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Hepatocyte permissiveness to Plasmodium infection is conveyed by a short and structurally conserved region of the CD81 large extracellular domain.

Yalaoui S, Zougbédé S, Charrin S, Silvie O, Arduise C, Farhati K, Boucheix C, Mazier D, Rubinstein E, Froissard P - PLoS Pathog. (2008)

Bottom Line: Still, the molecular mechanisms underlying sporozoite invasion are largely unknown.By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain.This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie-Paris6, UMR S511, Paris, France.

ABSTRACT
Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.

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The VVD (135–137)→AAA and DDD (137–139)→AAA mutants unable to support infection by P. yoelii sporozoites interact with CD9P-1 and EWI-2.CHO cells were transiently transfected with WT or mutant CD81 plasmids (in pEGFP-N3), together with a CD9P-1 (top) or a EWI-2 (bottom) cDNA. After 48 h, the cells were lysed with digitonin and immunoprecipitations with antibodies against CD81, CD9P-1 and EWI-2 were performed. After electrophoresis and transfer, the membranes were incubated with biotin-labelled mAbs to CD81 (TS81), CD9P-1 (1F11) and EWI-2 (8A12). The mutants are designed as follows: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA
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ppat-1000010-g007: The VVD (135–137)→AAA and DDD (137–139)→AAA mutants unable to support infection by P. yoelii sporozoites interact with CD9P-1 and EWI-2.CHO cells were transiently transfected with WT or mutant CD81 plasmids (in pEGFP-N3), together with a CD9P-1 (top) or a EWI-2 (bottom) cDNA. After 48 h, the cells were lysed with digitonin and immunoprecipitations with antibodies against CD81, CD9P-1 and EWI-2 were performed. After electrophoresis and transfer, the membranes were incubated with biotin-labelled mAbs to CD81 (TS81), CD9P-1 (1F11) and EWI-2 (8A12). The mutants are designed as follows: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA

Mentions: The two major CD81 partners identified so far in hepatocytic cells are CD9P-1 and EWI-2, two Ig domain molecules [25]. Because tetraspanins have been shown in some cases to modulate the function of the proteins to which they associate, it was important to check whether the mutants unable to support infection by P. yoelii sporozoites still associate with these molecules. CHO cells were transfected with CD9P-1 or EWI-2 and either WT CD81, the VVD (135–137)→AAA mutant or the DDD(137–139)→AAA mutant (all in fusion with EGFP), and their interactions was tested by immunoprecipitation after cell lysis with digitonin (a detergent suitable to visualize direct interactions within the tetraspanin web). In preliminary experiments TS81 was shown to recognize the endogenous hamster CD81 molecule expressed by CHO cells, while 1D6 did not, so 1D6 was used for further analysis. As shown in fig 7, CD9P-1 and EWI-2 were co-immunoprecipitated with WT CD81 and the two CD81 mutants, and reciprocally the CD81 mutants were co-immunoprecipitated with CD9P-1 and EWI-2. This shows that the inability of the two mutants to support infection by P. yoelii sporozoites is not due to a loss of interaction with these partner molecules and further indicates that these mutants have a correct conformation.


Hepatocyte permissiveness to Plasmodium infection is conveyed by a short and structurally conserved region of the CD81 large extracellular domain.

Yalaoui S, Zougbédé S, Charrin S, Silvie O, Arduise C, Farhati K, Boucheix C, Mazier D, Rubinstein E, Froissard P - PLoS Pathog. (2008)

The VVD (135–137)→AAA and DDD (137–139)→AAA mutants unable to support infection by P. yoelii sporozoites interact with CD9P-1 and EWI-2.CHO cells were transiently transfected with WT or mutant CD81 plasmids (in pEGFP-N3), together with a CD9P-1 (top) or a EWI-2 (bottom) cDNA. After 48 h, the cells were lysed with digitonin and immunoprecipitations with antibodies against CD81, CD9P-1 and EWI-2 were performed. After electrophoresis and transfer, the membranes were incubated with biotin-labelled mAbs to CD81 (TS81), CD9P-1 (1F11) and EWI-2 (8A12). The mutants are designed as follows: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279262&req=5

ppat-1000010-g007: The VVD (135–137)→AAA and DDD (137–139)→AAA mutants unable to support infection by P. yoelii sporozoites interact with CD9P-1 and EWI-2.CHO cells were transiently transfected with WT or mutant CD81 plasmids (in pEGFP-N3), together with a CD9P-1 (top) or a EWI-2 (bottom) cDNA. After 48 h, the cells were lysed with digitonin and immunoprecipitations with antibodies against CD81, CD9P-1 and EWI-2 were performed. After electrophoresis and transfer, the membranes were incubated with biotin-labelled mAbs to CD81 (TS81), CD9P-1 (1F11) and EWI-2 (8A12). The mutants are designed as follows: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA
Mentions: The two major CD81 partners identified so far in hepatocytic cells are CD9P-1 and EWI-2, two Ig domain molecules [25]. Because tetraspanins have been shown in some cases to modulate the function of the proteins to which they associate, it was important to check whether the mutants unable to support infection by P. yoelii sporozoites still associate with these molecules. CHO cells were transfected with CD9P-1 or EWI-2 and either WT CD81, the VVD (135–137)→AAA mutant or the DDD(137–139)→AAA mutant (all in fusion with EGFP), and their interactions was tested by immunoprecipitation after cell lysis with digitonin (a detergent suitable to visualize direct interactions within the tetraspanin web). In preliminary experiments TS81 was shown to recognize the endogenous hamster CD81 molecule expressed by CHO cells, while 1D6 did not, so 1D6 was used for further analysis. As shown in fig 7, CD9P-1 and EWI-2 were co-immunoprecipitated with WT CD81 and the two CD81 mutants, and reciprocally the CD81 mutants were co-immunoprecipitated with CD9P-1 and EWI-2. This shows that the inability of the two mutants to support infection by P. yoelii sporozoites is not due to a loss of interaction with these partner molecules and further indicates that these mutants have a correct conformation.

Bottom Line: Still, the molecular mechanisms underlying sporozoite invasion are largely unknown.By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain.This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie-Paris6, UMR S511, Paris, France.

ABSTRACT
Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.

Show MeSH
Related in: MedlinePlus