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Isolation of Novel Ligands for MAS-Related G Protein-Coupled Receptors X1 and X2, and Their Effect on Mast Cell Degranulation

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Isolation of Novel Ligands for MAS-Related G Protein-Coupled Receptors X1 and X2, and Their Effect on Mast Cell Degranulation D 1426 OULU 2017 D 1426 UNIVERSITY OF OULU P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA DMEDICA Toni Karhu Toni Karhu University Lecturer Tuomo Glumoff ISOLATION OF NOVEL LIGANDS University Lecturer Santeri Palviainen FOR MAS-RELATED G PROTEIN- COUPLED RECEPTORS X1 AND Postdoctoral research fellow Sanna Taskila X2, AND THEIR EFFECT ON Professor Olli Vuolteenaho MAST CELL DEGRANULATION University Lecturer Veli-Matti Ulvinen Planning Director Pertti Tikkanen Professor Jari Juga University Lecturer Anu Soikkeli Professor Olli Vuolteenaho UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE; Publications Editor Kirsti Nurkkala BIOCENTER OULU ISBN 978-952-62-1632-4 (Paperback) ISBN 978-952-62-1633-1 (PDF) ISSN 0355-3221 (Print) ISSN 1796-2234 (Online) ACTA UNIVERSITATIS OULUENSIS D Medica 1426 TONI KARHU ISOLATION OF NOVEL LIGANDS FOR MAS-RELATED G PROTEIN- COUPLED RECEPTORS X1 AND X2, AND THEIR EFFECT ON MAST CELL DEGRANULATION Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in Auditorium F202 of the Faculty of Medicine (Aapistie 5 B), on 15 September 2017, at 2 p.m. UNIVERSITY OF OULU, OULU 2017 Copyright © 2017 Acta Univ. Oul. D 1426, 2017 Supervised by Professor Karl-Heinz Herzig Reviewed by Professor Wolf-Georg Forssmann Professor Graham Dockray Opponent Professor Daniel Fourmy ISBN 978-952-62-1632-4 (Paperback) ISBN 978-952-62-1633-1 (PDF) ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online) Cover Design Raimo Ahonen JUVENES PRINT TAMPERE 2017 Karhu, Toni, Isolation of novel ligands for MAS-related G protein-coupled receptors X1 and X2, and their effect on mast cell degranulation. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; Biocenter Oulu Acta Univ. Oul. D 1426, 2017 University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland Abstract The mast cells are an integral part of the human immune system. They are important modulators of inflammatory and physiological processes. Mast cells exert their functions through degranulation and release of inflammatory mediators, such as histamine, proteases and cytokines. There are two main pathways leading to the mast cell activation, the immunoglobulin-dependent and the immunoglobulin-independent pathway. The latter pathway can be triggered by several non-immunological stimuli, and two novel receptors responsible for the activation have been identified, the MAS-related G protein-coupled receptor X1 (MRGPRX1) and X2. The MRGPRX1 and MRGPRX2 have two established functions: i) they trigger the degranulation of mast cells and ii) they are involved in pain perception and itch on a specific subset of sensory neurons. These receptors are not expressed in all of the populations of mast cells, only in the tryptase and chymase containing mast cells, contributing to the mast cell heterogeneity. Unlike most G protein-coupled receptors, the MRGPRX1 and MRGPRX2 are quite non-selective, binding an ever growing list of different ligands. Their ligands include endogenous neuropeptides, host defense peptides and protein fragments, as well as synthetic compounds such as different antibiotics. Their endogenous ligands could be a triggering signal in some mast cell-related diseases by degranulating mast cells and thereby inducing inflammation. Due to the non- selectivity of MRGPRX1 and MRGPRX2, they probably still have many hitherto unknown ligands. The aim of this study was to isolate novel endogenous ligands for the MRGPRX1 and MRGPRX2 from human tissues with the “reverse pharmacology approach” and to determine their potential to degranulate mast cells. The starting materials for the isolation, human platelets and plasma, contained MRGPRX1 and MRGPRX2 activating compounds. From the human plasma, three fragments of albumin able to activate the MRGPRX2 were isolated and sequenced. These fragments were dose-dependently activating the MRGPRX2 and degranulating mast cells. Two MRGPRX1 activating hemoglobin β-chain fragments were isolated from human platelets. These fragments were dose-dependently activating the MRGPRX1, but had no effect on mast cell degranulation. Keywords: isolation, mast cell, MRGPRX1, MRGPRX2 Karhu, Toni, Uusien ligandien eristäminen MRGPRX1 ja MRGPRX2 reseptoreille, ja niiden vaikutus syöttösolujen degranulaatioon. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Biocenter Oulu Acta Univ. Oul. D 1426, 2017 Oulun yliopisto, PL 8000, 90014 Oulun yliopisto Tiivistelmä Syöttösolut on tärkeä osa ihmisen immuunijärjestelmää. Ne ovat tärkeitä tulehdus- ja fysiologis- tenprosessien säätelijöitä. Syöttösolujen vaikutus välittyy degranulaation ja siinä vapautuvien tulehdusvälittäjäaineiden kautta. Vapautuviin aineisiin lukeutuu esim. histamiini ja lukuisia syto- kiinejä, sekä proteaaseja. Syöttösolujen aktivaatio voi tapahtua immunoglobuliineista riippuvaa tai immunoglobuliineista riippumatonta reittiä pitkin. Monet ei-immunologiset tekijät voivat lau- kaista jälkimmäisen reitin ja kaksi uutta tähän vaikuttavaa G-proteiinikytkentäistä reseptoria on löydetty, MAS-related G protein-coupled receptor X1 (MRGPRX1) ja X2. MRGPRX1:llä ja MRGPRX2:lla on kaksi tunnettua tehtävää: i) ne laukaisevat syöttösolujen degranulaation ja ii) ne osallistuvat kivun ja kutinan aistimiseen tietyissä tuntohermoissa. Näitä reseptoreita ei ilmennetä kaikissa syöttösoluissa, vaan ainoastaa tryptaasia ja kymaasia sisältä- vissä syöttösoluissa, ja täten osaltaan selittävät syöttösolujen monimuotoisuutta. Useimmista G- proteiinikytkentäisistä reseptoreista poiketen MRGPRX1 ja MRGPRX2 ovat laajakirjoisia, sito- en monia erilaisia ligandeja. Ligandeihin kuuluu endogeenisia neuropeptidejä, antimikrobiaali- sia peptidejä ja proteiinin fragmentteja, sekä synteettisiä yhdisteitä kuten erilaisia antibiootteja. Reseptoreiden endogeeniset ligandit voivat toimia laukaisijana jossain syöttösoluihin liittyvissä sairauksissa, degranuloidessaan syöttösoluja ja aiheuttaen paikallisen tulehdustilan. Reseptorei- den laajakirjoisuudesta johtuen niillä on oletettavasti monia vielä tuntemattomia ligandeja. Tämän tutkimuksen tarkoitus oli eristää uusia endogeenisiä ligandeja MRGPRX1:lle ja MRGPRX2:lle ihmisen kudoksista ”kääteisfarmakologista lähestymistapaa” hyödyntäen ja sel- vittää ligandien kyky syöttösolujen degranulaatioon. Lähtömateriaalina käytetyt ihmisen verihiutaleet ja plasma sisälsivät MRGPRX1:ta ja MRGPRX2:ta aktivoivia yhdisteitä. Plasmasta eristettiin ja sekvensoitiin kolme albumiinin frag- menttia, jotka aktivoivat MRGPRX2:ta. Nämä fragmentit aktivoivat MRGPRX2:ta ja degranu- loivat syöttösoluja annosriippuvaisesti. Kaksi MRGPRX1:tä aktivoivaa hemoglobiinin β-ketjun fragmenttia eristettiin ihmisen verihiutaleista. Nämä fragmentit tunnistettiin hemorfiineiksi ja ne aktivoivat MRGPRX1:tä annosriippuvaisesti, mutta eivät vaikuttaneet syöttösolujen degranulaa- tioon. Asiasanat: eristys, MRGPRX1, MRGPRX2, syöttösolu Science needs love. (Kazuhiko Tatemoto) 8 Acknowledgements This study was carried out in University of Oulu, Faculty of Medicine, Research Unit of Biomedicine and Biocenter Oulu. I sincerely wish to thank all those who have participated in this work. I wish to express my sincere gratitude to my supervisor Professor Karl-Heinz Herzig, MD, PhD, for giving me an opportunity to pursue my scientific career under his guidance. I appreciate all the support and guidance I have received during my PhD project. I am very grateful to my late supervisor Professor Kazuhiko Tatemoto, PhD, for initiating this project and introducing me to the science of peptide isolation. I am especially indebted to him for sharing and teaching me his passion and commitment to science in general during the brief time he was with us. He was a truly inspirational character and a great scientist. I want to thank the pre-examiners, Professor Graham Dockray, PhD, and Professor Wolf-Georg Forssmann, MD, PhD, for the evaluation of my thesis manuscript and for all the constructive criticism and valuable comments. I wish to thank all the co-authors for the scientific output and for making this thesis possible. I want the express my gratitude for Takayuki Naito, PhD, and Kiyotaka Akiyama, PhD, for the groundwork required for the study as well as providing the transfected cell lines. I am very thankful for Professor Olli Vuolteenaho, MD, PhD, for the peptide synthesis and Ulrich Bergmann, PhD, for the peptide sequencing. I wish to thank my follow-up group, Rajaram Venkatesan, PhD, Tiila-Riikka Kiema, PhD, and especially the chair Professor Lloyd Ruddock, PhD, for all the help and advice offered. I owe my warmest thanks to all my colleagues in the Research Unit of Biomedicine for generating a positive atmosphere to do science. Special thanks to Kari Mäkelä, PhD, for the friendship and all the enjoyable moments in and out of the office, Miia Kovalainen, PhD, for the substantial help with writing and reviewing, Shivaprakash J. Mutt, MSc, for assistance with the lab work and technicians Anna-Maija Koisti, Meeri Kröger and Anneli Rautio for the help with the extensive cell culture. I also want to thank the current and former colleagues, Laura Niiranen, MSc, Ghulan Raza, MSc, Remi Kamakura, MSc, Dominique Gagnon, PhD, Taina Lajunen, PhD, Marianne Haapea, PhD, Heli Ruotsalainen PhD, Katja Klausz PhD, Miika Heinonen PhD, Anna-Kaisa Purhonen PhD, Anne Huotari, MSc, Alicia Jurado Acosta, MSc, David Vicente,
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