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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.

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65Zn binding to  SPE-C. Recombinant toxins  were incubated with 100 μM  65Zn and blotted in triplicate to  nitrocellulose. After washing, the  strips were autoradiographed and  the individual dots counted.
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Figure 1: 65Zn binding to SPE-C. Recombinant toxins were incubated with 100 μM 65Zn and blotted in triplicate to nitrocellulose. After washing, the strips were autoradiographed and the individual dots counted.

Mentions: Recombinant SPE-C was tested for its ability to bind radioactive 65Zn in a zinc dot-blot assay alongside recombinant SEA, SEE, SEB, and SPE-A (Fig. 1). SPE-C bound 65Zn 100 times greater than background and 50-fold higher than either SEB or SPE-A (two toxins that do not require Zn2+ for superantigen function). SEA bound the highest levels of 65Zn in this assay, whereas both SPE-C and SEE bound approximately half this level. Given that bound 65Zn is determined at nonequilibrium conditions, these differences reflect a combination of both the equilibrium conditions, these differences reflect a combination of both the equilibrium affinity and the off-rate of zinc for the different toxin binding sites.


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)

65Zn binding to  SPE-C. Recombinant toxins  were incubated with 100 μM  65Zn and blotted in triplicate to  nitrocellulose. After washing, the  strips were autoradiographed and  the individual dots counted.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: 65Zn binding to SPE-C. Recombinant toxins were incubated with 100 μM 65Zn and blotted in triplicate to nitrocellulose. After washing, the strips were autoradiographed and the individual dots counted.
Mentions: Recombinant SPE-C was tested for its ability to bind radioactive 65Zn in a zinc dot-blot assay alongside recombinant SEA, SEE, SEB, and SPE-A (Fig. 1). SPE-C bound 65Zn 100 times greater than background and 50-fold higher than either SEB or SPE-A (two toxins that do not require Zn2+ for superantigen function). SEA bound the highest levels of 65Zn in this assay, whereas both SPE-C and SEE bound approximately half this level. Given that bound 65Zn is determined at nonequilibrium conditions, these differences reflect a combination of both the equilibrium conditions, these differences reflect a combination of both the equilibrium affinity and the off-rate of zinc for the different toxin binding sites.

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