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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...

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Autores principales: Velappan, Nileena, Mahajan, Avanika, Naranjo, Leslie, Velappan, Priyanka, Andrews, Nasim, Tiee, Nicholas, Chakraborti, Subhendu, Hemez, Colin, Gaiotto, Tiziano, Wilson, Bridget, Bradbury, Andrew
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748597/
https://www.ncbi.nlm.nih.gov/pubmed/31311408
http://dx.doi.org/10.1080/19420862.2019.1632113
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author Velappan, Nileena
Mahajan, Avanika
Naranjo, Leslie
Velappan, Priyanka
Andrews, Nasim
Tiee, Nicholas
Chakraborti, Subhendu
Hemez, Colin
Gaiotto, Tiziano
Wilson, Bridget
Bradbury, Andrew
author_facet Velappan, Nileena
Mahajan, Avanika
Naranjo, Leslie
Velappan, Priyanka
Andrews, Nasim
Tiee, Nicholas
Chakraborti, Subhendu
Hemez, Colin
Gaiotto, Tiziano
Wilson, Bridget
Bradbury, Andrew
author_sort Velappan, Nileena
collection PubMed
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 Y(218) or Y(224); 2) phosphorylation of the Y(228) 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 DNP(24)-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y(65)) 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.
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spelling pubmed-67485972019-09-25 Selection and characterization of FcϵRI phospho-ITAM specific antibodies Velappan, Nileena Mahajan, Avanika Naranjo, Leslie Velappan, Priyanka Andrews, Nasim Tiee, Nicholas Chakraborti, Subhendu Hemez, Colin Gaiotto, Tiziano Wilson, Bridget Bradbury, Andrew MAbs Report 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 Y(218) or Y(224); 2) phosphorylation of the Y(228) 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 DNP(24)-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y(65)) 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. Taylor & Francis 2019-07-16 /pmc/articles/PMC6748597/ /pubmed/31311408 http://dx.doi.org/10.1080/19420862.2019.1632113 Text en © 2019 The Author(s). Published with license by Taylor & Francis Group, LLC. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
spellingShingle Report
Velappan, Nileena
Mahajan, Avanika
Naranjo, Leslie
Velappan, Priyanka
Andrews, Nasim
Tiee, Nicholas
Chakraborti, Subhendu
Hemez, Colin
Gaiotto, Tiziano
Wilson, Bridget
Bradbury, Andrew
Selection and characterization of FcϵRI phospho-ITAM specific antibodies
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 Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748597/
https://www.ncbi.nlm.nih.gov/pubmed/31311408
http://dx.doi.org/10.1080/19420862.2019.1632113
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