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Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases
- Source :
- Biochemistry and Biophysics Reports; September 2018, Vol. 15 Issue: 1 p25-32, 8p
- Publication Year :
- 2018
-
Abstract
- Human α- and β-enolases are highly homologous enzymes, difficult to differentiate immunologically. In this work, we describe production, purification and properties of anti-α- and anti-β-enolase polyclonal antibodies. To raise antibodies, rabbits were injected with enolase isoenzymes that were purified from human kidney (α-enolase) and skeletal muscle (β-enolase). Selective anti-α- and anti-β-enolase antibodies were obtained by affinity chromatography on either α- or β-enolase-Sepharose columns. On Western blots, antibodies directed against human β-enolase, did not react with human α-isoenzyme, but recognized pig and rat β-enolase. To determine what makes these antibodies selective bioinformatic tools were used to predict conformational epitopes for both enolase isoenzymes. Three predicted epitopes were mapped to the same regions in both α- and β-enolase. Peptides corresponding to predicted epitopes were synthesized and tested against purified antibodies. One of the pin-attached peptides representing α-enolase epitope (the C-terminal portion of the epitope 3 - S262PDDPSRYISPDQ273) reacted with anti-α-enolase, while the other also derived from the α-enolase sequence (epitope 2 - N193VIKEKYGKDATN205) was recognized by anti-β-enolase antibodies. Interestingly, neither anti-α- nor anti-β-antibody reacted with a peptide corresponding to the epitope 2 in β-enolase (G194VIKAKYGKDATN206). Further analysis showed that substitution of E197with A in α-enolase epitope 2 peptide lead to 70% loss of immunological activity, while replacement of A198with E in peptide representing β-enolase epitope 2, caused 67% increase in immunological activity. Our results suggest that E197is essential for preserving immunologically active conformation in epitope 2 peptidic homolog, while it is not crucial for this epitope's antigenic activity in native β-enolase.
Details
- Language :
- English
- ISSN :
- 24055808
- Volume :
- 15
- Issue :
- 1
- Database :
- Supplemental Index
- Journal :
- Biochemistry and Biophysics Reports
- Publication Type :
- Periodical
- Accession number :
- ejs46368276
- Full Text :
- https://doi.org/10.1016/j.bbrep.2018.05.008