Limits...
The superantigen streptococcal pyrogenic exotoxin C (SPE-C) exhibits a novel mode of action.

Li PL, Tiedemann RE, Moffat SL, Fraser JD - J. Exp. Med. (1997)

Bottom Line: Despite this, SPE-C cross-links MHC class II to induce homotypic aggregation of class II-bearing B cells.Nondenaturing sodium dodecyl sulfate electrophoresis and size exclusion chromatography revealed that both wild-type and recombinant SPE-C exist in a stable dimer at neutral or alkaline pH.These data support a recent crystal structure of SPE-C and reveal yet another mechanism by which bacterial superantigens ligate and cross-link MHC class II.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, University of Auckland, 92019 Auckland, New Zealand.

ABSTRACT
Recombinant streptococcal pyrogenic exotoxin C (SPE-C) is a potent superantigen that stimulates Vbeta2-bearing human T cells, but is inactive in mice. SPE-C binds with high affinity to both human HLA-DR and murine I-E molecules, but not to murine I-A molecules in a zinc-dependent fashion. Competition binding studies with other recombinant toxins revealed that SPE-C lacks the generic low affinity major histocompatibility complex (MHC) class II alpha-chain binding site common to all other bacterial superantigens. Despite this, SPE-C cross-links MHC class II to induce homotypic aggregation of class II-bearing B cells. Nondenaturing sodium dodecyl sulfate electrophoresis and size exclusion chromatography revealed that both wild-type and recombinant SPE-C exist in a stable dimer at neutral or alkaline pH. These data support a recent crystal structure of SPE-C and reveal yet another mechanism by which bacterial superantigens ligate and cross-link MHC class II.

Show MeSH
Reversibility of SPE-C  dimer formation. Recombinant  SPE-C (2 mg/ml) was dialyzed  overnight in (A) 10 mM BisTris-Tris, pH 6.0, or (B) 10 mM BisTris-Tris, pH 9.0. Samples (20  μl) from each dialysis were incubated for 1 h at room temperature in 100 μl 50 mM BisTris-Tris–0.1 M NaCl at either pH  6.0, 7.0, 8.0, or 9.0, and then analyzed by size exclusion chromatography. The exact retention  time of each peak was determined with the Perceptive Biocad analysis software and is  shown beside each peak. Each  trace represents absorbance at  280 nm. The resulting figure was  generated from the Perceptive  Biocad program using the  stacked trace mode. The small  peak to the right of the pH 6.0  generated dimer is not monomer, but an SPE-C cleavage  product also seen to the right of  the pH 9.0 monomer peak.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199005&req=5

Figure 7: Reversibility of SPE-C dimer formation. Recombinant SPE-C (2 mg/ml) was dialyzed overnight in (A) 10 mM BisTris-Tris, pH 6.0, or (B) 10 mM BisTris-Tris, pH 9.0. Samples (20 μl) from each dialysis were incubated for 1 h at room temperature in 100 μl 50 mM BisTris-Tris–0.1 M NaCl at either pH 6.0, 7.0, 8.0, or 9.0, and then analyzed by size exclusion chromatography. The exact retention time of each peak was determined with the Perceptive Biocad analysis software and is shown beside each peak. Each trace represents absorbance at 280 nm. The resulting figure was generated from the Perceptive Biocad program using the stacked trace mode. The small peak to the right of the pH 6.0 generated dimer is not monomer, but an SPE-C cleavage product also seen to the right of the pH 9.0 monomer peak.

Mentions: Because the SPE-C used for SDS nondenaturing studies was recombinant material stored at high concentration (5 mg/ml), protein dimerization might simply be an artifact of high protein concentration and/or recombinant expression in E. coli. Crude S. pyogenes–derived SPE-C was therefore examined by nondenaturing SDS-PAGE and Western blotting using an affinity-purified anti–SPE-C rabbit anti-serum (Fig. 6). Culture supernatant from an overnight growth of S. pyogenes strain 2035 was first concentrated 1,000-fold by ammonium sulfate precipitation because SPE-C was undetectable in the raw culture medium. The concentration of wild-type SPE-C was estimated at <10 ng/ml in the raw S. pyogenes culture supernatant and ∼10 μg/ml in the crude concentrated sample. Electroblotting of the SPE-C dimer was not as efficient as the monomer, so the relative monomer/dimer ratios were misleading in these experiments. Nevertheless, SPE-C dimer was readily detected in the crude S. pyogenes culture supernatant at approximately the same proportions as in the recombinant SPE-C, despite differences in both purity and concentration. To analyze the effect of pH on SPE-C under more physiological conditions (i.e., in the absence of SDS), SPE-C was dialyzed overnight at either pH 6.0 or 9.0, and then incubated at varying pHs before separation by size exclusion on a Superose 12 HPLC column (Fig. 7). The pH 6.0 dialyzed sample separated exclusively in the dimer form (95% by peak integration), irrespective of the pH of the separating buffer (Fig. 7 A). In contrast, 50% of the pH 9.0 dialyzed sample dissociated to the monomer form when the pH was lowered to 7.0 or less (Fig. 7 B). The apparent molecular weight of both the dimer and monomer peaks appeared to increase with increasing pH. This manifested in a consistent decrease in retention time proportional to increasing pH.


