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Title: Selection and characterization of FcεRI phospho-ITAM specific antibodies

Abstract

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 utilized the FcεRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We determined 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 formore » 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.« less

Authors:
ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [3]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [3]
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  2. Univ. of New Mexico, Albuquerque, NM (United States)
  3. Specifica Inc., Los Alamos, NM (United States); Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Publication Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1558049
Report Number(s):
LA-UR-18-28041
Journal ID: ISSN 1942-0862
Grant/Contract Number:  
89233218CNA000001
Resource Type:
Accepted Manuscript
Journal Name:
mAbs
Additional Journal Information:
Journal Name: mAbs; Journal ID: ISSN 1942-0862
Country of Publication:
United States
Language:
English
Subject:
Biological Science; Phosphorylation and sequence specific antibodies, beta and gamma subunit of FceR1 receptor, allergy pathway; Phosphorylation state specific antibodies; phage display; yeast display; FcεR1 receptor subunits; post-translational modifications

Citation Formats

Velappan, Nileena, Mahajan, Avanika, Naranjo, Leslie, Velappan, Priyanka, Andrews, Nasim, Tiee, Nicholas, Chakraborti, Subhendu, Hemez, Colin, Gaiotto, Tiziano, Wilson, Bridget, and Bradbury, Andrew. Selection and characterization of FcεRI phospho-ITAM specific antibodies. United States: N. p., 2019. Web. doi:10.1080/19420862.2019.1632113.
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. United States. doi:10.1080/19420862.2019.1632113.
Velappan, Nileena, Mahajan, Avanika, Naranjo, Leslie, Velappan, Priyanka, Andrews, Nasim, Tiee, Nicholas, Chakraborti, Subhendu, Hemez, Colin, Gaiotto, Tiziano, Wilson, Bridget, and Bradbury, Andrew. Tue . "Selection and characterization of FcεRI phospho-ITAM specific antibodies". United States. doi:10.1080/19420862.2019.1632113. https://www.osti.gov/servlets/purl/1558049.
@article{osti_1558049,
title = {Selection and characterization of FcεRI phospho-ITAM specific antibodies},
author = {Velappan, Nileena and Mahajan, Avanika and Naranjo, Leslie and Velappan, Priyanka and Andrews, Nasim and Tiee, Nicholas and Chakraborti, Subhendu and Hemez, Colin and Gaiotto, Tiziano and Wilson, Bridget and Bradbury, Andrew},
abstractNote = {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 utilized the FcεRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We determined 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.},
doi = {10.1080/19420862.2019.1632113},
journal = {mAbs},
number = ,
volume = ,
place = {United States},
year = {2019},
month = {7}
}

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