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.

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 entsyymien 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 their careful revision of this thesis and valuable comments on it. M.Sc. Sandra Hänninen is acknowledged for her expert revision of the language. We are not able to do science on our own. I want to express my respect and thanks to other key persons, Professor Emeritus Ilmo Hassinen, Professor Taina Pihlajaniemi, Professor Seppo Vainio, Professor Leena Ala-Kokko, Docent Minna Männikkö, Docent Peppi Karppinen, Dr. Aki Manninen and Dr. Lauri Eklund, of the Department of Medical Biochemistry and Molecular Biology for providing excellent research facilities and atmosphere to make science. I want to thank all my co-authors for the pleasant collaboration and their essential contribution to this work. I want to express my deepest thanks to Dr. Kati Takaluoma for giving me hands-on introduction to the world of collagens and science itself. I also want to thank her for supervising my M.Sc. project and for enjoyable moments we have had together. M.D. Juha Lantto and M.D. Hanna- Leena Talsta are thanked for their contribution to these projects and nice moments in the office. I wish to express my gratitude to Professor Ruud Bank for his help with cross-links and Docent Raija Sormunen and Dr. Ilkka Miinalainen for their expertise and help with the electron microscope. This work would have not been possible without the outstanding technical assistance of Liisa Äijälä during all these years starting from lab basics to a novice. I also want to thank the whole staff at the department for creating the great atmosphere to work and for their given assistance along the way. Particularly Auli Kinnunen, Pertti Vuokila, Risto Helminen and Seppo

7 Lähdesmäki deserve my warmest acknowledgements for their professional and kind help. I am also really grateful for the people in the Biocenter Oulu Transgenic core facility and the Laboratory Animal Center for the excellent collaboration and good care of our mice. All former and present members of our research group are heartily thanked for being such wonderful colleagues. It has been a privilege to work with you and share all those memorable sunny and cloudy days during these years. You are the ones who have made our group so inspiring, supportive and warm. I couldn’t ask for more. I also wish to thank Johanna Korvala for sharing the unforgettable time preparing the theses and organizing the dissertation party. All of this would not be possible without the sincere and endless support of family and friends. I sincerely want you to know that you have always brought out the best in me. It’s not easy to find words to thank all of my friends who have been with me during all the good days but also during those days when I have needed you the most. Every day I wonder with smile on my face how lucky I am to have all of you in my life. And finally, I owe my deepest gratitude to my family, my parents and sisters and their families. Without your unconditional love, support and encouragement throughout all my life none of this would be possible. I love you. This work was supported by the Biocenter Oulu and the Emil Aaltonen Foundation.

Oulu, April 2012 Marjo Hyry

8 Abbreviations

AGE advanced glycation end-product bp base pair BS Bruck syndrome C carboxy CD circular dichroism cDNA complementary DNA C-P4H collagen prolyl 4-hydroxylase CRTAP cartilage-associated protein Da Dalton(s) deH-HLNL dehydro-hydroxylysinonorleucine deH-LNL dehydro-lysinonorleucine E embryonic day ECM extracellular matrix EDS Ehlers-Danlos syndrome ER endoplasmic reticulum FACIT fibril-associated collagens with interrupted triple helices FKBP10 FK506 binding protein 10 GGT hydroxylysyl glucosyltransferase GT hydroxylysyl galactosyltransferase HIF hypoxia-inducible factor HLKNL hydroxylysino-5-ketonorleucine HP hydroxylysyl pyridinoline HPLC reverse-phase high-pressure liquid chromatography Hyl hydroxylysine JMJD6 Jumonji domain-6 protein

Km Michaelis-Menten constant LH lysyl hydroxylase LKNL lysino-5-ketonorleucine LO lysyl oxidase LP lysyl pyridinoline MMP matrix metalloproteinase mRNA messenger RNA N amino NC noncollagenous OI osteogenesis imperfecta

9 P3H prolyl 3-hydroxylase PCR polymerase chain reaction PDI protein disulfide isomerase PLOD procollagen-lysine 2-oxoglutarate 5-dioxygenase; human gene Plod procollagen-lysine 2-oxoglutarate 5-dioxygenase; mouse gene Q-PCR real-time quantitative PCR RM Reichert’s membrane TEM transmission electron microscopy TGF-β transforming growth factor-β TIA T-cell-restricted intracellular antigen X, in -Gly-X-Y- any amino acid Y, in -Gly-X-Y- any amino acid

10 List of original articles

This thesis is based on the following articles, which are referred to in the text by their Roman numerals:

I Takaluoma K, Hyry M, Lantto J, Sormunen R, Bank RA, Kivirikko KI, Myllyharju J & Soininen R (2007) Tissue-specific changes in the hydroxylysine content and cross- links of collagens and alterations in fibril morphology in lysyl hydroxylase 1 knock- out mice. J Biol Chem 282(9): 6588–6596. II Hyry M, Lantto J & Myllyharju J (2009) Missense mutations that cause Bruck syndrome affect enzymatic activity, folding, and oligomerization of lysyl hydroxylase 2. J Biol Chem 284(45): 30917–30924. III Hyry M, Soininen R, Bank RA, Sormunen R, Miinalainen I, Talsta HL, Lantto J & Myllyharju J (2012) Lack of lysyl hydroxylase 2 in mouse leads to early lethality during embryonic development. Manuscript.

12 Contents

Abstract Tiivistelmä Acknowledgements 7 Acknowledgements 7 Abbreviations 9 List of original articles 11 Contents 13 1 Introduction 17 2 Review of the literature 19 2.1 Extracellular matrix ...... 19 2.1.1 The molecular multiplicity of the extracellular matrix and its connection to disorders ...... 19 2.2 Collagens ...... 20 2.2.1 The collagen family ...... 21 2.2.2 Collagen biosynthesis ...... 24 2.3 Collagen hydroxylysines and their function ...... 28 2.3.1 Glycosylation of hydroxylysines ...... 29 2.3.2 Collagen cross-links ...... 31 2.4 Lysyl hydroxylases ...... 36 2.4.1 Lysyl hydroxylase isoenzymes ...... 37 2.4.2 Molecular and catalytic properties ...... 42 2.5 Human diseases related to lysyl hydroxylases and animal models ...... 46 2.5.1 Ehlers-Danlos type VI ...... 46 2.5.2 Bruck syndrome 1 and 2 ...... 49 2.5.3 Fibrotic diseases and cancer ...... 53 2.5.4 A disease caused by mutations in LH3 and LH3 null mice ...... 55 2.5.5 Inactivation of LH in other animal models ...... 57 3 Outlines of the present study 59 4 Materials and methods 61 5 Results 63 5.1 LH1 knock-out mice have increased risk of lethal arterial ruptures (I) ...... 63 5.1.1 Generation of LH1 null mice and LH1 expression in adult mouse tissues ...... 63

13 5.1.2 LH1 null mice have no kyphoscoliosis but are hypotonic and have increased mortality due to arterial ruptures ...... 64 5.1.3 Abnormal collagen fibrils in LH1 null mice ...... 64 5.1.4 Decreased LH activity in LH1 null mice affects the hydroxylysine content in various tissues differently ...... 65 5.1.5 Abnormal hydroxylation of triple helical lysine residues affects the cross-linking pattern in the LH1 null mouse tissues ...... 66 5.2 Analysis of the molecular level consequences of Bruck syndrome mutations by the use of recombinant LH2 proteins (II) ...... 67 5.2.1 Expression of wild-type and recombinant LH2(long) polypeptides with Bruck syndrome mutations in insect cells and their purification ...... 67 5.2.2 The Bruck syndrome mutant R594H and G597V LH2(long) polypeptides have problems in dimerization and are prone to degradation ...... 68 5.2.3 Folding of the R594H and G597V mutant LH2(long) polypeptides is impaired and their proteolytic sensitivity is increased ...... 69 5.2.4 All Bruck syndrome mutations affect the activity of LH2 ...... 70 5.3 Lysyl hydroxylase 2 is essential for the embryonic development of mouse (III) ...... 71 5.3.1 LH2 null mice die after embryonic day 10.5 ...... 71 5.3.2 LH2 is expressed ubiquitously in the E10.5 embryos and in extraembryonic tissues and its lack seems to lead to certain changes in collagen expression ...... 72 5.3.3 Expression of the Plod1 and Plod3 genes is not upregulated in the Plod2gt/gt embryos ...... 73 5.3.4 LH2 is widely expressed in adult Plod2wt/gt tissues ...... 74 5.3.5 Changes in lysyl hydroxylation and cross-linking of collagen in adult Plod2wt/gt mice ...... 74 6 Discussion 77 6.1 LH1 null mice provide a tool to study tissue-specific consequences of the lack of LH1 activity ...... 77 6.2 Mutations causing Bruck syndrome lead to decreased LH2 activity, explaining the symptoms of diagnosed Bruck syndrome type 2 patients ...... 81

14 6.3 Inactivation of mouse Plod2 leads to lethality during early embryonic development ...... 83 7 Conclusions and future prospects 89 References 91 Original articles 111

16 1 Introduction

Collagens are the most abundant proteins in the human body and comprise the major structural backbone in the extracellular matrix (ECM) of tissues. Several collagen-specific enzymes are required in collagen biosynthesis, including lysyl hydroxylases (LHs). Hence, it is natural that changes in collagens or enzymes involved in collagen synthesis can lead to severe consequences. Collagen-related changes in the ECM cause many common conditions such as fibrosis, but they also cause rare disorders because of inherited mutations in ECM genes, such as osteogenesis imperfecta (OI) or the rarer Bruck syndrome (BS). Humans and mice have three different LH isoforms i.e. LH1, LH2 and LH3. Mutations in all three LH isoforms are known to cause severe connective tissue disorders in humans demonstrating their importance. LH1 mutations have long been known to cause the Ehlers-Danlos syndrome VIA (EDS VIA), which is characterized e.g. by progressive kyphoscoliosis and hypermobile joints. The discovery of LH2 was soon accompanied by identification of mutations in LH2 that cause the BS type 2. BS2 patients suffer from symptoms like fragile bones and congenital joint contractures. Recently, LH3 mutations have also been connected to two human disorders, and some years earlier a mouse LH3 knock- out was observed to be embryonically lethal, suggesting the importance of LHs also in mice. Hydroxylysine residues formed by LHs function in collagens as attachment sites for carbohydrate residues and certain residues participate in collagen cross- links. Moreover, the hydroxylation state of the lysines at the cross-link sites determines the cross-linking pattern and thus partly the mechanical properties of tissues. Although in vitro studies have revealed much information about LHs, the knowledge of in vivo functions of LH1 and LH2 has been restricted mostly to analyses of human patients. Our aim in this work was to generate LH1 and LH2 null mouse lines to obtain more detailed information of the in vivo functions of these enzymes. The mouse lines were also expected to provide more information about the EDS VIA and BS2 syndroms at the molecular level and about the intricate cross-link formation. In addition, we studied the effects of the known BS2 mutations on recombinant human LH2 polypeptides to understand the molecular pathology of the syndrome. Our results show that the LH1 null mouse line is valuable in the analysis of the in vivo function of LH1, as well as allowing better understanding of the pathology of EDS VIA. Surprisingly, our work demonstrates that LH2 is essential for the embryonic development of the mouse.

17 Our data also show that the mutations found in human BS2 patients decrease the enzyme activity in vitro by two different mechanisms, but do not lead to complete LH2 inactivation, which is likely to explain the survival of the BS2 patients. Both mouse lines also provide novel information on the roles of LH1 and LH2 in determining the collagen cross-linking pattern in tissues.

18 2 Review of the literature

2.1 Extracellular matrix

The extracellular matrix (ECM) is a complex environment that surrounds cells and enables the multicellularity of organisms. ECM consists of molecules that are secreted by the adjacent cells and thus the environment is tailored for the requirements of the cells and the tissue. ECM functions as a scaffold providing strength and support against physical stress, but it also has an essential role in cell behavior. It interacts with signaling molecules and growth factors, which regulate cellular events such as determination, differentiation, proliferation, migration and survival. The ECM is highly dynamic and it undergoes constant remodeling during life and particularly in many disease states. Failures in the renewal process underlie many pathological states like fibrosis, atherosclerosis and tumorigenesis. (For reviews, see Kielty & Grant 2002, Daley et al. 2008, Butcher et al. 2009, Tsang et al. 2010).

2.1.1 The molecular multiplicity of the extracellular matrix and its connection to disorders

The main components of the ECM are collagens and noncollagenous proteins such as proteoglycans, glycoproteins and elastins, but the different mechanical requirements of tissues determine the ratios of these proteins. Thus, the molecular diversity in the ECM is overwhelming and these structurally very different molecules are themselves responsible for the dynamic nature of the ECM. Many ECM proteins are large and complex consisting of several individually folded domains that are highly conserved between species. The synthesis, organization and interplay of the ECM components are strict. The synthesis is controlled by growth factors such as transforming growth factor-β (TGF-β) and the degradation by proteases like cysteine and serine proteases and matrix metalloproteinases (MMPs). It is clear that the activity of these molecules must be under precise control and misregulation can easily lead to severe consequences. (For reviews, see Daley et al. 2008, Badylak et al. 2009, Butcher et al. 2009, Hynes 2009, Järveläinen et al. 2009, Tsang et al. 2010). Recently it was suggested that in addition to signaling molecules and growth factors, basic features such as the

19 stiffness of the ECM also affect differentiation and cell behavior (Engler et al. 2006, Butcher et al. 2009). One minimal inherited or acquired change in one ECM component can lead to physicochemical and thus to mechanical and behavioral changes in the tissue or developing individual followed by a possible disorder or even lethality (Järveläinen et al. 2009, Levental et al. 2009). The rare Ehlers-Danlos syndrome (EDS) type VIA is a severe connective tissue disorder and an example of an inherited monogenic disorder. It is caused by mutations in one of the collagen- modifying enzymes, lysyl hydroxylase 1 (LH1, see 2.2.2 and 2.4). It is characterized by progressive kyphoscoliosis, hypermobile joints and muscle hypotonia, and patients have an increased risk of arterial ruptures. (For reviews, see Yeowell & Walker 2000, Steinmann et al. 2002). ECM changes are distinct also in common diseases that can be acquired or caused by a combination of acquired and genetic factors, not forgetting the interactions between the ECM and growth factors and other signaling molecules. In coronary heart disease, for instance, the ECM components participate in the formation of the atherosclerotic plaque. Changes in the ECM composition have been connected also to carcinogenesis and metastasis of the tumor cells. On the other hand, probably all diseases affect the quality and/or quantity of the ECM. Thus, exact knowledge about individual ECM molecules, their function and interplay with growth factors and signaling molecules is beneficial not only for the drug development and treatment of ECM disorders, but it can be utilized also in the treatment of a wide variety of other diseases. A deficiency in structural ECM proteins is challenging to treat, but the regulators provide a potential tool when their function is known. (Hynes 2009, Järveläinen et al. 2009, Levental et al. 2009, Van Agtmael & Bruckner-Tuderman 2010).

2.2 Collagens

Collagens are the major insoluble fibrous proteins in the ECM and the most abundant proteins in the human body. Thus, collagens have a remarkable role in the maintenance of various structures as well as in other essential ECM activities. Collagens are involved in many disorders with numerous mutations in collagen genes behind them. Collagen biosynthesis requires specific enzymes and mutations in these enzymes increase the number of collagen-related disorders. (For reviews, see Myllyharju & Kivirikko 2001, Myllyharju & Kivirikko 2004,

20 Gelse et al. 2003, Van Agtmael & Bruckner-Tuderman 2010, Marini et al. 2010, Parkin et al. 2011).

2.2.1 The collagen family

Humans have at least 28 different collagen types, which consist of over 40 different α chains (Myllyharju & Kivirikko 2004, Gordon & Hahn 2010). The heterogeneity of the collagen family members is also increased by alternative splicing of the primary transcripts coding for collagen polypeptides (Myllyharju & Kivirikko 2001, McAlinden et al. 2005, Seppinen & Pihlajaniemi 2011). All collagen molecules consist of three identical or different polypeptides called α chains, which are wrapped around each other to form a triple helix. The triple helical structure forms as a result of the repeating Gly-X-Y triplet, which represents a collagenous domain. As the smallest amino acid, the glycine residue is positioned in the central axis of the triple helix and it is the only amino acid that fits into that restricted space. In fact, the most harmful mutations in the collagen genes are often those that replace the glycine residue in the triple helical domain.

In the collagenous (Gly-X-Y)n sequence, the X and Y can be any amino acid except glycine, but often proline is in the X position and 4-hydroxyproline in the Y position. (Brodsky & Persikov 2005, Shoulders & Raines 2009, Gordon & Hahn 2010). All collagen molecules have at least one collagenous domain in their structure, but the amount and lengths of these parts vary between collagen types. Over 90% of type I collagen for instance is collagenous, while collagen XII contains only 10% of triple helix. Besides collagenous domains, collagens contain also noncollagenous (NC) parts which often have important functions. These domains may be involved in the binding of molecules or in the assembly of supramolecular structures, or they may have their own functions when released from the collagen, an example being endostatin. Endostatin is cleaved from the C-terminal NC1 domain of type XVIII collagen and is able to inhibit angiogenesis and tumor growth. Similar domains called restin and arresten are also present in collagens XV and IV, respectively. (Myllyharju & Kivirikko 2001, Gelse et al. 2003, Ricard-Blum & Ruggiero 2005, Ricard-Blum 2011). Collagenous domains are not unique to collagens. There are several proteins such as adiponectin, collectin, ficolin and macrophage receptor that contain a collagen-like domain in their structure, but they are not called collagens. (Myllyharju & Kivirikko 2004, Ricard-Blum 2011).

21 Collagens can be divided into fibrillar and non-fibrillar collagens and further into several subfamilies (see Fig. 1). The collagen superfamily consists of the fibril-forming collagens such as type I, II, III and V collagens, fibril-associated collagens with interrupted triple helices (FACITs) such as type IX and XII collagens, collagens that form hexagonal networks (type VIII and X collagens), network-forming collagens (type IV collagens), collagens that form beaded filaments (type VI collagen), collagens that form anchoring fibrils (type VII collagen), endostatin-producing collagens (type XV and XVIII collagens) and transmembrane collagens such as collagens XIII and XXV. (For reviews, see Myllyharju & Kivirikko 2004, Kadler et al. 2007, Gordon & Hahn 2010). Type XXVIII collagen is the most recently identified collagen in the superfamily. It cannot be placed clearly into any collagen subfamily mentioned above as it has properties typical of more than one of those groups. (Veit et al. 2006). Type XXIX collagen, reported in 2007, is likely a member of the collagen VI family rather than a new collagen type (Söderhäll et al. 2007, Fitzgerald et al. 2008).

