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Lysyl Hydroxylases 1 and 2 D 1159 OULU 2012 D 1159 UNIVERSITY OF OULU P.O.B. 7500 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA SERIES EDITORS DMEDICA Marjo Hyry ASCIENTIAE RERUM NATURALIUM Marjo Hyry Senior Assistant Jorma Arhippainen LYSYL HYDROXYLASES 1 AND 2 BHUMANIORA Lecturer Santeri Palviainen CHARACTERIZATION OF THEIR IN VIVO ROLES IN MOUSE AND THE MOLECULAR LEVEL CTECHNICA CONSEQUENCES OF THE LYSYL HYDROXYLASE 2 Professor Hannu Heusala MUTATIONS FOUND IN BRUCK SYNDROME DMEDICA Professor Olli Vuolteenaho ESCIENTIAE RERUM SOCIALIUM Senior Researcher Eila Estola FSCRIPTA ACADEMICA Director Sinikka Eskelinen GOECONOMICA Professor Jari Juga EDITOR IN CHIEF Professor Olli Vuolteenaho PUBLICATIONS EDITOR Publications Editor Kirsti Nurkkala UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE, INSTITUTE OF BIOMEDICINE, ISBN 978-951-42-9841-7 (Paperback) DEPARTMENT OF MEDICAL BIOCHEMISTRY AND MOLECULAR BIOLOGY; ISBN 978-951-42-9842-4 (PDF) BIOCENTER OULU; ISSN 0355-3221 (Print) CENTER FOR CELL-MATRIX RESEARCH ISSN 1796-2234 (Online) ACTA UNIVERSITATIS OULUENSIS D Medica 1159 MARJO HYRY LYSYL HYDROXYLASES 1 AND 2 Characterization of their in vivo roles in mouse and the molecular level consequences of the lysyl hydroxylase 2 mutations found in Bruck syndrome 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 F101 of the Department of Physiology (Aapistie 7), on 8 June 2012, at 10 a.m. UNIVERSITY OF OULU, OULU 2012 Copyright © 2012 Acta Univ. Oul. D 1159, 2012 Supervised by Professor Johanna Myllyharju Reviewed by Professor Heather Yeowell Professor Helen Skaer ISBN 978-951-42-9841-7 (Paperback) ISBN 978-951-42-9842-4 (PDF) ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online) Cover Design Raimo Ahonen JUVENES PRINT TAMPERE 2012 Hyry, Marjo, Lysyl hydroxylases 1 and 2. Characterization of their in vivo roles in mouse and the molecular level consequences of the lysyl hydroxylase 2 mutations found in Bruck syndrome University of Oulu Graduate School; University of Oulu, Faculty of Medicine, Institute of Biomedicine, Department of Medical Biochemistry and Molecular Biology; Biocenter Oulu; Center for Cell-Matrix Research, P.O. Box 5000, FI-90014 Oulu, Finland Acta Univ. Oul. D 1159, 2012 Oulu, Finland Abstract The extracellular matrix is not just a scaffold for cells and tissues, but rather a dynamic part of the human body. Characteristics of collagens, the major protein components of the extracellular matrix, are determined already during synthesis and mutations in genes encoding collagens, unbalance of regulators or dysfunction of collagen modifying enzymes, for instance, can lead to severe clinical complications. Certain hydroxylysine residues formed by lysyl hydroxylases (LHs) function in collagens as precursors of collagen cross-links that stabilize collagenous structures and thereby tissues. In humans, a deficiency of LH1, which is known to hydroxylate lysines in the helical regions of collagen polypeptides, causes Ehlers-Danlos syndrome VIA (EDS VIA). It is characterized e.g. by progressive kyphoscoliosis and hypermobile joints. Mutations in LH2, which is known to hydroxylate lysines in the telopeptides of collagen polypeptides, cause Bruck syndrome type 2 (BS2). BS2 patients suffer from fragile bones and congenital joint contractures, for instance, but the syndrome is usually not lethal. In this work we have generated and analyzed genetically modified LH1 and LH2 null mouse lines to study the in vivo functions and roles of these enzymes. Analyses concentrated also on collagen cross-links that were determined from several null or heterozygous mouse tissues. In the present work we also studied the effects of known BS2 mutations on recombinant human LH2 polypeptides to understand the molecular pathology of the syndrome. As an animal model for human EDS VIA, LH1 null mice had certain characteristics typical for EDS VIA, such as muscular hypotonia, but generally the symptoms were milder. Like EDS VIA patients, the mice have an increased risk of arterial ruptures and ultrastructural changes can be seen in the wall of the aorta, explained by inadequate helical lysine hydroxylation accompanied by a changed cross-linking state of tissues. Similarly, analysis of the LH2 null mouse line demonstrated the importance of the enzyme in cross-link formation. We showed that even a reduced amount of LH2 in adult mice changes the cross-linking pattern in tissues and a total lack of the enzyme leads to embryonic lethality. Furthermore, we demonstrated that LH2 is particularly important in tissue structures supporting blood vessels in the developing mouse embryo or in extraembryonic tissues. Finally, our in vitro studies with recombinant human LH2 polypeptides revealed that the known BS2 mutations severely affect the activity of the enzyme thus explaining the clinical symptoms of the patients, but the mutations do not lead to a total inactivation of the enzyme, which may be critical for the survival of patients. Keywords: Bruck syndrome, collagen, collagen cross-link, connective tissue disorder, Ehlers-Danlos syndrome type VIA, lysyl hydroxylase Hyry, Marjo, Lysyylihydroksylaasit 1 ja 2. