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Hepatic ABC transporters

Hepatic ABC transporters

Several ABC transporters of the human liver are responsible for the secretion of bile salts, lipids and cholesterol. Their interplay protects the biliary tree from the harsh detergent activity of bile salts by forming mixed micelles, which are composed of these three components. In detail, bile salt export pump (BSEP or ABCB11) translocates bile acids, while the multidrug resistance protein 3 (MDR3 or ABCB4) flops phosphosphatidylcholine lipids from the inner to the outer leaflet of the canalicular membrane of the hepatocyte. BSEP is responsible for bile salt transport, while MDR3 flops phosphatidylcholine from the inner to the outer membrane leaflet of the canalicular membrane. Finally, the heterodimeric ABC transporter ABCG5/G8 transports cholesterol the remaining component of the mixed micelles (Figure 1). Mutations in one of these transporters are associated with different kinds of severe liver diseases. For example, mutations in the BSEP gene lead to progressive familial intrahepatic cholestasis type 2 (PFIC2), which can only be cured by liver transplantation. Another example are mutations of MDR3 that lead to PFIC3

We are interested in the biochemical in vitro characterization of these ABC transporters. How do single nucleotide polymorphisms (SNPs) influence the activity of those transporters? What is the mode of action for MDR3? Why is MDR3 a specific PC floppase whereas the 80% identical homologue MDR1 (P-gp or ABCB1) is a major player in multidrug resistance? To answer all these questions we utilize heterologous expression to purify these transporters in mg quantity. Therefore, we have established a Saccharomyces cerevisiae based cloning strategy for BSEP and MDR3, because the cDNA is “toxic” for the cloning organism E. coli. This workflow enabled us to clone the cDNA in any expression vector and to integrate clinically relevant mutations in a short time frame.

Furthermore, we are able to express BSEP and MDR3 in the methylotrophic yeast Pichia pastoris and ferment it to high cell densities for purification with and without GFP fusion. We use different chromatography steps to purify those transporters to homogeneity and measure for example ATPase activity in detergent solution.

Beside the purification for e.g. BSEP we also established an uptake assay with BSEP expressing Pichia plasma membranes. With this assay we are able to screen e.g. clinically relevant mutations for transport defects and determine the kinetic values in this expression system. One has to stress that only mutants, in which trafficking was not impaired, were analyzed. So far, we investigated  positions 374 (G374S), 919 (T919Δ) and 1032 (G1032R). Additionally, we tried to obtain structure-function relations for 72 clinically relevant BSEP and 43 clinically relevant MDR3 mutations.

Following the same line, we successfully established a purification protocol for human MDR3. Mirroring its in vivo function, detergent-solubilized MDR3 showed a PC-lipid stimulated ATPase activity. Importantly, no other class of lipid stimulated this activity. This gives us an in vitro system in hand, with which we can analyze clinically relevant mutants such as the mutation within the extended X-loop of MDR3, Q1147E.

Relevant publications:

Prescher, M., T. Kroll & L. Schmitt, (2019) ABCB4/MDR3 in health and disease - at the crossroads of biochemistry and medicine. Biol Chem. in press.

Ellinger, P., J. Stindt, C. Droge, K. Sattler, C. Stross, S. Kluge, D. Herebian, S.H.J. Smits, M. Burdelski, S. Schulz-Jurgensen, A. Ballauff, J. Schulte Am Esch, E. Mayatepek, D. Haussinger, R. Kubitz & L. Schmitt, (2017) Partial external biliary diversion in bile salt export pump deficiency: Association between outcome and mutation. World J Gastroenterol 23: 5295-5303.

Droge, C., M. Bonus, U. Baumann, C. Klindt, E. Lainka, S. Kathemann, F. Brinkert, E. Grabhorn, E.D. Pfister, D. Wenning, A. Fichtner, D.N. Gotthardt, K.H. Weiss, P. McKiernan, R.D. Puri, I.C. Verma, S. Kluge, H. Gohlke, L. Schmitt, R. Kubitz, D. Haussinger & V. Keitel, (2017) Sequencing of FIC1, BSEP and MDR3 in a large cohort of patients with cholestasis revealed a high number of different genetic variants. J Hepatol 67: 1253-1264.

Stindt, J., S. Kluge, C. Droge, V. Keitel, C. Stross, U. Baumann, F. Brinkert, A. Dhawan, G. Engelmann, R. Ganschow, P. Gerner, E. Grabhorn, A.S. Knisely, K.A. Noli, I. Pukite, R.W. Shepherd, T. Ueno, L. Schmitt, C. Wiek, H. Hanenberg, D. Haussinger & R. Kubitz, (2016) Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency. Hepatology 63: 524-537.

Przybylla, S., J. Stindt, D. Kleinschrodt, J. Schulte Am Esch, D. Haussinger, V. Keitel, S.H. Smits & L. Schmitt, (2016) Analysis of the Bile Salt Export Pump (ABCB11) Interactome Employing Complementary Approaches. PLoS One 11: e0159778.

Kluth, M., J. Stindt, C. Droge, D. Linnemann, R. Kubitz & L. Schmitt, (2015) A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3. J Biol Chem 290: 4896-4907.

Stindt, J., P. Ellinger, K. Weissenberger, C. Droge, D. Herebian, E. Mayatepek, B. Homey, S. Braun, J. Schulte am Esch, M. Horacek, A. Canbay, L. Schmitt, D. Haussinger & R. Kubitz, (2013) A novel mutation within a transmembrane helix of the bile salt export pump (BSEP, ABCB11) with delayed development of cirrhosis. Liver international 33: 1527-1535.

Ellinger, P., M. Kluth, J. Stindt, S.H. Smits & L. Schmitt, (2013) Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11) and MDR3 (ABCB4) expressed in the yeast Pichia pastoris. PLoS One 8: e60620.