22 a. Fibril-forming collagens, types I, II, III, V, XI, XXIV and XXVII b. FACIT and related collagens, types IX, XII, Genes: COL1A1, COL1A2; COL2A1; COL3A1; COL5A1, COL5A2, COL5A3, XIV, XVI, XIX, XX, XXI, XXII and XXVI COL5A4; COL11A1, COL11A2; COL24A1; COL27A1 Genes: COL9A1, COL9A2, COL9A3 ; COL12A1; COL14A1; Triple helical region COL16A1; COL19A1; COL20A1; COL21A1; COL22A1; COL26A1 N pro- C propeptide peptide IX Type II fibril 300 nm GAG 100 nm GAG

c. Collagens forming hexagonal networks, types VIII and X XII and XIV Genes: COL8A1, COL8A2; COL10A1 Type I fibril

VIII X 100 nm 100 nm 100 nm 100 nm

d. The family of type IV collagens e. Type VI collagen forming beaded filaments Genes: COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6 Genes: COL6A1, COL6A2, COL6A3 VI Dimer Tetramer Beaded filament 7S Dimer

100 nm 7S 200 nm 100 nm 100 nm Tetramer

f. Type VII collagen forming anchoring fibrils Gene: COL7A1 Genes: COL15A1; COL18A1 Genes: COL13A1; COL17A1; COL23A1; VII COL25A1

100 nm

Dimer 200 nm GAG

Anchoring fibril Basement Anchoring membrane plaque

ficolin ectodysplasin

Fig. 1. Members of the collagen superfamily and their known supramolecular assemblies. Reprinted from Myllyharju & Kivirikko 2004 with permission of the publisher.

Fibrillar collagens

Fibril-forming (fibrillar) collagens form the most abundant subfamily of collagens and this group includes seven different collagen types. Quantitatively the most common types are type I, II and III collagens, whereas types V, XI, XXIV and XXVII are minor forms. (Gelse et al. 2003, Myllyharju & Kivirikko 2004, Ricard-Blum & Ruggiero 2005). The fibril diameter of the collagen molecules

23 varies from 12 nm to over 500 nm and depends on the tissue and the stage of development (Kadler et al. 2007). Tissues that must withstand high stress levels contain a higher proportion of collagen fibers with a large diameter. Thus, diameter increases at the expense of flexibility. (Bailey et al. 1998). Fibrillar collagens, excluding types XXIV and XXVII, share an about 1000 amino acid residues long domain which has a perfect collagenous (Gly-X-Y)n sequence without any interruptions (Gordon & Hahn 2010). In addition, they all have N- and C-terminal propeptides, which are cleaved before fibril formation, and may have also some smaller NC domains. However, the cleavage of the N propeptide is not always essential. It can also be partially cleaved or entirely intact such as in the early embryonic form of type II collagen (IIA). The role of N propeptide in the mature collagen is not known, but the globular N-terminal end of the N propeptides may have binding functions. One common structure in the globular N-terminal end is a cysteine-rich domain, which is shown to be able to bind growth factors such as certain bone morphogenetic proteins and TGF-β1. (Fleischmajer et al. 1990, Ng et al. 1993, Zhu et al. 1999, Larraín et al. 2000 Ricard-Blum & Ruggiero 2005). Fibrillar collagens provide stability and integrity to the tissues and they are ubiquitously found in tissues such as bone, cartilage and skin. Type I collagen is usually a heterotrimer consisting of two α1(I) chains and one α2(I) chain. It forms about 90% of the organic mass of bone and together with type V collagen it is responsible for the structural backbone of bone. Type I collagen is also the major collagen type in many other tissues such as tendon, skin and ligaments. Although type I collagen is the most common collagen type in many tissues, the tissues can be mechanically very different. In skin, for example, type I collagen forms compounds with type III collagen providing tensile strength for the tissue, and in bone, load bearing ability as well as tensional and torsional properties are accompanied by calcification. (Gelse et al. 2003). The stability of the collagenous structures is also affected by the type of cross-links predominating between collagen molecules (Knott & Bailey 1998, Bank et al. 1999).

2.2.2 Collagen biosynthesis

Collagen biosynthesis from messenger RNA (mRNA) to an active protein is a complex process characterized by many co- and post-translational modifications that are unique only for collagens and proteins containing collagen-like domains (see Fig. 2). The biosynthesis of fibril-forming collagens is best known and thus

24 used often as an example of collagen synthesis in general. Collagen biosynthesis can be divided to intracellular and extracellular stages. Intracellular steps include the synthesis and modification of a procollagen molecule from a gene transcript, whereas extracellular steps convert procollagen molecules to collagen molecules and furthermore to more extensive final structures. (Kielty & Grant 2002, Gelse et al. 2003).

Synthesis & co- and post-translational modifications

-OH Collagen prolyl 4-hydroxylase -OH -OH -OH Prolyl 3-hydroxylase Nucleus -OH -OH -OH Lysyl hydroxylase -O-Gal -OH -O-Gal-Glc -O-Gal-Glc -OH -O-Gal Collagen glycosyltransferases

Endoplasmic OH

reticulum - O-Gal-Glc Cytopl asm - S Assembly of 3 procollagen chains & - - - S OH OH OH S disulfide bonding OH OH - - S Protein disulfide isomerase

Assembly of triple helix

Extracellular space Cleavage of propeptides N and C proteinases

Assembly into collagen fibrils & cross-link formation Lysyl oxidase

Fig. 2. Main steps in the biosynthesis of fibril-forming collagens. Modified from Myllyharju & Kivirikko 2004.

Intracellular co- and post-translational modifications

Collagen polypeptide chains are translated in the rough endoplasmic reticulum (ER) as pre-pro-α-chains. The growing polypeptide chain is directed into the lumen of the ER according to an N-terminal signal sequence. The signal sequence is removed after translocation into the lumen by a signal peptidase, and the produced pro-α-chain is immediately available for further co- and post- translational modifications. These modifications are performed within the cisternae of ER and they consist mainly of hydroxylations and glycosylations. Collagen prolyl 4-hydroxylases (C-P4Hs), prolyl 3-hydroxylases (P3Hs) and lysyl

25 hydroxylases (LHs) are members of the 2-oxoglutarate dioxygenase family, and they catalyze hydroxylations of certain proline and lysine residues in procollagen chains to 4-hydroxyproline, 3-hydroxyproline and hydroxylysine, respectively (see Fig. 2). Glycosylations of some of the hydroxylysyl residues to galactosylhydroxylysine and glucosylgalactosyl-hydroxylysine are catalyzed by collagen galactosyltransferase and glugosyltransferase, respectively. (Kielty & Grant 2002, Canty & Kadler 2005, Myllyharju 2005). 4-Hydroxyproline residues are essential for the thermal stability of the collagen triple helix. Collagen α-chains that are not properly hydroxylated by C- P4H do not hold together in a triple helix conformation in physiological conditions. (Kivirikko & Pihlajaniemi 1998, Myllyharju & Kivirikko 2001). The role of 3-hydroxyproline was unknown for a long time until a decreased prolyl 3- hydroxylation in CRTAP (cartilage-associated protein) deficiency was shown to be connected to osteogenesis imperfecta (OI) (Morello et al. 2006, Marini et al. 2010). 3-Hydroxyproline residues are now suggested to have a role in the assembly of collagen fibrils (see below, Weis et al. 2010). Hydroxylysine residues are needed for the formation of covalent collagen cross-links and they serve as sites for glycosylation (Kielty & Grant 2002). In addition to the collagenous domain, hydroxylysine residues are found in the short noncollagenous telopeptide regions at the ends of the collagen molecule (Kivirikko et al. 1992). LHs and hydroxylysines will be discussed in more detail later (see 2.3 and 2.4). C-P4Hs, P3Hs and LHs require the cofactors Fe2+, 2-oxoglutarate, molecular oxygen, and ascorbate (vitamin C) for the enzymatic reaction, and the activity of these enzymes is required for the formation of functionally normal collagens (Myllyharju & Kivirikko 2004, Marini et al. 2010). In humans the lack of vitamin C, for instance, leads to inadequate hydroxylations of collagens, the retaining of collagen chains within the ER, and thus to poor connective tissue renewal which is characteristic of scurvy, for instance (Canty & Kadler 2005). Intracellular modifications of collagen also consist of reactions that are common for many noncollagenous proteins, such as glycosylation of certain asparagine residues and formation of disulfide bonds (Myllyharju & Kivirikko 2004, Kielty & Grant 2002, Gelse et al. 2003).

Trimerization and triple helix formation of collagens

During post-translational modifications three newly synthesized pro-α-chains connect via their C propeptide areas to form an attachment point, the nucleus. The

26 association is guided according to specific recognition sequences. (Lees et al. 1997, Kielty & Grant 2002). The trimeric structure is stabilized by formation of interchain disulfide bonds between the C propeptides. This reaction is catalyzed by the multifunctional protein disulfide isomerase (PDI). (Kielty & Grant 2002, Myllyharju & Kivirikko 2004). PDI also has another stabilizing feature when it acts as a chaperone and binds to newly synthesized collagen polypeptide chains preventing their aggregation (Wilson et al. 1998, Myllyharju & Kivirikko 2001). Collagen biosynthesis also requires other molecular chaperones such as the essential collagen-specific chaperone Hsp47. Hsp47 inhibits procollagen aggregation by binding procollagen in the ER and facilitating triple helix formation. (Canty & Kadler 2005, Ishida & Nagata 2011). Triple helix formation begins when C propeptides have associated and the nucleus is formed, the cis peptide bonds involving proline are converted to the trans form by the peptidyl-prolyl cis-trans isomerase, and when each pro-α-chain contains about 100 4-hydroxyproline residues. Triple helix formation propagates from the C terminus to the N terminus like a zipper. (Myllyharju & Kivirikko 2001, Canty & Kadler 2002, Kielty & Grant 2002). Generally the triple helix structure prevents further hydroxylations, but one of the three LH isoenzymes, LH3, has been recently reported to modify also native triple helical collagen molecules in the extracellular space (Kielty & Grant 2002, Salo et al. 2006a). Although the enzymatic modifications of collagen polypeptides described above are necessary, overmodification is harmful. A delay in triple helix formation can lead to intracellular overmodification of the collagen polypeptides followed by a perturbation of the final triple helix structure (Torre-Blanco et al. 1992, Raghunath et al. 1994). The triple helical procollagen molecules are transported from the ER to the extracellular space across the Golgi stacks probably via cisternal maturation (Myllyharju & Kivirikko 2001, Kielty & Grant 2002, Canty & Kadler 2005).

Extracellular stage

The assembly of collagen molecules into fibrils is thought to be an extracellular step in collagen biosynthesis. However, the assembly may begin already during the transportation from the ER. (Canty & Kadler 2005). A key point in the fibril formation is the cleavage of the globular N and C propeptides of the procollagen molecules. This is carried out by two specific metalloproteinases, a procollagen N proteinase and a procollagen C proteinase, respectively. The produced collagen

27 molecules then assemble spontaneously into a fibrillar structure in the case of fibril-forming collagens. Finally, collagen cross-links are formed within and between collagen molecules to stabilize the formed structures. (Myllyharju & Kivirikko 2001, Kielty & Grant 2002, Canty & Kadler 2005). This final cross- linking step begins with an oxidative deamination of the telopeptide lysine or hydroxylysine residue by lysyl oxidase (LO), which is followed by certain spontaneous reactions (Knott & Bailey 1998, Mäki 2009). In tissues the fibrils are usually organized further into higher-order three-dimensional structures (Canty & Kadler 2005).

Specific features in the biosynthesis of non-fibrillar collagens

Although the basic features of the biosynthesis of non-fibrillar collagens are identical to those described above with fibrillar collagens, distinct differences also exist. Final macromolecular assemblies determine the direction of the synthesis. Some collagen types are subjected to additional modifications like additions of glycosaminoglycan side chains. It is also assumed that the triple helix of transmembrane collagens may be propagated from the N to C terminus. Many collagens retain an N- or C-terminal noncollagenous domain or both in their structures, and thus these domains are not cleaved during extracellular modifications. (Myllyharju & Kivirikko 2001, Kielty & Grant 2002).

2.3 Collagen hydroxylysines and their function

Hydroxylysine (Hyl) residues are usually found in the Y position of the repeating X-Y-Gly triplet sequence in collagens and the collagenous domains of several noncollagenous proteins. The repeating Gly-X-Y sequences are written here as X-Y-Gly due to the hydroxylation properties of collagen hydroxylases, the minimum sequence requirement for hydroxylation by e.g. LH being X-Lys-Gly. Hydroxylysine residues are also found in X-Hyl-Ser and X-Hyl-Ala sequences in the telopeptide regions, i.e. in the ends of the α chains of some fibril-forming collagens. (Kivirikko et al. 1992, Myllyharju 2005). A hydroxylysine residue has been discovered in an X-Hyl-Ala sequence also in the NC1 domain of type IV collagen, where it was suggested to participate in the formation of an S- hydroxylysyl-methionine cross-link, a sulfilimine bond, with a particular methionine residue (Vanacore et al. 2005, Vanacore et al. 2009). In addition to collagenous polypeptides, a single hydroxylysine residue is known to be present

28 e.g. in anglerfish somatostatin-28 (Kivirikko et al. 1992). Recently hydroxylysine residues were found in histone peptide fragments and in proteins contributing to RNA splicing, where the hydroxylation reaction is suggested to be catalyzed by the Jumonji domain-6 protein (JMJD6) and not by LHs (Chang et al. 2007, Webby et al. 2009). The lysyl hydroxylation is usually not complete and some lysine residues located in the Y position of X-Y-Gly triplets or in telopeptides remain unhydroxylated. The hydroxylysine content of collagens depends on the collagen type and tissue as well as the physiological and pathological state of the tissue. (Kivirikko et al. 1992, Myllyharju 2005). Generally the highest hydroxylation content is observed in type IV collagen, where almost 90% of lysine residues in the helical area are hydroxylated, whereas in collagen III, for instance, the value is less than 20% (Kivirikko et al. 1992). The significance of hydroxylysine residues can be seen in various diseases. The extent of hydroxylation is decreased in Bruck Syndrome (Bank et al. 1999, van der Slot et al. 2003) and EDS VIA (Steinmann et al. 2002) and increased in fibrotic diseases (Brinckmann et al. 1996, Brinckmann et al. 1999, van der Slot et al. 2003). Hydroxylysine residues have at least two major functions in collagen molecules. They act as attachment sites for either galactose or glucosylgalactose carbohydrate residues. However, the purpose of these glycosylations is not fully understood. Hydroxylysine residues are also essential in the formation of intermolecular stabilizing covalent collagen cross-links and thus in supporting the structure of the collagen molecule and its supramolecular assemblies. (Kivirikko & Pihlajaniemi 1998, Kielty & Grant 2002).

2.3.1 Glycosylation of hydroxylysines

Carbohydrate units linked to hydroxylysine residues of collagen molecules are either monosaccharide galactoses or disaccharide glucosylgalactoses (Myllyharju 2005). As the hydroxylation of lysine residues varies between collagen types, tissue distribution and physiological state, similarly the extent of the hydroxylysine glycosylation as well as the ratio of galactose and glucosylgalactose vary (Kivirikko & Myllylä 1979, Kivirikko et al. 1992). Type IV and VI collagens contain the highest amount of glycosylated hydroxylysines with almost complete glycosylation (Kivirikko & Myllylä 1979, Chu et al. 1988), and recently the inadequate glycosylation of these collagen types was reported to prevent their normal assembly and secretion (Sipilä et al. 2007).

29 Two different transferase activities are required during the two glycosylation steps. In the first step the formation of a bond between the hydroxylysine residue and galactose moiety is catalyzed by hydroxylysyl galactosyltransferase (GT). Some of the formed galactosylhydroxylysines are further modified by galactosyl hydroxylysyl glucosyltransferase (GGT) to form a glucosylgalactosyl hydroxylysine residue. (Myllyharju 2005). LH3, but not LH1 or LH2, is now known to have both collagen glycosyltransferase activities in addition to its LH activity (Heikkinen et al. 2000, Rautavuoma et al. 2002, Wang et al. 2002a). It was speculated earlier that in addition to LH3 there are likely to be additional collagen glycosyltransferases in tissues, especially as the GT activity of LH3 is low (Rautavuoma et al. 2002, Rautavuoma et al. 2004) and two glycosyltransferase GLT25 family members, GLT25 domain 1 and 2 (GLT25D1 and GLT25D2), were recently observed to have strong collagen GT activity (Schegg et al. 2009). GLT25D1 is localized in the ER like LHs (Liefhebber et al. 2010). It has been suggested that the GLT25D1 could be responsible for the GT reactions for example in bone type I collagen (Sricholpech et al. 2011).

The function of glycosylated hydroxylysines

Although the hydroxylysine glycosylation of collagens was observed decades ago, its exact biological role is still not fully understood (Kivirikko & Myllylä 1979). However, studies of type I and II collagens have revealed that the carbohydrate residues may affect the collagen fibril diameter and collagen fibrillogenesis (Torre-Blanco et al. 1992, Notbohm et al. 1999, Brinckmann et al. 1999, Sricholpech et al. 2011), and an older study of type I collagen suggests that the carbohydrate moieties could prevent excess cross-link formation (Yamauchi et al. 1982). Nevertheless, glycosylated hydroxylysines have been shown to participate in the formation of cross-links (Henkel et al. 1976, Hanson & Eyre 1996, Brinckmann et al. 1999). The importance of glycosylated hydroxylysine residues is also demonstrated by studies with LH3 knock-out mice. The mice die during embryonic development due to lack of hydroxylysine glycosylation activity and subsequent impaired production of type IV collagen (Rautavuoma et al. 2004, Ruotsalainen et al. 2006). As indicated earlier, the under-glycosylation of highly glycosylated collagen types IV and VI affected their assembly and secretion (Sipilä et al. 2007). Cellular studies have also shown that glycosylation catalyzed by LH3 is important for the organization of the ECM as well as cell growth and viability (Risteli et al. 2009, Wang et al. 2009). Mutation studies of mannose-

30 binding lectins that contain collagenous domains have also revealed that the glycosylation of hydroxylysines is important in the folding and secretion of the protein (Heise et al. 2000). However, just as collagens have numerous different functions, the role of hydroxylysine-linked carbohydrates is presumably more diverse than discussed above. For example, at least in type II collagen glycosylated hydroxylysine residues can act as antigens and induce autoimmune responses such as autoimmune arthritis (Myers et al. 2004), and they are also suggested to affect the binding of type IV collagen to the melanoma receptor (Lauer-Fields et al. 2003).

