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. 2016 Aug 25;11(8):e0161607.
doi: 10.1371/journal.pone.0161607. eCollection 2016.

A Comprehensive Analysis of Plasmodium Circumsporozoite Protein Binding to Hepatocytes

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Free PMC article

A Comprehensive Analysis of Plasmodium Circumsporozoite Protein Binding to Hepatocytes

Jinghua Zhao et al. PLoS One. .
Free PMC article

Abstract

Circumsporozoite protein (CSP) is the dominant protein on the surface of Plasmodium sporozoites and plays a critical role in the invasion by sporozoites of hepatocytes. Contacts between CSP and heparin sulfate proteoglycans (HSPGs) lead to the attachment of sporozoites to hepatocytes and trigger signaling events in the parasite that promote invasion of hepatocytes. The precise sequence elements in CSP that bind HSPGs have not been identified. We performed a systematic in vitro analysis to dissect the association between Plasmodium falciparum CSP (PfCSP) and hepatocytes. We demonstrate that interactions between PfCSP and heparin or a cultured hepatoma cell line, HepG2, are mediated primarily by a lysine-rich site in the amino terminus of PfCSP. Importantly, the carboxyl terminus of PfCSP facilitates heparin-binding by the amino-terminus but does not interact directly with heparin. These findings provide insights into how CSP recognizes hepatocytes and useful information for further functional studies of CSP.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. GFP-PfCSP interacts with heparin.
A, Domain structures of full-length and recombinant PfCSP. The termini are indicated. SP, signal peptide; NTD, N-terminal domain; CTD, C-terminal domain; RI, region I; RII+, region II plus; RIII, region III; GPI, GPI anchor sequence. B, Sequence alignment of residues preceding the region I in the NTD of CSP. Residues in PfCSP are numbered, and residues mutated are highlighted in bold. Mutations that affect heparin binding are colored in red and mutation that does not is colored in cyan. Basic residues in other CSPs that are near region I are colored in orange. The region I is highlighted by a yellow box. Peptides that have been tested for heparin binding are underlined. C,D, Heparin binding of GFP-PfCSP (C) and GFP alone (D). ~150 μg of purified protein was applied to the heparin column, and samples were analyzed by SDS-PAGE, followed by Coomassie staining. Domain structure of the protein used is shown on the left. I, input; FT, flow-through. E-N, as in C, but with GFP-tagged CSP mutants. Arrowhead indicates a contaminant. All data were confirmed by at least three independent experiments using three independently purified batches of proteins. Data shown are from a representative experiment.
Fig 2
Fig 2. Comparison of recombinant CSP from bacteria and yeast.
A, Heparin binding of GFP, GFP-PfCSP, GFP-PfCSPΔN, and GFP-αTSR purified from P. pastoris. Samples for GFP-PfCSP are analyzed by immunoblotting (IB) using antibodies against GFP. All other samples are analyzed by Coomassie staining. A degraded product of GFP-PfCSP is indicated by arrowhead. B, Gel filtration analysis of GFP-PfCSPΔN purified from either E. coli or P. pastoris. Fractions are analyzed by SDS-PAGE and coomassie staining. C, as in B, but with GFP-αTSR.
Fig 3
Fig 3. Homotypic interactions between CSP termini.
A, B, Purified GFP-PfCSPΔC or GFP alone was incubated with HA-αTSR. Immunoprecipitation (IP) was performed using anti-GFP (A) or anti-HA (B) antibodies. The samples were analyzed by SDS-PAGE and immunoblotting (IB) with anti-HA or anti-GFP antibodies. C, as in A, but with GFP-PfCSP instead of GFP. D, as in B, but with GFP-PfCSP instead of GFP.
Fig 4
Fig 4. Attachment of CSP to hepatocytes.
A, Purified GFP-PfCSP or mutant proteins were incubated with HepG2 cells. Binding of the proteins to the cells was quantified by using flow cytometry to detect GFP fluorescence. Analyzed data are shown on the right. The data are representative of at least four repetitions. B, Purified GFP-PfCSP or mutant proteins were incubated with HepG2 cells. GFP on the cell surface was detected by live cell imaging. Nuclei were stained with Hoechst 33258 for identification of individual cells. Approximately 300 cells were counted for each sample. The data represent at least three repetitions.

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Grant support

Q. Wang is supported by startup funds from Tianjin Medical University. J. Hu is supported by the National Natural Science Foundation of China (grant 31225006), and an International Early Career Scientist grant from Howard Hughes Medical Institute.
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