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Substance P analogs devoid of key residues fail to activate human mast cells via MRGPRX2

Mast cells play an important role in disease pathogenesis by secreting immunomodulatory molecules. Mast cells are primarily activated by the crosslinking of their high affinity IgE receptors (FcεRI) by antigen bound immunoglobulin (Ig)E antibody complexes. However, mast cells can also be activated b...

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Autores principales: Raj, Shammy, Hlushak, Stepan, Arizmendi, Narcy, Kovalenko, Andriy, Kulka, Marianna
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10203606/
https://www.ncbi.nlm.nih.gov/pubmed/37228611
http://dx.doi.org/10.3389/fimmu.2023.1155740
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author Raj, Shammy
Hlushak, Stepan
Arizmendi, Narcy
Kovalenko, Andriy
Kulka, Marianna
author_facet Raj, Shammy
Hlushak, Stepan
Arizmendi, Narcy
Kovalenko, Andriy
Kulka, Marianna
author_sort Raj, Shammy
collection PubMed
description Mast cells play an important role in disease pathogenesis by secreting immunomodulatory molecules. Mast cells are primarily activated by the crosslinking of their high affinity IgE receptors (FcεRI) by antigen bound immunoglobulin (Ig)E antibody complexes. However, mast cells can also be activated by the mas related G protein-coupled receptor X2 (MRGPRX2), in response to a range of cationic secretagogues, such as substance P (SP), which is associated with pseudo-allergic reactions. We have previously reported that the in vitro activation of mouse mast cells by basic secretagogues is mediated by the mouse orthologue of the human MRGPRX2, MRGPRB2. To further elucidate the mechanism of MRGPRX2 activation, we studied the time-dependent internalization of MRGPRX2 by human mast cells (LAD2) upon stimulation with the neuropeptide SP. In addition, we performed computational studies to identify the intermolecular forces that facilitate ligand-MRGPRX2 interaction using SP. The computational predictions were tested experimentally by activating LAD2 with SP analogs, which were missing key amino acid residues. Our data suggest that mast cell activation by SP causes internalization of MRGPRX2 within 1 min of stimulation. Hydrogen bonds (h-bonds) and salt bridges govern the biding of SP to MRGPRX2. Arg1 and Lys3 in SP are key residues that are involved in both h-bonding and salt bridge formations with Glu164 and Asp184 of MRGPRX2, respectively. In accordance, SP analogs devoid of key residues (SP1 and SP2) failed to activate MRGPRX2 degranulation. However, both SP1 and SP2 caused a comparable release of chemokine CCL2. Further, SP analogs SP1, SP2 and SP4 did not activate tumor necrosis factor (TNF) production. We further show that SP1 and SP2 limit the activity of SP on mast cells. The results provide important mechanistic insight into the events that result in mast cell activation through MRGPRX2 and highlight the important physiochemical characteristics of a peptide ligand that facilitates ligand-MRGPRX2 interactions. The results are important in understanding activation through MRGPRX2, and the intermolecular forces that govern ligand-MRGPRX2 interaction. The elucidation of important physiochemical properties within a ligand that are needed for receptor interaction will aid in designing novel therapeutics and antagonists for MRGPRX2.
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spelling pubmed-102036062023-05-24 Substance P analogs devoid of key residues fail to activate human mast cells via MRGPRX2 Raj, Shammy Hlushak, Stepan Arizmendi, Narcy Kovalenko, Andriy Kulka, Marianna Front Immunol Immunology Mast cells play an important role in disease pathogenesis by secreting immunomodulatory molecules. Mast cells are primarily activated by the crosslinking of their high affinity IgE receptors (FcεRI) by antigen bound immunoglobulin (Ig)E antibody complexes. However, mast cells can also be activated by the mas related G protein-coupled receptor X2 (MRGPRX2), in response to a range of cationic secretagogues, such as substance P (SP), which is associated with pseudo-allergic reactions. We have previously reported that the in vitro activation of mouse mast cells by basic secretagogues is mediated by the mouse orthologue of the human MRGPRX2, MRGPRB2. To further elucidate the mechanism of MRGPRX2 activation, we studied the time-dependent internalization of MRGPRX2 by human mast cells (LAD2) upon stimulation with the neuropeptide SP. In addition, we performed computational studies to identify the intermolecular forces that facilitate ligand-MRGPRX2 interaction using SP. The computational predictions were tested experimentally by activating LAD2 with SP analogs, which were missing key amino acid residues. Our data suggest that mast cell activation by SP causes internalization of MRGPRX2 within 1 min of stimulation. Hydrogen bonds (h-bonds) and salt bridges govern the biding of SP to MRGPRX2. Arg1 and Lys3 in SP are key residues that are involved in both h-bonding and salt bridge formations with Glu164 and Asp184 of MRGPRX2, respectively. In accordance, SP analogs devoid of key residues (SP1 and SP2) failed to activate MRGPRX2 degranulation. However, both SP1 and SP2 caused a comparable release of chemokine CCL2. Further, SP analogs SP1, SP2 and SP4 did not activate tumor necrosis factor (TNF) production. We further show that SP1 and SP2 limit the activity of SP on mast cells. The results provide important mechanistic insight into the events that result in mast cell activation through MRGPRX2 and highlight the important physiochemical characteristics of a peptide ligand that facilitates ligand-MRGPRX2 interactions. The results are important in understanding activation through MRGPRX2, and the intermolecular forces that govern ligand-MRGPRX2 interaction. The elucidation of important physiochemical properties within a ligand that are needed for receptor interaction will aid in designing novel therapeutics and antagonists for MRGPRX2. Frontiers Media S.A. 2023-05-09 /pmc/articles/PMC10203606/ /pubmed/37228611 http://dx.doi.org/10.3389/fimmu.2023.1155740 Text en Copyright © 2023 Raj, Hlushak, Arizmendi, Kovalenko and Kulka https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Raj, Shammy
Hlushak, Stepan
Arizmendi, Narcy
Kovalenko, Andriy
Kulka, Marianna
Substance P analogs devoid of key residues fail to activate human mast cells via MRGPRX2
title Substance P analogs devoid of key residues fail to activate human mast cells via MRGPRX2
title_full Substance P analogs devoid of key residues fail to activate human mast cells via MRGPRX2
title_fullStr Substance P analogs devoid of key residues fail to activate human mast cells via MRGPRX2
title_full_unstemmed Substance P analogs devoid of key residues fail to activate human mast cells via MRGPRX2
title_short Substance P analogs devoid of key residues fail to activate human mast cells via MRGPRX2
title_sort substance p analogs devoid of key residues fail to activate human mast cells via mrgprx2
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10203606/
https://www.ncbi.nlm.nih.gov/pubmed/37228611
http://dx.doi.org/10.3389/fimmu.2023.1155740
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