2.3.2 Collagen cross-links

Collagen cross-links are essential in the determination of the physical and chemical properties of collagens and their significance is demonstrated in many diseases (Bank et al. 1999, Brinckmann et al. 1999, Steinmann et al. 2002, Eyre & Wu 2005, Levental et al. 2009). Cross-links can be formed between similar collagen molecules but also between different collagen types (Eyre & Wu 2005). Two opinions exist whether one cross-link is formed between two or three collagen molecules (Bailey et al. 1998). Cross-links can be separated into enzyme-mediated or non-enzymatic according to the initiation of the cross-link formation (Reiser et al. 1992). Undesirable, occasional sugar-related cross-links derived from the non-enzymatic pathway are connected to aging and diabetes, for instance, while the enzymatic cross-linking initiated by LO is essential for proper tissue function (Reiser et al. 1992, Eyre & Wu 2005, Robins 2007, Mäki 2009). At least fibril-forming collagen types I, II, III, V and XI as well as certain other collagen types are cross-linked by a mechanism requiring LO enzyme. The formation of these cross-links can follow two basic pathways, the lysine aldehyde (allysine) or hydroxylysine aldehyde (hydroxyallysine) pathway, where certain lysine or hydroxylysine residues, respectively, function as precursors. (Knott & Bailey 1998, Eyre & Wu 2005). At least types I, II and III of the fibrillar collagens have four lysine residues that participate in the cross-link formation. Two of the residues are located in the triple helical area and two in the telopeptide regions, one residue in each telopeptide. (Knott & Bailey 1998). The alternatively-spliced form of LH2, LH2(long), is the LH that hydroxylates the telopeptide lysines in collagens, while LH1 is mainly responsible for helical lysine hydroxylation (Uzawa et al. 1999, Eyre et al. 2002, Mercer et al. 2003, van der Slot et al. 2003, Pornprasertsuk et al. 2004, Takaluoma et al. 2007). The

31 hydroxylation of lysine residues in the telopeptide area determines whether the allysine or hydroxyallysine pathway is utilized for cross-linking, the hydroxylation state of the helical lysine determines the final form for cross-link, and LO completes the procedure (Knott & Bailey 1998, Eyre & Wu 2005). Thus, LHs and LO are the regulatory enzymes in the cross-linking pathway. Many general and connective tissue-specific factors such as TGF-β, connective tissue growth factor, tumor necrosis factor-α, vitamin B6, and the active form of vitamin D regulate the activity of these enzymes and thus also cross-linking. (Saito & Marumo 2010). The cross-linking pathways initiated by LO are illustrated in Fig. 3. Allysine pathway Hydroxyallysine pathway

tLys tHyl

LO LO

Allysine Hydroxyallysine

+ hLys + hHyl + hLys + hHyl

a s deH-LNL deH-HLNL LKNL HLKNL

D r Aldimines Ketoimines

+ hHis + Hydroxyallysine / HLKNL

i l Lysyl Hydroxylysyl

s HHL ? LP HP

s pyrrole pyrrole

Fig. 3. Lysyl oxidase-mediated collagen cross-linking. Modified from Eyre & Wu 2005.

32 Lysyl oxidase-mediated cross-linking

Enzymatic cross-linking begins with an oxidative deamination of a telopeptide lysine or hydroxylysine residue by LO (Knott & Bailey 1998). The formed aldehydes allysine or hydroxyallysine, respectively, are further modified spontaneously during subsequent steps of the two separate pathways (Knott & Bailey 1998, Eyre & Wu 2005) (see Fig. 3). In allysine pathway the telopeptide lysine residue reacts with a triple helical lysine or hydroxylysine residue yielding a divalent cross-link dehydro- lysinonorleucine (deH-LNL) or dehydro-hydroxylysinonorleucine (deH-HLNL), respectively (Knott & Bailey 1998). The deH-HLNL of these immature aldimines can react further with a helical histidine residue to form a mature, trifunctional cross-link histidinohydroxylysinonorleucine (HHL), which is common in the skin for instance (Eyre & Wu 2005, Robins 2007). In hydroxyallysine pathway the telopeptide hydroxylysine residue can react with a triple helical lysine or hydroxylysine residue to form a lysino-5-ketonorleucine (LKNL) or a hydroxylysino-5-ketonorleucine (HLKNL) cross-link, respectively (Knott & Bailey 1998). Depending on the theory, these ketoimines are further incorporated with a lysine aldehyde or a hydroxylysine aldehyde or two ketoimines are condensed to form mature pyridinoline and pyrrole cross-links (Knott & Bailey 1998, Eyre & Wu 2005, Robins 2007). Thus, the pyridinolines lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) are formed from the reaction between LKNL or HLKNL, respectively, and hydroxyallysine or another HLKNL (Knott & Bailey 1998, Eyre & Wu 2005). Both cross-links are common in stiff tissues, but LP is predominant in calcified tissues and HP in cartilage, for instance. Lysyl pyrrole and hydroxylysyl pyrrole are formed in turn from LKNL or HLKNL, respectively, and allysine or deH-HLNL. (Bailey et al. 1998, Knott & Bailey 1998, Eyre & Wu 2005). Recently, a new maturation product, named arginoline, was detected from the cartilage collagens. The arginoline cross-link is formed from the ketoimine by an oxidation reaction followed by an addition of free arginine. It is a functionally divalent but nonreducible cross-link. About half of the mature cross-links in the type II collagen are arginoline cross-links instead of HP. (Eyre et al. 2010). A hydroxylysine residue is also involved in the covalent sulfilimine bond found in collagen IV. This cross-link is formed between a hydroxylysine and a methionine. (Vanacore et al. 2005, Vanacore et al. 2009).

33 Cross-links in tissues

The cross-linking pattern as well as the degree of it seem to be more tissue- specific than collagen type-specific (Knott & Bailey 1998, Eyre & Wu 2005). Cross-links in skin and other soft tissues are mainly products from the allysine pathway, whereas cross-links derived from the hydroxyallysine pathway predominate in tissues that require strength and stiffness such as bone, dentin, cartilage and tendon (Knott & Bailey 1998, Kielty & Grant 2002, Eyre & Wu 2005). In addition, physiological processes affect the cross-linking state as seen in the relatively fast cervix remodelling during pregnancy, for instance (Akins et al. 2011). In skin the level of lysine hydroxylation is low and the main form of the divalent cross-link is deH-HLNL, which can mature later to HHL (Knott & Bailey 1998, Avery & Bailey 2005, Robins 2007). Despite the high physical requirements of bone, only a small amount of mature pyridinoline cross-links exist in bone collagens, but the existing cross-links are formed from lysines that are hydroxylated, i.e. ketoimines and pyridinolines (Knott & Bailey 1998). This is suggested to be caused by the increased proportion of pyrrole cross-links (Bailey et al. 1998). In addition, bone tissue is under constant remodelling with the half- life of collagen being low when compared to the slowly renewing cartilage, where lysine residues are highly hydroxylated and collagen cross-links well matured (Knott & Bailey 1998, Avery & Bailey 2005, Eyre et al. 2010). The half-life of type II collagen predominating in cartilage is estimated to be about 100 years (Kielty & Grant 2002, Avery & Bailey 2005). The cross-linking pattern with a large amount of divalent cross-links in bone is suggested to have an important role in the correct mineralization (Yamauchi & Katz 1993, Knott et al. 1997, Bank et al. 1999, Pornprasertsuk et al. 2005), but mineralization is also suggested to affect the maturation of collagen cross-links (Knott & Bailey 1998). The nucleation of calcium apatite crystals begins in areas adjacent to cross-linking sites, for instance (Yamauchi & Katz 1993). In addition, the level of telopeptide hydroxylation and cross-linking may also affect the collagen fibril diameter (Brinckmann et al. 1999, Bailey 2002, Pornprasertsuk et al. 2005). Studies of osteoporotic bones have revealed over-hydroxylated collagens leading to increased immature cross-linking, finer collagen fibrils, increased collagen synthesis as well as degradation, and reduced calcification, which in turn lead to the increased fragility of bone (Bailey 2002, Pornprasertsuk et al. 2005). Recently, decreased levels of LH2, mature HP and LP cross-links and two matrix proteins in mouse cervical tissue were accompanied by increased

34 collagen fibril diameter (Akins et al. 2011). In addition to enzymatic collagen cross-links, the role of non-enzymatic, sugar related cross-links is also emphasized in the determination of bone quality (see below) (Saito & Marumo 2010). Cross-links can be determined from tissue samples, but the analysis of collagen turnover products from urine and blood is also a useful method to detect some connective tissue disorders (Knott & Bailey 1998, Eyre & Wu 2005, Saito & Marumo 2010). Pyridinolines and ketoimines originate mainly from mineralized tissues and are thus valuable markers of the quality of these tissues and ongoing collagen turnover in them (Knott & Bailey 1998). However, the analysis is not always that straightforward and pyridinoline cross-links in samples are not always from bone collagen. For example, growing children get higher values due to the breakdown of mineralized cartilage in growth plates rather than bone. (Eyre & Wu 2005).

Age-related, non-enzymatic cross-links

Lysine residues are also involved in the non-enzymatic cross-links between collagen molecules in advanced glycation end-products (AGEs), such as glucosepane and pentosidine (Saito & Marumo 2010). AGEs are formed spontaneously by a reaction between reducing sugars and proteins of the body (Susic 2007, Saito & Marumo 2010). The formation requires time so that it affects long-lived proteins of the body, such as collagen (Susic 2007). As collagens mature after enzymatic cross-linking, the metabolic turnover decreases and allows for non-enzymatic cross-linking (Bailey et al. 1998). In contrast to the enzyme- mediated cross-links that are a prerequisite for normal tissue function, AGE- mediated cross-links are connected to aging and they are harmful for tissues (Robins 2007). These cross-links are suggested to have a role in diabetes mellitus, cardiovascular diseases and osteoporosis, for instance (Susic 2007, Saito & Marumo 2010). They increase the stiffness of the tissue and affect the resistance of the tissue to normal enzymatic degradation (Susic 2007). In bone AGEs make collagen fibers brittle and they may also affect the activity of osteoblasts and osteoclasts. AGE-related cross-links can be determined from tissue samples and, just as turnover products of enzymatic cross-links, also e.g. pentosidine can be analyzed from serum or urine samples. Serum and urinary levels of pentosidine do not directly reflect the content in bone but may correlate to fracture risk. (Saito & Marumo 2010).

35 2.4 Lysyl hydroxylases

Hydroxylysine residues in polypeptides are derived from lysine residues by enzymatic reaction and not from free hydroxylysines (Kivirikko & Pihlajaniemi 1998). The reaction involves an oxidative decarboxylation of 2-oxoglutarate that is catalyzed by LH, which was first purified to homogeneity from chick embryos and human placental tissue and cloned from chick (Turpeenniemi-Hujanen et al. 1980, Turpeenniemi-Hujanen et al. 1981, Myllylä et al. 1991, Kivirikko & Pihlajaniemi 1998). At present, three LH isoforms, LH1, LH2 and LH3, have been characterized from vertebrates including human (Hautala et al. 1992a, Valtavaara et al. 1997, Valtavaara et al. 1998, Passoja et al. 1998a), mouse (Ruotsalainen et al. 1999), rat (Armstrong & Last 1995, Mercer et al. 2003) and zebrafish (Schneider & Granato 2006, Schneider & Granato 2007). However, only a single LH is found in the nematode Caenorhabditis elegans (Norman & Moerman 2000) and the fly Drosophila melanogaster (Bunt et al. 2010). LHs together with C-P4Hs and P3Hs belong to the 2-oxoglutarate-dependent dioxygenase family and the genes encoding LHs are called PLODs (protocollagen-lysine, 2-oxoglutarate, 5-dioxygenase) (Kivirikko & Pihlajaniemi 1998, Kielty & Grant 2002). Recently a Jumonji domain-6 protein (JMJD6) was also reported to have lysyl hydroxylation activity (Webby et al. 2009). Like collagen-modifying hydroxylases, JMJD6 is suggested to be an iron- and 2-oxoglutarate-dependent dioxygenase that belongs to the subfamily of Jumonji C domain-containing hydroxylases (Chang et al. 2007). These enzymes in turn are connected to diseases such as cancer (for a review, see Kampranis & Tsichlis 2009). The latest data suggest that JMJD6 hydroxylates lysine residues in histones and in proteins contributing to RNA splicing (Webby et al. 2009) and that the enzyme may also bind and modify single-stranded RNA (Hong et al. 2010). LHs are localized to the lumen of the ER (Myllylä et al. 1991, Kellokumpu et al. 1994) even though they do not contain any retention sequence typical for proteins remaining in the ER (Myllylä et al. 1991, Hautala et al. 1992a). In addition, any cycling or retrieval from post-ER compartments is not involved (Suokas et al. 2003). LH1 and LH3 have been shown to be associated with the ER membrane (Kellokumpu et al. 1994, Salo et al. 2006a). LH1 contains a distinct C- terminal segment that is suggested to be important both for its localization into the lumen of the ER as well as its association with ER membranes (Kellokumpu et al. 1994, Suokas et al. 2000, Suokas et al. 2003). Thus, the retention within the

36 ER and the association with the ER membrane are found to be coupled phenomena as both characteristics are due to the C-terminal segment (Suokas et al. 2000). This C-terminal region shows high sequence homology in all three LH isoforms (Valtavaara et al. 1998, Passoja et al. 1998a), and it is assumed that all three LH isoenzymes utilize the same mechanism in their localization to the ER (Suokas et al. 2000, Suokas et al. 2003), which is also supported by the latest data concerning LH3 (Wang et al. 2012). In addition to the ER, the active form of LH3 is found in the extracellular space and the localization seems to have tissue- specificity. The intracellular form is preferred at least in the kidney, whereas the extracellular form is prevalent in the liver. In addition, LH3 is detected in the serum. (Salo et al. 2006a).

2.4.1 Lysyl hydroxylase isoenzymes

The existence of LH isoenzymes and especially separate forms for the hydroxylation of the helical and telopeptide regions was debated for a long time (Barnes et al. 1974, Ihme et al. 1984, Royce & Barnes 1985, Gerriets et al. 1993). The presence of hydroxylysine residues in distinct amino acid sequences in helical and telopeptide regions, for instance, led to the suggestion that different forms of LHs may exist (Barnes et al. 1974, Royce & Barnes 1985). Moreover, EDS VIA patients were reported to have immense variability in the lysine hydroxylation levels between various tissues and collagen types, values ranging from total lack of hydroxylation to normal levels (Ihme et al. 1984), and highly purified LH was reported to be able to hydroxylate essentially completely the triple helical lysines in type I protocollagen, while telopeptide lysines were unhydroxylated (Royce & Barnes 1985). Finally, two new isoforms, LH2 (Valtavaara et al. 1997) and LH3 (Valtavaara et al. 1998, Passoja et al. 1998a), were identified and later LH2 was recognized to have two different splicing forms (Yeowell & Walker 1999a). PLOD1, PLOD2 and PLOD3, the genes encoding human LH1, LH2 and LH3 (Hautala et al. 1992a, Szpirer et al. 1997, Valtavaara et al. 1998), respectively, as well as the corresponding mouse genes (Plod1, Plod2 and Plod3) are located in different chromosomes (Sipilä et al. 2000). According to a phylogenetic study, it has been suggested that the mouse Plod1 and Plod2 isoforms are derived from one gene by two gene duplications, Plod3 being the ancestral gene (Ruotsalainen et al. 1999). At the amino acid level all three human LH isoforms share about 50% identity (Valtavaara et al. 1998). If individual isoforms are compared (LH1-LH2, LH1-LH3, LH2-LH3) the identities

37 are even higher, ranging from about 60% to over 75% in both human and mouse (Valtavaara et al. 1997, Valtavaara et al. 1998, Passoja et al. 1998a, Ruotsalainen et al. 1999). Between species, individual mouse LHs are about 90% identical to the corresponding human LHs at the amino acid level (Ruotsalainen et al. 1999). The most concerved areas are located in the C-terminal region, where the amino acids essential for LH activity are located, and in the central region of the molecule (Pirskanen et al. 1996, Valtavaara et al. 1997, Passoja et al. 1998a, Passoja et al. 1998b, Valtavaara et al. 1998, Rautavuoma et al. 2002).

Lysyl hydroxylase 1

LH1 was the first LH isoenzyme cloned from chick and human and it was named LH before another isoform, LH2, was discovered (Hautala et al. 1992a, Valtavaara et al. 1997). The gene coding for human LH1, PLOD1, is located on chromosome 1 and it contains 19 exons (Hautala et al. 1992a, Heikkinen et al. 1994). PLOD1 encodes a polypeptide of 727 amino acids including a signal peptide of 18 residues (Hautala et al. 1992a). In mouse the gene is localized to chromosome 4 (Sipilä et al. 2000). Interestingly, introns 9 and 16 of PLOD1 show extensive homology because of many Alu sequences (Heikkinen et al. 1994). These sequences are suggested to be involved in many human gene rearrangements and therefore in some hereditary diseases (Deininger & Batzer 1999). In the case of PLOD1, Alu sequences have been connected to one of the most common mutations causing EDS VIA (Heikkinen et al. 1994, Pousi et al. 1994, Heikkinen et al. 1997, Deininger & Batzer 1999).

