Related Experiment Videos
Conformational dependence of Anaplasma marginale major surface protein 5 surface-exposed B-cell epitopes
D Munodzana1, T F McElwain, D P Knowles
1Laboratory Diagnostics and Research Branch, Central Veterinary Laboratory, Harare, Zimbabwe.
Abstract:
The Anaplasma marginale outer membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940-2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization with A. marginale outer membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denatured A. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to the A. marginale surface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.
Insights
The study shows that the Anaplasma marginale outer membrane protein 5 (MSP5) immunodominant epitope requires specific protein structures for antibody recognition. Maintaining these conformations is crucial for effective antibody responses against Anaplasma marginale.
Area of Science:
- Veterinary Immunology
- Bacteriology
- Molecular Biology
Background:
- The outer membrane of Anaplasma marginale contains immunogenic major surface proteins (MSPs) forming complex structures.
- Effective antibody induction against surface-exposed MSP epitopes may depend on maintaining MSP secondary to quaternary structures.
Purpose of the Study:
- To investigate the conformational requirements of immunodominant epitopes on MSP5, a key surface protein of Anaplasma marginale.
- To determine if antibody recognition of MSP5 epitopes is dependent on their native structure.
Main Methods:
- Epitope mapping using recombinant full-length and truncated MSP5 proteins.
- Assessing antibody binding using monoclonal antibodies (MAbs) and polyclonal antisera.
- Disulfide bond reduction and chemical modification to evaluate epitope structure.
Main Results:
- The immunodominant epitope recognized by MAb ANAF16C1 on MSP5 requires both amino- and carboxy-terminal regions, indicating conformational dependence.
- Disulfide bonds involving N-terminal cysteines are essential for this epitope's conformation.
- Antibody recognition of MSP5 by sera from protected animals was conformationally dependent, requiring native protein structure.
Conclusions:
- The secondary and tertiary structures of MSP epitopes are critical targets for the immune system.
- Immunogens designed to preserve the native conformation of MSPs are likely to elicit more effective antibody responses against Anaplasma marginale.