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Transmembrane Domain Oligomerization Propensity determined by ToxR Assay
Published on: May 26, 2011
Evidence that lipopolysaccharide and pertussis toxin bind to different domains on the same p73 receptor on murine
1Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66160.
Insights
This study shows that Bordetella pertussis toxin (PT) binding to cells reduces lipopolysaccharide (LPS) binding to the p73 receptor. This suggests p73 recognizes both LPS and PT at distinct sites, impacting bacterial endotoxin recognition.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Lipopolysaccharide (LPS) is a key bacterial endotoxin recognized by mammalian cells.
- A specific LPS-binding protein, p73, has been identified on lymphoreticular cells.
- Bordetella pertussis toxin (PT) interacts with LPS in fluid phase, with lysozyme (LZM) competing for binding.
Purpose of the Study:
- To investigate the effect of cell-bound PT on LPS binding to the p73 receptor.
- To determine if PT's interaction with LPS in fluid phase is responsible for its effect on LPS-p73 binding.
- To elucidate the binding sites of LPS and PT on the p73 molecule.
Main Methods:
- Utilized a photoactivable, radioiodinated LPS derivative as a photo-cross-linking probe.
- Studied the binding of LPS probe to p73 on murine splenocytes in the presence of cell-bound PT.
- Investigated the role of PT's A-protomer and LPS-LZM interactions in these binding events.
Main Results:
- Cell-bound PT significantly reduced the binding of the LPS probe to the p73 receptor on splenocytes.
- This reduction was independent of the PT A-protomer and concentration-dependent.
- Lysozyme did not affect LPS probe binding to p73 or the observed reduction by PT.
- Cell-bound PT was radiolabeled by the LPS probe, indicating a direct interaction.
Conclusions:
- Cell-bound PT influences LPS binding to p73, but not through fluid-phase LPS-PT interactions.
- PT and LPS likely bind to distinct sites on the p73 molecule.
- The p73 protein may serve as a common recognition molecule for both LPS and PT.
Abstract:
In previous studies, we used a photoactivable, radioiodinated lipopolysaccharide (LPS) derivative to define and characterize a specific bacterial endotoxic LPS-binding protein (p73) on mammalian lymphoreticular cells, including B and T lymphocytes and macrophages. More recently, using the same methodology, we characterized a specific interaction of LPS with the S2 subunit of Bordetella pertussis pertussis toxin (PT) in the fluid phase (M.-G. Lei and D. C. Morrison, J. Biol. Chem., 268:1488-1493, 1993). Furthermore, we showed that lysozyme (LZM) but not polymyxin B can compete with PT for binding to LPS in the fluid phase, a result suggesting that these two molecules compete for the same binding site on LPS. In this report, we demonstrate that the binding of PT to murine splenocytes (cell-bound PT) reduces the ability of the LPS photo-cross-linking probe to bind to the p73 receptor. The reduction can also be demonstrated with the PT B oligomer, a result indicating that the observed reduction of LPS binding to the p73 receptor by PT is A-protomer (S1-subunit) independent. More importantly, our studies document that cell-bound PT can be radiolabelled by the LPS probe, coincident with the observed reduction in p73 photoaffinity labelling. The preferential interaction of LPS with the PT S2 subunit in the fluid phase was, however, not observed with cell-bound PT. The reduction in radiolabelling of the p73 receptor by the LPS probe and in radiolabelling of cell-bound PT was shown to be concentration dependent. The data presented here document, however, that LZM does not reduce the ability of the LPS probe to bind to the p73 receptor on mouse splenocytes, nor does the presence of LZM bound to LPS influence the observed reduction in photoaffinity labelling of p73 by the LPS probe or radiolabelling of cell-bound PT by the LPS probe. Collectively, these results support the concept that the ability of LPS to interact with PT in the fluid phase is not responsible for the ability of cell-bound PT to influence the binding of the LPS probe to the p73 receptor. Thus, it is suggested that PT and LPS bind to different sites on the p73 molecule and that this same p73 protein may recognize both LPS and PT.
