongly upregulated during keratinocyte and HaCaT cell differentiation. These findings support the notion that ABCB1 and ABCG1 are potentially implicated in cholesterol transport, whereas ABCC1, ABCC3, and ABCC4 are candidate regulators of the translocation of sulfated lipids during stratum corneum keratinization. Our results suggest specific biologic functions for members of the ABC transporter family in epidermal lipid reorganization during terminal keratinocyte differentiation.

ATP-Binding Cassette Transporters↗

PubMed2003

Real-time reverse transcription-PCR expression profiling of the complete human ATP-binding cassette transporter superfamily in various tissues.

BACKGROUND: ATP-binding cassette (ABC) transporters are involved in many physiologic processes, such as lipid transport, sterol homeostasis, immune mechanisms, and drug transport, and cause various human inherited diseases. Thus, the analysis of ABC transporter mRNA expression profiles for basic research, especially in the field of lipid metabolism, for clinical diagnosis, and for monitoring of drug effects is of great interest. METHODS: We have developed a rapid, accurate, and highly sensitive real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of all 47 currently known members of the ABC transporter superfamily. Our expression analysis is based on relative quantification using a calibration curve method. With our assay, expression monitoring of a large number of RNA samples in a 384-well format with only 50 ng of total RNA is possible. RESULTS: In contrast to previous expression analyses of single ABC genes, our method allows the rapid and complete analysis of all ABC transporters in given RNA samples. We used our newly established expression panel to study the gene expression of all human ABC transporters in 20 different human tissues. As a result, we identified tissues with high transcriptional activity for ABC transporters. These organs are mainly involved in secretory function (adrenal gland), metabolic function (liver), barrier function (lung, trachea, small intestine), and tropic function (placenta, uterus). CONCLUSIONS: Our RT-PCR assay allows rapid, high-throughput transcriptional profiling of the complete ABC transporter superfamily and thus provides a new enabling tool for research, clinical diagnosis of disease, and drug testing and development.

ATP-Binding Cassette Transporters↗

PubMed2003

Pharmacogenomics and pharmacogenetics of cholesterol-lowering therapy.

Cholesterol-lowering therapy is the central approach in the primary and secondary prevention of cardiovascular disease, the leading cause of death in industrialized countries. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are currently the most potent and widely used cholesterol-lowering drugs. Large-scale clinical trials unequivocally demonstrated the efficacy of statin treatment in reducing the risk of cardiovascular events. In general, HMG-CoA reductase inhibitors are well tolerated, although in a minority of patients severe adverse effects like myopathy or rhabdomyolysis may develop. The incidence of this potentially life-threatening side effects increases with co-adminstration of drugs that are metabolized via the same pharmacokinetic pathways or at high-dose statin therapy. The recent focus on the pleiotropic effects of statins that are more frequently observed at higher doses and the conclusion drawn from the large statin trials that low-density lipoprotein (LDL)-cholesterol is "the lower the better", may need careful consideration in individuals at risk of adverse drug reactions. On the other hand, not all patients respond to statin therapy with a reduction in coronary heart disease (CHD) risk. It is therefore of interest to develop diagnostic test systems, which would allow to identify patients at increased risk of adverse drug reactions or patients with a lack of therapeutic effect. Beside exogenous factors, genetic variability determines the response of an individual to drug therapy and the analysis of genetic variants affecting pharmacokinetic or pharmacodynamic aspects of drug therapy is the subject of pharmacogenomics. This review summarizes current knowledge of the pharmacology and the pharmacogenomics of statin therapy.

Cholesterol, LDL↗

PubMed2002

Molecular and functional interaction of the ATP-binding cassette transporter A1 with Fas-associated death domain protein.

