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Modification of cysteine residues in vitro and in vivo affects the immunogenicity and antigenicity of major histocompatibility complex class I-restricted viral determinants.

Chen W, Yewdell JW, Levine RL, Bennink JR - J. Exp. Med. (1999)

Bottom Line: Similar enhancing effects were obtained by substituting cysteine with alanine or serine in the synthetic peptides, demonstrating that sulfhydryl modification of cysteine is responsible for the impaired antigenicity and immunogenicity of NP39-47 and NP218-226.We found similar effects for two widely studied, cysteine-containing peptides from lymphocytic choriomeningitis virus.These findings demonstrate that posttranslational modifications affect the immunogenicity and antigenicity of cysteine-containing viral peptides and that this must be considered in studying the status of such peptides in immunodominance hierarchies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

ABSTRACT
In studying the subdominant status of two cysteine-containing influenza virus nuclear protein (NP) determinants (NP39-47 and NP218-226) restricted by H-2Kd, we found that the antigenicity of synthetic peptides was enhanced 10-100-fold by treatment with reducing agents, despite the fact that the affinity for Kd was not enhanced. Reducing agents also markedly enhanced the immunogenicity of cysteine-containing peptides, as measured by propagation of long-term T cell lines in vitro. Similar enhancing effects were obtained by substituting cysteine with alanine or serine in the synthetic peptides, demonstrating that sulfhydryl modification of cysteine is responsible for the impaired antigenicity and immunogenicity of NP39-47 and NP218-226. We found similar effects for two widely studied, cysteine-containing peptides from lymphocytic choriomeningitis virus. The major modifications of cysteine-containing synthetic peptides are cysteinylation and dimerization occurring through cysteine residues. We demonstrate that both of these modifications occur in cells synthesizing a cytosolic NP218-226 minigene product and, further, that T cells specific for cysteinylated NP218-226 are induced by influenza virus infection in mice, demonstrating that this modification occurs in vivo. These findings demonstrate that posttranslational modifications affect the immunogenicity and antigenicity of cysteine-containing viral peptides and that this must be considered in studying the status of such peptides in immunodominance hierarchies.

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Effects of reducing agents on the antigenicity and Kd binding  of synthetic NP218–226. Synthetic peptides were diluted in I-10 with or  without 200 μM of the indicated reducing agents and added to assay wells  containing 51Cr-labeled target cells and TCD8+ specific for NP218–226 (A) or  NP147–155 (B), and lysis was determined by microcytotoxicity assay.
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Figure 3: Effects of reducing agents on the antigenicity and Kd binding of synthetic NP218–226. Synthetic peptides were diluted in I-10 with or without 200 μM of the indicated reducing agents and added to assay wells containing 51Cr-labeled target cells and TCD8+ specific for NP218–226 (A) or NP147–155 (B), and lysis was determined by microcytotoxicity assay.

Mentions: Cysteine readily forms disulfide bonds at neutral or slightly basic pH in the presence of O2 at atmospheric tension, and oxidation to the disulfide is stimulated by trace amounts of iron salts that are present in tissue culture media. The disulfide can be reduced to the original thiol form by exposure to reducing agents. To determine whether disulfide formation affected peptide antigenicity, synthetic NP218–226 was added to cells in the presence of dithiothreitol or TCEP, and cells were tested for lysis by NP218–226-specific TCD8+. Either of these reducing agents increased peptide potency by ∼10-fold (Fig. 3 A). Reducing agents did not affect the potency of noncysteine-containing peptides, including NP147–155 (Fig. 3 B), an LCMV peptide (described below), or the cysteine→ serine- or cysteine→ alanine-substituted NP39–47 peptides (not shown). Enhancement of cysteine peptide recognition is, as expected, dependent on the concentration of reducing agent, with TCEP being more effective on a molar basis than dithiothreitol (not shown). The optimal concentration for TCEP was 200 μM (used in additional experiments), as higher concentrations (1 mM) sometimes increased spontaneous release values in 51Cr-release assays. The effect of TCEP on NP218–226 antigenicity is particularly impressive when considered in view of the 100–1,000-fold decrease in peptide binding to Kd in the presence of TCEP (described below).


