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 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 bothmore »
- Authors:
-
[1];
[2];
[3];
[1];
[1];
[1];
[1];
[1];
[1];
[2];
[3]
- Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
- Univ. of New Mexico, Albuquerque, NM (United States)
- 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 Volume: 11; Journal Issue: 7; Journal ID: ISSN 1942-0862
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 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 = 7,
volume = 11,
place = {United States},
year = {2019},
month = {7}
}
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Works referenced in this record:
Mechanism-based corrector combination restores ΔF508-CFTR folding and function
journal, May 2013
- Okiyoneda, Tsukasa; Veit, Guido; Dekkers, Johanna F.
- Nature Chemical Biology, Vol. 9, Issue 7
Immunohistochemical assessment of protein phosphorylation state: the dream and the reality
journal, July 2008
- Mandell, James W.
- Histochemistry and Cell Biology, Vol. 130, Issue 3
Optimal Screening of Surface-Displayed Polypeptide Libraries
journal, February 1998
- Boder, E. T.; Wittrup, K. D.
- Biotechnology Progress, Vol. 14, Issue 1
Distinct Functions of the FcϵR1 γ and β Subunits in the Control of FcϵR1-mediated Tyrosine Kinase Activation and Signaling Responses in RBL-2H3 Mast Cells
journal, February 1995
- Wilson, Bridget S.; Kapp, Nicholas; Lee, Rebecca J.
- Journal of Biological Chemistry, Vol. 270, Issue 8
Deep sequencing in library selection projects: what insight does it bring?
journal, August 2015
- Glanville, J.; D’Angelo, S.; Khan, Ta
- Current Opinion in Structural Biology, Vol. 33
Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels
journal, May 2000
- Wang, Fei; Zeltwanger, Shawn; Hu, Shenghui
- The Journal of Physiology, Vol. 524, Issue 3
Beyond natural antibodies: the power of in vitro display technologies
journal, March 2011
- Bradbury, Andrew R. M.; Sidhu, Sachdev; Dübel, Stefan
- Nature Biotechnology, Vol. 29, Issue 3
A Novel High-Throughput Screen Reveals Yeast Genes That Increase Secretion of Heterologous Proteins
journal, December 2006
- Wentz, A. E.; Shusta, E. V.
- Applied and Environmental Microbiology, Vol. 73, Issue 4
Phosphorylation of Cell Surface Receptors: A Mechanism for Regulating Signal Transduction Pathways
journal, February 1988
- Sibley, David R.; Benovic, Jeffrey L.; Caron, Marc G.
- Endocrine Reviews, Vol. 9, Issue 1
Dysregulation of Protein Phosphorylation/Dephosphorylation in Alzheimer's Disease: A Therapeutic Target
journal, January 2006
- Gong, Cheng-Xin; Liu, Fei; Grundke-Iqbal, Inge
- Journal of Biomedicine and Biotechnology, Vol. 2006
Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker
journal, November 2012
- Ferrara, Fortunato; Naranjo, Leslie A.; Kumar, Sandeep
- PLoS ONE, Vol. 7, Issue 11
Directed evolution for the development of conformation-specific affinity reagents using yeast display
journal, September 2005
- Weaver-Feldhaus, Jane M.; Miller, Keith D.; Feldhaus, Michael J.
- Protein Engineering, Design and Selection, Vol. 18, Issue 11
The Receptor with High Affinity for Immunoglobulin E
journal, April 1986
- Metzger, H.; Alcaraz, G.; Hohman, R.
- Annual Review of Immunology, Vol. 4, Issue 1
The Receptor with High Affinity for IgE
journal, February 1992
- Metzger, Henry
- Immunological Reviews, Vol. 125, Issue 1
Antibody validation
journal, March 2010
- Bordeaux, Jennifer; Welsh, Allison W.; Agarwal, Seema
- BioTechniques, Vol. 48, Issue 3
C-terminal Repeat Domain Kinase I Phosphorylates Ser2 and Ser5 of RNA Polymerase II C-terminal Domain Repeats
journal, March 2004
- Jones, Janice C.; Phatnani, Hemali P.; Haystead, Timothy A.
- Journal of Biological Chemistry, Vol. 279, Issue 24
Zinc carnosine works with bovine colostrum in truncating heavy exercise–induced increase in gut permeability in healthy volunteers
journal, June 2016
- Davison, Glen; Marchbank, Tania; March, Daniel S.
