Selection and characterization of FcεRI phospho-ITAM specific antibodies
Post-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids (e.g., anti-phosphotyrosine), which are often used to assay these changes, require isolation of the spe...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2019-10-01
|
| Series: | mAbs |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2019.1632113 |
| _version_ | 1828150257508155392 |
|---|---|
| author | Nileena Velappan Avanika Mahajan Leslie Naranjo Priyanka Velappan Nasim Andrews Nicholas Tiee Subhendu Chakraborti Colin Hemez Tiziano Gaiotto Bridget Wilson Andrew Bradbury |
| author_facet | Nileena Velappan Avanika Mahajan Leslie Naranjo Priyanka Velappan Nasim Andrews Nicholas Tiee Subhendu Chakraborti Colin Hemez Tiziano Gaiotto Bridget Wilson Andrew Bradbury |
| author_sort | Nileena Velappan |
| collection | DOAJ |
| description | Post-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids (e.g., anti-phosphotyrosine), which are often used to assay these changes, require isolation of the specific protein prior to analysis and do not identify the specific residue that has been modified (in the case that multiple amino acids have been modified). Phosphorylation state-specific antibodies (PSSAs) developed to recognize post-translational modifications within a specific amino acid sequence can be used to study the timeline of modifications during a signal cascade. We used the FcεRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We selected three β-subunit antibodies that recognized: 1) phosphorylation of tyrosines Y218 or Y224; 2) phosphorylation of the Y228 tyrosine; and 3) phosphorylation of all three tyrosines. We used these antibodies to study the receptor activation timeline of FcεR1 in rat basophilic leukemia cells (RBL-2H3) upon stimulation with DNP24-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y65) of the receptor and applied this antibody to evaluate receptor activation. Recognition patterns of these antibodies show different timelines for phosphorylation of tyrosines in both β and γ subunits. Our methodology provides a strategy to select antibodies specific to post-translational modifications and provides new reagents to study mast cell activation by the high-affinity IgE receptor, FcεRI. |
| first_indexed | 2024-04-11T21:41:46Z |
| format | Article |
| id | doaj.art-10edd85e6c9b4e6fb382d9d01fc06caf |
| institution | Directory Open Access Journal |
| issn | 1942-0862 1942-0870 |
| language | English |
| last_indexed | 2024-04-11T21:41:46Z |
| publishDate | 2019-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj.art-10edd85e6c9b4e6fb382d9d01fc06caf2022-12-22T04:01:33ZengTaylor & Francis GroupmAbs1942-08621942-08702019-10-011171206121810.1080/19420862.2019.1632113Selection and characterization of FcεRI phospho-ITAM specific antibodiesNileena Velappan0Avanika Mahajan1Leslie Naranjo2Priyanka Velappan3Nasim Andrews4Nicholas Tiee5Subhendu Chakraborti6Colin Hemez7Tiziano Gaiotto8Bridget Wilson9Andrew Bradbury10Biosecurity and Public Health, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USADepartment of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, USASpecifica Inc., Los Alamos, NM, USABiosecurity and Public Health, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USABiosecurity and Public Health, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USABiosecurity and Public Health, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USABiosecurity and Public Health, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USABiosecurity and Public Health, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USABiosecurity and Public Health, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USADepartment of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, USASpecifica Inc., Los Alamos, NM, USAPost-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids (e.g., anti-phosphotyrosine), which are often used to assay these changes, require isolation of the specific protein prior to analysis and do not identify the specific residue that has been modified (in the case that multiple amino acids have been modified). Phosphorylation state-specific antibodies (PSSAs) developed to recognize post-translational modifications within a specific amino acid sequence can be used to study the timeline of modifications during a signal cascade. We used the FcεRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We selected three β-subunit antibodies that recognized: 1) phosphorylation of tyrosines Y218 or Y224; 2) phosphorylation of the Y228 tyrosine; and 3) phosphorylation of all three tyrosines. We used these antibodies to study the receptor activation timeline of FcεR1 in rat basophilic leukemia cells (RBL-2H3) upon stimulation with DNP24-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y65) of the receptor and applied this antibody to evaluate receptor activation. Recognition patterns of these antibodies show different timelines for phosphorylation of tyrosines in both β and γ subunits. Our methodology provides a strategy to select antibodies specific to post-translational modifications and provides new reagents to study mast cell activation by the high-affinity IgE receptor, FcεRI.https://www.tandfonline.com/doi/10.1080/19420862.2019.1632113Phosphorylation state-specific antibodiesphage displayyeast displayFcεR1 receptor subunitspost-translational modifications |
| spellingShingle | Nileena Velappan Avanika Mahajan Leslie Naranjo Priyanka Velappan Nasim Andrews Nicholas Tiee Subhendu Chakraborti Colin Hemez Tiziano Gaiotto Bridget Wilson Andrew Bradbury Selection and characterization of FcεRI phospho-ITAM specific antibodies mAbs Phosphorylation state-specific antibodies phage display yeast display FcεR1 receptor subunits post-translational modifications |
| title | Selection and characterization of FcεRI phospho-ITAM specific antibodies |
| title_full | Selection and characterization of FcεRI phospho-ITAM specific antibodies |
| title_fullStr | Selection and characterization of FcεRI phospho-ITAM specific antibodies |
| title_full_unstemmed | Selection and characterization of FcεRI phospho-ITAM specific antibodies |
| title_short | Selection and characterization of FcεRI phospho-ITAM specific antibodies |
| title_sort | selection and characterization of fcεri phospho itam specific antibodies |
| topic | Phosphorylation state-specific antibodies phage display yeast display FcεR1 receptor subunits post-translational modifications |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2019.1632113 |
| work_keys_str_mv | AT nileenavelappan selectionandcharacterizationoffceriphosphoitamspecificantibodies AT avanikamahajan selectionandcharacterizationoffceriphosphoitamspecificantibodies AT leslienaranjo selectionandcharacterizationoffceriphosphoitamspecificantibodies AT priyankavelappan selectionandcharacterizationoffceriphosphoitamspecificantibodies AT nasimandrews selectionandcharacterizationoffceriphosphoitamspecificantibodies AT nicholastiee selectionandcharacterizationoffceriphosphoitamspecificantibodies AT subhenduchakraborti selectionandcharacterizationoffceriphosphoitamspecificantibodies AT colinhemez selectionandcharacterizationoffceriphosphoitamspecificantibodies AT tizianogaiotto selectionandcharacterizationoffceriphosphoitamspecificantibodies AT bridgetwilson selectionandcharacterizationoffceriphosphoitamspecificantibodies AT andrewbradbury selectionandcharacterizationoffceriphosphoitamspecificantibodies |