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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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SPE-C and SEA induce homotypic aggregation in LG-2  cells. Freshly split LG-2 cells were incubated with 1 μg/ml of recombinant toxin in 24-well culture plates and monitored for 24 h. Percent aggregation was determined by counting the number of cells not bound to  an aggregated clump.
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Figure 4: SPE-C and SEA induce homotypic aggregation in LG-2 cells. Freshly split LG-2 cells were incubated with 1 μg/ml of recombinant toxin in 24-well culture plates and monitored for 24 h. Percent aggregation was determined by counting the number of cells not bound to an aggregated clump.

Mentions: SEA and anti–MHC class II mAbs such as LB3.1 rapidly induce homotypic aggregation of LG-2 cells. In addition, both these MHC class II ligands induce the expression of proinflammatory cytokines such as IL-1 and TNF-α in MHC class II–bearing APCs (20, 27). This activity is a direct function of two separate binding sites for MHC class II molecules on SEA and presumably reflects the ability to cross-link MHC class II. Mutation of either site in SEA or papain cleavage of LB3.1 into F(ab) fragments abolishes the ability to induce homotypic aggregation. Purified recombinant SPE-C was tested in the LG-2 homotypic aggregation assay and found to be as potent as SEA (Fig. 4) suggesting that SPE-C was also capable of cross-linking MHC class II. In contrast, neither recombinant SEB nor SPE-A displayed any aggregation activity consistent with the presence of only a single MHC class II binding site on these molecules. Given that the previous experiments suggested the α-chain binding site was absent in SPE-C, two hypotheses were put forward to account for the presumed class II cross-linking activity of SPE-C. The first was that SPE-C binds to MHC class II via the zinc-mediated β-chain site and another unidentified site. The second hypothesis was that SPE-C might form dimers in solution with two identical β-chain binding sites. To test the second hypothesis, recombinant SPE-C was examined by nondenaturing SDS-PAGE in an attempt to observe SPE-C oligomers. Homodimers were clearly visible under both reducing and nonreducing conditions, representing >60% of the total amount of SPE-C loaded (Fig. 5). These dimers were absent when the sample was denatured in nonreducing conditions (not shown). One feature of these dimers was their discrete nature, even in the presence of 1% SDS and 10 mM dithiothreitol. No smearing between dimer and monomer was observed. Another feature of the SPE-C dimers was their dependence on neutral or alkaline pH for formation. At conditions below pH 6.0, virtually no dimers were detected, whereas at pH 7.0, >60% of the SPE-C was observed in dimer form. Neither EDTA nor iodoacetic acid (a sulfhydryl alkylating reagent) affected dimer stability, indicating the absence of covalently disulfide linked homodimers.


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)

SPE-C and SEA induce homotypic aggregation in LG-2  cells. Freshly split LG-2 cells were incubated with 1 μg/ml of recombinant toxin in 24-well culture plates and monitored for 24 h. Percent aggregation was determined by counting the number of cells not bound to  an aggregated clump.
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Related In: Results  -  Collection

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Figure 4: SPE-C and SEA induce homotypic aggregation in LG-2 cells. Freshly split LG-2 cells were incubated with 1 μg/ml of recombinant toxin in 24-well culture plates and monitored for 24 h. Percent aggregation was determined by counting the number of cells not bound to an aggregated clump.
Mentions: SEA and anti–MHC class II mAbs such as LB3.1 rapidly induce homotypic aggregation of LG-2 cells. In addition, both these MHC class II ligands induce the expression of proinflammatory cytokines such as IL-1 and TNF-α in MHC class II–bearing APCs (20, 27). This activity is a direct function of two separate binding sites for MHC class II molecules on SEA and presumably reflects the ability to cross-link MHC class II. Mutation of either site in SEA or papain cleavage of LB3.1 into F(ab) fragments abolishes the ability to induce homotypic aggregation. Purified recombinant SPE-C was tested in the LG-2 homotypic aggregation assay and found to be as potent as SEA (Fig. 4) suggesting that SPE-C was also capable of cross-linking MHC class II. In contrast, neither recombinant SEB nor SPE-A displayed any aggregation activity consistent with the presence of only a single MHC class II binding site on these molecules. Given that the previous experiments suggested the α-chain binding site was absent in SPE-C, two hypotheses were put forward to account for the presumed class II cross-linking activity of SPE-C. The first was that SPE-C binds to MHC class II via the zinc-mediated β-chain site and another unidentified site. The second hypothesis was that SPE-C might form dimers in solution with two identical β-chain binding sites. To test the second hypothesis, recombinant SPE-C was examined by nondenaturing SDS-PAGE in an attempt to observe SPE-C oligomers. Homodimers were clearly visible under both reducing and nonreducing conditions, representing >60% of the total amount of SPE-C loaded (Fig. 5). These dimers were absent when the sample was denatured in nonreducing conditions (not shown). One feature of these dimers was their discrete nature, even in the presence of 1% SDS and 10 mM dithiothreitol. No smearing between dimer and monomer was observed. Another feature of the SPE-C dimers was their dependence on neutral or alkaline pH for formation. At conditions below pH 6.0, virtually no dimers were detected, whereas at pH 7.0, >60% of the SPE-C was observed in dimer form. Neither EDTA nor iodoacetic acid (a sulfhydryl alkylating reagent) affected dimer stability, indicating the absence of covalently disulfide linked homodimers.

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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