- The American Journal of Clinical Nutrition, Vol. 104, Issue 2
Correction of Both NBD1 Energetics and Domain Interface Is Required to Restore ΔF508 CFTR Folding and Function
journal, January 2012
- Rabeh, Wael M.; Bossard, Florian; Xu, Haijin
- Cell, Vol. 148, Issue 1-2
An Ultra-specific Avian Antibody to Phosphorylated Tau Protein Reveals a Unique Mechanism for Phosphoepitope Recognition
journal, November 2012
- Shih, Heather H.; Tu, Chao; Cao, Wei
- Journal of Biological Chemistry, Vol. 287, Issue 53
Exploiting recombination in single bacteria to make large phage antibody libraries
journal, January 2000
- Sblattero, Daniele; Bradbury, Andrew
- Nature Biotechnology, Vol. 18, Issue 1
Issues associated with the use of phosphospecific antibodies to localise active and inactive pools of GSK-3 in cells
journal, January 2011
- Campa, Victor M.; Kypta, Robert M.
- Biology Direct, Vol. 6, Issue 1
The winding path of protein methylation research: milestones and new frontiers
journal, May 2017
- Murn, Jernej; Shi, Yang
- Nature Reviews Molecular Cell Biology, Vol. 18, Issue 8
Induction of 15-Lipoxygenase Expression by IL-13 Requires Tyrosine Phosphorylation of Jak2 and Tyk2 in Human Monocytes
journal, November 1998
- Roy, Biswajit; Cathcart, Martha K.
- Journal of Biological Chemistry, Vol. 273, Issue 48
Dysregulation of Met receptor tyrosine kinase activity in invasive tumors
journal, April 2002
- Danilkovitch-Miagkova, Alla; Zbar, Berton
- Journal of Clinical Investigation, Vol. 109, Issue 7
Recombinant renewable polyclonal antibodies
journal, January 2015
- Ferrara, Fortunato; D’Angelo, Sara; Gaiotto, Tiziano
- mAbs, Vol. 7, Issue 1
Molecular regulation of mast cell activation
journal, June 2006
- Rivera, J.; Gilfillan, A.
- Journal of Allergy and Clinical Immunology, Vol. 117, Issue 6
RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73
journal, January 2018
- Papaspyropoulos, Angelos; Bradley, Leanne; Thapa, Asmita
- Nature Communications, Vol. 9, Issue 1
Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.
journal, August 1988
- Huston, J. S.; Levinson, D.; Mudgett-Hunter, M.
- Proceedings of the National Academy of Sciences, Vol. 85, Issue 16
Phosphorylation State-Specific Antibodies
journal, November 2003
- Mandell, James W.
- The American Journal of Pathology, Vol. 163, Issue 5
Monoclonal antibodies to phosphotyrosine
journal, May 1988
- Glenney, John R.; Zokas, Liza; Kamps, Mark P.
- Journal of Immunological Methods, Vol. 109, Issue 2
Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents
journal, October 2015
- Bradbury, Andrew R. M.; Plückthun, Andreas
- Protein Engineering Design and Selection, Vol. 28, Issue 10
Reproducibility crisis: Blame it on the antibodies
journal, May 2015
- Baker, Monya
- Nature, Vol. 521, Issue 7552
Molecular Dissection of the FcRβ Signaling Amplifier
journal, August 2004
- On, Marina; Billingsley, James M.; Jouvin, Marie-Hélène
- Journal of Biological Chemistry, Vol. 279, Issue 44
An assessment of histone-modification antibody quality
journal, December 2010
- Egelhofer, Thea A.; Minoda, Aki; Klugman, Sarit
- Nature Structural & Molecular Biology, Vol. 18, Issue 1
Systematic evaluation of quantotypic peptides for targeted analysis of the human kinome
journal, August 2014
- Worboys, Jonathan D.; Sinclair, John; Yuan, Yinyin
- Nature Methods, Vol. 11, Issue 10
Post-translational modifications and activation of p53 by genotoxic stresses: p53 post-translational modifications
journal, May 2001
- Appella, Ettore; Anderson, Carl W.
