Human NTCP Research Articles

Structural basis for hepatitis B virus restriction by a viral receptor homologue

Macaque restricts hepatitis B virus (HBV) infection because its receptor homologue, NTCP (mNTCP), cannot bind preS1 on viral surface. To reveal how mNTCP loses the viral receptor function, we here solve the cryo-electron microscopy structure of mNTCP. Superposing on the human NTCP (hNTCP)-preS1 complex structure shows that Arg158 of mNTCP causes steric clash to prevent preS1 from embedding onto the bile acid tunnel of NTCP. Cell-based mutation analysis confirms that only Gly158 permitted preS1 binding, in contrast to robust bile acid transport among mutations. As the second determinant, Asn86 on the extracellular surface of mNTCP shows less capacity to restrain preS1 from dynamic fluctuation than Lys86 of hNTCP, resulting in unstable preS1 binding. Additionally, presence of long-chain conjugated-bile acids in the tunnel induces steric hindrance with preS1 through their tailed-chain. This study presents structural basis in which multiple sites in mNTCP constitute a molecular barrier to strictly restrict HBV.

The feasibility of establishing a hamster model for HBV infection: in vitro evidence.

Chronic hepatitis B virus (HBV) infection remains a significant public health burden with no cure currently available. The research to cure HBV has long been hampered by the lack of immunocompetent small animal models capable of supporting HBV infection. Here, we set out to explore the feasibility of the golden Syrian hamster as an immunocompetent small rodent model for HBV infection. We first started with in vitro assessments of the HBV replication cycle in primary hamster hepatocytes (PHaHs) by adenoviral HBV (Ad-HBV) transduction. Our results demonstrated that PHaHs support HBV reverse transcription and subsequent cccDNA formation via the intracellular recycling pathway. Next, with luciferase reporter assays, we confirmed that PHaHs support the activities of all HBV major promoters. Then, we transduced PHaHs with an adenoviral vector expressing HBV receptor human Na+/taurocholate cotransporting polypeptide NTCP (Ad-huNTCP), followed by HBV inoculation. While the untransduced PHaHs did not support HBV infection, Ad-huNTCP-transduced PHaHs supported de novo cccDNA formation, viral mRNA transcription, and expression of viral antigens. We then humanized the amino acid (aa) residues of hamster NTCP (haNTCP) critical for HBV entry, aa84-87 and aa157-165, and transfected HepG2 cells with constructs expressing wild-type haNTCP and humanized-haNTCP, H84R/P87N and H84R/P87N/G157K/M160V/M165L, respectively, followed by HBV inoculation. The results showed that the humanization of H84R/P87N alone was sufficient to support HBV infection at a level comparable to that supported by huNTCP. Taken together, the above in vitro evidence supports the future direction of humanizing haNTCP for HBV infection in vivo.IMPORTANCEOne of the biggest challenges in developing an HBV cure is the lack of immunocompetent animal models susceptible to HBV infection. Developing such models in mice has been unsuccessful due to the absence of a functional HBV receptor, human NTCP (huNTCP), and the defect in supporting viral cccDNA formation. In search of alternative models, we report herein multiple lines of in vitro evidence for developing a golden Syrian hamster model for HBV infection. We demonstrate that the primary hamster hepatocytes (PHaHs) support HBV replication, transcription, and cccDNA formation, and PHaHs are susceptible to de novo HBV infection in the presence of huNTCP. Furthermore, expressing hamster NTCP with two humanized residues critical for HBV entry renders HepG2 cells permissive to HBV infection. Thus, our work lays a solid foundation for establishing a gene-edited hamster model that expresses humanized NTCP for HBV infection in vivo.

