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postgraduate thesis: The novel venom peptide P17 induces chemotactic recruitment and differentiation of monocytes into macrophages via alternative mast cell activation
Title | The novel venom peptide P17 induces chemotactic recruitment and differentiation of monocytes into macrophages via alternative mast cell activation |
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Authors | |
Advisors | |
Issue Date | 2020 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Duraisamy, K.. (2020). The novel venom peptide P17 induces chemotactic recruitment and differentiation of monocytes into macrophages via alternative mast cell activation. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | Peptides are highly recognized as potential therapeutics for their selective, potent and safe nature. Peptide P17, a peptide isolated from the venom of the ant Tetramorium bicarinatum, is known to induce an alternative phenotype of human monocyte-derived macrophages through activating unknown GPCRs. In this study, we screened this peptide (single dose concentration of 0.1 µM) with 314 Tango-GPCRs from Addgene to identify a receptor it is binding to. Among the 32 positive hits obtained, we found that human MRGPRX2 is activated by peptide P17 in a dose-dependent manner. This activation has been confirmed in wild-type MRGPRX2-transfected CHO cells by a dose-dependent release of calcium through Fluo-4NW calcium assay, and by a single dose of peptide P17 (2.5 µM) through confocal live cell imaging. In the Fluo-4NW calcium assay, P17 dose dependently increased the [Ca2+]i which plateaued at micromolar concentration of the peptide P17. Various effects of P17 have also been tested in human immune cells that expresses MRGPRX2 inherently. From in silico studies, it was observed that peptide P17 was able to interact with Tyr89, Phe172, Ser173, Asp174, Gly175, Trp250, Lys251 and Ser253 aa residues of the of the receptor. Residue Lys8 of peptide P17 specifically formed a cation-π interaction with the Phe172 of MRGPRX2. Essential amino acids in P17, that are responsible for the activation of the receptor are found through alanine scanning and sequence downsizing. [Ala2]P17, [Ala8]P17, [Ala10]P17 and [Ala12]P17 were partial agonists, while [Ala4]P17 and [Ala7]P17 were more potent than P17 in the activation of the receptor. Through site directed mutagenesis, we found that Phe172 in MRGPRX2 is essential for the activation of the receptor by P17. Thereafter, we have showed P17 induces calcium and β-hexosaminidase release in human LAD2 mast cells. Treatment by the MRGPRX2 antagonist quercetin and shRNA-mediated MRGPRX2 knockdown led to a reduction in P17-evoked % β-hexosaminidase release. In addition, [Ala8]P17 acted as an antagonist for P17 as it abrogated P17 mediated % β-arrestin recruitment and % β-hexosaminidase release. Cytokine array consisting 120 cytokine markers indicated that P17 is able to induce various cytokine releases in LAD2 cells including MCP-1 and MIP1-α. EGTA and quercetin reduced the P17-induced MCP-1 and MIP1-α releases. Interestingly, P17 also activates LAD2 cells to recruit THP-1 monocytes in trans-well migration assay, whereas shRNA treated MRGPRX2-impaired LAD2 cells were not able to recruit monocytes. In addition, P17-evoked LAD2 cells were also able to stimulate differentiation of THP-1 monocytes into macrophages, as indicated by the enhanced expressions of macrophage markers CD11a, CD11b, CD11c, ICAM-1, CD14 and TNF-α. Elevated levels of CD11b in differentiated THP-1 monocytes were also detected by flow cytometry. Pretreatment with quercetin prior to P17 treatment in LAD2 cells, reduced the expression of macrophage markers in THP-1 cells. Finally, Evans blue extravasation in paws, immunohistochemical and immunofluorescent staining in ears, all show P17 mediated monocyte recruitment in mice. |
Degree | Doctor of Philosophy |
Subject | Peptides Monocytes Macrophages |
Dept/Program | Biological Sciences |
Persistent Identifier | http://hdl.handle.net/10722/308580 |
DC Field | Value | Language |
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dc.contributor.advisor | Chow, BKC | - |
dc.contributor.advisor | Zhang, J | - |