Modification of cysteine residues in vitro and in vivo affects the immunogenicity and antigenicity of major histocompatibility complex class I-restricted viral determinants.

Chen W, Yewdell JW, Levine RL, Bennink JR - J. Exp. Med. (1999)

Effects of reducing agents on the antigenicity and Kd binding  of synthetic NP218–226. Synthetic peptides were diluted in I-10 with or  without 200 μM of the indicated reducing agents and added to assay wells  containing 51Cr-labeled target cells and TCD8+ specific for NP218–226 (A) or  NP147–155 (B), and lysis was determined by microcytotoxicity assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193077&req=5

Figure 3: Effects of reducing agents on the antigenicity and Kd binding of synthetic NP218–226. Synthetic peptides were diluted in I-10 with or without 200 μM of the indicated reducing agents and added to assay wells containing 51Cr-labeled target cells and TCD8+ specific for NP218–226 (A) or NP147–155 (B), and lysis was determined by microcytotoxicity assay.
Mentions: Cysteine readily forms disulfide bonds at neutral or slightly basic pH in the presence of O2 at atmospheric tension, and oxidation to the disulfide is stimulated by trace amounts of iron salts that are present in tissue culture media. The disulfide can be reduced to the original thiol form by exposure to reducing agents. To determine whether disulfide formation affected peptide antigenicity, synthetic NP218–226 was added to cells in the presence of dithiothreitol or TCEP, and cells were tested for lysis by NP218–226-specific TCD8+. Either of these reducing agents increased peptide potency by ∼10-fold (Fig. 3 A). Reducing agents did not affect the potency of noncysteine-containing peptides, including NP147–155 (Fig. 3 B), an LCMV peptide (described below), or the cysteine→ serine- or cysteine→ alanine-substituted NP39–47 peptides (not shown). Enhancement of cysteine peptide recognition is, as expected, dependent on the concentration of reducing agent, with TCEP being more effective on a molar basis than dithiothreitol (not shown). The optimal concentration for TCEP was 200 μM (used in additional experiments), as higher concentrations (1 mM) sometimes increased spontaneous release values in 51Cr-release assays. The effect of TCEP on NP218–226 antigenicity is particularly impressive when considered in view of the 100–1,000-fold decrease in peptide binding to Kd in the presence of TCEP (described below).

Bottom Line: Similar enhancing effects were obtained by substituting cysteine with alanine or serine in the synthetic peptides, demonstrating that sulfhydryl modification of cysteine is responsible for the impaired antigenicity and immunogenicity of NP39-47 and NP218-226.We found similar effects for two widely studied, cysteine-containing peptides from lymphocytic choriomeningitis virus.These findings demonstrate that posttranslational modifications affect the immunogenicity and antigenicity of cysteine-containing viral peptides and that this must be considered in studying the status of such peptides in immunodominance hierarchies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

ABSTRACT
In studying the subdominant status of two cysteine-containing influenza virus nuclear protein (NP) determinants (NP39-47 and NP218-226) restricted by H-2Kd, we found that the antigenicity of synthetic peptides was enhanced 10-100-fold by treatment with reducing agents, despite the fact that the affinity for Kd was not enhanced. Reducing agents also markedly enhanced the immunogenicity of cysteine-containing peptides, as measured by propagation of long-term T cell lines in vitro. Similar enhancing effects were obtained by substituting cysteine with alanine or serine in the synthetic peptides, demonstrating that sulfhydryl modification of cysteine is responsible for the impaired antigenicity and immunogenicity of NP39-47 and NP218-226. We found similar effects for two widely studied, cysteine-containing peptides from lymphocytic choriomeningitis virus. The major modifications of cysteine-containing synthetic peptides are cysteinylation and dimerization occurring through cysteine residues. We demonstrate that both of these modifications occur in cells synthesizing a cytosolic NP218-226 minigene product and, further, that T cells specific for cysteinylated NP218-226 are induced by influenza virus infection in mice, demonstrating that this modification occurs in vivo. These findings demonstrate that posttranslational modifications affect the immunogenicity and antigenicity of cysteine-containing viral peptides and that this must be considered in studying the status of such peptides in immunodominance hierarchies.

Show MeSH
Related in: MedlinePlus