Unique epitope–antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface display
ABSTRACTCarbonic anhydrase (CA)-IX is an extracellular enzyme that is essential in the adaptation of tumor cells to their increasingly more hypoxic and acidic microenvironment. Within the family of carbonic anhydrases, CA-IX is unique in that it is the only CA with an N-terminal intrinsically disord...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2023-12-01
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| Series: | mAbs |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2023.2248672 |
| _version_ | 1797356291047292928 |
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| author | Feng Ni Cunle Wu Ping Xu Ping Wang Yves Fortin Melanie Arbour Luke Masson Denis L’Abbé Andrea Acel Mylene Gosselin Anne E.G. Lenferink |
| author_facet | Feng Ni Cunle Wu Ping Xu Ping Wang Yves Fortin Melanie Arbour Luke Masson Denis L’Abbé Andrea Acel Mylene Gosselin Anne E.G. Lenferink |
| author_sort | Feng Ni |
| collection | DOAJ |
| description | ABSTRACTCarbonic anhydrase (CA)-IX is an extracellular enzyme that is essential in the adaptation of tumor cells to their increasingly more hypoxic and acidic microenvironment. Within the family of carbonic anhydrases, CA-IX is unique in that it is the only CA with an N-terminal intrinsically disordered region (IDR) containing a proteoglycan (PG)-like domain. This PG-like IDR has been described to be instrumental in CA-IX’s enzyme activity, as well as tumor cell motility and invasion. We have characterized the antibody–epitope interactions of two novel and unique antibodies (11H9 and 12H8) that are specific for the human CA-IX’s IDR. Binding interactions of these antibodies to the intact IDR were studied by surface plasmon resonance and high-resolution nuclear magnetic resonance (NMR) spectroscopy, while the specific epitopes were determined by both NMR and yeast surface display (YSD). Our data show that 12H8 binds to the N-terminus of CA-IX, while 11H9 has a high affinity for an epitope located in the central region of the IDR containing three GEEDLP repeats in a manner that is different from the previously described M75 antibody. Titration NMR spectroscopy using CA-IX’s entire IDR in addition identified a secondary epitope of 11H9 at the beginning of the PG-like domain that remains exposed and available for further binding events after the engagement at its primary epitope at the center of the PG-like domain. Transverse relaxation optimized NMR spectroscopy of 11H9-F(Ab) in complex with the CA-IX IDR outlines structural rigidification of a linear epitope, while the rest of the IDR remains largely unstructured upon complex formation. This study illustrates how high-resolution NMR and YSD are used as complementary tools for a comprehensive characterization of antibody–epitope interactions involving intrinsically unstructured antigen domains with highly repetitive sequences. |
| first_indexed | 2024-03-08T14:24:30Z |
| format | Article |
| id | doaj.art-bef68df9eb5940de9d22b6603ddf4189 |
| institution | Directory Open Access Journal |
| issn | 1942-0862 1942-0870 |
| language | English |
| last_indexed | 2024-03-08T14:24:30Z |
| publishDate | 2023-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj.art-bef68df9eb5940de9d22b6603ddf41892024-01-13T11:27:51ZengTaylor & Francis GroupmAbs1942-08621942-08702023-12-0115110.1080/19420862.2023.2248672Unique epitope–antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface displayFeng Ni0Cunle Wu1Ping Xu2Ping Wang3Yves Fortin4Melanie Arbour5Luke Masson6Denis L’Abbé7Andrea Acel8Mylene Gosselin9Anne E.G. Lenferink10Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, CanadaABSTRACTCarbonic anhydrase (CA)-IX is an extracellular enzyme that is essential in the adaptation of tumor cells to their increasingly more hypoxic and acidic microenvironment. Within the family of carbonic anhydrases, CA-IX is unique in that it is the only CA with an N-terminal intrinsically disordered region (IDR) containing a proteoglycan (PG)-like domain. This PG-like IDR has been described to be instrumental in CA-IX’s enzyme activity, as well as tumor cell motility and invasion. We have characterized the antibody–epitope interactions of two novel and unique antibodies (11H9 and 12H8) that are specific for the human CA-IX’s IDR. Binding interactions of these antibodies to the intact IDR were studied by surface plasmon resonance and high-resolution nuclear magnetic resonance (NMR) spectroscopy, while the specific epitopes were determined by both NMR and yeast surface display (YSD). Our data show that 12H8 binds to the N-terminus of CA-IX, while 11H9 has a high affinity for an epitope located in the central region of the IDR containing three GEEDLP repeats in a manner that is different from the previously described M75 antibody. Titration NMR spectroscopy using CA-IX’s entire IDR in addition identified a secondary epitope of 11H9 at the beginning of the PG-like domain that remains exposed and available for further binding events after the engagement at its primary epitope at the center of the PG-like domain. Transverse relaxation optimized NMR spectroscopy of 11H9-F(Ab) in complex with the CA-IX IDR outlines structural rigidification of a linear epitope, while the rest of the IDR remains largely unstructured upon complex formation. This study illustrates how high-resolution NMR and YSD are used as complementary tools for a comprehensive characterization of antibody–epitope interactions involving intrinsically unstructured antigen domains with highly repetitive sequences.https://www.tandfonline.com/doi/10.1080/19420862.2023.2248672Antibodiesantibody–antigen interactionscarbonic anhydrase IXepitope mappingF(ab)intrinsically disordered proteins |
| spellingShingle | Feng Ni Cunle Wu Ping Xu Ping Wang Yves Fortin Melanie Arbour Luke Masson Denis L’Abbé Andrea Acel Mylene Gosselin Anne E.G. Lenferink Unique epitope–antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface display mAbs Antibodies antibody–antigen interactions carbonic anhydrase IX epitope mapping F(ab) intrinsically disordered proteins |
| title | Unique epitope–antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface display |
| title_full | Unique epitope–antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface display |
| title_fullStr | Unique epitope–antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface display |
| title_full_unstemmed | Unique epitope–antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface display |
| title_short | Unique epitope–antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface display |
| title_sort | unique epitope antibody interactions in the intrinsically disordered proteoglycan like domain of human carbonic anhydrase ix defined by high resolution nmr combined with yeast surface display |
| topic | Antibodies antibody–antigen interactions carbonic anhydrase IX epitope mapping F(ab) intrinsically disordered proteins |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2023.2248672 |
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