Lysyl hydroxylase 2

The human PLOD2 gene coding for LH2 is localized to chromosome 3 (Szpirer et al. 1997) and the corresponding mouse gene Plod2 to chromosome 9 (Sipilä et al. 2000). Mutations in the PLOD2 gene cause Bruck syndrome (BS) type 2 (see 2.5.2., van der Slot et al. 2003, Ha-Vinh et al. 2004). LH2 is the only LH isoform that is known to have different splicing variants (Yeowell & Walker 1999a). The two different forms are named LH2(short) and LH2(long) or LH2a and LH2b, respectively (Yeowell & Walker 1999a, Walker et al. 2005). LH2(long) was characterized two years later than the short form (Valtavaara et al. 1997, Yeowell & Walker 1999a). In the short form exon 13A between exons 13 and 14 is spliced out, yielding a product of 737 amino acids that includes an N-terminal signal

38 peptide (Valtavaara et al. 1997, Yeowell & Walker 1999a). Exon 13A is 63 bp long corresponding to 21 amino acids, and thus the long form is 758 amino acids long (Yeowell & Walker 1999a). The amino acids encoded by the additional exon 13A in LH2(long) are suggested to change the peptide binding properties of LH2 (Risteli et al. 2004). The expression of LH2 splice variants shows tissue specificity (Yeowell & Walker 1999a, Salo et al. 2006b). In humans LH2(long) is expressed in all tissues studied, while the short form is expressed in certain tissues and only together with the long form (Yeowell & Walker 1999a, Walker et al. 2005). The LH2 splicing pattern is suggested to be connected to the specificity for collagen telopeptide lysine hydroxylation (Mercer et al. 2003, van der Slot et al. 2003). LH2, and particularly LH2(long), is reported to be the collagen telopeptide LH responsible for the initiation of the formation of pyridinoline cross-links (Uzawa et al. 1999, Mercer et al. 2003, van der Slot et al. 2003, Pornprasertsuk et al. 2004, van der Slot et al. 2004, Takaluoma et al. 2007). The role of the short form is unknown. In fibrotic diseases such as systemic sclerosis, the splicing of LH2 is changed so that LH2(long) expression is increased (van der Slot et al. 2003, van der Slot et al. 2004). Accordingly, overaccumulation of collagen in fibrotic conditions is accompanied by overhydroxylation of the collagen telopeptides and an increased amount of pyridinoline cross-links (Trojanowska et al. 1998, Brinckmann et al. 1999, Brinckmann et al. 2001, van der Slot et al. 2004). The prevalence and clinical significance of fibrosis have led to increasing investigation of the factors regulating splicing of the LH2 mRNA. Early studies of LH2 splicing with a protein synthesis inhibitor cycloheximide demonstrated that the splicing pattern can be changed by affecting the synthesis of a protein factor necessary for the splicing (Walker et al. 2005). Phylogenetic and sequence analyses have revealed that introns flanking exon 13A of the LH2 transcript and exon 13A itself contain certain regulatory splicing sequences (Yeowell & Walker 1999a, Yeowell et al. 2009, Seth & Yeowell 2010, Seth et al. 2011). A branch point sequence, suggested to participate in the exon inclusion, is followed by a polypyrimidine tract that could contribute to the exclusion of exon 13A (Yeowell & Walker 1999a, Yeowell et al. 2009). In addition, a sequence similar to a U-rich element that is known to function as a binding site for TIA (T-cell-restricted intracellular antigen) proteins is detected downstream of exon 13A (Förch et al. 2000, Del Gatto-Konczak et al. 2000, Le Guiner et al. 2001, Yeowell et al. 2009). The binding of TIA proteins to U-rich elements has been connected to the inclusion of alternatively spliced exons in general (Förch et al. 2000, Del Gatto-

39 Konczak et al. 2000, Le Guiner et al. 2001). Actually, two TIA proteins, TIA-1 and TIAL1, are shown to be essential for efficient inclusion of the alternatively spliced exon 13A in LH2(long) mRNA and thus for the regulation of LH2(long) expression (Yeowell et al. 2009). The intron upstream of exon 13A also contains highly conserved motifs that bind Fox proteins (Baraniak et al. 2006, Seth & Yeowell 2010). These proteins are known to regulate alternative splicing (Baraniak et al. 2006), and in the splicing of LH2 Fox-2 has also been shown to be required for the inclusion of exon 13A and thus production of the LH2(long) mRNA (Seth & Yeowell 2010). Recently exon 13A itself was also reported to contain elements affecting the alternative splicing. Exon 13A is suggested to contain at least one enhancer and two silencer elements of exonic splicing that could bind regulatory proteins. (Seth et al. 2011).

Lysyl hydroxylase 3

The PLOD3 gene encoding human LH3 is located on chromosome 7 (Valtavaara et al. 1998). The gene contains 19 exons and the size of the LH3 polypeptide is 738 amino acids including a 24-residue signal peptide (Valtavaara et al. 1998, Rautavuoma et al. 2000). The gene contains Alu sequences in the introns like that of LH1, but the Alu sequences are shorter (Rautavuoma et al. 2000). In mouse the gene is localized to chromosome 5 (Sipilä et al. 2000). LH3 is a multifunctional enzyme possessing GT and GGT activities in addition to its LH activity (Heikkinen et al. 2000, Rautavuoma et al. 2002, Wang et al. 2002a, Rautavuoma et al. 2004, Ruotsalainen et al. 2006, Salo et al. 2006a). The glycosyltransferase activities of LH3 are localized in the N-terminus in contrast to the LH activity which exists in the C-terminus of the enzyme (Pirskanen et al. 1996, Passoja et al. 1998b, Heikkinen et al. 2000, Rautavuoma et al. 2002, Wang et al. 2002a, Wang et al. 2002b). LH3 differs from other LH isoforms also in its cellular localization. In addition to its location in the lumen of the ER, it is found as an active enzyme in the extracellular space and in association with collagenous proteins (Kellokumpu et al. 1994, Salo et al. 2006a). Thus, in addition to the general conception that LHs modify collagen polypeptides only in the ER lumen before the formation of the triple helical structure, it has been suggested that LH3 could modify also native collagen molecules in the extracellular space, which furthermore indicates a possible role for LH3 in ECM remodeling (Kielty & Grant 2002, Salo et al. 2006a). Actually, cellular studies have suggested that glycosylation activity in the extracellular

40 space could dramatically affect cell morphology and cell behavior, although it depends on the cell type (Wang et al. 2009). However, LH3 is suggested to prefer collagen polypeptide chains rather than native triple helical collagen molecules as substrates (Sricholpech et al. 2011). The enzyme is reported to be secreted from cells via two pathways by using a Golgi complex or by bypassing it (Wang et al. 2012). LH3 is also found from serum samples indicating that the GGT activity present in serum could be derived from LH3 (Salo et al. 2006a).

Expression of different lysyl hydroxylase isoforms

Expression levels of LH isoenzymes in human and mouse have been studied at the mRNA and protein levels. LHs are widely expressed in different tissues and developmental stages. Straightforward conclusions about any specific LH isoenzyme expression patterns are essentially impossible as the results vary markedly between different studies. The expression patterns of LH isoforms in certain adult human and mouse tissues are shown in Table 1.

Table 1. Expression of lysyl hydroxylase isoenzymes in selected adult human and mouse tissues.

Tissue LH1 LH2 LH3 human mouse human mouse human mouse Blood vessels yes2 n.d. yes7 n.d. n.d. yes9 Brain yes1 yes6,8 yes3,7 yes6,8,10 yes4,5 yes6,10 Cartilage yes2 n.d. yes7 n.d. n.d. yes9 Heart yes1 yes6,8 yes3 yes6,8,10 yes4,5 yes6 Kidney n.d. no8/yes6 yes3,7 yes6,8,10 yes4,5 yes6,9,10 Liver yes1 yes6,8 yes3,7 no6,8,10 yes4,5 yes6,9,10 Lung yes1,2 yes6,8 yes3,7 no8/yes6,10 yes4,5 yes6,9,10 Pancreas n.d. n.d. yes3 yes10 yes4,5 yes10 Placenta yes1,2 n.d. yes3,7 n.d. yes4,5 yes9 Skeletal muscle yes1,8 yes6,8 yes3,8 no8/yes6,10 yes4,5 yes6,10 1 (Heikkinen et al. 1994); 2 (Yeowell et al. 1994); 3 (Valtavaara et al. 1997); 4 (Passoja et al. 1998a); 5 (Valtavaara et al. 1998); 6 (Ruotsalainen et al. 1999); 7 (Yeowell & Walker 1999a); 8 (Hjalt et al. 2001); 9 (Rautavuoma et al. 2004); 10 (Salo et al. 2006b); n.d. = not determined.

In rat tissues mRNA expression of all LH isoforms including both LH2 splicing variants was detected in almost all tissue types examined, indicating that LHs do not have tissue-specific expression in the rat (Mercer et al. 2003). In humans, both LH2 variants are expressed in the cartilage, frontal lobe, kidney, liver, spleen

41 and placenta (Yeowell & Walker 1999a). LH2(long) is expressed in all tissues studied so far and it is the main transcript in all tissues studied except the kidney and spleen (Yeowell & Walker 1999a). In mouse the short form of LH2 is predominant in the testis, brain and kidney (Salo et al. 2006b). Thus, the expression of the two human and mouse LH2 splicing variants is suggested to be tissue-specific (Yeowell & Walker 1999a, Salo et al. 2006b). All LH isoforms have been shown to be expressed with similar tissue distributions in the mouse embryos from embryonic day (E) 7 onwards, the first time point analyzed. LH1 expression was high throughout the analyzed period, E7-E17, but LH2 was expressed strongly only at E7 and remained low after that. LH3 was highly expressed at E7 and E15, the expression level being lower at the other time points. (Salo et al. 2006b). Earlier data from LH3 null mice with the Plod3 gene disrupted with a β-galactosidase reporter demonstrated intense X-gal staining in heterozygous and null embryos at E8.5-9.5, the staining being localized also to placental blood vessels (Rautavuoma et al. 2004). At E9.5 and E11.5 all LH isoforms are expressed in the dorsal aorta, somites, tail bud, brain and at sites of the developing maxilla and mandible of branchial arch as well as in the limb bud at E11.5. Expression of the two LH2 splicing forms is suggested to be developmentally regulated during mouse embryogenesis as the short form predominates until E11.5 and the long form after that. (Salo et al. 2006b). However, in humans, only the long form is present in fetal skin cell lines, but both forms are seen in embryonic kidney cells (Yeowell & Walker 1999a, Walker et al. 2005).

2.4.2 Molecular and catalytic properties

An active form of LHs is a homodimer, the size of one monomer being about 85000 Da (Turpeenniemi-Hujanen et al. 1980, Turpeenniemi-Hujanen et al. 1981, Myllylä et al. 1988, Rautavuoma et al. 2002). The dimerization of human LH3 has been reported to be dependent on amino acids 541-547, but amino acids from other sites may also contribute (Heikkinen et al. 2011). However, dimerization seems not to be a prerequisite for the gycosylation activity of LH3 (Heikkinen et al. 2011). LHs consist of three domains (Rautavuoma et al. 2002) and the C- terminal domain contains essential amino acids required for the LH activity (Pirskanen et al. 1996, Passoja et al. 1998b). The glycosylation activity of LH3 is located in its N-terminal domain (Rautavuoma et al. 2002, Wang et al. 2002a, Wang et al. 2002b).

42 Reaction mechanism and catalytically important amino acids

As a member of the 2-oxoglutarate dioxygenase family, LHs, as well as C-P4Hs and P3Hs, require Fe2+, 2-oxoglutarate, molecular oxygen and ascorbate for the enzymatic reaction, and the reaction mechanism of all these collagen 2+ hydroxylases resembles each other. Fe , 2-oxoglutarate, O2 and the peptide substrate are bound in this order during the enzyme reaction. Reaction products are released in sequence as well, although Fe2+ remains usually unreleased between catalytic cycles. The substrate is released first, followed by CO2 and succinate. (Myllyharju 2005). A simplified representation of the hydroxylation of a lysine by LH is shown in Fig. 4. O O NH CH C NH CH C HOOC COOH Fe2+ CH2 O CH2 ++++O2 CO2 CH2 Ascorbate CH2 HOOC CH2 HOOC HC OH

CH22 NH CH22 NH

Lysine 2-Oxoglutarate Hydroxylysine Succinate

Fig. 4. A schematic representation of the hydroxylation of lysine by lysyl hydroxylase.

In the lysyl hydroxylation reaction, molecular oxygen binds to the Fe2+ in the catalytic site of LH. One oxygen molecule of the O2 is transferred to the hydroxyl group formed in the lysine, while another oxygen atom is incorporated into the succinate produced in the reaction. (Kivirikko et al. 1992, Kivirikko & Pihlajaniemi 1998). Ascorbate is essential for lysyl hydroxylation, although it is not consumed stoichiometrically in the complete hydroxylation reaction. Collagen hydroxylases can perform many catalytic cycles in the absence of ascorbate, but it is needed as an obligatory element to reactivate the enzymes. This is because the enzymes occasionally catalyze uncoupled decarboxylation of 2-oxoglutarate, where ascorbate is consumed stoichiometrically. (Myllylä et al. 1984). The substrate binding site of LH is suggested to be an open, hydrophilic pocket where acidic amino acids have a significant role in binding (Risteli et al. 2004). At the amino acid level individual human and mouse LH isoforms are about 60–75% identical, and when all isoforms are compared together the identity is about 50% (Valtavaara et al. 1997, Valtavaara et al. 1998, Passoja et al. 1998a Ruotsalainen et al. 1999). The C-terminal region is especially highly conserved in LHs, containing several critical amino acids that are found in other 2-oxoglutarate

43 dioxygenase family members as well (Pirskanen et al. 1996, Myllyharju & Kivirikko 1997, Valtavaara et al. 1997, Passoja et al. 1998a, Passoja et al. 1998b, Valtavaara et al. 1998, Vranka et al. 2004). It is assumed that Fe2+ is bound to LH with three linkages (Kivirikko & Pihlajaniemi 1998) via the amino acids histidine 638, aspartate 640 and histidine 690 in processed human LH1 (Pirskanen et al. 1996). Many divalent cations, such as Zn2+, function as competitive inhibitors with respect to Fe2+ (Kivirikko & Pihlajaniemi 1998). In addition, arginine 700 (in processed human LH1) is a positively charged residue that ionically binds the C5 carboxyl group of 2-oxoglutarate (Hanauske-Abel & Günzer 1982, Passoja et al. 1998b). LHs also contain several conserved cysteine residues and seven of them are shown to be important for the enzyme activity of LH1 (Yeowell et al. 2000a). There is only a limited amount of data available about catalytically important amino acids for the glycosyltransferase activity of LH3. However, indications about critical amino acids have been obtained by comparing the cDNA sequences of the human and mouse LH3s to the only LH of Caenorhabditis elegans, which is reported to possess both LH and glycosylation activity (Norman & Moerman 2000, Wang et al. 2002a, Wang et al. 2002b). 29 amino acids are conserved in these sequences of which cysteine 144 and leucine 208 in the human sequence were shown to be important for the LH3 GGT activity in all three species (Wang et al. 2002b). The cysteine but not the leucine residue is also important for the GT activity of the enzyme (Wang et al. 2002a). In addition, a conserved motif at amino acids 187–191 in the human sequence containing important aspartate residues is suggested to be a potential binding site for Mn2+ in the GGT active site (Wang et al. 2002a, Wang et al. 2002b). The aspartate-containing short site may represent a DxD motif that is commonly found in many glycosyltransferases (Unligil et al. 2000, Gastinel et al. 2001, Wang et al. 2002b).

Substrate specificity

The hydroxylysine content of collagens varies between collagen types, tissues and normal physiological states of the tissue but also in certain disorders. These observations have indicated some kind of substrate specificity as well as tissue specificity of the LH isoenzymes. (Ihme et al. 1984, Kivirikko et al. 1992, Bank et al. 1999, Eyre et al. 2002). The minimal sequence requirement for the function of LHs is the X-Lys-Gly triplet (Kivirikko et al. 1992). All isoforms have been shown to hydroxylate synthetic peptides representing collagenous domains of

44 type I and IV collagens (Takaluoma et al. 2007) and they seem not to have strict sequence requirements (Risteli et al. 2004, Takaluoma et al. 2007). In addition, all isoenzymes are able to hydroxylate lysines in the triple helical domain of recombinant type I procollagen produced in insect cells, but LH2 is the only isoform capable of hydroxylating the telopeptide lysines in this system (Takaluoma et al. 2007). However, overexpression studies with LH2 in fibroblasts demonstrated that LH2 may not be able to hydroxylate lysines in the triple helical area (Wu et al. 2006). Hydroxylysine residues in collagen telopeptides are found in X-Hyl-Ser and X-Hyl-Ala sequences, which differ from the collagenous X-Hyl-Gly sequence (Myllyharju 2005). However, these X-Hyl-Ser and X-Hyl-Ala sequences seem not to be the only requirement for the hydroxylation by LH2 as in in vitro studies the enzyme was not able to hydroxylate a short synthetic peptide representing the type I collagen telopeptide, but rather required a longer substrate as demonstrated in the insect cell coexpression experiments with a procollagen I chain (Takaluoma et al. 2007). Despite the lack of a strict substrate specificity, several observations suggest preferential roles for each LH isoenzyme. LH2 hydroxylates the telopeptide lysines, but may also hydroxylate helical lysines (Uzawa et al. 1999, Mercer et al. 2003, van der Slot et al. 2003, Pornprasertsuk et al. 2004, Takaluoma et al. 2007), LH1 hydroxylates helical lysines (Eyre et al. 2002, Steinmann et al. 2002) and LH3 glycosylates hydroxylysines (Heikkinen et al. 2000, Rautavuoma et al. 2002, Wang et al. 2002a, Rautavuoma et al. 2004, Ruotsalainen et al. 2006, Salo et al. 2006a), but its exact role in lysyl hydroxylation is not known. LH3 is capable of hydroxylating helical lysines of type I procollagen at least in a recombinant overexpression system (Takaluoma et al. 2007), and it is suggested to be responsible for the hydroxylation of lysine residues that are further glycosylated by the same enzyme (Sipilä et al. 2007). LH1 has been suggested to have some telopeptide hydroxylation activity (Mercer et al. 2003, Walker et al. 2004a), but opposite results have also been obtained (Royce & Barnes 1985, Takaluoma et al. 2007). In addition, analysis of EDS VIA patients with null LH1 mutations have revealed some hydroxylation of collagenous domains indicating that LH2 or LH3 or both could function as helical LHs as well (Eyre et al. 2002). LH3 is the only isoenzyme that is reported as likely to be able to modify also native collagen molecules in the extracellular space (Salo et al. 2006a). However, in vitro studies on skin and tendon collagens have suggested that the native type I collagen is not the most appropriate substrate for LH3, which rather prefers collagen polypeptide chains (Sricholpech et al. 2011).

