Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases
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 purif...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2018-09-01
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| Series: | Biochemistry and Biophysics Reports |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405580818300438 |
| _version_ | 1819212419801546752 |
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| author | Jadwiga Pietkiewicz Regina Danielewicz Iwona S. Bednarz-Misa Ireneusz Ceremuga Jerzy Wiśniewski Magdalena Mierzchala-Pasierb Agnieszka Bronowicka-Szydełko Edmund Ziomek Andrzej Gamian |
| author_facet | Jadwiga Pietkiewicz Regina Danielewicz Iwona S. Bednarz-Misa Ireneusz Ceremuga Jerzy Wiśniewski Magdalena Mierzchala-Pasierb Agnieszka Bronowicka-Szydełko Edmund Ziomek Andrzej Gamian |
| author_sort | Jadwiga Pietkiewicz |
| collection | DOAJ |
| description | 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 E197 with A in α-enolase epitope 2 peptide lead to 70% loss of immunological activity, while replacement of A198 with E in peptide representing β-enolase epitope 2, caused 67% increase in immunological activity. Our results suggest that E197 is 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. Keywords: Enolase purification, Mass spectrometry, Epitope prediction, Specific antibodies, Cross-reactivity |
| first_indexed | 2024-12-23T06:42:40Z |
| format | Article |
| id | doaj.art-04d3c77dbaa74536b393cd667a5c248c |
| institution | Directory Open Access Journal |
| issn | 2405-5808 |
| language | English |
| last_indexed | 2024-12-23T06:42:40Z |
| publishDate | 2018-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biochemistry and Biophysics Reports |
| spelling | doaj.art-04d3c77dbaa74536b393cd667a5c248c2022-12-21T17:56:40ZengElsevierBiochemistry and Biophysics Reports2405-58082018-09-01152532Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolasesJadwiga Pietkiewicz0Regina Danielewicz1Iwona S. Bednarz-Misa2Ireneusz Ceremuga3Jerzy Wiśniewski4Magdalena Mierzchala-Pasierb5Agnieszka Bronowicka-Szydełko6Edmund Ziomek7Andrzej Gamian8Department of Medical Biochemistry, Wroclaw Medical University,Chalubinskiego 10, 50-368 Wroclaw, Poland; Corresponding author.Department of Medical Biochemistry, Wroclaw Medical University,Chalubinskiego 10, 50-368 Wroclaw, PolandDepartment of Medical Biochemistry, Wroclaw Medical University,Chalubinskiego 10, 50-368 Wroclaw, PolandDepartment of Medical Biochemistry, Wroclaw Medical University,Chalubinskiego 10, 50-368 Wroclaw, PolandDepartment of Medical Biochemistry, Wroclaw Medical University,Chalubinskiego 10, 50-368 Wroclaw, PolandDepartment of Medical Biochemistry, Wroclaw Medical University,Chalubinskiego 10, 50-368 Wroclaw, PolandDepartment of Medical Biochemistry, Wroclaw Medical University,Chalubinskiego 10, 50-368 Wroclaw, PolandWroclaw Research Center, Stablowicka 147, 50-066 Wroclaw, PolandDepartment of Medical Biochemistry, Wroclaw Medical University,Chalubinskiego 10, 50-368 Wroclaw, Poland; Wroclaw Research Center, Stablowicka 147, 50-066 Wroclaw, PolandHuman 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 E197 with A in α-enolase epitope 2 peptide lead to 70% loss of immunological activity, while replacement of A198 with E in peptide representing β-enolase epitope 2, caused 67% increase in immunological activity. Our results suggest that E197 is 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. Keywords: Enolase purification, Mass spectrometry, Epitope prediction, Specific antibodies, Cross-reactivityhttp://www.sciencedirect.com/science/article/pii/S2405580818300438 |
| spellingShingle | Jadwiga Pietkiewicz Regina Danielewicz Iwona S. Bednarz-Misa Ireneusz Ceremuga Jerzy Wiśniewski Magdalena Mierzchala-Pasierb Agnieszka Bronowicka-Szydełko Edmund Ziomek Andrzej Gamian Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases Biochemistry and Biophysics Reports |
| title | Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases |
| title_full | Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases |
| title_fullStr | Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases |
| title_full_unstemmed | Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases |
| title_short | Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases |
| title_sort | experimental and bioinformatic approach to identifying antigenic epitopes in human α and β enolases |
| url | http://www.sciencedirect.com/science/article/pii/S2405580818300438 |
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