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After release from the oocyst, sporozoites invade the mosquito salivary glands [1–3]. All constructs were confirmed by DNA sequencing. For more information about PLOS Subject Areas, click When the cells detached from the plate, medium was added to terminate the reaction. Arrowhead indicates a contaminant. Fractions containing target proteins were detected by SDS-PAGE or Western blot. LAU + Smart WAFT.
To address the possibilities that incorrect folding of the NTD in PfCSPΔC prevents interaction with HA-αTSR, and that the NTD and CTD interact inter-molecularly, HA-αTSR was incubated with GFP-PfCSP prior to immunoprecipitation with anti-GFP or anti-HA. Protein Glycosylation in the Malaria Parasite. Purified proteins were diluted with binding buffer (10 mM Tris pH 7.4) and loaded onto a heparin affinity column (Heparin HP 1 ml; GE Healthcare) equilibrated in the same buffer.
CSP sequences enriched in basic residues have previously been scanned for heparin binding activity. The circumsporozoite protein (CSP) contains a putative C-terminal GPI anchoring signal, and it is widely presumed that this protein is GPI anchored.
Alternatively, the αTSR domain may bind to molecules other than HSPGs on the surface of hepatocytes to promote secondary attachment to hepatocytes and invasion. The αTSR domain has also been shown to be accessible to antibodies only when the NTD is cleaved off at region I, suggesting that the CTD is masked by the NTD prior to cleavage [14, 25]. Sequence elements of CSP that mediate primary and secondary interactions to HSPGs are still unclear. Agarose was resuspended in 500 μl binding buffer and 20 μg purified HA-αTSR added to incubate for 2 h at 4°C.
We thank Alicia Prater for proofreading, and Yajuan Wan and Zishan Yang for help with the FACS experiments.
These results are consistent with in vitro data demonstrating that the purified TSR domain from Plasmodium falciparum CSP (PfCSP) does not bind heparin [8]. Is the Subject Area "Heparin" applicable to this article?
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PpGFP-PfCSPΔN and EcGFP-PfCSPΔN proteins have a tendency to oligomerize and elute over the same wide range (Fig 2B). Because CSP is known to recognize heparin, we initially determined the retention of the protein on a heparin column while washing with a salt gradient. here. 1-800-355-5354. https://doi.org/10.1371/journal.pone.0161607, Editor: Friedrich Frischknecht, University of Heidelberg Medical School, GERMANY, Received: February 2, 2016; Accepted: August 9, 2016; Published: August 25, 2016. Subsequent cleavage of CSP in region I, unmasks the TSR domain. For E. coli expression, the wild-type CS and all mutants were inserted into the EcoRI/NotI sites of pET-28a. Copyright © 1999 Elsevier Science Ltd. All rights reserved. C, as in B, but with GFP-αTSR.
GPI. By continuing you agree to the use of cookies. Data shown are from a representative experiment. Results: The STN was the predominant site of DBS implantation before and after CSP 468 publication (93.2% of cases, and 60.4%, respectively), but GPi targeting increased significantly following CSP 468 publication (from 5.3% to 37.4%; p < .001). Affiliation Important: Static pressure requirements are significantly affected by the amount of make-up air supplied to an area.
Swift CSP.
To further analyze the activities of the NTD of CSP in a cellular context, we incubated various GFP-tagged CSPs with HepG2 cells and monitored their interactions using live cell imaging.
Incubation of HepG2 cells with GFP-PfCSP, but not GFP alone, significantly increased the GFP signal on the HepG2 cells (Fig 4A). Give us a call at 1-800-355-5354. Copyright © 2020 Elsevier B.V. or its licensors or contributors.
These results suggest that the CTD can stabilize the NTD. It has been widely believed that O-glycosylation is the major carbohydrate modification in the intraerythrocytic stage of P. falciparum and that the parasite has no N-glycosylation capacity.
Customer Service. More. Following the attachment of sporozoites to hepatocytes, proteolysis at a site in region I exposes the CTD. Taken together, the results indicate that the lysine cluster in proximity to region I is most critical for heparin binding.
A well experienced team with a high quality services, I recommend. Our data provide important insights into how CSP contributes to the attachment of Plasmodium sporozoites to liver cells. This website builds under HTML5, please use a browser above IE 9.0 version or Chrome.
Several basic regions of CSP have been proposed to engage the negatively charged heparin.
The codon-optimized wild-type CS gene encoding P. falciparum 3D7 CS (residues 19–127 and 256–374) was fused with a GFP tag at the N-terminus and a His tag at the C-terminus.
Yes The data are representative of at least four repetitions. https://doi.org/10.1016/S0169-4758(99)01412-X.
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.
Gel filtration analysis showed that the GFP-PfCSPΔN and GFP-αTSR from yeast (PpGFP-PfCSPΔN and PpGFP-αTSR, respectively) and E. coli (EcGFP-PfCSPΔN and EcGFP-αTSR) behave similarly (Fig 2B and 2C). Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, and Tianjin Key Laboratory of Protein Sciences, Tianjin, 300071, China, The circumsporozoite protein (CSP) belongs to a diverse group of plasmodial cell surface proteins that are anchored to the cell membrane by a myristoylated glycosylphosphatidylinositol (GPI) glycolipid 1, 2.GPI‐lipidation is a multistep process that begins with the preassembly of the GPI membrane anchor moiety. The TSR domain has cell adhesive properties [15], and is thought to mediate the secondary attachment between sporozoites and hepatocytes, thus leading to the invasion of hepatocytes by sporozoites [14]. Department of Immunology, School of Basic Medical Sciences, Tianjin Medical University, and Tianjin Key Laboratory of Cellular and Molecular Immunology, Tianjin, 300070, China. Here, we conduct a systematic analysis of the interaction between CSP and heparin or hepatocytes using GFP-tagged recombinant CS proteins. However, most of these studies did not examine the attachment of sporozoites to hepatocytes and focused on either sporozoite exit from the dermis or sporozoite invasion of hepatocytes.
Recent structural studies have revealed that region III and the TSR fold into a single “αTSR” domain (also referred to as the C-terminal domain, CTD) [8]. The deadline for SWIFT customers to submit their results is before the end of 2017. Substitutions of additional lysines, specifically K88, K90, and K92 individually or in combination, also decreased CSP binding to heparin (Fig 1F, 1G and 1H).
Consistent with our observation, mutant parasites lacking region I but containing the lysine cluster exhibited normal attachment to Hepa1-6 cells [14]. To determine exactly which domain(s) of CSP interacts with liver cells, we incubated different recombinant CS proteins with HepG2 cells and measured their association by flow cytometry using the GFP tag of the recombinant CS proteins for quantification. B.O.S Framework. Though variation exists in the amino acid sequence across species, the overall domain structure is well conserved. One possibility is that the repeat region, which is largely missing in the recombinant proteins, plays a coordinative role.
Mutations that affect heparin binding are colored in red and mutation that does not is colored in cyan. All point mutations and truncations were generated by overlap PCR using wild-type CS as the template.
Fractions are analyzed by SDS-PAGE and coomassie staining.
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