45 2.5 Human diseases related to lysyl hydroxylases and animal models

The kyphoscoliotic form of EDS (EDS VI) was for a long time the only disorder connected to LHs. Studies of the syndrome have provided important information about the function of LHs and the importance of lysyl hydroxylation in collagens (Yeowell & Walker 2000, Steinmann et al. 2002). Currently mutations are found in all LH isoforms leading to severe connective tissue disorders (Yeowell & Walker 2000, van der Slot et al. 2003, Salo et al. 2008). In addition, overexpression of the long form of LH2 is connected to fibrosis (van der Slot et al. 2003, van der Slot et al. 2004).

2.5.1 Ehlers-Danlos type VI

The kyphoscoliotic type of EDS or EDS VI is an autosomal recessive, inheritable connective tissue disorder. EDS VI patients are clinically characterized by articular hypermobility, kyphoscoliosis, hyperextensible skin, bad scarring, easy bruising, and muscular hypotonia. (Yeowell & Walker 2000, Steinmann et al. 2002). Patients suffer also from ophthalmological complications and have an increased risk of spontaneous arterial ruptures (Wenstrup et al. 1989, Steinmann et al. 2002). Two different forms of EDS VI have been identified. In EDS VIA the biochemical basis for this disease is a deficiency of LH1 activity, caused by mutations in the PLOD1 gene. Therefore, the amount of triple helical hydroxylysine is decreased and thus collagen cross-linking is changed (see Figs. 3 and 5). (Yeowell & Walker 2000, Steinmann et al. 2002). EDS VIB patients have the same phenotype as EDS VIA patients, but the LH activity is normal (Steinmann et al. 2002, Walker et al. 2004b) and currently the molecular pathology of this form is unknown. Moreover, a mutation in the zinc transporter gene was recently reported to cause EDS VI-like symptoms with abnormal collagen hydroxylation and urinary pyridinoline levels but with normal LH and C-P4H activities in cell extracts (Giunta et al. 2008).

Genetic background of Ehlers-Danlos syndrome type VIA

Various different mutations in the PLOD1 gene have been identified from EDS VIA patients. Mutations consist of a single base pair (bp) mutation to large duplications and deletions. (Yeowell & Walker 2000, Steinmann et al. 2002). The

46 most common mutation connected to Alu-Alu recombination is a large duplication of seven exons (Pousi et al. 1994, Heikkinen et al. 1997, Brinckmann et al. 1998, Yeowell et al. 2000b, Giunta et al. 2005a). Another predominant form is a nonsense mutation in exon 14 (Yeowell & Walker 1997, Walker et al. 1999, Yeowell & Walker 1999b, Pousi et al. 2000, Yeowell et al. 2000b). Several compound heterozygote mutations have been reported as well (Yeowell & Walker 2000). The first found compound heterozygote patient also confirmed the autosomal recessive inheritance of the syndrome (Ha et al. 1994). LH1 has several alternative splicing pathways that can either bypass or compensate for mutations and thus cells may sustain partial LH1 activity (Yeowell & Walker 2000). According to one study small amounts of differentially spliced LH1 transcripts are also detected from normal cells (Heikkinen et al. 1999), although LH2 is considered to be the only LH isoform having splicing variants (Yeowell & Walker 1999a).

Molecular biology

The LH activity in patients with EDS VIA is markedly reduced (Yeowell & Walker 2000, Steinmann et al. 2002). However, activity levels vary significantly between tissues, individuals and even between assays performed (Steinmann et al. 2002). There are differences in hydroxylation levels also between collagen types as type I and III collagens are severely affected, while collagen types II and V are basically normally hydroxylated in EDS VIA patients, generally (Ihme et al. 1984, Eyre et al. 2002). Although the collagen is normally secreted from cells, the decreased hydroxylation and thus glycosylation of collagens can be seen as increased electrophoretic mobility in SDS-PAGE analysis (Jarish et al. 1998, Walker et al. 1999, Giunta et al. 2005a, Giunta et al. 2005b). In addition, the solubility rate of tissue collagens in denaturing solvents is suggested to be related to the level of hydroxylation (Steinmann et al. 2002). The degree of collagen hydroxylation is suggested to correlate with the clinical status of the EDS VIA patient (Ihme et al. 1984). The skin for instance is severely affected in the patients, while in cartilage the consequences are milder (Eyre & Glimcher 1972, Pinnell et al. 1972, Eyre et al. 2002). The LH activity determined from skin fibroblasts of patients is usually less than 25% of controls (Yeowell & Walker 2000, Steinmann et al. 2002). In EDS VIA patients the lysine residues in collagen telopeptides are normally hydroxylated, while the lysines in the triple helical area are underhydroxylated, affecting the collagen cross-linking

47 and thus the characteristics of collagenous structures (see Fig. 5) (Steinmann et al. 2002, Eyre & Wu 2005). Although the pyridinoline levels in skin are generally low, in normal skin the mature HP cross-links are suggested to be four times more prevalent than LP cross-links, whereas in EDS VIA patients HP cross-links are absent (see Figs. 3 and 5) (Pasquali et al. 1995, Pasquali et al. 1997, Knott & Bailey 1998). The changed excretion pattern of LP and HP cross-links derived from degraded collagens especially from tissues such as bone increases the LP/HP ratio in the urine, which can be determined and used as a diagnostic marker (Pasquali et al. 1994, Pasquali et al. 1995, Steinmann et al. 1995). In EDS VIB patients the hydroxylation level of helical lysines in skin fibroblasts is decreased almost as much as in EDS VIA (Steinmann et al. 2002, Uzawa et al. 2003). Interestingly, some of the EDS VIB patients are reported to have decreased LH2 or LH3 mRNA levels (Walker et al. 2004b).

Diagnosis and treatment of Ehlers-Danlos syndrome type VIA

During childhood EDS VIA patients are usually suspected to have a neuromuscular disease because of the hypotonia and delayed gross motor development, and the correct diagnosis is thus often obtained late (Steinmann et al. 2002, Giunta et al. 2005a). In addition to clinical symptoms, the disease can be diagnosed by measuring LH activity in cultured fibroblasts or LP/HP ratio in the urine, as indicated earlier (Pasquali et al. 1994, Pasquali et al. 1995, Steinmann et al. 1995). One study has introduced a method to confirm the diagnosis from RNA and DNA samples (Giunta et al. 2005a) and successful prenatal exclusions of the syndrome are also reported (Yeowell & Walker 1999b, Yeowell et al. 2000b). Although the syndrome is not treatable, some patients have had some benefit from pharmacological doses of ascorbate (vitamin C) (Dembure et al. 1987). It has been shown to increase the collagen synthesis and LH activity both in normal and EDS VIA fibroblasts (Dembure et al. 1987, Yeowell et al. 1995, Pasquali et al. 1997, Yeowell et al. 1997). In addition, ascorbate is shown to affect also the cross-link content in fibroblasts (Pasquali et al. 1997).

48 hHyl

m r tHyl

tHyl

B r

tLys u

tLys y n

hLys d

m e

V hHyl

S tHyl

E tHyl

Fig. 5. Simplified illustration of collagen cross-linking in a stiff tissue, such as bone, in a normal situation and in Ehlers-Danlos syndrome type VIA and Bruck syndrome.

2.5.2 Bruck syndrome 1 and 2

Bruck syndrome (BS) is an extremely rare, autosomal recessive connective tissue disorder with features typical for osteogenesis imperfecta (OI). About 30 patients have been reported to suffer from BS. BS patients typically have osteoporosis, fragile bones, congenital contractures of joints, progressive kyphoscoliosis and short stature, but they usually have normal dentinogenesis and no hearing loss. BS can be best distinguished from OI by the presence of congenital joint contractures and the absence of hearing loss. (Viljoen et al. 1989, Brenner et al. 1993, McPherson & Clemens 1997, Brady & Patton 1997, Breslau-Siderius et al. 1998, Blacksin et al. 1998, Leroy et al. 1998, Bank et al. 1999, van der Slot et al. 2003, Ha-Vinh et al. 2004, Datta et al. 2005, Berg et al. 2005, Mokete et al. 2005, Yapicioğlu et al. 2009, Shaheen et al. 2010, Kelley et al. 2011). Typical features

49 of OI include increased bone fragility and short stature, impaired hearing, dentinogenesis imperfecta and hypermobile joints (for a review, see Basel & Steiner 2009). Most OI cases are caused by mutations in the genes coding for the pro-α1 or pro-α2 chains of type I collagen, but mutations are detected also in members of the protein complex responsible for collagen prolyl 3-hydroxylation and recently also from a SERPINH1 gene that encodes the collagen chaperone Hsp47 (Basel & Steiner 2009, Christiansen et al. 2010).

Genetic background

BS is a genetically heterogeneous disease. Mutation analysis of BS families has led to two different linkages. BS type 1 (BS1) is linked to chromosome 17 and BS type 2 (BS2) to the PLOD2 gene in chromosome 3. (Bank et al. 1999, van der Slot et al. 2003, Ha-Vinh et al. 2004). Three different point mutations have been found so far from exon 17 of the PLOD2 gene that encodes LH2 (van der Slot et al. 2003, Ha-Vinh et al. 2004), while the genetic background and the molecular mechanism of BS1 have been elusive for a long time. Recently, mutations in FKBP10, a gene encoding FK506 binding protein 10 (FKBP10, also known as FKBP65) were suggested to cause BS1 or a subtype of OI (Alanay et al. 2010, Alanay & Krakow, 2010, Shaheen et al. 2010, Kelley et al. 2011). The FKBP10 gene is located in chromosome 17, but not at the location suggested earlier to cause BS1 (Bank et al. 1999, Kelley et al. 2011).

Molecular biology

BS subtypes can be distinguished according to the genetic defect (Bank et al. 1999, van der Slot et al. 2003). BS2 is caused by a defective telopeptide LH, LH2(long) (Bank et al. 1999, van der Slot et al. 2003, Ha-Vinh et al. 2004, Takaluoma et al. 2007), while the molecular basis of BS1 is still unknown. In BS patients the lysine residues in the triple helical area of type I collagen are normally hydroxylated, while the telopeptide lysines are underhydroxylated (see Fig. 5) (Bank et al. 1999). The hydroxylation state of the telopeptide lysines affects the cross-linking profile of collagen molecules (Eyre & Wu 2005). In the bone of the patients, for instance, the amount of telopeptide hydroxylysine- derived mature pyridinoline cross-links, HP and LP, in type I collagen is reduced (see Figs. 3 and 5). In contrast, the amount of lysinonorleucine, a derivative of an immature, difunctional cross-link originated from unhydroxylated telopeptide

50 lysines via the allysine route, is reported to be increased 5-fold. Interestingly, cross-linking of type I collagen has tissue-specific features as in contrast to the bone, the type I collagen in the ligament of a BS1 patient is normally cross-linked and thus hydroxylated. (Bank et al. 1999). The decreased number of stable pyridinoline cross-links and increased collagen turnover in bone can be analyzed from urine samples (Ha-Vinh et al. 2004). Morphological analyses of severely affected bone tissue of BS patients have revealed wormian bones in the skull, cystic changes at old fracture sites and a mixed callus as well as normal or immature mineralization in bone and osteoids that vary in thickness (Viljoen et al. 1989, Brenner et al. 1993, McPherson & Clemens 1997, Brady & Patton 1997, Blacksin et al. 1998, Leroy et al. 1998, Ha-Vinh et al. 2004, Datta et al. 2005, Mokete et al. 2005). At the cell level osteoblasts have swollen mitochondria and dilated ER and the diameter of collagen fibrils in osteoids vary (Brenner et al. 1993). The cross-linking pattern is suggested to have an important role in the correct mineralization of calcified tissues (Yamauchi & Katz 1993, Knott et al. 1997, Bank et al. 1999, Pornprasertsuk et al. 2005). Unsurprisingly, the mineral content in BS bone is shown to be reduced and the size of hydroxyapatite crystals is increased (Brenner et al. 1993). Thus, it is suggested that the fragility of BS bones is likely to be due to a changed cross-linking pattern together with inadequate mineralization (Bank et al. 1999). Biochemical studies show also increased extractability of type I collagen in BS bone tissue (Brenner et al. 1993). However, the amounts of type I and III collagens are reported to be normal and also their structure and synthesis are seemingly normal (Brenner et al. 1993, McPherson & Clemens 1997, Breslau-Siderius et al. 1998, Leroy et al. 1998, Bank et al. 1999, Kelley et al. 2011), which differs from OI where collagens are usually overmodified due to a delay in the triple helix formation (Engel & Prockop 1991, Raghunath et al. 1994).

The gene FKBP10 and Bruck syndrome

The mutations in the FKBP10 gene, connected to BS1, include a homozygous 1 bp duplication, a 35 bp deletion and two different compound heterozygous mutations, all of them located in the coding region of FKBP10, while no mutations have been found from the PLOD2 gene in these patients (Kelley et al. 2011). FKBP10 is a member of immunophilins that are known to function as intracellular receptors capable of binding and mediating the action of certain immunosuppressant drugs (Coss et al. 1995; for a review, see Göthel & Marahiel

51 1999). Like other immunophilins, FKBP10 is suggested to have some peptidylprolyl cis-trans isomerase activity, which can be important in the folding of collagens and tropoelastin monomers (Coss et al. 1995, Davis et al. 1998, Ishikawa et al. 2008). Interestingly, mutations in another FK506 binding protein, FKBP14 (FK506 binding protein 14) are reported to cause a new variant of EDS, a disorder where patients share many clinical features particularly of EDS VIA and Ullrich congenital muscular dystrophy. In contrast to EDS VIA, these patients have normal urinary LP/HP ratio. (Baumann et al. 2012). Like LHs, FKBP10 is located in the lumen of the ER and it acts as a chaperone of folded and unfolded collagen (Myllylä et al. 1991, Kellokumpu et al. 1994, Davis et al. 1998, Patterson et al. 2000, Ishikawa et al. 2008). It is also suggested to have a role in the chaperoning of tropoelastin (Davis et al. 1998, Patterson et al. 2000). Studies with FKBP10 and collagen together have suggested that FKBP10 could function as a complex with other proteins to process collagen (Zeng et al. 1998, Ishikawa et al. 2008). Collagens from patients with a mutation in FKBP10 are not overmodified (Alanay et al. 2010). This was observed also with an OI patient who had a mutation in SERPINH1 (Christiansen et al. 2010), when typically in OI the overmodification is quite common (Marini et al. 2010). In contrast to the other FKBP family members that are expressed ubiquitously, FKBP10 mRNA is detected in the mouse lung, spleen, heart, brain and testis (Coss et al. 1995). Another study shows that the expression of FKBP10 in adult mice is absent or minimal, but in developing mice the expression was seen in the smooth muscle layer of the airways and pulmonary vessels and at the mRNA level in tissues such as aorta and kidney, indicating developmental regulation of the protein (Patterson et al. 2000). However, the possible connection between FKBP10 and LH2 is still unknown.

Treatment of Bruck syndrome

There is no treatment for BS. However, bisphosphonate cyclic pamidronate has been used as a pharmacotherapy against bone fragility (Ha-Vinh et al. 2004, Mokete et al. 2005, Andiran et al. 2008, Yapicioğlu et al. 2009, Shaheen et al. 2010). Bisphosphonates are suggested to function as inhibitors of bone resorption by interfering with the function of osteoclasts (for a review, see Russell 2006). In a reported BS patient the treatment affected fracture incidence and bone mineral density (Andiran et al. 2008). However, a long-term follow-up study of a BS

52 patient, not receiveing treatment, revealed that the prevalence of fractures decreases after maturation of the bone (Mokete et al. 2005).

2.5.3 Fibrotic diseases and cancer

Fibrotic diseases

Inflammatory reaction in the tissue is a normal response to damage caused by numerous factors from bacterial infections to mechanical damage. The programmed phases of inflammation protect the tissue and enable recovery. Sometimes impaired regulation of the inflammatory reaction causing a chronic inflammation can lead to fibrosis, which is characterized by excessive accumulation of ECM and especially collagen. (Rubin & Farber 1999, Trojanowska et al. 1998, Hunzelmann & Brinckmann 2011). In soft tissues collagen cross-links are normally formed from unhydroxylated telopeptide lysines, but in fibrotic lesions the cross-linking pattern of collagen is altered (Brinckmann et al. 1996, Knott & Bailey 1998, Brinckmann et al. 1999, Brinckmann et al. 2001). The increased amount of pyridinoline cross-links (see Fig. 3) found in fibrotic tissue is caused by overhydroxylation of the telopeptide lysine residues accompanied by the LO reaction (van der Slot et al. 2003, van der Slot et al. 2004, Levental et al. 2009, Saito & Marumo 2010). The resulting collagenous structures are highly resistant to normal remodelling and degenerative procedures such as degradation by MMPs (Trojanowska et al. 1998). Collagen matrices having an increased amount of pyridinoline cross-links are shown to be less susceptible to MMP-1 degradation, for instance (van der Slot-Verhoeven et al. 2005). As LH2(long) is the only LH isoform significantly upregulated in fibrosis and it is known to be a telopeptide LH, it has been suggested to be responsible for overhydroxylation of the telopeptide lysines and thus increased pyridinoline cross-link formation (van der Slot et al. 2003, van der Slot et al. 2004, Takaluoma et al. 2007). Although overexpression of all LH isoforms is shown to increase collagen synthesis (Mercer et al. 2003), overexpression of only LH2 was shown to both increase the collagen synthesis and change the cross-linking pattern (Wu et al. 2006). Thus, LH2(long) is an important factor in fibrosis and prevention of its upregulation together with LO is important from the clinical point of view. The role of LH2(short) seems to be insignificant in fibrotic conditions (van der Slot et al. 2003, van der Slot et al. 2004). Although fibrosis and increased pyridinoline

53 cross-links are connected, direct connection of this phenomenon to the resistance of collagenous structures to remodeling is not confirmed. As a consequence of the inflammatory response the fibrotic process is connected to many inflammatory cytokines and growth factors (Trojanowska et al. 1998, Hunzelmann & Brinckmann 2011). TGF-β is an important and common inflammatory factor and it is shown to increase the expression of LH2(long) and thus the formation of pyridinoline cross-links (Trojanowska et al. 1998, van der Slot et al. 2005, Hunzelmann & Brinckmann 2011). In addition, TGF-β is known to increase generally the expression of ECM proteins and inhibitors of MMPs (Hunzelmann & Brinckmann 2011). Besides TGF-β, other cytokines such as interleukin 4 and activin A are also shown to induce LH2(long) expression (van der Slot et al. 2005, Brinckmann et al. 2005). However, the effect of at least TGF- β is presumably not direct, but it is mediated by other profibrotic factors that are rapidly upregulated via TGF-β induction (van der Slot et al. 2005). Thus, knowledge about factors contributing to increased expression of the LH2(long) form, including knowledge about the inflammatory response and alternative splicing, are important in the prevention of fibrosis.

