Limits...
Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein.

Barabas S, Gary R, Bauer T, Lindner J, Lindner P, Weinberger B, Jilg W, Wolf H, Deml L - PLoS Pathog. (2008)

Bottom Line: Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules.Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro.The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, University of Regensburg, Germany.

ABSTRACT
Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I-dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8(+) cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

Show MeSH

Related in: MedlinePlus

Deletion of a C-terminal arginine-rich domain within BZLF1 with homology to PTD does not abrogate the capacity of uBZLF1 to activate specific T cells.(A) A truncated BZLF1 protein, engineered by deletion of the arginine-rich domain (BZLF1Δ176–189) was expressed in E. coli, purified and treated with urea as described in materials and methods. (B) The purity of BZLF1 and BZLF1Δ176–189 was assessed by Coomassie staining. (C) PBMC of healthy EBV-seropositive donors were stimulated for 20 h with 10 µg/ml uBZLF1, BZLF1Δ176–189 or the HIV control peptide E10F and the frequencies of IFN-γ producing cells were assayed by ELISpot. The data show mean spot forming cell (SFC) values±standard deviation (s.d.) of 5 replicate stimulations and are a representative of results performed with PBMC of 3 different individuals.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2572144&req=5

ppat-1000198-g003: Deletion of a C-terminal arginine-rich domain within BZLF1 with homology to PTD does not abrogate the capacity of uBZLF1 to activate specific T cells.(A) A truncated BZLF1 protein, engineered by deletion of the arginine-rich domain (BZLF1Δ176–189) was expressed in E. coli, purified and treated with urea as described in materials and methods. (B) The purity of BZLF1 and BZLF1Δ176–189 was assessed by Coomassie staining. (C) PBMC of healthy EBV-seropositive donors were stimulated for 20 h with 10 µg/ml uBZLF1, BZLF1Δ176–189 or the HIV control peptide E10F and the frequencies of IFN-γ producing cells were assayed by ELISpot. The data show mean spot forming cell (SFC) values±standard deviation (s.d.) of 5 replicate stimulations and are a representative of results performed with PBMC of 3 different individuals.

Mentions: During the last decade short arginine-rich protein-transduction domains (PTD) have been described to mediate polypeptide translocation across cell membranes. Sequence alignments revealed the presence of an arginine-rich domain within the C-terminal region of BZLF1 exhibiting similarities to a known PTD domain of the Antennapedia (Antp) homeoprotein (Fig. 3A). To examine the role of this PTD-like domain in the translocation of uBZLF1 into APC, we expressed a deletion mutant (BZLF1Δ176–189), lacking the putative PTD in E. coli (Fig. 3B). We stimulated PBMC of HLA B8-positive EBV-seropositive healthy donors with either 10 µg/ml uBZLF1 or truncated uBZLF1Δ176–189 and determined the number of specifically restimulated, IFN-γ secreting T cells by ELISpot. PBMC stimulated with HIV Gag-derived peptide E10F served as negative control. In these experiments, uBZLF1 and uBZLF1Δ176–189 showed no significant differences in their capacity to reactivate BZLF1-specific T cells (Fig. 3C), indicating that the predicted PTD within BZLF1 plays no significant role in urea-mediated protein translocation into the endogenous processing pathway of APC.


Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein.

Barabas S, Gary R, Bauer T, Lindner J, Lindner P, Weinberger B, Jilg W, Wolf H, Deml L - PLoS Pathog. (2008)

Deletion of a C-terminal arginine-rich domain within BZLF1 with homology to PTD does not abrogate the capacity of uBZLF1 to activate specific T cells.(A) A truncated BZLF1 protein, engineered by deletion of the arginine-rich domain (BZLF1Δ176–189) was expressed in E. coli, purified and treated with urea as described in materials and methods. (B) The purity of BZLF1 and BZLF1Δ176–189 was assessed by Coomassie staining. (C) PBMC of healthy EBV-seropositive donors were stimulated for 20 h with 10 µg/ml uBZLF1, BZLF1Δ176–189 or the HIV control peptide E10F and the frequencies of IFN-γ producing cells were assayed by ELISpot. The data show mean spot forming cell (SFC) values±standard deviation (s.d.) of 5 replicate stimulations and are a representative of results performed with PBMC of 3 different individuals.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572144&req=5

ppat-1000198-g003: Deletion of a C-terminal arginine-rich domain within BZLF1 with homology to PTD does not abrogate the capacity of uBZLF1 to activate specific T cells.(A) A truncated BZLF1 protein, engineered by deletion of the arginine-rich domain (BZLF1Δ176–189) was expressed in E. coli, purified and treated with urea as described in materials and methods. (B) The purity of BZLF1 and BZLF1Δ176–189 was assessed by Coomassie staining. (C) PBMC of healthy EBV-seropositive donors were stimulated for 20 h with 10 µg/ml uBZLF1, BZLF1Δ176–189 or the HIV control peptide E10F and the frequencies of IFN-γ producing cells were assayed by ELISpot. The data show mean spot forming cell (SFC) values±standard deviation (s.d.) of 5 replicate stimulations and are a representative of results performed with PBMC of 3 different individuals.
Mentions: During the last decade short arginine-rich protein-transduction domains (PTD) have been described to mediate polypeptide translocation across cell membranes. Sequence alignments revealed the presence of an arginine-rich domain within the C-terminal region of BZLF1 exhibiting similarities to a known PTD domain of the Antennapedia (Antp) homeoprotein (Fig. 3A). To examine the role of this PTD-like domain in the translocation of uBZLF1 into APC, we expressed a deletion mutant (BZLF1Δ176–189), lacking the putative PTD in E. coli (Fig. 3B). We stimulated PBMC of HLA B8-positive EBV-seropositive healthy donors with either 10 µg/ml uBZLF1 or truncated uBZLF1Δ176–189 and determined the number of specifically restimulated, IFN-γ secreting T cells by ELISpot. PBMC stimulated with HIV Gag-derived peptide E10F served as negative control. In these experiments, uBZLF1 and uBZLF1Δ176–189 showed no significant differences in their capacity to reactivate BZLF1-specific T cells (Fig. 3C), indicating that the predicted PTD within BZLF1 plays no significant role in urea-mediated protein translocation into the endogenous processing pathway of APC.

Bottom Line: Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules.Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro.The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, University of Regensburg, Germany.

ABSTRACT
Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I-dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8(+) cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

Show MeSH
Related in: MedlinePlus