ATP-binding cassette transporter A1 (ABCA1) is a major regulator of cellular cholesterol and phospholipid homeostasis. Its function has not been fully characterized and may depend on the association with additional proteins. To identify ABCA1-interacting proteins a human liver yeast two-hybrid library was screened with the 144 C-terminal amino acids of ABCA1. Fas-associated death domain protein (FADD) was identified to bind to ABCA1, and this interaction was confirmed by pull-down assays and co-immunoprecipitations. Recombinant expression of a dominant negative form of FADD or the C terminus of ABCA1 in the human hepatoma cell line HepG2 markedly reduced the transfer of phospholipids to apoA-I. This indicates that the binding of additional proteins, one of them being full-length FADD, is required for ABCA1 function. The association of FADD with ABCA1 provides an unexpected link between high density lipoprotein metabolism and an adaptor molecule mainly described in death receptor signal transduction.

ATP Binding Cassette Transporter 1↗

PubMed2002

Molecular structure of a novel cholesterol-responsive A subclass ABC transporter, ABCA9.

We recently identified a novel ABC A subclass transporter, ABCA6, in human macrophages. Here, we report the molecular cloning of an additional ABC A subfamily transporter from macrophages denoted ABCA9. The identified coding sequence is 4.9 kb in size and codes for a 1624 amino acid protein product. In accordance with the proposed nomenclature, the novel transporter was designated ABCA9. The putative full-length ABC transporter polypeptide consists of two transmembrane domains and two nucleotide binding folds and thus conforms to the group of full-size ABC transporters. We identified alternative ABCA9 mRNA variants in human macrophages that predict the existence of three truncated forms of the novel transporter. Among the human ABC A subfamily transporters, ABCA9 exhibits the highest amino acid sequence homology with ABCA8 (72%) and ABCA6 (60%), respectively. The striking amino acid sequence similarity between these transporter molecules supports the notion that they represent an evolutionary more recently emerged subgroup within the family of ABC A transporters, which we refer to as "ABCA6-like transporters." ABCA9 mRNA is ubiquitously expressed with the highest mRNA levels in heart, brain, and fetal tissues. Analysis of the genomic structure revealed that the ABCA9 gene consists of 39 exons that are located within a genomic region of approximately 85 kb size on chromosome 17q24.2. In human macrophages, ABCA9 mRNA is induced during monocyte differentiation into macrophages and suppressed by cholesterol import indicating that ABCA9, like other known ABC A subfamily transporters, is a cholesterol-responsive gene. Based on this information, ABCA9 is likely involved in monocyte differentiation and macrophage lipid homeostasis.

ATP-Binding Cassette Transporters↗

PubMed2002

The carboxyterminus of the ATP-binding cassette transporter A1 interacts with a beta2-syntrophin/utrophin complex.

Recent work identified ABCA1 as the major regulator of plasma HDL-cholesterol responsible for the removal of excess choline-phospholipids and cholesterol from peripheral cells and tissues. ABCA1 function may depend on the association with heteromeric proteins and to identify these candidates a human liver yeast two-hybrid library was screened with the carboxyterminal 144 amino acids of ABCA1. Beta2-syntrophin was found to interact with ABCA1 and the C-terminal five amino acids of ABCA1 proned to represent a perfect tail for binding to syntrophin PDZ domains. Immunoprecipitation further confirmed the association of ABCA1 and beta2-syntrophin and in addition utrophin, known to couple beta2-syntrophin and its PDZ ligands to the F-actin cytoskeleton, was identified as a constituent of this complex. ABCA1 in the plasmamembrane of human macrophages was found to be partially associated with Lubrol rafts and effluxed choline-phospholipids involve these microdomains. Beta2-syntrophin does not colocalize in these rafts indicating that beta2-syntrophin may participate in the retaining of ABCA1 in cytoplasmic vesicles and for the targeting of ABCA1 to plasmamembrane microdomains when ABCA1 is released from beta2-syntrophin.

ATP Binding Cassette Transporter 1↗

PubMed2002

Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues.