Treatment of fibrosis

Certain organophosphate compounds inhibit the function of LH and have been earlier suggested to be valuable for testing as antifibrotic drugs (Samimi & Last 2001). An antihypertension drug minoxidil has been suggested to be a potential drug for fibrosis as it decreases LH activity (Murad & Pinnell 1987, Hautala et al. 1992b). However, minoxidil has consequently been found to be ineffective as it decreases LH1 expression rather than LH2 or LH3 expression, and thus changes the LP/HP ratio but does not decrease the total amount of pyridinoline cross-links (Zuurmond et al. 2005).

Fibrosis, hypoxia and cancer

In the chronic phase of inflammation, fibrosis is accompanied by hypoxia (Rubin & Farber 1999, Hunzelmann & Brinckmann 2011). In hypoxic conditions C-P4H, LH1 and LH2, but not LH3, have been shown to be upregulated leading to increased accumulation of collagen (Lal et al. 2001, Denko et al. 2003, Hofbauer et al. 2003, Scheurer et al. 2004, Brinckmann et al. 2005, van Vlimmeren et al. 2010), while the expression of collagen polypeptides themselves has been

54 reported to be either upregulated, downregulated or not affected (Hofbauer et al. 2003, Scheurer et al. 2004, Brinckmann et al. 2005, van Vlimmeren et al. 2010). Hypoxia is characteristic also in cancer, and ECM remodeling and changes in tissue stiffness are also characteristic in tumor formation (for reviews, see Butcher et al. 2009, Semenza 2011). Moreover, a fibrotic tumor predicts malignancy (Colpaert et al. 2003, Levental et al. 2009). Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that has been reported to influence cancer progression (Semenza 2011). Mouse and human PLOD1 and PLOD2 genes have been shown to have a putative hypoxia responsive element in their promoter region suggesting that the regulation of these genes may be mediated by HIF (Hofbauer et al. 2003, Scheurer et al. 2004, Brinckmann et al. 2005). In fact, PLOD2 expression is elevated in cancer cells (Lal et al. 2001, Dong et al. 2005, Evens et al. 2010, Rajkumar et al. 2011, Noda et al. 2012), and the elevation is reported to be associated with increased HIF-1α gene expression (Evens et al. 2010). Upregulation of several collagen-modifying enzymes including LO in hypoxia could indicate their important role in the ECM remodeling during early phases of tumor angiogenesis, for instance (Denko et al. 2003, Scheurer et al. 2004, Levental et al. 2009). HIF-1α-induced upregulation of PLOD2 in cancer is suggested to be associated with poor prognosis (Dong et al. 2005).

2.5.4 A disease caused by mutations in LH3 and LH3 null mice

LH3 was for a long time the only LH isoform that was not connected to any human diseases, although mice having an inactivated Plod3 gene were known to die during embryogenesis (Rautavuoma et al. 2004, Ruotsalainen et al. 2006). Recently, however, LH3 mutations were connected to two different connective tissue disorders. A patient with severe symptoms overlapping several connective tissue disorders was shown to have mutations in the PLOD3 gene (Salo et al. 2008). In addition, one epidermolysis bullosa family was connected to PLOD3 mutations (Savolainen et al. 1981, Risteli et al. 2009).

Human disorders connected to LH3

As indicated earlier, some connective tissue disorders can be detected based on the collagen turnover products in urine or serum samples (Knott & Bailey 1998, Eyre & Wu 2005, Saito & Marumo 2010). The first patient with a defective LH3 enzyme was also detected via cross-link analysis from urine. A glycosylated

55 derivate of the pyridinoline cross-link was absent in the sample and the amount of glucosylgalactosyl hydroxylysine was decreased. (Salo et al. 2008). As LH3 is known to have collagen glycosylation activity (Heikkinen et al. 2000, Rautavuoma et al. 2002, Wang et al. 2002a, Rautavuoma et al. 2004, Ruotsalainen et al. 2006, Salo et al. 2006a) besides the two GT25 family members that possess collagen GT activity (Schegg et al. 2009), the PLOD3 gene of the patient was analyzed and found to have a compound heterozygote mutation (Salo et al. 2008). One of the mutations was located in the region responsible for the glycosylation activities and the other in the LH activity region (Pirskanen et al. 1996, Heikkinen et al. 2000, Rautavuoma et al. 2002, Wang et al. 2002a, Wang et al. 2002b, Salo et al. 2008). However, despite the locations of the mutations, both reduced all three activities i.e. LH, GT and GGT. The mutations lead to a decreased concentration of the LH3 protein and reduced LH and glycosylation activities, which are suggested to be the molecular basis for the disorder by affecting collagen synthesis. (Salo et al. 2008). Based on these findings and the fact that LH3 is known to be essential for the development of the mouse (Rautavuoma et al. 2004, Ruotsalainen et al. 2006), it is not surprising that the patient has severe symptoms such as osteopenia, joint contractures, arterial ruptures, skin blistering, deafness and myopia that are all connected to many different collagen disorders (Salo et al. 2008). In addition, the reported patient had a stillborn sibling who was reported to have several abnormalities. According to autopsy, the intrauterine death at 28 weeks of gestation was suggested to be caused by cerebral vascular rupture. (Salo et al. 2008). In addition to the LH3 compound heterozygous patient, a large family diagnosed to have an epidermolysis bullosa simplex was discovered to transcribe only one LH3 allele (Savolainen et al. 1981, Risteli et al. 2009). The patients were reported to develop blisters extremely easily and blisters were frequently observed particularly in the hands and feet, which are regularly under mechanical stress. The healing occurred without scarring. The first blisters were detected at birth or shortly after that, but the symptoms usually became milder with age. (Savolainen et al. 1981). The decreased amount of LH3 in these patients led to decreased glycosylation activity, which in turn was suggested to affect the organization of the ECM (Savolainen et al. 1981, Risteli et al. 2009). Four polymorphic changes in the non-coding region of PLOD3 were detected, but the reason for the allelic silencing remained unknown. However, as a heterozygous deficiency it demonstrated the importance of the correct amount of LH3 activity in humans. (Risteli et al. 2009).

56 LH3 knock-out mice

The currently unnamed, severe ECM disorder caused by LH3 mutations, reveals the importance of this enzyme for humans, but LH3 has been demonstrated to be essential also in the mouse (Rautavuoma et al. 2004, Ruotsalainen et al. 2006). Mouse embryos having an inactivated Plod3 gene die during early development at E9.5 (Rautavuoma et al. 2004, Ruotsalainen et al. 2006). The deficiency of LH3 was found to be critical for the synthesis of type IV collagen and thus for the stability of basement membranes (Rautavuoma et al. 2004, Ruotsalainen et al. 2006). In addition to the defect in the embryos, a possible role of abnormal function of placental vasculature or the Reichert’s membrane (RM) in the developmental retardation was not excluded (Rautavuoma et al. 2004, Ruotsalainen et al. 2006). The embryonic lethality of LH3 knock-out mice was determined to be caused by the deficient glycosylation activity rather than LH activity of the LH3 enzyme (Rautavuoma et al. 2004, Ruotsalainen et al. 2006). At least the GGT activity in mouse LH3 is critical for mouse development, indicating that LH3 may be the main molecule responsible for GGT activity in mouse (Ruotsalainen et al. 2006). Studies with LH3 knock-out embryos and fibroblasts suggested that the deficient glycosylation of type IV collagen leads to delayed processing, intracellular accumulation, and delayed secretion as well as increased intracellular degradation of type IV collagen (Rautavuoma et al. 2004, Sipilä et al. 2007). In addition, the lack of glycosylation activity but not LH activity of LH3 seemed to prevent the assembly and thus secretion of type VI collagen. Although the lack of LH activity of LH3 does not prevent the secretion of type VI collagen, it affects the distribution of type IV and VI collagens in the examined adult mouse tissues. (Sipilä et al. 2007). Recent studies with cells have revealed that the impaired glycosylation activity of LH3 affects fibrillogenesis of the bone type I collagen as well. Furthermore, GGT activity is suggested to be the main function of LH3 in osteoblasts. (Sricholpech et al. 2011).

2.5.5 Inactivation of LH in other animal models

The nematode Caenorhabditis elegans and the fly Drosophila melanogaster have only a single LH, while the zebrafish Danio rerio, like other vertebrates, has all three forms including both LH2 splicing variants (Norman & Moerman 2000, Schneider & Granato 2006, Schneider & Granato 2007, Bunt et al. 2010). The

57 single C. elegans LH possesses both lysyl hydroxylase and collagen glucosyltransferase activity and thus resembles the vertebrate LH3 (Wang et al. 2002a, Wang et al. 2002b). The D. melanogaster LH also contains the residues that have been shown to be necessary for the glycosyltransferase activity of vertebrate LH3 (Bunt et al. 2010). In C. elegans and D. melanogaster the absence of the only LH leads to lethality during embryogenesis (Norman & Moerman 2000, Bunt et al. 2010). As in LH3 knock-out mice (Rautavuoma et al. 2004, Sipilä et al. 2007), the enzyme is required in the processing and secretion of type IV collagen in these organisms (Norman & Moerman 2000, Bunt et al. 2010). Similarly, LH3 is essential for collagen secretion in D. rerio (Schneider & Granato 2006).

58 3 Outlines of the present study

Collagens are the main structural components of the ECM in the human body and LH isoenzymes have an important role in their synthesis. Mutations in the PLOD1 and PLOD2 genes are known to cause the rare and severe connective tissue disorders EDS VIA and BS2 in humans, respectively, emphasizing the importance of these enzymes in a healthy individual. Moreover, earlier studies on the lack of LH3 in mouse had revealed novel information of its importance during the embryonic development. Later, LH3 mutations have also been connected to human disorders. The Plod3 knock-out mouse line demonstrated that LH1 and LH2 are not able to compensate for the lack of LH3 activity, but otherwise the exact in vivo roles of LH1 and LH2 have remained mostly elusive. Thus, we wanted to generate Plod1 and Plod2 knock-out mouse lines to study the expression patterns and in vivo roles of these isoenzymes, as well as potential compensatory roles of the other LH isoenzymes. Moreover, the mouse lines were also expected to serve as the first animal models for EDS VIA and BS, enabling the study of tissues that were impossible and unethical to study from the human patients. The first BS mutation was linked to chromosome 17, but the pathological mechanism remained unknown. Later, three different mutations were found in the PLOD2 gene located in chromosome 3. The BS form connected to LH2 mutations was thereafter called BS2. Although studies of BS2 patients provided clinical support for the theory that LH2 is the telopeptide LH, the mutations did not change any of the catalytically critical amino acids of the enzyme and the molecular mechanism of the disorder remained unknown. We therefore decided to produce the BS2 mutant and wild-type human LH2(long) polypeptides in insect cells, purify and analyze them to understand the molecular pathology of the BS2 syndrome in more detail.

60 4 Materials and methods

The materials and methods used in this thesis are summarized in the table below. Detailed information with references can be found in the original articles I-III.

Table 2. Methods.

Level Method Used in DNA Cloning techniques I PCR I, II, III Southern Blot analysis I, III Site-directed mutagenesis II RNA RNA isolation I, III RT-PCR I, III Quantitative real-time PCR III Protein Expression of recombinant proteins in insect cells II Purification and characterization of recombinant proteins II SDS-PAGE, non-denaturing PAGE and Western blotting II LH activity assay using 2-oxo[1-14C]glutarate II LH activity assay with [14C]lysine-labelled protocollagen I Metal chelate affinity chromatography II Gel filtration II N-terminal sequencing II Amino acid analysis I, III Circular dichroism spectroscopy II Cell and tissue Cell culture I, II Preparation and staining of paraffin sections I, III Immunohistochemistry and immunofluorescence I, III Transmission electron microscopy I, III Collagen cross-link analysis I, III Other Generation of mouse lines I, III

62 5 Results

5.1 LH1 knock-out mice have increased risk of lethal arterial ruptures (I)

5.1.1 Generation of LH1 null mice and LH1 expression in adult mouse tissues

Mouse lines lacking LH1 activity were generated to study the role of LH1 in vivo and to generate a mouse model for EDS VIA. The Plod1 gene was disrupted by a homologous recombination of a targeting construct to exon 2 (Figure 1A in I). The in-frame insertion of the LacZNeo cassette led to the deletion of exons 3–6. Two mouse lines having the same phenotype were generated from ES cells and backcrossed into the C57BL/6 mouse line. RT-PCR downstream from the site of recombination confirmed the inactivation of the Plod1 gene (Figure 1C in I). The genotypes of the offspring obtained by cross-breeding of the heterozygous mice followed the Mendelian inheritance, but both null lines lacked sufficiently strong β-galactosidase staining in tissues to study the expression of the Plod1 gene (data not shown). Therefore, an additional LH1 null mouse line with a gene-trap insertion cassette containing a β-galactosidase reporter in intron 12 of the Plod1 gene was generated and backcrossed into the C57BL/6 mouse line (not shown). The LH1 gene-trap mouse line with functional β-galactosidase expression in tissues had identical phenotypic characteristics with the two other Plod1-/- mouse lines. The gene-trap mouse line was used for analysis of Plod1 expression by X- gal staining. X-gal staining, representing the Plod1 promoter-driven β-galactosidase expression, was localized to cells that produce fibrillar collagens, such as fibroblasts in the lung, skin and tendon (Figure 2A–B and 2D in I). In skin the papilla of hair follicles and arrector pili muscles were also stained (Figure 2B in I). In cornea the staining was seen in keratocytes (Figure 2F in I). Intense staining in Plod1-/- mouse tissues was observed in the heart, where the strongest staining was seen in atrial auricles (Figure 2C in I), while in the heart muscle the staining was weaker (not shown). In the skeletal muscle the staining was observed mostly in the epimysium (not shown). Moreover, strong LH1 expression was observed in the bone and cartilage, where the staining was seen in osteoblasts and the periosteum and in chondrocytes (Figure 2E in I).

63 5.1.2 LH1 null mice have no kyphoscoliosis but are hypotonic and have increased mortality due to arterial ruptures

LH1 null mice were fertile and most of them had a normal lifespan. The mice were not observed to suffer from kyphoscoliosis, which is a characteristic feature of the EDS VIA syndrome (Steinmann et al. 2002), but they were passive and hypotonic during handling and they also appeared to be slower than wild-type mice. In walking tests on a rod or on the wall of a plastic mouse cage, LH1 null mice had notable difficulties to move normally and they got tired easily, while the wild-type littermates had no difficulties in running fast (Figure 3 in I). These features were more distinct when the mice became older and their weight increased. Although most of the Plod1-/- mice had a normal lifespan, 9% of the females and 17% of the males were found dead in their cages in the morning. Premature deaths occurred suddenly during the most active night-time. In almost all of these mice autopsy revealed a hemorrhage in the thoracic or abdominal cavity (Table 1 in I). In addition, histological analyses showed aortic ruptures (Figure 4C in I), and in one case there were also signs of a previous non-lethal injury (Figure 4D in I). The ruptures were seen in the tunica media, between the external layers of the elastic lamellae, while the elastic lamellae themselves were normal (Figure 4C–D and 4F in I). Healthy Plod1-/- mice showed no distinct histological changes in the aorta when compared to the wild-type mice (Figure 4A–B in I). X-gal staining was observed in the cells of the tunica media and adventitia of the aorta in the LH1 gene-trap mouse line (Figure 4B in I). These cells appeared to be less organized in LH1 null mice. Echocardiographic analysis indicated that the structure and function of the heart is normal (not shown). However, transmission electron microscopy (TEM) analyses of healthy Plod1-/- mouse aortic walls revealed degenerative changes in smooth muscle cells, such as vacuolization of the cells and swollen mitochondria (Figure 4F in I). Some completely degenerated cells were also seen (Figure 4F in I).

5.1.3 Abnormal collagen fibrils in LH1 null mice

In addition to kyphoscoliosis, typical characteristics of EDS VIA include hyperextensible, fragile skin and abnormal scarring, for instance (Steinmann et al. 2002). The Plod1-/- mice appeared to have normal skin, and no hyperextensibility or abnormal scarring was observed. Histological analyses of the skin were also

64 normal (not shown). However, TEM analysis from the skin revealed alterations in collagen fibrils. The mean collagen fibril diameter in the skin of Plod1-/- mice was increased when compared to that of wild-type (91 nm and 62 nm, respectively) and also the variation between fibril diameters was larger in LH1 null mice (Figure 5E–H in I). Similar observations were made from aorta samples, where mean fibril diameter in the Plod1-/- samples was 67 nm and in the wild-type samples 44 nm (Figure 5A–D in I). In addition to larger variability in the fibril diameters, LH1 null samples were also observed to have fibrils with irregular shape (Figure 5B and 5F in I). In the aorta, also the diameter within a fibril varied in the Plod1-/- samples (not shown) and the null skin samples contained degrading fibrils (Figure 5F in I). Collagen fibrils in the cornea were normal (not shown).