Identification of amino acids restricting HBV receptor function in porcine NTCP

With 254 million chronically infected patients, hepatitis B virus (HBV) continues to be a severe health threat. While animal models play a crucial role in developing new therapies, the availability of preclinical HBV models is very limited. Therefore, novel in vivo infection models are urgently needed. The bona fide HBV receptor, sodium-taurocholate cotransporting polypeptide (NTCP), determines HBV’s species and cell-type specificity. Recent studies have indicated that the expression of human NTCP is the only limiting factor for HBV infection in selected species, such as macaques or pigs. Here, we confirm HBV infection of pig hepatocytes expressing human NTCP and show that porcine NTCP does not support HBV binding. By gradually humanizing porcine NTCP and site-directed mutagenesis, we identified amino acids 158 and 167 in porcine NTCP, limiting HBV interaction. In a proof-of-concept experiment, we showed that the expression of porcine NTCP with humanized amino acids 157-167 renders primary porcine hepatocytes fully susceptible to HBV. These results pave the way for generating transgenic pigs with humanized porcine chimeric NTCP as a novel, fully immunocompetent infection model for developing and validating new curative HBV therapies.

Joint host-pathogen genomic analysis identifies hepatitis B virus mutations associated with human NTCP and HLA class I variation

Evolutionary changes in the hepatitis B virus (HBV) genome could reflect its adaptation to host-induced selective pressure. Leveraging paired human exome and ultra-deep HBV genome-sequencing data from 567 affected individuals with chronic hepatitis B, we comprehensively searched for the signatures of this evolutionary process by conducting "genome-to-genome" association tests between all human genetic variants and viral mutations. We identified significant associations between an East Asian-specific missense variant in the gene encoding the HBV entry receptor NTCP (rs2296651, NTCP S267F) and mutations within the receptor-binding region of HBV preS1. Through in silico modeling and invitro preS1-NTCP binding assays, we observed that the associated HBV mutations are in proximity to the NTCP variant when bound and together partially increase binding affinity to NTCP S267F. Furthermore, we identified significant associations between HLA-A variation and viral mutations in HLA-A-restricted Tcell epitopes. We used in silico binding prediction tools to evaluate the impact of the associated HBV mutations on HLA presentation and observed that mutations that result in weaker binding affinities to their cognate HLA alleles were enriched. Overall, our results suggest the emergence of HBV escape mutations that might alter the interaction between HBV PreS1 and its cellular receptor NTCP during viral entry into hepatocytes and confirm the role of HLA class I restriction in inducing HBV epitope variations.

Molecular mechanisms of Na+-driven bile acid transport in human NTCP

Human Na+ taurocholate co-transporting protein (hNTCP) is a key bile salt transporter to maintain enterohepatic circulation and is responsible for the recognition of hepatitis B and D viruses. Despite landmark cryoelectron microscopy studies revealing open-pore and inward-facing states of hNTCP stabilized by antibodies, the transport mechanism remains largely unknown. To address this knowledge gap, we used molecular dynamics and enhanced sampling metadynamics simulations to elucidate the intrinsic mechanism of hNTCP-mediated taurocholate acid (TCA) transport driven by Na+ binding. We uncovered three TCA-binding modes, including one that closely matched the limited cryoelectron microscopy density observed in the open-pore hNTCP. We also captured several key hNTCP conformations in the substrate transport cycle, particularly including an outward-facing, substrate-bound state. Furthermore, we provided thermodynamic evidence supporting that changes in the Na+-binding state drive the TCA transport by exploiting the amphiphilic nature of the substrate and modulating the protein environment, thereby enabling the TCA molecule to flip through. Understanding these mechanistic details of Na+-driven bile acid transport may aid in the development of hNTCP-targeted therapies for liver diseases.

Structural basis of hepatitis B virus receptor binding.

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.

Ergosterol peroxide blocks HDV infection as a novel entry inhibitor by targeting human NTCP receptor

