ABC-genes have been identified in each genomes that has been sequenced so far; in human the ABC-gene family is composed of 48 members (protein coding genes) and some “additional” members (pseudogenes). The high number of ABC-genes demonstrates their important, however mostly unknown, physiological role. According to the current hypothesis, different members have a wide expression pattern and partly overlapping functions, which account for the relatively complex phenotypes upon their loss of function.
We have embarked upon a research program that involves a wide spectrum of experimental strategies aimed to elucidate the molecular basis of pseudoxanthoma elasticum and sitosterolemia. Both diseases are caused by mutations in ABC-transporter genes: that of ABCC6 and ABCG5/G8, respectively. Investigating the molecular basis of monogenic inherited diseases provides invaluable information about normal human physiology. Such basic research usually does not result in therapeutic interventions directly but may open new avenues in such directions.
Figure 1
Similar to the general strategy in case of ABC transporters the basic aim in our research of disease associated ABC proteins is to identify and characterize transported substrates. One of the unique features of the proposed strategy is that potential physiological substrate candidates identified by functional in vitro assays will direct us to metabolic pathways for further candidates and such compounds will also be tested as inducers/inhibitors of gene expression (see part: Expressional regulation of ABC transporter genes). Similarly, compounds modulating gene expression will be tested as potential substrates of the transporters. Discovery of the physiological substrates of the transporters will help to understand their physiological role in the given tissue. In case of human ABCC6 protein another important goal of the research project is to establish genotype-phenotype relationships of the PXE-associated missense mutant forms of ABCC6 in order to initiate further effort to correct these mutations. For this purpose we utilize various heterologous expression systems, and experimental strategies of cell biology additional to transport biochemistry.
Pseudoxanthoma elasticum (PXE)
Figure 2
Pseudoxanthoma elasticum is a heritable disorder, affecting several different elastic tissues, including the skin, the eye and the arterial system. Calcification of elastic fibers within the extracellular matrix in these tissues contributes to the development of the disease. It was an unexpected finding that mutations in a gene coding for an ABC transporter protein, ABCC6/MRP6 result in the development of PXE, traditionally thought of as a connective tissue disorder. While a spectrum of mutations within the ABCC6 gene is clearly responsible for PXE, the functional relationship between altered ABCC6 gene products and the PXE phenotype is still unknown. Similarly, very little is known about the transcriptional regulation of the ABCC6 gene.
Functional characterization of ABCC6 and PXE-associated mutants
Figure 3
To date, 186 mutations in the human ABCC6 gene, associated with PXE, have been identified. This high heterogeneity of PXE alleles in the population is comparable to that of other autosomal diseases. The most prevalent mutations are missense mutations (94 mutations) (see fig.2). In the case of several ABC transporters, including several MRPs, expression of the gene products in insect (e.g. Spodoptera frugiperda, Sf9) cells, using recombinant baculoviruses, proved to be an efficient tool for analyzing various aspects of transporter function. Human ABCC6 protein has been cloned and expressed in Sf9 insect cells first by our group in 2002 (see fig.3).
Figure 4
It has been demonstrated that ABCC6 is a primary active transporter of organic anions (see fig.4) and also revealed that the loss of ATP-dependent transport resulted in PXE phenotype through direct influence on the transport activity of three missense mutants of ABCC6/MRP6 protein we studied (see fig.3 and 5) [
1]. It has been also shown that the human ABCC6 protein is exclusively localized to the basolateral membrane of kidney-derived epithelial cells (MDCKII) (see fig.6) [
2] This finding is in harmony with that of Scheffer et al [
3], in which human ABCC6 was found on the basolateral surface of hepatocytes.
Figure 5
Different activities are compartmentalized into different organelles in eukaryotic cells, which possess mechanisms for transporting the newly synthesized polypeptides from the site of synthesis to the site (compartment) of their activity. Mutation in the protein can lead to an “escape” from the intracellular traffic system and the compromised intramolecular traffick can be the molecular basis of several inherited disease phenotype. For elucidating whether this mechanism can contribute to the PXE phenotype we are also investigating the subcellular localization and maturation of PXE-causing mutants expressed in different mammalian cell lines.
Sitosterolemia
Figure 6
Sitosterolemia is a rare heritable disorder of lipid metabolism. Patients suffer from premature coronary artery disease and atherosclerosis. Under normal circumstances, the human body retains approximately 50% of dietary cholesterol and less than 5% of dietary plant sterols, suggesting that the body is able to discriminate between these subtly different compounds. ABCG5 and ABCG8 seem to play a crucial role in this molecular recognition and in the elimination of plant sterols. Mutations in either one of the two genes were found to be responsible for the development of this disease.
Functional characterization of ABCG5/G8 and sitosterolemia missense mutants
Figure 7
In order to elucidate the molecular basis of sitosterolemia we utilize various heterologous expression systems, and experimental strategies of activity measurements and cell biology. We could achieve high level of expression of the ABCG5 and ABCG8 proteins, in various combinations, in Sf9 cells. When the function of the proteins was examined in isolated membrane preparations, we found a distinct, vanadate sensitive ATPase activity only when the two proteins were co-expressed to form a heterodimer. This ATPase activity, but not that of the individually expressed ABCG5 or
Figure 8
ABCG8 proteins, was significantly stimulated by the addition of certain androgen hormones and analogues, while unaffected by many sterols or sterol derivatives. Interestingly, the androgen-stimulated ABCG5/ABCG8 ATPase activity was effectively inhibited by progesterone [
4]. Thus, we have a functional assay to study the interaction of the heterodimer ABCG5/ABCG8 with its potential substrates and modulators.
Our preliminary results suggest that ABCG5/ABCG8 ATPase activity closely correlates with the physiological transport function of this heterodimer. Our results may provide a new aspect of biochemical and functional characterization of the transport activity or regulation of ABCG5/ABCG8.
References:
- Iliás A, Urbán Z, Seidl TL, Le Saux O, Sinkó E, Boyd CD, Sarkadi B and Váradi A (2002)
Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6).
J Biol Chem 277, 16860-7 [PubMed] - Sinkó E, Iliás A, Ujhelly O, Homolya L, Scheffer GL, Bergen AA, Sarkadi B and Váradi A (2003)
Subcellular localization and N-glycosylation of human ABCC6, expressed in MDCKII cells.
Biochem Bioph Res Co 308, 263-9 [PubMed] - Scheffer GL, Hu X, Pijnenborg AC, Wijnholds J, Bergen AA and Scheper RJ (2002)
MRP6 (ABCC6) detection in normal human tissues and tumors.
Lab Invest 82, 515-8 [PubMed] - Müller M, Klein I, Kopácsi S, Remaley AT, Rajnavölgyi E, Sarkadi B and Váradi A (2006)
Co-expression of human ABCG5 and ABCG8 in insect cells generates an androstan stimulated membrane ATPase activity.
FEBS Lett 580, 6139-44 [PubMed]
