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Analysis of functional regions of YPM, a superantigen derived from gram-negative bacteria
Y Ito1, G Seprényi, J Abe
1Department of Allergy and Immunology, and Department of Child Ecology, National Children's Medical Research Center, Tokyo, Japan. tkohsaka@nch.go.jp
Abstract:
The bacterial superantigens, staphylococcal enterotoxins and streptococcal pyrogenic exotoxins, are grouped in a family by the conservation of amino acid sequence and polypeptide folding patterns. In the case of Yersinia pseudotuberculosis-derived mitogen (YPM), however, there is no noticeable homology with this family, although many of the in vitro functional features conform to the criteria for a superantigen. To study the mode of action of YPM at the molecular level, we first generated a number of YPM point mutants with reduced T-cell proliferative activity using random mutagenesis and localized the amino acid positions involved in either major histocompatibility complex class II or T-cell receptor Vbeta-interaction. Plotting the elucidated positions on the hydrophilicity profile suggested that they reside mostly on the outer portion of the molecule. We also report that the two cysteines positioned almost at opposing ends of the YPM molecule are connected by an S-S bond the destruction of which causes fatal damage. Finally, we obtained evidence that YPM partially competes with staphylococcal enterotoxin E for human leukocyte antigen-DR binding. This raises the question of whether these different types of superantigens have acquired the same function by genetic convergence or originated from a common ancestral gene.
Insights
Yersinia pseudotuberculosis-derived mitogen (YPM) functions as a superantigen despite lacking homology to known families. Its molecular interactions reveal key residues and a critical disulfide bond, suggesting convergent evolution of superantigen function.
Area of Science:
- Immunology
- Microbiology
- Structural Biology
Background:
- Bacterial superantigens, like staphylococcal enterotoxins and streptococcal pyrogenic exotoxins, share conserved sequences and structures.
- Yersinia pseudotuberculosis-derived mitogen (YPM) exhibits superantigen activity but lacks homology to known superantigen families.
Purpose of the Study:
- To elucidate the molecular mechanism of YPM's superantigen activity.
- To identify key amino acid residues and structural features critical for YPM function.
Main Methods:
- Random mutagenesis to generate YPM point mutants with reduced T-cell proliferation.
- Localization of critical amino acid positions involved in MHC class II and TCR Vbeta interaction.
- Analysis of disulfide bond formation and its impact on YPM stability.
- Competition assays for binding to human leukocyte antigen-DR (HLA-DR).
Main Results:
- Identified amino acid positions critical for YPM interaction with MHC class II and TCR Vbeta, predominantly located on the molecule's surface.
- Discovered a crucial disulfide bond connecting two cysteines, essential for YPM's structural integrity and function.
- Demonstrated partial competition between YPM and staphylococcal enterotoxin E for HLA-DR binding.
Conclusions:
- YPM's functional superantigen activity is mediated by surface-exposed residues and a vital disulfide bond.
- The findings raise questions about the evolutionary origins of superantigen function, possibly through genetic convergence or a common ancestor.