The ATP-binding cassette transporter 1 (ABCA1) has recently been identified as a key regulator of high-density lipoprotein (HDL) metabolism, which is defective in familial HDL-deficiency syndromes such as Tangier disease. ABCA1 functions as a facilitator of cellular cholesterol and phospholipid efflux, and its expression is induced during cholesterol uptake in macrophages. To assess the role of macrophage ABCA1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr(-/-)) mice that are selectively deficient in leukocyte ABCA1 (ABCA1(-/-)) by using bone marrow transfer (ABCA1(-/-) --> LDLr(-/-)). Here we demonstrate that ABCA1(-/-) --> LDLr(-/-) chimeras develop significantly larger and more advanced atherosclerotic lesions compared with chimeric LDLr(-/-) mice with functional ABCA1 in hematopoietic cells. Targeted disruption of leukocyte ABCA1 function did not affect plasma HDL cholesterol levels. The amount of macrophages in liver and spleen and peripheral blood leukocyte counts is increased in the ABCA1(-/-) --> LDLr(-/-) chimeras. Our results provide evidence that leukocyte ABCA1 plays a critical role in the protection against atherosclerosis, and we identify ABCA1 as a leukocyte factor that controls the recruitment of inflammatory cells.

ATP Binding Cassette Transporter 1↗

PubMed2002

Identification of sterol-independent regulatory elements in the human ATP-binding cassette transporter A1 promoter: role of Sp1/3, E-box binding factors, and an oncostatin M-responsive element.

The ATP-binding cassette transporter A1 (ABCA1) shows a differentiation-, cAMP-, and sterol-dependent up-regulation in human monocytes. As part of an ongoing study, we investigated the proximal promoter regions that are highly conserved between the human and murine ABCA1 genes. Using reporter gene assays, we show here that a TATA box 24 bp upstream of the transcription initiation site is essential for promoter activity in RAW 264.7 and HepG2 cells, whereas further enhancement of transcriptional activity is mediated by the -175 bp promoter region. Gel shift assays revealed in vitro binding of Sp1 to a -91 GnC motif as well as binding of Sp1 and Sp3 to a -157 GnC promoter region. In co-transfection experiments using Drosophila S2 cells, we demonstrate that Sp3 competes with Sp1 for binding to the -157 GnC motif and acts as a repressor. On the other hand, overexpression of Sp1 increased ABCA1 mRNA expression in HeLa cells and enhanced cellular cholesterol and phospholipid efflux in RAW 246.7 macrophages. We also show here that the conserved E-box at position -140 binds upstream stimulatory factors 1 and 2 and hepatic nuclear factor 1alpha and that mutagenesis of the E-box enhanced constitutive ABCA1 expression in RAW 264.7 cells, implying a role for this element in silencing ABCA1 expression. Besides the functional importance for basal gene expression, we have identified that the core promoter region (-175 to +224) is also responsible for the induction of ABCA1 by the cytokine oncostatin M, resulting in a rapid increase in ABCA1 mRNA levels in HepG2 cells. Interestingly, this oncostatin M-induced expression is not dependent on the currently known sequence motifs in the ABCA1 promoter. In conclusion, a functional complex of cis-elements within the proximal human ABCA1 promoter associated with the transcription factors Sp1/3, upstream stimulatory factors 1 and 2, and hepatic nuclear factor 1alpha has been characterized, which allows a subtle tissue-specific regulation of ABCA1 gene expression.

ATP-Binding Cassette Transporters↗

PubMed2002

Frontal lobe atrophy due to a mutation in the cholesterol binding protein HE1/NPC2.