The superantigen streptococcal pyrogenic exotoxin C (SPE-C) exhibits a novel mode of action.

Li PL, Tiedemann RE, Moffat SL, Fraser JD - J. Exp. Med. (1997)

Reversibility of SPE-C  dimer formation. Recombinant  SPE-C (2 mg/ml) was dialyzed  overnight in (A) 10 mM BisTris-Tris, pH 6.0, or (B) 10 mM BisTris-Tris, pH 9.0. Samples (20  μl) from each dialysis were incubated for 1 h at room temperature in 100 μl 50 mM BisTris-Tris–0.1 M NaCl at either pH  6.0, 7.0, 8.0, or 9.0, and then analyzed by size exclusion chromatography. The exact retention  time of each peak was determined with the Perceptive Biocad analysis software and is  shown beside each peak. Each  trace represents absorbance at  280 nm. The resulting figure was  generated from the Perceptive  Biocad program using the  stacked trace mode. The small  peak to the right of the pH 6.0  generated dimer is not monomer, but an SPE-C cleavage  product also seen to the right of  the pH 9.0 monomer peak.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199005&req=5

Figure 7: Reversibility of SPE-C dimer formation. Recombinant SPE-C (2 mg/ml) was dialyzed overnight in (A) 10 mM BisTris-Tris, pH 6.0, or (B) 10 mM BisTris-Tris, pH 9.0. Samples (20 μl) from each dialysis were incubated for 1 h at room temperature in 100 μl 50 mM BisTris-Tris–0.1 M NaCl at either pH 6.0, 7.0, 8.0, or 9.0, and then analyzed by size exclusion chromatography. The exact retention time of each peak was determined with the Perceptive Biocad analysis software and is shown beside each peak. Each trace represents absorbance at 280 nm. The resulting figure was generated from the Perceptive Biocad program using the stacked trace mode. The small peak to the right of the pH 6.0 generated dimer is not monomer, but an SPE-C cleavage product also seen to the right of the pH 9.0 monomer peak.
Mentions: Because the SPE-C used for SDS nondenaturing studies was recombinant material stored at high concentration (5 mg/ml), protein dimerization might simply be an artifact of high protein concentration and/or recombinant expression in E. coli. Crude S. pyogenes–derived SPE-C was therefore examined by nondenaturing SDS-PAGE and Western blotting using an affinity-purified anti–SPE-C rabbit anti-serum (Fig. 6). Culture supernatant from an overnight growth of S. pyogenes strain 2035 was first concentrated 1,000-fold by ammonium sulfate precipitation because SPE-C was undetectable in the raw culture medium. The concentration of wild-type SPE-C was estimated at <10 ng/ml in the raw S. pyogenes culture supernatant and ∼10 μg/ml in the crude concentrated sample. Electroblotting of the SPE-C dimer was not as efficient as the monomer, so the relative monomer/dimer ratios were misleading in these experiments. Nevertheless, SPE-C dimer was readily detected in the crude S. pyogenes culture supernatant at approximately the same proportions as in the recombinant SPE-C, despite differences in both purity and concentration. To analyze the effect of pH on SPE-C under more physiological conditions (i.e., in the absence of SDS), SPE-C was dialyzed overnight at either pH 6.0 or 9.0, and then incubated at varying pHs before separation by size exclusion on a Superose 12 HPLC column (Fig. 7). The pH 6.0 dialyzed sample separated exclusively in the dimer form (95% by peak integration), irrespective of the pH of the separating buffer (Fig. 7 A). In contrast, 50% of the pH 9.0 dialyzed sample dissociated to the monomer form when the pH was lowered to 7.0 or less (Fig. 7 B). The apparent molecular weight of both the dimer and monomer peaks appeared to increase with increasing pH. This manifested in a consistent decrease in retention time proportional to increasing pH.

Bottom Line: Despite this, SPE-C cross-links MHC class II to induce homotypic aggregation of class II-bearing B cells.Nondenaturing sodium dodecyl sulfate electrophoresis and size exclusion chromatography revealed that both wild-type and recombinant SPE-C exist in a stable dimer at neutral or alkaline pH.These data support a recent crystal structure of SPE-C and reveal yet another mechanism by which bacterial superantigens ligate and cross-link MHC class II.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, University of Auckland, 92019 Auckland, New Zealand.

ABSTRACT
Recombinant streptococcal pyrogenic exotoxin C (SPE-C) is a potent superantigen that stimulates Vbeta2-bearing human T cells, but is inactive in mice. SPE-C binds with high affinity to both human HLA-DR and murine I-E molecules, but not to murine I-A molecules in a zinc-dependent fashion. Competition binding studies with other recombinant toxins revealed that SPE-C lacks the generic low affinity major histocompatibility complex (MHC) class II alpha-chain binding site common to all other bacterial superantigens. Despite this, SPE-C cross-links MHC class II to induce homotypic aggregation of class II-bearing B cells. Nondenaturing sodium dodecyl sulfate electrophoresis and size exclusion chromatography revealed that both wild-type and recombinant SPE-C exist in a stable dimer at neutral or alkaline pH. These data support a recent crystal structure of SPE-C and reveal yet another mechanism by which bacterial superantigens ligate and cross-link MHC class II.

Show MeSH