Site-directed chemical modification of horse cytochrome c results in changes in antigenicity due to local and long-range conformational perturbations.
Abstract: Comparative binding studies with peptide fragments of the whole antigen, or with evolutionarily related intact proteins with varying degrees of sequence homology, have been used extensively to map antigenic sites on proteins to the resolution of single amino acid residues. These methods are limited, however, since high affinity antibodies will often not react with peptides and evolutionarily related proteins are available for only a few antigens. In this study we use site-directed chemical modification of horse cytochrome c to identify residues involved in the binding sites of four monoclonal antibodies specific for this protein. Thus, we have N-formylated the single tryptophan found in horse cytochrome c at position 59 and N-carbethoxylated one of the histidyl residues, which was determined to be at position 26 by the analysis of proteolytic cleavage fragments of the modified protein using liquid secondary ion-mass spectrometry on triple quadropole or tandem quadropole Fourier transform instruments. We discuss the impact of these modifications on the antigenicity of horse cytochrome c with regard to the conformational perturbations introduced by such modifications and with reference to our previous studies on the binding sites of these antibodies using other methodologies (Jemmerson, R., and Paterson, Y. (1986) BioTechniques 4, 18-31).
Publication Date: 1987-08-25 PubMed ID: 2442150
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- U.S. Gov't
- P.H.S.
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research explores how the strategic chemical modification of proteins, specifically horse cytochrome c, can result in changes in the antigenicity, potentially useful in identifying specific binding sites of monoclonal antibodies.
Research Context
- The study is built on the premise that mapping antigenic sites on proteins, down to the level of single amino acids, is often done through comparative binding studies involving peptide fragments of the whole antigen or with proteins that have evolved with varying degrees of sequence homology.
- There were limitations to these traditional methods, such as high affinity antibodies not reacting with peptides, and the lack of availability of evolutionarily related proteins for certain antigens.
Research Methodology
- As a solution, the researchers used a method called site-directed chemical modification, which specifically targets horse cytochrome c protein in this study.
- Two modifications were made to the horse cytochrome c protein: the tryptophan at position 59 was N-formylated and a histidyl residue at position 26 was N-carbethoxylated.
- The position of the modified histidyl residue was verified using proteolytic cleavage and advanced techniques in mass spectrometry.
Research Findings and Discussions
- The main focus of this study was to examine the effect of these modifications on the antigenicity of the horse cytochrome c protein.
- The research discussed the structural alterations induced by these modifications and how those changes could affect the protein’s ability to bind to the specific monoclonal antibodies.
- The results were also interpreted in the context of the team’s previous work on the binding sites of these antibodies using other methodologies.
Conclusion
- This insight on how site-directed chemical modifications affect the antigenicity of the protein could be instrumental in further understanding of the binding sites of monoclonal antibodies.
- It can potentially pave the way for new methodologies in mapping antigenic sites on proteins, beyond the limitations of currently used techniques.
Cite This Article
APA
Cooper HM, Jemmerson R, Hunt DF, Griffin PR, Yates JR, Shabanowitz J, Zhu NZ, Paterson Y.
(1987).
Site-directed chemical modification of horse cytochrome c results in changes in antigenicity due to local and long-range conformational perturbations.
J Biol Chem, 262(24), 11591-11597.
Publication
Researcher Affiliations
MeSH Terms
- Animals
- Binding, Competitive
- Chromatography, High Pressure Liquid
- Cytochrome c Group / metabolism
- Diethyl Pyrocarbonate
- Epitopes / metabolism
- Horses
- Lasers
- Mass Spectrometry
- Models, Molecular
- Peptide Fragments / metabolism
- Protein Conformation
- Stereoisomerism
- Tryptophan / metabolism
Grant Funding
- AI 19499 / NIAID NIH HHS
- AI 21486 / NIAID NIH HHS
- GM 31841 / NIGMS NIH HHS
Citations
This article has been cited 4 times.- Wadhwa S, Jain A, Woodward JG, Mumper RJ. Lipid nanocapsule as vaccine carriers for his-tagged proteins: evaluation of antigen-specific immune responses to HIV I His-Gag p41 and systemic inflammatory responses.. Eur J Pharm Biopharm 2012 Feb;80(2):315-22.
- Watson DS, Platt VM, Cao L, Venditto VJ, Szoka FC Jr. Antibody response to polyhistidine-tagged peptide and protein antigens attached to liposomes via lipid-linked nitrilotriacetic acid in mice.. Clin Vaccine Immunol 2011 Feb;18(2):289-97.
- Duquesnoy RJ. A structurally based approach to determine HLA compatibility at the humoral immune level.. Hum Immunol 2006 Nov;67(11):847-62.
- Van Regenmortel MH. Protein antigenicity.. Mol Biol Rep 1992 Jun;16(3):133-8.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
