Mutational Analysis of the GXXXG/A Motifs in the Human Na+/Taurocholate Co-Transporting Polypeptide NTCP on Its Bile Acid Transport Function and Hepatitis B/D Virus Receptor Function

Homodimerization is essential for plasma membrane sorting of the liver bile acid transporter NTCP and its function as Hepatitis B/D Virus (HBV/HDV) receptor. However, the protein domains involved in NTCP dimerization are unknown. NTCP bears two potential GXXXG/A dimerization motifs in its transmembrane domains (TMDs) 2 and 7. The present study aimed to analyze the role of these GXXXG/A motifs for the sorting, function, and dimerization of NTCP. The NTCP mutants G60LXXXA64L (TMD2), G233LXXXG237L (TMD7) and a double mutant were generated and analyzed for their interaction with wild-type NTCP using a membrane-based yeast-two hybrid system (MYTH) and co-immunoprecipitation (co-IP). In the MYTH system, the TMD2 and TMD7 mutants showed significantly lower interaction with the wild-type NTCP. In transfected HEK293 cells, membrane expression and bile acid transport activity were slightly reduced for the TMD2 mutant but were completely abolished for the TMD7 and the TMD2/7 mutants, while co-IP experiments still showed intact protein-protein interactions. Susceptibility for in vitro HBV infection in transfected HepG2 cells was reduced to 50% for the TMD2 mutant, while the TMD7 mutant was not susceptible for HBV infection at all. We conclude that the GXXXG/A motifs in TMD2 and even more pronounced in TMD7 are important for proper folding and sorting of NTCP, and so indirectly affect glycosylation, homodimerization, and bile acid transport of NTCP, as well as its HBV/HDV receptor function.

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Selective hepatitis B and D virus entry inhibitors from the group of pentacyclic lupane-type betulin-derived triterpenoids

Scientific Reports ◽

10.1038/s41598-020-78618-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Michael Kirstgen ◽

Kira Alessandra Alicia Theresa Lowjaga ◽

Simon Franz Müller ◽

Nora Goldmann ◽

Felix Lehmann ◽

...

Keyword(s):

Bile Acid ◽

Hepatitis B ◽

Treatment Options ◽

Receptor Function ◽

Acid Transport ◽

Virus Binding ◽

Virus Receptor ◽

Bile Acid Transport ◽

Entry Inhibitors

AbstractCurrent treatment options against hepatitis B and D virus (HBV/HDV) infections have only limited curative effects. Identification of Na+/taurocholate co-transporting polypeptide (NTCP) as the high-affinity hepatic receptor for both viruses in 2012 enables target-based development of HBV/HDV cell-entry inhibitors. Many studies already identified appropriate NTCP inhibitors. However, most of them interfere with NTCP’s physiological function as a hepatic bile acid transporter. To overcome this drawback, the present study aimed to find compounds that specifically block HBV/HDV binding to NTCP without affecting its transporter function. A novel assay was conceptualized to screen for both in parallel; virus binding to NTCP (measured via binding of a preS1-derived peptide of the large HBV/HDV envelope protein) and bile acid transport via NTCP. Hits were subsequently validated by in vitro HDV infection studies using NTCP-HepG2 cells. Derivatives of the birch-derived pentacyclic lupane-type triterpenoid betulin revealed clear NTCP inhibitory potency and selectivity for the virus receptor function of NTCP. Best performing compounds in both aspects were 2, 6, 19, and 25. In conclusion, betulin derivatives show clear structure–activity relationships for potent and selective inhibition of the HBV/HDV virus receptor function of NTCP without tackling its physiological bile acid transport function and therefore are promising drug candidates.

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Sorting of rat liver and ileal sodium-dependent bile acid transporters in polarized epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1998.275.5.g1045 ◽

1998 ◽

Vol 275 (5) ◽

pp. G1045-G1055 ◽

Cited By ~ 8

Author(s):

An-Qiang Sun ◽

Meenakshisundaram Ananthanarayanan ◽

Carol J. Soroka ◽

Sundararajah Thevananther ◽

Benjamin L. Shneider ◽

...

Keyword(s):

Bile Acid ◽

Amino Acid ◽

Mdck Cells ◽

Cytoplasmic Tail ◽

Apical Surface ◽

Acid Transport ◽

Transport Activity ◽

Wild Type ◽

Bile Acid Transport ◽

Apical Domain

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that ∼75% of each protein was delivered to the basolateral surface compared with ∼90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 ± 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.

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Long-term trans-inhibition of the hepatitis B and D virus receptor NTCP by taurolithocholic acid

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00263.2020 ◽

2020 ◽

Author(s):

Kira Alessandra AT Lowjaga ◽

Michael Kirstgen ◽

Simon F Müller ◽

Nora Goldmann ◽

Felix Lehmann ◽

...

Keyword(s):

Plasma Membrane ◽

Bile Acid ◽

Hepatitis B ◽

Inhibitory Effect ◽

Hek293 Cells ◽

Transport Function ◽

Receptor Function ◽

Secondary Bile Acid ◽

Entry Inhibitors

Human hepatic bile acid transporter Na+/taurocholate co-transporting polypeptide (NTCP) represents the liver-specific entry receptor for the Hepatitis B and D Viruses (HBV/HDV). Chronic hepatitis B and D affect several million people worldwide, but treatment options are limited. Recently, HBV/HDV entry inhibitors targeting NTCP have emerged as promising novel drug candidates. Nevertheless, the exact molecular mechanism that NTCP uses to mediate virus binding and entry into hepatocytes is still not completely understood. It is already known that human NTCP mRNA expression is down-regulated under cholestasis. Furthermore, incubation of rat hepatocytes with the secondary bile acid taurolithocholic acid (TLC) triggers internalization of the rat Ntcp protein from the plasma membrane. In the present study, the long-term inhibitory effect of TLC on transport function, HBV/HDV receptor function and membrane expression of human NTCP were analyzed in HepG2 and HEK293 cells stably overexpressing NTCP. Even after short pulse preincubation, TLC had a significant long-lasting inhibitory effect on the transport function of NTCP, but the NTCP protein was still present at the plasma membrane. Furthermore, binding of the HBV/HDV myr-preS1 peptide and susceptibility for in vitro HDV infection were significantly reduced by TLC preincubation. We hypothesize that TLC rapidly accumulates in hepatocytes and mediates long-lasting trans-inhibition of the transport and receptor function of NTCP via a particular TLC binding site at an intracellularly accessible domain of NTCP. Physiologically, this trans-inhibition might protect hepatocytes from toxic overload of bile acids. Pharmacologically, it provides an interesting novel NTCP target site for potential long-acting HBV/HDV entry inhibitors.

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