Hepatitis D virus (HDV), which co-infects or superinfects patients with hepatitis B virus, is estimated to affect 74 million people worldwide. Chronic hepatitis D is the most severe form of viral hepatitis and can result in liver cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Currently, there are no efficient HDV-specific drugs. Therefore, there is an urgent need for novel HDV therapies that can achieve a functional cure or even eliminate the viral infection. In the HDV life cycle, agents targeting the entry step of HDV infection preemptively reduce the intrahepatic viral RNA. Human sodium taurocholate co-transporting polypeptide (hNTCP), a transporter of bile acids on the plasma membrane of hepatocytes, is an essential entry receptor of HDV and is a promising molecular target against HDV infection. Here, we investigated the effect of ergosterol peroxide (EP) on HDV infection in vitro and in vivo. EP inhibited HDV infection of hNTCP-expressing dHuS-E/2 hepatocytes by interrupting the early fusion/endocytosis step of HDV entry. Furthermore, molecular modeling suggested that EP hinders LHBsAg binding to hNTCP by blocking access to S267 and V263. In addition, we generated hNTCP-expressing transgenic (Tg) C57BL/6 mice using the Cre/loxP system for in vivo study. EP reduced the liver HDV RNA level of HDV-challenged hNTCP-Cre Tg mice. Intriguingly, EP downregulated the mRNA level of liver IFN-γ. We demonstrate that EP is a bona fide HDV entry inhibitor that acts on hNTCP and has the potential for use in HDV therapies.

Hepatitis delta virus RNA decline post-inoculation in human NTCP transgenic mice is biphasic.

Chronic infection with hepatitis B and delta viruses (HDV) is the most serious form of viral hepatitis due to more severe manifestations of an accelerated progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma. We characterized early HDV kinetics post-inoculation and incorporated mathematical modeling to provide insights into host-HDV dynamics. We analyzed HDV RNA serum viremia in 192 immunocompetent (C57BL/6) and immunodeficient (NRG) mice that did or did not transgenically express the HDV receptor-human sodium taurocholate co-transporting polypeptide (hNTCP). Kinetic analysis indicates an unanticipated biphasic decline consisting of a sharp first-phase and slower second-phase decline regardless of immunocompetence. HDV decline after re-inoculation again followed a biphasic decline; however, a steeper second-phase HDV decline was observed in NRG-hNTCP mice compared to NRG mice. HDV-entry inhibitor bulevirtide administration and HDV re-inoculation indicated that viral entry and receptor saturation are not major contributors to clearance, respectively. The biphasic kinetics can be mathematically modeled by assuming the existence of a non-specific-binding compartment with a constant on/off-rate and the steeper second-phase decline by a loss of bound virus that cannot be returned as free virus to circulation. The model predicts that free HDV is cleared with a half-life of 35 minutes (standard error, SE: 6.3), binds to non-specific cells with a rate of 0.05 per hour (SE: 0.01), and returns as free virus with a rate of 0.11 per hour (SE: 0.02). Characterizing early HDV-host kinetics elucidates how quickly HDV is either cleared or bound depending on the immunological background and hNTCP presence. IMPORTANCE The persistence phase of HDV infection has been studied in some animal models; however, the early kinetics of HDV in vivo is incompletely understood. In this study, we characterize an unexpectedly HDV biphasic decline post-inoculation in immunocompetent and immunodeficient mouse models and use mathematical modeling to provide insights into HDV-host dynamics.

In vitro cell culture models to study hepatitis B and D virus infection.

Chronic infection with the hepatitis B virus (HBV) and hepatitis D virus (HDV) can cause a major global health burden. Current medication regimens can repress viral replication and help to control disease progression, but a complete cure is hardly achieved due to the difficulties to eradicate viral templates (cccDNA and integrates). To develop novel curative antiviral therapies for HBV/HDV infection, it is vital to precisely understand the details of the molecular biology of both viruses and the virus-host interactions. One important prerequisite for gaining this aim is the availability of suitable in vitro models that support HBV/HDV infection, replicate both viruses via their authentic template and allow to adequately study host cell responses. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) receptor as the most crucial host factor promoted HBV/HDV research to a new era. Recently, the structure of human NTCP was solved, gaining a deeper understanding of HBV recognition as the bona fide receptor. After decades of continuous efforts, new progress has been achieved in the development of cell culture models supporting HBV/HDV study. This review summarizes the cell culture models currently available, discusses the advantages and disadvantages of each model, and highlights their future applications in HBV and HDV research.

Effect of mTOR inhibitors on sodium taurocholate cotransporting polypeptide (NTCP) function in vitro.