This is the first description of slowly progressive Niemann-Pick disease type C (NPC) without the typical lysosomal storage in bone marrow and viscera in two descendants of a group of 17th century French-Canadians. The index patient was a married 43-year-old woman with onset of dementia in her thirties, later followed by the development of ataxia and athetoid movements. Her autopsy disclosed frontal lobe atrophy, neurolysosomal storage with oligolamellar inclusion and tau-positive neurofibrillary tangles. Of the 119 family members screened, only a married 42-year-old sister displayed symptoms of a dementia. Both women displayed vertical supranuclear ophthalmoplegia; expressive aphasia; concrete, stimulus-bound, perseverative behavior; and impaired conceptualization and planning. Cultured fibroblasts showed decreased cholesterol esterification and positive filipin staining, but no mutation was detected in coding or promoter regions of the NPC1 gene using conformation sensitive gel electrophoresis and sequencing. Sequencing showed a homozygous gene mutation that is predicted to result in an amino acid substitution, V39M, in the cholesterol binding protein HE1 (NPC2). Adult-onset NPC2 with lysosomal storage virtually restricted to neurons represents a novel phenotypic and genotypic variant with diffuse cognitive impairment and focal frontal involvement described for the first time.

Adult↗

PubMed2002

Mutations in the human ATP-binding cassette transporters ABCG5 and ABCG8 in sitosterolemia.

Phytosterolemia or Sitosterolemia is a rare autosomal recessive disorder characterized by highly elevated plasma levels of plant sterols and cholesterol as a consequence of hyperabsorption and impaired biliary secretion of sterols. The disease is caused by mutations in two half size ATP-binding cassette transporters, ABCG5 and ABCG8. We have analyzed the genomic sequence of ABCG5 and ABCG8 in five well-characterized patients with Sitosterolemia. In the first patient we found a heterozygous mutation in exon 8 of the ABCG5 gene leading to a premature termination of the protein (Arg408Ter). This German patient is the first European showing a mutation of the ABCG5 gene. In a second patient we found a novel heterozygous mutation in exon 5 of ABCG8 (c.584T>A; Leu195Gln). Both patients were heterozygous for the identified mutation, but no mutation could be identified on the other chromosome. In three further analyzed patients we found mutations in exons 7, 9 and 11 of the ABCG8 gene, respectively, of which two result in a premature termination signal for translation products. One of these patients was compound heterozygous (Trp361Ter and Arg412Ter), the other was homozygous for Trp361Ter. The third patient was homozygous for an amino acid exchange (Gly574Arg). In conclusion this report describes one novel mutation affecting a highly conserved amino acid and two previously identified mutations in the ABCG8 gene. In addition, we identified for the first time a mutation in the ABCG5 gene of a European Sitosterolemia patient.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2002

Cloning and characterization of a novel apolipoprotein A-I binding protein, AI-BP, secreted by cells of the kidney proximal tubules in response to HDL or ApoA-I.

Apolipoprotein A-I (apoA-I) is the major apolipoprotein of high-density lipoproteins (HDL) and has an important role in the regulation of the stability, lipid transport, and metabolism of HDL particles. To identify novel proteins that are involved in HDL metabolism, we used mature apoA-I (amino acids 25-267) as a bait for the screening of a human liver two-hybrid cDNA library. Among the identified genes, several encoded known proteins, including serum amyloid A(2a) (SAA(2a)), apoC-I, and phosphodiesterase HCAM1 (PDE1A), found to interact with apoA-I. In addition, we have cloned a novel 29 kDa apoA-I interacting protein, which we named AI-BP (apoA-I binding protein). The AI-BP encoding gene, APOA1BP, which is located on chromosome 1q21, is composed of six exons and five introns and spans 2.5 kb. Northern blot analysis demonstrated ubiquitous expression of the APOA1BP mRNA with the highest expression in kidney, heart, liver, thyroid gland, adrenal gland, and testis. AI-BP protein is not detectable in serum of healthy probands, but serum samples of patients with septic syndromes may contain elevated levels of AI-BP. Significant amounts of AI-BP protein are found in cerebrospinal fluid and urine of healthy probands. The stimulation of cells derived from the kidney proximal tubules with apoA-I or HDL induces a concentration-dependent secretion of AI-BP indicating an important role for AI-BP, in the renal tubular degradation or resorption of apoA-I.

Amino Acid Sequence↗