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta, Biolääketieteen laitos, Lääketieteellinen biokemia ja molekyylibiologia; Biocenter Oulu; Center for Cell-Matrix Research, PL 5000, 90014 Oulu Acta Univ. Oul. D 1159, 2012 Oulu Tiivistelmä Solunulkoinen matriksi ei ole ainoastaan soluja ja kudoksia tukeva rakenne, vaan se on dynaa- minen osa ihmiskehoa. Kollageenien, solunulkoisen matriksin yleisimpien proteiinien ominai- suudet määräytyvät jo kollageenien synteesivaiheessa ja mutaatiot kollageeneja koodittavissa geeneissä, säätelytekijöiden epätasapaino tai esimerkiksi kollageeneja muokkaavien entsyymien toimintahäiriöt voivat johtaa vaikeisiin kliinisiin komplikaatioihin. Tietyt lysyylihydroksylaasi- en (LH) muodostamat hydroksilysiinitähteet toimivat kollageeneissa kollageeniristisidosten esi- asteina. Ristisidokset vakauttavat kollageenirakenteita ja siten myös kudoksia. LH1 hydroksyloi lysiinejä kollageenipolypeptidien kolmoiskierteisellä alueella ja ihmisellä entsyymin puutos aiheuttaa tyypin VIA Ehlers-Danlosin syndrooman (EDS VIA), jossa potilailla on esimerkiksi etenevää kyfoskolioosia ja yliliikkuvat nivelet. Mutaatiot LH2-entsyymissä, joka hydroksyloi lysiinejä kollageenipolypeptidien telopeptidialueilla, aiheuttavat tyypin 2 Bruckin syndrooman (BS2). BS2-potilaat kärsivät mm. luiden hauraudesta ja niveljäykkyydestä, mutta syndrooma ei yleensä ole letaali. Tässä työssä loimme ja analysoimme geneettisesti muunnellut LH1 ja LH2 hiirilinjat, joiden kyseinen LH-geeniaktiivisuus on hiljennetty. Linjojen avulla halusimme tutkia näiden entsyymi- en toimintaa ja merkitystä in vivo. Analyysit keskittyivät myös kollageeniristisidoksiin, joita tut- kittiin useista poistogeenisten tai heterotsygoottisten hiirten kudoksista. Ymmärtääksemme BS2:n molekyylipatologiaa, tutkimme tässä työssä myös tunnettujen BS2-mutaatioiden vaiku- tuksia ihmisen LH2-rekombinanttiproteiinissa. EDS VIA:n eläinmallina LH1 poistogeenisillä hiirillä on joitakin ominaisuuksia, kuten lihas- hypotonia, jotka ovat tyypillisiä EDS VIA:lle, mutta yleisesti oireet ovat lievempiä. Kuten EDS VIA-potilailla, hiirillä on kohonnut valtimoiden repeytymisriski ja aortan seinämän ultraraken- teessa voidaankin havaita muutoksia. Oireita voidaan selittää riittämättömällä kollageenien kol- moiskierteisen alueen lysiinien hydroksylaatiolla, joka muuttaa kollageenien ristisidostilaa kudoksissa. Myös LH2-hiirilinjan analysointi osoitti kyseisen entsyymin tärkeyden ristisidosten muodostamisessa. Jo alentunut LH2:n määrä aikuisissa hiirissä muuttaa kudosten kollageeniris- tisidoksia ja täydellinen entsyymin puuttuminen johtaa sikiön kuolemaan. Lisäksi osoitimme, että LH2 on erityisen tärkeä kudosrakenteissa, jotka tukevat kehittyvän hiiren sikiön tai sikiön ulkopuolisten kudosten verisuonia. In vitro-tutkimukset ihmisen LH2-rekombinanttiproteiinilla paljastivat, että tunnetut BS2-mutaatiot vaikuttavat erittäin haitallisesti entsyymin toimintaan, mikä selittää potilaiden kliiniset oireet, mutta mutaatiot eivät kuitenkaan aiheuta entsyymin täy- dellistä inaktivaatiota, mikä voi olla kriittistä potilaiden selviytymisen kannalta. Asiasanat: Bruckin syndrooma, kollageeni, kollageeniristisidos, lysyylihydroksylaasi, sidekudossairaus, tyypin VIA Ehlers-Danlosin syndrooma Acknowledgements This research was carried out at the Institute of Biomedicine, Department of Medical Biochemistry and Molecular Biology, University of Oulu, during years 2005–2012. I wish to express my deepest gratitude to my excellent supervisor, Professor Johanna Myllyharju, who gave me the opportunity to start my scientific career and prepare my thesis in her group. I have been privileged to have her as a supervisor as she has provided all the guidance, support, trust and encouragement that the project required. Academy Professor Emeritus Kari Kivirikko deserves my most sincere thanks for his amazing attitude towards science and for his endless optimism, which truly gives inspiration and basis for the scientific career as well as life itself. I want to thank warmly Docent Raija Soininen for her enthusiasm, trust, encouragement, and all the effort in the mouse projects. I am grateful to Professor Emeritus Heather Yeowell and Professor Helen Skaer for
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