The sodium taurocholate cotransporting polypeptide (NTCP; gene name SLC10A1) is the primary hepatic basolateral uptake transporter for conjugated bile acids and the entry receptor for the hepatitis B and D virus (HBV/HDV). Regulation of human NTCP remains a knowledge gap due to significant species differences in substrate and inhibitor selectivity and plasma membrane expression. In the present study, various kinase inhibitors were screened for inhibition of NTCP function and taurocholate (TCA) uptake using NTCP-transfected HuH-7 cells. This study identified everolimus, an mTOR inhibitor and macrocyclic immunosuppressive drug, as an NTCP inhibitor with modest potency (IC50 = 6.7-8.0µM). Further investigation in differentiated HuH-7 cells expressing NTCP and NTCP-overexpressing Flp-In T-REx 293 cells revealed that the mechanism of action of everolimus on NTCP is direct inhibition and mTOR-independent. Structural analogs of everolimus inhibited NTCP-mediated TCA uptake, however, functional analogs did not affect NTCP-mediated TCA transport, providing further evidence for direct inhibition. This work contributes to the growing body of literature suggesting that NTCP-mediated bile acid uptake may be inhibited by macrocyclic peptides, which may be further exploited to develop novel medications against HBV/HDV.

Unlocking the secrets to human NTCP structure

Sodium taurocholate co-transporting polypeptide (NTCP) is encoded by the solute carrier family 10 member 1 (SLC10A1) gene and is predominantly expressed on the sinusoidal membrane of hepatocytes with the function of hepatic uptake of bile salt, steroid hormones, thyroid hormones, and various bile-acid-conjugated drugs. Almost 10 years ago, NTCP was identified as a functional cellular entry receptor of hepatitis B and hepatitis D viruses (HBV/HDV, respectively),1 which are among the major etiological factors leading to cirrhosis, liver failure, and hepatocellular carcinoma and affect around 250 million people worldwide.

Conversion of hepatitis B virus relaxed circular to covalently closed circular DNA is supported in murine cells

Background & AimsHBV has a narrow host restriction, with humans and chimpanzees representing the only known natural hosts. The molecular correlates of resistance in species that are commonly used in biomedical research, such as mice, are currently incompletely understood. Expression of human NTCP (hNTCP) in mouse hepatocytes enables HBV entry, but subsequently covalently closed circular (cccDNA) does not form in most murine cells. It is unknown if this blockade in cccDNA formation is due to deficiency in repair of relaxed circular DNA (rcDNA) to cccDNA.MethodsHere, we deployed both in vivo and in vitro virological and biochemical approaches to investigate if murine cells contain a complete set of repair factors capable of converting HBV rcDNA to cccDNA.ResultsWe demonstrate that HBV cccDNA does form in murine cell culture or in mice when recombinant rcDNA without a protein adduct is directly introduced into cells. We further show that the murine orthologues of core components in DNA lagging strand synthesis, required for the repair of rcDNA to cccDNA in human cells, can support this crucial step in the HBV life cycle. It is worth noting that recombinant HBV rcDNA substrates, either without a protein adduct or containing neutravidin to mimic HBV polymerase, were used in our study; it remains unclear if the HBV polymerase removal processes are the same in mouse and human cells.ConclusionsCollectively, our data suggest that the HBV life cycle is blocked post entry and likely before the repair stage in mouse cells, which yields critical insights that will aid in the construction of a mouse model with inbred susceptibility to HBV infection.Lay summaryHepatitis B virus (HBV) is only known to infect humans and chimpanzees in nature. Mouse models are often used in modeling disease pathogenesis and preclinical research to assess the efficacy and safety of interventions before they are then tested in human participants. However, because mice are not susceptible to HBV infection it is difficult to accurately model human infection (and test potential treatments) in mouse models. Herein, we have shown that mice are able to perform a key step in the HBV life cycle, tightening the net around the possible reason why HBV can not efficiently infect and replicate in mice.

Structure of the bile acid transporter and HBV receptor NTCP

Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually1,2. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes3. However, the molecular basis for the virus-transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs.