- European Journal of Biochemistry, Vol. 268, Issue 10
Cell Signaling by Receptor Tyrosine Kinases
journal, June 2010
- Lemmon, Mark A.; Schlessinger, Joseph
- Cell, Vol. 141, Issue 7
Impact of Single-chain Fv Antibody Fragment Affinity on Nanoparticle Targeting of Epidermal Growth Factor Receptor-expressing Tumor Cells
journal, August 2007
- Zhou, Yu; Drummond, Daryl C.; Zou, Hao
- Journal of Molecular Biology, Vol. 371, Issue 4
Downstream Signaling Molecules Bind to Different Phosphorylated Immunoreceptor Tyrosine-based Activation Motif (ITAM) Peptides of the High Affinity IgE Receptor
journal, November 1996
- Kimura, Teruaki; Kihara, Hidetoshi; Bhattacharyya, Siba
- Journal of Biological Chemistry, Vol. 271, Issue 44
The FcϵRIβ Immunoreceptor Tyrosine-based Activation Motif Exerts Inhibitory Control on MAPK and IκB Kinase Phosphorylation and Mast Cell Cytokine Production
journal, September 2004
- Furumoto, Yasuko; Nunomura, Satoshi; Terada, Tomoyoshi
- Journal of Biological Chemistry, Vol. 279, Issue 47
The origins of protein phosphorylation
journal, May 2002
- Cohen, Philip
- Nature Cell Biology, Vol. 4, Issue 5
Autoimmune-associated PTPN22 R620W Variation Reduces Phosphorylation of Lymphoid Phosphatase on an Inhibitory Tyrosine Residue
journal, June 2010
- Fiorillo, Edoardo; Orrú, Valeria; Stanford, Stephanie M.
- Journal of Biological Chemistry, Vol. 285, Issue 34
Reproducibility: Standardize antibodies used in research
journal, February 2015
- Bradbury, Andrew; Plückthun, Andreas
- Nature, Vol. 518, Issue 7537
Of ITIMs, ITAMs, and ITAMis: revisiting immunoglobulin Fc receptor signaling
journal, October 2015
- Getahun, Andrew; Cambier, John C.
- Immunological Reviews, Vol. 268, Issue 1
Enhanced Secretion of Heterologous Proteins from Yeast by Overexpression of Ribosomal Subunit RPP0
journal, June 2008
- Wentz, A. E.; Shusta, E. V.
- Biotechnology Progress, Vol. 24, Issue 3
Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays
journal, January 2004
- Panse, Sören; Dong, Liying; Burian, Antje
- Molecular Diversity, Vol. 8, Issue 3
Roles for the High Affinity IgE Receptor, FcRI, of Human Basophils in the Pathogenesis and Therapy of Allergic Asthma: Disease Promotion, Protection or Both?
journal, November 2010
- Youssef, Lama A.
- The Open Allergy Journal, Vol. 3, Issue 1
The discovery of ubiquitin-dependent proteolysis
journal, October 2005
- Wilkinson, K. D.
- Proceedings of the National Academy of Sciences, Vol. 102, Issue 43
Deciphering the structure and function of FcεRI/mast cell axis in the regulation of allergy and anaphylaxis: a functional genomics paradigm
journal, December 2011
- Manikandan, Jayapal; Kothandaraman, Narasimhan; Hande, Manoor Prakash
- Cellular and Molecular Life Sciences, Vol. 69, Issue 12
Detailed specificity analysis of antibodies binding to modified histone tails with peptide arrays
journal, February 2011
- Bock, Ina; Dhayalan, Arunkumar; Kudithipudi, Srikanth
- Epigenetics, Vol. 6, Issue 2
Nature-inspired design of motif-specific antibody scaffolds
journal, August 2013
- Koerber, James T.; Thomsen, Nathan D.; Hannigan, Brett T.
- Nature Biotechnology, Vol. 31, Issue 10
Editorial: Antibody Can Get It Right: Confronting Problems of Antibody Specificity and Irreproducibility
journal, September 2014
- Schonbrunn, Agnes
- Molecular Endocrinology, Vol. 28, Issue 9
Optimal Aggregation of FcεRI with a Structurally Defined Trivalent Ligand Overrides Negative Regulation Driven by Phosphatases
journal, May 2014
- Mahajan, Avanika; Barua, Dipak; Cutler, Patrick
- ACS Chemical Biology, Vol. 9, Issue 7
Tyrosine Sulfation as a Protein Post-Translational Modification
journal, January 2015
- Yang, Yuh-Shyong; Wang, Chen-Chu; Chen, Bo-Han
- Molecules, Vol. 20, Issue 2
Monophosphorylation of CD79a and CD79b ITAM Motifs Initiates a SHIP-1 Phosphatase-Mediated Inhibitory Signaling Cascade Required for B Cell Anergy
journal, November 2011
- O'Neill, Shannon K.; Getahun, Andrew; Gauld, Stephen B.
- Immunity, Vol. 35, Issue 5
Antibodies: validate recombinants once
journal, April 2015
- Bradbury, Andrew M.; Plückthun, Andreas
- Nature, Vol. 520, Issue 7547
Dysregulation of Met receptor tyrosine kinase activity in invasive tumors
journal, April 2002
- Danilkovitch-Miagkova, Alla; Zbar, Berton
- Journal of Clinical Investigation, Vol. 109, Issue 7