5.1.4 Decreased LH activity in LH1 null mice affects the hydroxylysine content in various tissues differently

One diagnostic marker of EDS VIA is decreased LH activity in cultured skin fibroblasts (Steinmann et al. 2002). Thus, LH activity was determined from homogenized skin samples of the LH1 null mice and also from homogenized aorta, which was found to be a structurally weak tissue in the mutant mice as well as in EDS VIA patients (Steinmann et al. 2002). The method where [14C]-lysine labelled non-hydroxylated protocollagen is used as a substrate (Kivirikko & Myllylä 1982) determines total LH activity in samples comprising the activities of LH1, LH2 and LH3. LH activity in the skin samples of Plod1-/- mice was decreased to 36% of that in the wild-type samples (Table 2 in I). No reports are available about the LH activity in the aorta of EDS VIA patients. It was thus quite surprising that the residual LH activity in the Plod1-/- aorta was as high as 44% of that in the wild-type sample (Table 2 in I). The activity in the skin of the LH1 heterozygous mice was about 55% and in the aorta about 60% of that in the wild- type samples (Table 2 in I). Total hydroxylysine content determined by reverse-phase high-pressure liquid chromatography (HPLC) was decreased in all the Plod1-/- tissues studied when compared to the wild-type tissues (Table 3 in I). The hydroxylysine content, expressed as per triple helix, varies markedly between tissues as each tissue contains different amounts of different collagen types and different collagen types contain variable amounts of hydroxylated lysines. The highest amount of hydroxylysine in wild-type samples was observed in the lung (55 residues / triple helix) (Table 3 in I). Also in the Plod1-/- lung samples the level was relatively high,

65 being about 85% of that of the wild-type (Table 3 in I). The lowest amount of hydroxylysine in the wild-type samples was detected in the skin with 21 residues per triple helix (Table 3 in I). In Plod1-/- skin the corresponding value was only 22% of that of the wild-type, being the most dramatically affected tissue studied (Table 3 in I). The hydroxylysine content was markedly decreased also in the Plod1-/- tail tendon and cornea, being 24% and 30% of the wild-type values, respectively, while the aorta, femur and heart auricle were less affected (hydroxylysine content 63%, 75% and 69%, respectively, of the wild-type values) (Table 3 in I).

5.1.5 Abnormal hydroxylation of triple helical lysine residues affects the cross-linking pattern in the LH1 null mouse tissues

As changes in the hydroxylation of certain lysine residues in collagens affect cross-link formation and as in EDS VIA patients the cross-linking pattern is changed (Steinmann et al. 2002), mature cross-links were studied from wild-type and Plod1-/- mice. Similar to the hydroxylysine levels, also the LP and HP content varies markedly between tissues (Table 4 in I). The highest levels of both mature cross-links in the wild-type mouse were observed in the aorta, the amounts of HP and LP being 0.71 and 0.26 residues per triple helix, respectively (Table 4 in I). The amount of HP was decreased in all LH1 null tissues studied, and the most dramatic change was seen in the aorta, where the HP content was 28% of that of the wild-type sample (Table 4 in I). The lowest HP and LP levels in wild-type tissues were observed in the tail tendon (0.051 and 0.006 residues / triple helix, respectively) and cornea (0.051 and 0.016 residues / triple helix) (Table 4 in I). In the cornea of LH1 null mice the HP amount was only 33% of that of the wild-type, but in the tail tendon the relative amount was 59% (Table 4 in I). In Plod1-/- lung the amount of HP was decreased markedly (34% of the wild-type), while the femur was slightly less affected (47% of the wild-type) (Table 4 in I). Although only certain hydroxylysine residues in collagens participate in the cross-link formation, it was interesting that there was no correlation between the changes in hydroxylysine and HP levels (Tables 3 and 4 in I). For example, in the tail tendon the hydroxylysine level was 24% of the wild-type tissue, but the amount of HP was as high as 59% of the wild-type value and in the lung the corresponding percentages were 86% and 34%, respectively. In human skin the levels of mature LP and HP cross-links are generally low (Knott & Bailey 1998, Avery & Bailey 2005) and they were not detectable in the mouse skin either.

66 As in the EDS VIA patients, the LP level in the Plod1-/- mouse tissues was increased in comparison to the corresponding wild-type tissues (Table 4 in I). The increase varied from about 5-fold to about 60-fold when compared to the wild- type (Table 4 in I). As a consequence of the decreased HP levels and increased LP levels, the LP/HP ratio was also increased dramatically in the LH1 null tissues (Table 4 in I). In wild-type tissues the LP/HP ratio varied between 0.12 and 0.39, whereas the values in the Plod1-/- mouse tissues ranged from 3.8 to 12.5, the smallest increase being observed in the aorta and the highest in the tail tendon (Table 4 in I). Also the total amount of pyridinoline cross-links (HP + LP) was increased in all LH1 null mouse tissues studied (Table 4 in I). The increase varied between tissues, being lowest in the aorta (about 140% of the wild-type) and highest in the tail tendon, where the rise was about 720% of the wild-type sample.

5.2 Analysis of the molecular level consequences of Bruck syndrome mutations by the use of recombinant LH2 proteins (II)

5.2.1 Expression of wild-type and recombinant LH2(long) polypeptides with Bruck syndrome mutations in insect cells and their purification

BS2 is caused by mutations in LH2 (van der Slot et al. 2003). To study the cause of BS2 at the molecular level, the currently known point mutations leading to LH2 amino acid substitutions R594H, G597V, and T604I were introduced into the pAcgp67A-LH2(long) baculovirus transfer vector by site-directed mutagenesis (Figure 1 in II) (Krol et al. 1996, Rautavuoma et al. 2002). The baculoviral signal peptide gb67 directs the synthesized protein for secretion into the culture medium and the N-terminal histidine tag enables selective purification. Wild-type and the three BS mutant LH2(long) polypeptides were expressed in High five insect cells and the secreted recombinant proteins were purified from the culture medium 72 h after infection by metal chelate affinity chromatography followed by histidine elution. As the final yield of the purified LH2 mutant polypeptides R594H and G597V was low, we decided to analyze the behavior of the mutant polypeptides during purification by taking aliquots from different purification steps for analysis by SDS-PAGE (Figure 2 in II). Wild-type and T604I LH2(long) bound effectively to the affinity column, whereas a significant amount of the mutants R594H and

67 G597V were present in the flow-through samples (Figure 2A–B in II). In addition, the bound R594H and G597V LH2(long) polypeptides detached easily from the column during washing steps indicating that the binding mediated by the histidine tag is weak (Figure 2C–D in II). The R594H and G597V mutant polypeptides that remained bound to the affinity column after the washing procedure eluted immediately after the addition of the elution buffer, whereas the T604I mutant and wild-type polypeptides required a longer presence of histidine to detach from the column (Figure 3 in II). These results indicate that in the T604I mutant and wild-type LH2(long) the histidine tag is readily exposed, while the R594H and G597V mutations may change the structure of the protein so that the tag becomes partly hidden.

5.2.2 The Bruck syndrome mutant R594H and G597V LH2(long) polypeptides have problems in dimerization and are prone to degradation

To analyze the purified recombinant wild-type and mutant LH2(long) proteins, fractions containing the purest samples were pooled, concentrated and the histidine was removed by PD-10 columns. Analyses by SDS-PAGE under reducing conditions and nondenaturing PAGE revealed that in contrast to the wild-type and T604I mutant polypeptides, the R594H and G597V mutants coeluted with additional polypeptides (Figure 4A and 4C in II). An anti-LH antibody identified some of these additional polypeptides in Western blot analysis (Figure 4B and 4D in II). LHs are dimeric proteins (Kivirikko et al. 1992, Rautavuoma et al. 2002) that dissociate into a monomeric form in reducing SDS-PAGE (Rautavuoma et al. 2002). Western blot analysis showed that the R594H and G597V mutants formed aggregates that were not dissociated into monomers in SDS-PAGE like the wild-type and T604I polypeptides (Figure 4B in II). The antibody recognized also two other bands in the R594H and G597V samples with a molecular weight smaller than that of the LH2 monomer, indicating that these two mutant forms were prone to degradation (Figure 4B in II). Identification of the other coeluting bands by N-terminal sequencing was unsuccessful and thus these polypeptides were regarded as impurities (data not shown). As indicated earlier, LH1 is shown to be a homodimeric protein in tissues (Kivirikko et al. 1992) and also recombinant LH isoforms produced in insect cells are shown to form dimers (Rautavuoma et al. 2002). Analysis of the mutant

68 recombinant polypeptides by nondenaturing PAGE revealed that the T604I mutant assembled into dimers normally when compared to the wild-type, while oligomerization of the R594H and G597V mutants was abnormal (Figure 4C–D in II). The R594H and G597V polypeptides formed oligomers that had either a slower or faster mobility than the dimers formed by the wild-type or T604I mutant LH2(long) polypeptides, and they also formed aggregates that were unable to enter the gel properly (Figure 4C–D in II).

5.2.3 Folding of the R594H and G597V mutant LH2(long) polypeptides is impaired and their proteolytic sensitivity is increased

To understand the consequences of the BS point mutations on the folding of LH2, the wild-type and mutant LH2(long) polypeptides were analyzed by far-UV circular dichroism (CD) spectroscopy. The CD spectra of the wild-type and T604I mutant LH2(long) were essentially identical and typical for folded, mainly α-helical proteins with a positive maximum at 193 nm and negative minima at 208 and 222 nm (Figure 5 in II). In accordance with the purification, SDS-PAGE, native PAGE and Western analyses, marked changes in the folding of the mutants R594H and G597V were observed. The R594H and G597V LH2(long) polypeptides both had only one negative minimum at about 207 nm (Figure 5 in II), indicating a dramatic change in the folding of these two mutant forms. As the results from the above analyses indicated impaired structural quality of two of the mutant LH2(long) polypeptides, the proteolytic sensitivity of wild- type and mutant forms were studied by thermolysin digestion. LH polypeptides have been previously shown to have two protease-sensitive regions that are digested by thermolysin generating three domains with molecular weights of 16, 30 and 37 kDa (Rautavuoma et al. 2002). Digestion of the wild-type and T604I mutant polypeptides produced these three thermolysin-resistant domains with molecular weights similar to those reported earlier (Rautavuoma et al. 2002) (Figure 6 in II), but digestion of the R594H and G597V mutants led to more extensive proteolysis, supporting the above results that folding of these mutants is not normal (Figure 6 in II).

69 5.2.4 All Bruck syndrome mutations affect the activity of LH2

To understand the effect of the BS2 point mutations on the activity of LH2, LH activity of the recombinant wild-type and mutant enzymes was analyzed by an assay based on the hydroxylation-coupled decarboxylation of 14 2-oxo[1- C]glutarate with a synthetic peptide (Ile-Lys-Gly)3 as a substrate (Kivirikko & Myllylä 1982). The wild-type LH2(long) was used in the activity assays as an internal control. The results of the assays showed that the activity of all the three mutant LH2 enzymes is decreased. Under the assay conditions used, typical activity values obtained with the wild-type LH2(long) were about 6000 dpm, whereas the activity of the T604I mutant was only about 500 dpm and those of the R594H and G597V mutants less than 300 dpm. Although the activity of the T604I mutant was decreased to less than 10% of that of the wild-type, the Km value of this mutant for the peptide substrate was normal (Table 1 in II). However, changes in the Km values for the reaction cosubstrates may also have an effect on the enzyme 2+ activity. The Km values of the T604I mutant for ascorbate and Fe were normal when compared to the wild-type, whereas the Km for 2-oxoglutarate was increased by about 10-fold (Table 1 in II). When 2-oxoglutarate was used in a saturating concentration in the reaction, the activity of the T604I mutant increased slightly, being about 30% of the wild-type (data not shown). The Km values of the R594H and G597V mutants for the peptide substrate were markedly increased when compared to the wild-type (Table 1 in II). Because of the folding problems and extremely high Km values for the substrate, the catalytic properties of the R594H and G597V mutants were not studied further. LH2(long) is reported to hydroxylate the telopeptide lysines in collagens (Uzawa et al. 1999, Mercer et al. 2003, van der Slot et al. 2003, Pornprasertsuk et al. 2004, van der Slot et al. 2004, Takaluoma et al. 2007). Previously it was shown that LH2(long) is the only LH isoenzyme capable of hydroxylating the N telopeptide lysine (Lys9) in a full-length pro-α1(I) chain of type I collagen when coexpressed in insect cells (Takaluoma et al. 2007). As impaired 2-oxoglutarate binding was the only characteristic that clearly distinguished the T604I mutant LH2(long) from the wild-type, we tested whether the mutant is nevertheless able to hydroxylate the telopeptide Lys9 of type I collagen in insect cell coexpression experiments. Full-length proα1(I) chains were coexpressed with C-P4H and wild- type or T604I mutant LH2(long). The produced recombinant type I procollagen homotrimers were digested with pepsin and the triple helical collagen I

70 homotrimers were further purified and analyzed by N-terminal sequencing. The wild-type LH2(long) hydroxylated the telopeptide Lys9 as described previously (Takaluoma et al. 2007), whereas the hydroxylysine peak was absent in the reverse-phase HPLC profile of the collagen I homotrimer purified after coexpression with the T604I mutant LH2(long) (Figure 7A–B in II). Thus, the T604I mutant LH2(long) was not able to hydroxylate the telopeptide Lys9 residue during coexpression, revealing the importance of proper binding of reaction cosubstrates in the LH reaction.

5.3 Lysyl hydroxylase 2 is essential for the embryonic development of mouse (III)

5.3.1 LH2 null mice die after embryonic day 10.5

To study the in vivo role of LH2, we generated three different mouse lines carrying a gene-trap insertion cassette with a β-galactosidase reporter in the Plod2 gene that disrupts the gene function (the LH2:KST067 gene-trap targeting strategy is shown in Figure 1A in III). All three lines showed the same main phenotype i.e. embryonic lethality, and thus we chose one mouse line originating from the targeted ES cell line LH2:KST067 for further analysis. The lack of the production of LH2 mRNA with a sequence from exon 5 onwards was verified by RT-PCR (Figure 1B in III). The heterozygous Plod2wt/gt (wt stands for a wild-type and gt for a gene-trapped allele) mice appeared to be healthy and fertile and had a normal lifespan, but genotyping (Figure 1C in III) revealed that intercrosses of the heterozygous mice yielded only wild-type and heterozygous offspring. Analysis of the embryos at different ages from heterozygous breedings revealed that the Plod2gt/gt embryos developed seemingly normally until E10.5, when the LH2 null embryos constituted 23% of the population (n = 165 and 724, respectively) (Table 2 in III). Thus, the amounts of the Plod2gt/gt embryos at E10.5 were close to the expected Mendelian ratio. At E11.5 the proportion of LH2 null embryos was about 14% (n = 326) and at E12.5 only 6% (n = 85) (Table 2 in III). After E12.5 living Plod2gt/gt embryos were not observed. Older embryos between E11.5–12.5 were collected when the backcrossing was still ongoing. Thus, the given percentages could be even lower in the backcrossed lines. The Plod2gt/gt embryos developed apparently normally until E10.5 but died suddenly after this. The size and appearance of the LH2 null embryos at 10.5 was

71 seemingly normal when compared to the wild-type and heterozygous littermates (Figure 2A in III), but pooled blood was frequently observed inside the fetal membranes (Figure 2B–C in III) and sometimes possibly between the visceral yolk sac and amnion (data not shown), while the heart was still beating. After the first observation of the accumulated blood, 21% of the Plod2gt/gt embryos and 2% of the Plod2wt/gt embryos were found to have this phenomenon, while wild-type embryos were all normal (Table 3 in III).

5.3.2 LH2 is expressed ubiquitously in the E10.5 embryos and in extraembryonic tissues and its lack seems to lead to certain changes in collagen expression

X-Gal staining, representing the Plod2 promoter-driven β-galactosidase expression was extensive in E10.5 Plod2gt/gt embryos (Figure 3A–D in III). Staining was observed ubiquitously in the mesenchymal tissue (Figure 3A in III). The expression was intense also in somites, the auditory vesicle and amnion, for instance (Figure 3A–D in III). In placenta, X-gal staining was observed in the maternal decidua and on the embryonic side in the chorionic plate and labyrinth layer, where the expression was seen in the mesenchymal tissue surrounding embryonic vessels (Figure 3E–F in III). Parietal endoderm cells producing the Reichert’s membrane, the parietal layer of the yolk sac, were strongly stained in Plod2gt/gt embryos, but the expression was not seen in the membrane (Figure 4B in III). It has been shown earlier that the collagen α1(IV)2α2(IV) null mice die between E10.5–E11.5 because of deficient basement membrane assembly and finally of the failure of the Reichert's membrane (Pöschl et al. 2004). No obvious differences between the Plod2gt/gt and wild-type embryos were seen in light microscopy or TEM analysis of the Reichert’s membrane or in the type IV collagen immunofluorescence staining (Figure 4A–F in III). However, according to TEM analysis, we were not able to exclude a weakened adhesion of the Reichert’s membrane to placenta in Plod2gt/gt embryos (data not shown) and also the normal detachment of parietal endoderm cells was sometimes questioned (Figure 4D in III). In other extraembryonic tissues, pronounced X-gal staining was observed in the mesodermal part of the visceral yolk sac, but the endodermal side was unstained (Figure 5B–C in III). Morphological analysis of the visceral yolk sac of Plod2gt/gt embryos revealed that some areas of the mesodermal part of the tissue

72 appeared loose in comparison to wild-type (Figure 5A–C in III). The loosened mesodermal tissue was also surrounding vitelline vessels, and thus it cannot be excluded that in some of the hemorrhage cases this site may have collapsed followed by a rupture in the vitelline vessel that could have led to the bleeding between the yolk sac and amnion. In immunofluorescence staining of extraembryonic tissues, type III and type V collagens showed differential expression patterns (Figure 6A–D in III). Type III collagen was seen in the endodermal side of the visceral yolk sac (Figure 6A–B in III), while type V collagen was observed in the mesodermal part of the tissue (Figure 6C–D in III). In the Plod2gt/gt yolk sac the staining of both collagen types seems weaker than that of the Plod2wt/wt tissue although the distribution was normal (Figure 6A–D in III). Staining of type I collagen was seen in the mesodermal part of the visceral yolk sac, but differences between genotypes were not observed (data not shown). Type I, IV and V collagens but not type III collagen were expressed also in the chorionic plate of the placenta, but no obvious differences were seen between the Plod2gt/gt and Plod2wt/wt embryos (data not shown). As the phenotype of the mouse line indicated a cardiovascular defect, anti-CD31 staining was performed to study the structure of large vessels of the embryos, but the endothelium of the aorta appeared intact (Figure 7A–B in III). However, TEM analysis revealed that in the Plod2gt/gt aorta the overall structure of the endothelium seemed looser when compared to Plod2wt/wt embryos (Figure 7C–D in III). The mesenchymal cell network under the endothelium was sparse and the cells appeared elongated and showed numerous thin protrusions in Plod2gt/gt embryos, while in the Plod2wt/wt embryos the mesenchymal cells were larger and they were located more closely to the endothelium. LH2 is expressed in the mesodermal tissue surrounding the aorta (Figure 3A in III). As blood pressure increases during development this area must sustain higher mechanical loads and provide support for the large vessels. Thus, collagen fibrils were studied by TEM from this mesodermal area and some fibrils were seen in both Plod2wt/wt and Plod2gt/gt samples (Figure 7E–F in III).

