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

Abstract

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.

Tyrosine 146 of the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) Is Essential for Its Hepatitis B Virus (HBV) Receptor Function and HBV Entry into Hepatocytes

“…Although, during revision of the manuscript, three independent cryo-EM structures of human NTCP were published [13][14][15], partly with bound viral myr-preS1 peptide, the precise molecular interactions between the host-derived NTCP receptor and the HBV surface proteins are still not fully understood. Experimental approaches, such as protein engineering and computational biology methods, are powerful tools in exploring motifs within the NTCP protein that are essential for regulation of its HBV affinity during infection [3,9,10,12,23,24,32,33]. In this study, we identified a novel NTCP tyrosine-enriched motif 139YIYSRGIY146 that is significantly involved in the regulation of NTCP-HBV preS1 interactions and, thus, in the control of NTCP receptor function (Figure 7A,B).…”

“…It was demonstrated that N5A/N11A and N5Q/N11Q double mutants lacking both N-glycosylation sites of NTCP were transport negative for bile acids and showed markedly diminished NTCP-driven HBV entry into hepatocytes. As N-glycosylation generally alters the dimerization properties of proteins [22], and NTCP dimerization normally occurs early in the sorting process of the protein towards the plasma membrane [23][24][25], changes in the glycosylation status of NTCP could fundamentally affect its proper sorting to the plasma membrane, where NTCP fulfills its transporter and receptor functions.…”

Multitasking Na+/Taurocholate Cotransporting Polypeptide (NTCP) as a Drug Target for HBV Infection: From Protein Engineering to Drug Discovery

“…Single mutations, deletions, and fragment replacements within the NTCP/Ntcp sequence have identified numerous regions and residues that define the protein structure, topology and its functionality. As depicted in Table 1 , those studies were mainly focused on: (i) Na + -dependent bile acid uptake and substrate specificity [ 20 , 33 , 40 , 46 , 48 , 64 , 65 , 66 , 67 , 68 ], (ii) HBV/HDV receptor function [ 9 , 13 , 33 , 34 , 40 , 63 , 68 , 69 , 70 , 71 , 72 ], (iii) cellular localization and protein trafficking [ 33 , 39 , 40 , 64 , 65 , 73 , 74 ], (iv) posttranslational modifications [ 33 , 34 , 39 ], (v) oligomerization [ 63 , 71 , 75 , 76 ], and (vi) the presence of conserved motifs or functional domains [ 13 , 39 , 62 , 64 , 67 , 71 , 77 ].…”

“…The largest number of them examined residues that participate in bile acid transport. According to previous reports, amino acids located at the N-terminal part of NTCP/Ntcp, such as D24, C44, G60, A64, Q68, S105, N106, D115, S119 have been reported to markedly regulate the bile acid transport function [ 66 , 71 , 75 , 78 ]. Moreover, strong evidence suggests that transmembrane domain VIII, which is localized in the “core” domain, is essentially involved in the bile acid transport activity.…”

“…This includes a confirmation of identified virus-binding motifs as well as the recognition of novel NTCP regions required for virus docking and internalization upon infection. Moreover, given an increasing number of NTCP-interacting partners and the NTCP-self oligomerization that have recently been identified as key players during HBV infection [ 19 , 63 , 71 , 76 ], it is fundamental to evaluate their detailed contribution to structural changes of the NTCP/ligand complex. To functionally address all aforementioned NTCP-related topics, the purification of NTCP protein under native conditions is desperately needed.…”