Design of Dimeric Bile Acid Derivatives as Potent and Selective Human NTCP Inhibitors.

Dimeric bile acid derivatives (DBADs) were developed and tested for their anti-HBV and anti-HDV activities as sodium taurocholate cotransporting polypeptide (NTCP) inhibitors. DBADs exhibited strong and persistent potency of NTCP inhibition, whereas diverse linkers and constitutions showed distinct inhibition features. Motif aa157-165 on NTCP was shown to be a possible binding site of DBADs; therefore, we determined DBADs' selectivity among NTCPs from different species. A cyclized DBAD scaffold DBA-41 exhibited a high affinity to human NTCP (hNTCP). Intraperitoneal administration of DBA-41 to hNTCP-tg mice induced serum total bile acid elevation. DBA-41 may serve as a biological tool to study NTCP physiological function.

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells.

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293Toverexpressing human NTCP, HNF4α, RXRα and PPARα) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.

Cell Culture Models and Animal Models for HBV Study.

Highly representative and relevant cell and mouse models are required for HBV study, including uncovering its lifecycle, investigation of the viral-host interaction, and development and evaluation of the novel antiviral therapy. During the past 40years, both HBV cell culture models and animal models have evolved over several generations, each with significant improvement for specific purposes. In one aspect, HBV cell culture models experienced the original noninfection model including HBV plasmid DNA transfection and HBV genome integrated stable cells such as HepG2.2.15 which constitutively produces HBV virus and HepAD38 cells and its derivatives which drug-regulated HBV production. As for HBV infection models, HepaRG cells once dominated the HBV infection field for over a decade, but its complicated and labor-extensive cell differentiation procedures discouraged primary researchers from stepping in the field. The identification of human NTCP as HBV receptor evoked great enthusiasm of the whole HBV field, and its readily adaptive characteristic makes it popular in many HBV laboratories. Recombinant cccDNA (rc-cccDNA) emerged recently aiming to tackle the very basic question of how to eventually eradicate cccDNA without HBV real virus infection. In the other aspect, HBV transgenic mouse was firstly generated in the 1990s, which was helpful to decipher HBV production in vivo. However, the HBV transgenic mice were naturally immune tolerant to HBV viral products. Subsequently, a series of nonintegrated HBV mouse models were generated through plasmid hydrodynamic tail vein injection and viral vector-mediated delivery approaches, and HBV full life cycle was incomplete as cccDNA was not formed from HBV relaxed circular DNA (rcDNA). Human NTCP transgenic mouse still could not support productive HBV infection, and humanized mouse liver with human hepatocytes which supported whole HBV life cycle still dominates HBV infection in vivo, a value but expensive model until now. Other methods to empower mouse to carry HBV cccDNA were also exploited. In this chapter, we summarized the advantages and disadvantages of each model historically and provided protocols for HBV infection in HepG2-NTCP cells, HBV rc-cccDNA transfection in HepG2 cells, and HBV infection in NRG-Fah-/- liver humanized mouse.

Phosphodiesterase-induced cAMP degradation restricts hepatitis B virus infection.

Hepatitis B virus (HBV) entry into hepatocytes is mediated via a high-affinity interaction between the preS1 glycoprotein and sodium/bile acid cotransporting polypeptide (NTCP). To date, in vitro model systems rely on high multiplicities of infection to achieve infection of cell lines overexpressing human NTCP. This study investigates a novel regulatory pathway for NTCP trafficking to the cell surface, induced by DMSO-mediated cellular differentiation. DMSO rapidly induces high cell surface expression of NTCP and results in increased susceptibility of cells to HBV infection. Additionally, DMSO treatment induces actin, as well as Tubulin reshaping within the cells. We show that direct disruption of the actin and Tubulin network directly enhances NTCP expression and the subsequent susceptibility of cells to HBV infection. DMSO induces these changes via alterations in the levels of cyclic (c)AMP, which participates in the observed actin rearrangements. Blocking of phosphodiesterases (PDEs), which degrade accumulated cAMP, had the same effect as DMSO differentiation and demonstrates that DMSO prevents phosphodiesterase-mediated cAMP degradation. This identifies adenylate cyclase as a novel target for blocking the entry of HBV via targeting the cell surface accumulation of NTCP.This article is part of the theme issue ‘Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses’.