dc.contributor.author | Duraisamy, Karthi | - |
dc.date.accessioned | 2021-12-02T02:31:59Z | - |
dc.date.available | 2021-12-02T02:31:59Z | - |
dc.date.issued | 2020 | - |
dc.identifier.citation | Duraisamy, K.. (2020). The novel venom peptide P17 induces chemotactic recruitment and differentiation of monocytes into macrophages via alternative mast cell activation. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/308580 | - |
dc.description.abstract | Peptides are highly recognized as potential therapeutics for their selective, potent and safe nature. Peptide P17, a peptide isolated from the venom of the ant Tetramorium bicarinatum, is known to induce an alternative phenotype of human monocyte-derived macrophages through activating unknown GPCRs. In this study, we screened this peptide (single dose concentration of 0.1 µM) with 314 Tango-GPCRs from Addgene to identify a receptor it is binding to. Among the 32 positive hits obtained, we found that human MRGPRX2 is activated by peptide P17 in a dose-dependent manner. This activation has been confirmed in wild-type MRGPRX2-transfected CHO cells by a dose-dependent release of calcium through Fluo-4NW calcium assay, and by a single dose of peptide P17 (2.5 µM) through confocal live cell imaging. In the Fluo-4NW calcium assay, P17 dose dependently increased the [Ca2+]i which plateaued at micromolar concentration of the peptide P17. Various effects of P17 have also been tested in human immune cells that expresses MRGPRX2 inherently. From in silico studies, it was observed that peptide P17 was able to interact with Tyr89, Phe172, Ser173, Asp174, Gly175, Trp250, Lys251 and Ser253 aa residues of the of the receptor. Residue Lys8 of peptide P17 specifically formed a cation-π interaction with the Phe172 of MRGPRX2. Essential amino acids in P17, that are responsible for the activation of the receptor are found through alanine scanning and sequence downsizing. [Ala2]P17, [Ala8]P17, [Ala10]P17 and [Ala12]P17 were partial agonists, while [Ala4]P17 and [Ala7]P17 were more potent than P17 in the activation of the receptor. Through site directed mutagenesis, we found that Phe172 in MRGPRX2 is essential for the activation of the receptor by P17. Thereafter, we have showed P17 induces calcium and β-hexosaminidase release in human LAD2 mast cells. Treatment by the MRGPRX2 antagonist quercetin and shRNA-mediated MRGPRX2 knockdown led to a reduction in P17-evoked % β-hexosaminidase release. In addition, [Ala8]P17 acted as an antagonist for P17 as it abrogated P17 mediated % β-arrestin recruitment and % β-hexosaminidase release. Cytokine array consisting 120 cytokine markers indicated that P17 is able to induce various cytokine releases in LAD2 cells including MCP-1 and MIP1-α. EGTA and quercetin reduced the P17-induced MCP-1 and MIP1-α releases. Interestingly, P17 also activates LAD2 cells to recruit THP-1 monocytes in trans-well migration assay, whereas shRNA treated MRGPRX2-impaired LAD2 cells were not able to recruit monocytes. In addition, P17-evoked LAD2 cells were also able to stimulate differentiation of THP-1 monocytes into macrophages, as indicated by the enhanced expressions of macrophage markers CD11a, CD11b, CD11c, ICAM-1, CD14 and TNF-α. Elevated levels of CD11b in differentiated THP-1 monocytes were also detected by flow cytometry. Pretreatment with quercetin prior to P17 treatment in LAD2 cells, reduced the expression of macrophage markers in THP-1 cells. Finally, Evans blue extravasation in paws, immunohistochemical and immunofluorescent staining in ears, all show P17 mediated monocyte recruitment in mice. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | Peptides | - |
dc.subject.lcsh | Monocytes | - |
dc.subject.lcsh | Macrophages | - |
dc.title | The novel venom peptide P17 induces chemotactic recruitment and differentiation of monocytes into macrophages via alternative mast cell activation | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Doctor of Philosophy | - |
dc.description.thesislevel | Doctoral | - |
dc.description.thesisdiscipline | Biological Sciences | - |
dc.description.nature | published_or_final_version | - |
dc.date.hkucongregation | 2021 | - |
dc.identifier.mmsid | 991044360599003414 | - |