Selection and characterization of FcϵRI phospho-ITAM specific antibodies
| Autor: | Priyanka Velappan, Andrew Bradbury, Colin Hemez, Nasim Andrews, Nicholas Tiee, Subhendu Chakraborti, Bridget S. Wilson, Leslie Naranjo, Avanika Mahajan, Nileena Velappan, Tiziano Gaiotto |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Phage display
Immunology FcϵR1 receptor subunits Yeast display Antibodies Cell Line 03 medical and health sciences 0302 clinical medicine Report Yeasts post-translational modifications Immunology and Allergy Animals Phosphorylation state-specific antibodies yeast display Tyrosine Phosphorylation Receptor Peptide sequence 030304 developmental biology chemistry.chemical_classification 0303 health sciences Receptors IgE FCER1 3. Good health Amino acid Basophils Rats chemistry Biochemistry 030220 oncology & carcinogenesis phage display Cell Surface Display Techniques Antibodies Phospho-Specific Protein Processing Post-Translational |
| Zdroj: | mAbs |
| ISSN: | 1942-0870 1942-0862 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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