Tanshinone IIA reverses rifampicin-induced liver injury via activating NRF2-mediated regulation of human NTCP transporter expression

Tanshinone IIA reverses rifampicin-induced liver injury via activating NRF2-mediated regulation of human NTCP transporter expression

Characterisation of the hepatitis B virus cross-species transmission pattern via Na+/taurocholate co-transporting polypeptides from 11 New World and Old World primate species.

The hepatic Na+/taurocholate co-transporting polypeptide (NTCP in man, Ntcp in animals) is the high-affinity receptor for the hepatitis B (HBV) and hepatitis D (HDV) viruses. Species barriers for human HBV/HDV within the order Primates were previously attributed to Ntcp sequence variations that disable virus-receptor interaction. However, only a limited number of primate Ntcps have been analysed so far. In the present study, a total of 11 Ntcps from apes, Old and New World monkeys were cloned and expressed in vitro to characterise their interaction with HBV and HDV. All Ntcps showed intact bile salt transport. Human NTCP as well as the Ntcps from the great apes chimpanzee and orangutan showed transport-competing binding of HBV derived myr-preS1-peptides. In contrast, all six Ntcps from the group of Old World monkeys were insensitive to HBV myr-preS1-peptide binding and HBV/HDV infection. This is basically predetermined by the amino acid arginine at position 158 of all studied Old World monkey Ntcps. An exchange from arginine to glycine (as present in humans and great apes) at this position (R158G) alone was sufficient to achieve full transport-competing HBV myr-preS1-peptide binding and susceptibility for HBV/HDV infection. New World monkey Ntcps showed higher sequence heterogeneity, but in two cases with 158G showed transport-competing HBV myr-preS1-peptide binding, and in one case (Saimiri sciureus) even susceptibility for HBV/HDV infection. In conclusion, amino acid position 158 of NTCP/Ntcp is sufficient to discriminate between the HBV/HDV susceptible group of humans and great apes (158G) and the non-susceptible group of Old World monkeys (158R). In the case of the phylogenetically more distant New World monkey Ntcps amino acid 158 plays a significant, but not exclusive role.

Age-dependent glycosylation of the sodium taurocholate cotransporter polypeptide: From fetal to adult human livers.

Sodium taurocholate cotransporter polypeptide (NTCP), mainly expressed on the sinusoidal membrane of hepatocytes, is one of the major transporters responsible for liver bile acid (BA) re‐uptake. NTCP transports conjugated BA from the blood into hepatocytes and is crucial for correct enterohepatic circulation. Studies have shown that insufficient hepatic clearance of BA correlates with elevated serum BA in infants younger than 1 year of age. In the current study, we investigated human NTCP messenger RNA and protein expression by using reverse‐transcription quantitative polymerase chain reaction and immunoblotting in isolated and cryopreserved human hepatocytes from two different age groups, below and above 1 year of age. Here, we show that NTCP messenger RNA expression is not modulated whereas NTCP protein posttranslational glycosylation is modulated in an age‐dependent manner. These results were confirmed by quantification analysis of NTCP 55‐kDa N‐glycosylated bands, which showed significantly less total NTCP protein in donors below 1 year of age compared to donors older than 1 year. NTCP tissue localization was also analyzed by means of immunofluorescence. This revealed that NTCP cellular localization in fetal samples was mainly perinuclear, suggesting that NTCP is not glycosylated, while its postnatal localization on the plasma membrane is age dependent compared to multidrug resistant protein 2, which is apical starting in fetal life. Conclusion: After birth, the NTCP age‐dependent maturation process requires approximately 1 year to complete NTCP glycosylation in human hepatocytes. Therefore, NTCP late posttranslational glycosylation appears to be important for correct NTCP membrane localization, which might explain physiologic cholestasis in neonatal life and might play a central role for HBV infection after birth. (Hepatology Communications 2018;2:693‐702)