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Fusion expression of Occludin extracellular loops and an ?-helical bundle: A new research model for tight junction.

ABSTRACT: Tight junctions (TJs) are the outermost structures of intercellular junctions and are highly specialized membrane domains involved in many important cellular processes. However, most TJ proteins are four-time transmembrane proteins and are difficult to express in their correct soluble form, which limits their functional study and therapeutic application. Human occludin (OCLN) is a major component of TJs and an essential co-receptor for hepatitis C virus (HCV) cell entry. To explore expression strategy for recombinant TJ proteins possessing integrated and functional extracellular loops, OCLN was here used as a model molecule, and several prokaryotic fusion constructs were designed by docking OCLN extracellular loops (ECLs) to HIV-1 gp41 NHR and CHR six-helical bundle (6HV1); then their biophysical features and anti-HCV activity were evaluated. The proteins were successfully expressed and purified in E. coli, and the double-loop constructs (D1ECL1S+D2ECL2 as a representative) were found to have more potent HCV neutralizing activity than single-loop constructs at non-cytotoxic concentrations. Circular dichroism studies indicate that D1ECL1S+D2ECL2 adopt stable ?-helical folds consistent with design. Thermal denaturation assay indicated that D1ECL1S+D2ECL2 is highly stable at 80°C (melting temperature, Tm, of 89.08 ± 2.0°C) and comparable in stability to the 6HV1 scaffold. Moreover, the time-of-addition experiment revealed that D1ECL1S+D2ECL2 predominantly functioned during the early stages of HCV entry. Taken together, these findings provide a novel strategy for recombinant TJ protein expression in vitro, which may shed light on functional and structural studies for TJs and may provide a new avenue to drug development.

SUBMITTER: Chi X

PROVIDER: S-EPMC5407606 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Fusion expression of Occludin extracellular loops and an α-helical bundle: A new research model for tight junction.

Chi Xiaojing X Zhao Xia X Wang Wei W Niu Yuqiang Y Cheng Min M Liu Xiuying X Cui Sheng S Yang Wei W

PloS one 20170427 4

Tight junctions (TJs) are the outermost structures of intercellular junctions and are highly specialized membrane domains involved in many important cellular processes. However, most TJ proteins are four-time transmembrane proteins and are difficult to express in their correct soluble form, which limits their functional study and therapeutic application. Human occludin (OCLN) is a major component of TJs and an essential co-receptor for hepatitis C virus (HCV) cell entry. To explore expression st ...[more]

PMID: 28448574

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Project description:Porcine epidemic diarrhea virus (PEDV), the causative agent of porcine epidemic diarrhea, has caused huge economic losses in pig-producing countries. Although PEDV was long believed to replicate in the intestinal epithelium by using aminopeptidase N as a receptor, the mechanisms of PEDV infection are not fully characterized. In this study, we found that PEDV infection of epithelial cells results in disruption of the tight junctional distribution of occludin to its intracellular location. Overexpression of occludin in target cells makes them more susceptible to PEDV infection, whereas ablation of occludin expression by use of small interfering RNA (siRNA) in target cells significantly reduces their susceptibility to virus infection. However, the results observed with occludin siRNA indicate that occludin is not required for virus attachment. We conclude that occludin plays an essential role in PEDV infection at the postbinding stages. Furthermore, we observed that macropinocytosis inhibitors blocked occludin internalization and virus entry, indicating that virus entry and occludin internalization are closely coupled. However, the macropinocytosis inhibitors could not impede virus replication once the virus had entered host cells. This suggests that occludin internalization by macropinocytosis or a macropinocytosis-like process is involved in the virus entry events. Immunofluorescence confocal microscopy showed that PEDV was trapped at cellular junctional regions upon macropinocytosis inhibitor treatment, indicating that occludin may serve as a scaffold in the vicinity of virus entry. Collectively, these data show that occludin plays an essential role in PEDV infection during late entry events. Our observation may provide novel insights into PEDV infection and related pathogenesis.IMPORTANCE Tight junctions are highly specialized membrane domains whose main function is to attach adjacent cells to each other, thereby forming intercellular seals. Here we investigate, for the first time, the role of the tight junction protein occludin in PEDV infection. We observed that PEDV infection induced the internalization of occludin. By using genetic modification methods, we demonstrate that occludin plays an essential role in PEDV infection. Moreover, PEDV entry and occludin internalization seem to be closely coupled. Our findings reveal a new mechanism of PEDV infection.

Dataset Information

Fusion expression of Occludin extracellular loops and an ?-helical bundle: A new research model for tight junction.

Publications

Fusion expression of Occludin extracellular loops and an α-helical bundle: A new research model for tight junction.

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