5.3.3 Expression of the Plod1 and Plod3 genes is not upregulated in the Plod2gt/gt embryos

To study whether inactivation of the Plod2 gene leads to upregulation of Plod1 and Plod3 gene expression, we performed Q-PCR and RT-PCR analyses from 10.5 days old embryos. Plod2 expression followed the expected levels with about

73 50% expression in the Plod2wt/gt embryos relative to wild-type, whereas expression was not detected in the Plod2gt/gt embryos (Figure 8A–B in III). The LH mRNA levels were determined from single embryos and results varied markedly between individuals in the same genotype group. At E10.5 the development of embryos is fast and even minor age differences may cause large differences in the mRNA levels. According to our LH1 null mouse study, the other two LH isoenzymes are suggested to at least partially compensate for the LH1 deficiency (Article I in this thesis). However, the mRNA levels of Plod1 and Plod3 genes in the Plod2wt/gt and Plod2gt/gt embryos did not show any significant increase, thus suggesting that they cannot compensate for the decreased LH2 level (Figure 8A–B in III). In any case, the LH1 and LH3 could not compensate for the telopeptide LH activity of LH2.

5.3.4 LH2 is widely expressed in adult Plod2wt/gt tissues

Plod2 was expressed in almost all the adult tissues studied. In the bone, X-gal staining was observed in the endosteum, periosteum and round osteocytes (Figure 9A–B in III). The pattern was similar in both bone types analyzed, the femur and calvaria. Staining was also intense in the cartilage (Figure 9C in III), tendon (data not shown), and in the smooth muscle layers of the uterus (Figure 9D in III) and intestine (not shown). In blood vessels LH2 was expressed moderately in endothelial cells (Figure 9E–F in III). In the tunica media the expression was localized to smooth muscle cells located between the elastic lamellae (Figure 9E– F in III). X-gal staining was also seen in the cells of tunica adventitia (Figure 9E– F in III). In the eye, LH2 was expressed in the anterior lens epithelium as well as in the ganglion cell layer and in the inner nuclear layer of the retina (data not shown). In the skin the expression was strongest in the hair bulbs (data not shown).

5.3.5 Changes in lysyl hydroxylation and cross-linking of collagen in adult Plod2wt/gt mice

In BS2 patients the collagen cross-linking is altered due to abnormal function of LH2, therefore we wanted to study the collagen cross-linking from E10.5 embryos. However, maturation of collagen cross-links requires time. Therefore, the collagen cross-link analysis from embryos turned out to be unsuccessful as the mature cross-links had not yet formed at E10.5 (data not shown). We thus decided

74 to study whether any differences in collagen content, collagen hydroxylations, and cross-linking can be seen in adult heterozygous Plod2wt/gt mouse tissues relative to wild-type. The amounts of collagen and 4-hydroxyproline were unchanged in all adult Plod2wt/gt tissues studied when compared to wild-type control mice (Figure 10A– B in III). The highest collagen levels in wild-type tissues were observed in the tail tendon and skin, where the collagen content was 58% and 52% of the dry weight of the tissue, respectively (Figure 10B in III). The total hydroxylysine content, expressed per triple helix, varies markedly between tissues as collagen types contain variable amounts of hydroxylated lysines and tissues contain different amounts of different collagen types. LH2(long) is responsible for the hydroxylation of telopeptide lysines that participate in cross-link formation and there are a maximum of two of these lysines per collagen molecule (Knott & Bailey 1998, van der Slot et al. 2003, Takaluoma et al. 2007). The triple helical area contains additional lysine residues that can be hydroxylated most probably by all three LH isoenzymes (Takaluoma et al. 2007). Two of these lysine residues are involved in cross-links (Knott & Bailey 1998). Thus, changes in the hydroxylation levels of the telopeptide lysines do not dramatically affect the total lysine hydroxylation level. In addition, the Plod2wt/gt mice have one functional Plod2 allele left. The highest amount of total hydroxylysine in wild-type samples was observed in the lung (51 residues / triple helix) and the lowest in the skin (18 residues / triple helix) (Figure 10D in III). In the Plod2wt/gt mouse tissues the total hydroxylysine content was changed significantly only in the calvaria and femur, which also normally contain a large amount of pyridinoline cross-links. The amount of hydroxylysine was decreased to 85% in calvaria and to 80% in femur when compared to the corresponding wild-type tissues (Figure 10D in III). As changes in the hydroxylation of the telopeptide lysine residues in collagens affect the cross-links in BS patients (Bank et al. 1999), mature pyridinoline cross-links were studied from the wild-type and Plod2wt/gt mice. Total hydroxylysine levels but also the LP and HP contents varied markedly between different tissues (Figure 10D–F in III). The highest levels of both mature cross- links in wild-type mouse were observed in the lung (LP 0.57 and HP 0.51 residues / triple helix) and aorta (LP 0.12 and HP 0.38 residues / triple helix) (Figure 10E– F in III), although the total amount of collagen was low in these tissues (Figure 10B in III). The lowest levels of mature cross-links in wild-type were detected in the tail tendon (LP 0.0015 and HP 0.0078 residues / triple helix) (Figure 10E–F in

75 III). The change in the LP content between Plod2wt/wt and Plod2wt/gt was significant only in the calvaria, where the level was decreased in the Plod2wt/gt sample to 51% relative to the wild-type (Figure 10E in III). In contrast, the HP levels in the Plod2wt/gt mouse tissues were markedly more decreased. The decrease in the HP levels was significant in all the Plod2wt/gt tissues studied except the aorta (Figure 10F in III), which was most severely affected in the Plod1-/- mouse (Article I of this thesis). The most dramatic change in the HP was seen in the calvaria, where the HP content was 33% (reduced from 0.1 to 0.04 residues / triple helix) of the wild-type. In addition to calvaria, the skin had only 45% (reduced from 0.01 to 0.005 residues / triple helix) and femur 65% (reduced from 0.4 to 0.26 residues / triple helix) of the wild-type HP levels. Slightly minor changes were observed in the tail tendon (70% of the wild-type value of 0.008 residues / triple helix) and lung (83% of the wild-type value of 0.51 residues / triple helix). Interestingly, in general the amount of HP cross-links was markedly more affected than the LP cross-links in the Plod2wt/gt tissues, although the only difference between the LP and HP is in the hydroxylation state of the helical lysine. However, LH2 is suggested to have also helical lysyl hydroxylase activity (Takaluoma et al. 2007, Article I in this thesis). As long-lived proteins, collagens are susceptible to non-enzymatic, unwanted cross-links involving reducing sugars (Saito & Marumo 2010). As lysine residues are participating in these advanced glycation end-products (AGEs), the effect of reduced lysyl hydroxylation on the amount of pentosidine, one of the AGEs, was also studied in the Plod2wt/gt tissues. Some changes were seen in the femur, for instance, where the pentosidine level was slightly increased in the Plod2wt/gt mice, but the results were not statistically significant (Figure 10C in III).

76 6 Discussion

6.1 LH1 null mice provide a tool to study tissue-specific consequences of the lack of LH1 activity

We have generated a Plod1-/- mouse line to study the function of LH1 in vivo. In addition, we expected that the mouse line could also serve as a disease model for the human EDS VIA. This is the first reported animal model for a defect in the gene coding for LH1, and the created mouse line enables for the first time detailed examination of the effects of the lack of this enzyme in various organs and tissues which is difficult, impossible, or unethical to do in the case of the EDS VIA patients. EDS VIA patients suffer already at birth from kyphoscoliosis, muscular hypotonia and joint laxity, for instance (Steinmann et al. 2002). The LH1 null mice are viable, their breeding is normal, and they are not observed to suffer from kyphoscoliosis. However, they do have muscular hypotonia which becomes more obvious when the mice are older and have increased weight. Although the Plod1-/- mice appear to be flaccid during handling and have muscle weakness and gait abnormalities in situations where strength and coordination is needed, they do not have an obvious skeletal phenotype like the EDS VIA patients. This can be due to differences in the size and anatomy of the mouse and human as well as the upright posture of humans that exposes the skeleton to forces different from those in the mice. In EDS VIA patients the kyphoscoliosis is also progressive, which is thought to be due to a supplementary effect of the muscle hypotonia and joint laxity as the vertebral bodies, for instance, are shown to be normal (Steinmann et al. 2002). Muscular hypotonia, which is connected to a basic ECM-forming enzyme, is an interesting phenotype which could also be exploited in studies of other muscular disorders and especially the role of connective tissue in these diseases. Mutations in highly glycosylated and thus hydroxylated type VI collagen cause Ullrich congenital muscular dystrophy and the milder Bethlem myopathy (for a review, see Lampe & Bushby 2005). In addition to muscular dystrophy, the disorders are characterized by joint hyperlaxity and contractures. A null mouse for type VI collagen develops a muscular phenotype with variation in the muscle fiber diameter and fiber necrosis (Bonaldo et al. 1998). Interestingly, similar to the Plod1-/- mouse a null mouse line for type VI collagen has recently been shown also to possess abnormalities in collagen fibril diameter and fibril

77 assembly in tendons (Izu et al. 2011). Moreover, the mechanical properties of tendons were decreased. In contrast to Plod1-/- mice, however, the collagen fibril diameter in the collagen VI null mice was generally decreased and not increased (Izu et al. 2011). EDS VIA patients have soft, hyperextensible skin that is prone to abnormal scarring. In addition, the patients bruise easily. The skin of the Plod1-/- mice appeared to be normal, but detailed analysis revealed that the collagen fibrils were abnormal. The diameter of the fibrils varied in the Plod1-/- skin samples and their shape was in part irregular when compared to the wild-type skin. In addition to cross-link formation, hydroxylysine residues are known to function as attachment sites for carbohydrate residues, which in turn affect the fibril diameter and fibrillogenesis of collagen molecules (Myllyharju 2005), supporting these findings for the Plod1-/- skin collagens. The hydroxylysine content in EDS VIA skin is reduced typically to about 5% of that of healthy controls (Steinmann et al. 2002), whereas in Plod1-/- mouse skin the hydroxylysine content was over 20%. In addition, the residual LH activity in Plod1-/- mouse skin homogenates was quite high, 35% of the wild-type, while in EDS VIA patient fibroblasts the values vary from 5% to 25% of the controls (Yeowell & Walker 2000). Thus, the lack of an obvious skin phenotype in the Plod1-/- mice is likely to be because of the higher LH activity and hydroxylysine levels in the mutant mouse skin. These differences might also be due to possible variable expressions of the LH isoenzymes in the Plod1-/- mouse and EDS VIA skin. A life-threatening complication in EDS VIA patients is an arterial rupture. The arteries of the Plod1-/- mice were also found to be structurally weak as some of the LH1 null mice died during the night because of aortic rupture. This feature was almost twice as prevalent in male as in female mice. In histological analysis the aortic wall of apparently healthy Plod1-/- mice appeared to be generally normal. Moreover, heart function was also normal. In contrast, in necropsy samples of the animals that had died suddenly and had hemorrhages in the thoracic or abdominal cavity, dissections in the aorta were observed. As in the skin, detailed analysis of the aorta by TEM revealed abnormal collagen fibrils and also degrading fibrils even in the apparently healthy looking Plod1-/- mice. Degenerative changes were also observed in the smooth muscle cells of the Plod1-/- aortas. These observations are interesting because the hydroxylysine level (63% of wild-type) and residual LH activity (44% of wild-type) in the Plod1-/- aorta are in fact higher than in the skin (22% and 36%, respectively), which appeared to be asymptomatic. However, mechanical requirements in the aorta are

78 high due to high blood pressure and the weakened structures in the tissue are probably predisposing it to ruptures. In addition to the skin and aorta, the hydroxylysine content was also decreased in other Plod1-/- tissues studied. It is suggested that LH1 is mainly responsible for the hydroxylation of lysine residues in the triple helical region of collagens, while LH2(long) exclusively hydroxylates the telopeptides (Eyre et al. 2002, van der Slot et al. 2003, Takaluoma et al. 2007). The exact role of LH3 in lysyl hydroxylation is still unknown as for example the embryonic lethality of LH3 null mice has been shown to be due to the lack of its collagen glycosyltransferase and not LH activity (Rautavuoma et al. 2004, Ruotsalainen et al. 2006). Although the Plod1-/- mice do not have functional LH1, in certain tissues, such as the lung, the hydroxylysine level is as high as 86% of the wild- type, while in the skin the level was only 22%. The differential hydroxylation content in tissues most likely reflects tissue specificities of the LH isoenzymes, differences in their amounts in different tissues, and probably also differential compensatory mechanisms of the LHs. At the mRNA level all LH isoforms are expressed in almost all mouse and human tissues analyzed (Valtavaara et al. 1997, Valtavaara et al. 1998, Passoja et al. 1998a, Ruotsalainen et al. 1999, Yeowell & Walker 1999a, Rautavuoma et al. 2004). At the protein level the data is still limited. Our data also clearly suggest that in addition to LH1, the other two isoforms, LH2 and LH3, are able to hydroxylate helical lysines to variable extents in different tissues in vivo. The mechanical strength of tissues depends in part on the type and amount of the existing collagen cross-links. The type of the cross-link, in turn, depends on the hydroxylation state of certain triple helical and telopeptide lysines. Two different routes exist for cross-link formation. The hydroxyallysine pathway leads to the formation of pyridinoline cross-links HP and LP that predominate in tissues such as bone and cartilage. LP is formed between two telopeptide hydroxylysine molecules and a helical lysine, whereas in the HP cross-link all participating lysines are hydroxylated. The allysine pathway, which leads to mechanically weaker bonds, is common in the skin and cornea, for instance. (Knott & Bailey 1998, Eyre & Wu 2005). As the cross-linking profile in tissues of EDS VIA patients is changed, they have increased urinary LP levels and decreased HP levels (Pasquali et al. 1994, Pasquali et al. 1995, Steinmann et al. 1995). As was expected, the amount of HP cross-links was decreased in all the Plod1-/- mouse tissues studied, while the amount of LP cross-links was increased. This supports the earlier suggestion that LH1 is the main LH that hydroxylates helical lysines.

79 The total amount of HP + LP was increased in the Plod1-/- mice because of increased LP that could indicate a response to the changed cross-linking state. Both pyridinolines require telopeptide lysine hydroxylation and thus, at least LH2(long) upregulation could be behind this response. However, the maturation of cross-links to LP and HP requires time and it is not known whether the rate is the same for both pyridinoline cross-links; this may also influence the results. Surprisingly, there was no correlation between the overall hydroxylation level and the amount of cross-links. Although the total hydroxylysine amount in the aorta of the Plod1-/- mouse was rather high (63% of wild-type), the amount of HP was only 28% of that of the wild-type, while in the tail tendon the total hydroxylysine content was low (24% of wild-type), but the HP value was still 59% of the control, for instance. These results clearly show that LH2 and/or LH3 are able to hydroxylate the triple helical cross-link site lysines to some extent in various tissues when LH1 is not functional. Moreover, the results demonstrate again the complex roles and interplay between the LH isoenzymes in tissues. Changes in lysyl hydroxylation not only influence the mechanical strength of tissues via cross-links but also individual collagen fibrils as was discussed earlier. The diameter and shape of collagen molecules in the Plod1-/- skin and aorta were changed, but not in cornea. Many factors may contribute to these characteristics, but currently they are not well known. However, glycosylation of hydroxylysine residues is known to affect fibrillogenesis and fibril diameter (Torre-Blanco et al. 1992, Notbohm et al. 1999, Sricholpech et al. 2011). In the Plod1-/- mice the amount of hydroxylysine residues available for glycosylation is decreased, which is likely to affect the glycosylation level as well. Moreover, collagen I fibrils contain small amounts of type III and V collagens, which in turn are shown to regulate the thickness of fibrils (Kielty & Grant 2002). EDS patients that have a vascular or classical form of the syndrome have mutations in these minor collagens and they have abnormal collagen I fibrils (Steinmann et al. 2002). The null mouse lines for these collagen types are also shown to have abnormal collagen fibrils (Liu et al. 1997, Wenstrup et al. 2006), as well as mouse lines lacking small leucine-rich proteoglycans such as biglycan, decorin, fibromodulin and lumican (Danielson et al. 1997, Chakravarti et al. 1998, Svensson et al. 1999, Corsi et al. 2002). Thus, it is possible that deficient lysyl hydroxylation and glycosylation of fibrillar collagens in the Plod1-/- mice may interfere with proper binding of other proteins, which in turn may lead to abnormal collagen fibrils. Moreover, the Plod1-/- mice show that the abnormal collagen fibrils observed in

80 different EDS types can also be caused by a defective collagen-modifying enzyme and not just by mutations in the collagen molecules themselves.

6.2 Mutations causing Bruck syndrome lead to decreased LH2 activity, explaining the symptoms of diagnosed Bruck syndrome type 2 patients

BS has been linked to lysyl hydroxylation of telopeptides because of the decreased amount of pyridinoline cross-links in bone collagen (Bank et al. 1999). Three different mutations found from the PLOD2 gene led to the suggestion that these mutations cause BS2 and that the LH2 is the telopeptide LH (van der Slot et al. 2003, Ha-Vinh et al. 2004). As these mutations do not change any of the amino acids that are known to be catalytically critical, the molecular mechanism causing the disorder has been unknown. In this study we produced the BS mutant and wild-type human LH2(long) polypeptides in insect cells and purified and analyzed them to understand the molecular pathology of the syndrome. Our data show that all three known BS2 mutations lead to marked reduction of LH2 activity. The most significant decrease in the activity was observed in the mutants R594H and G597V, whose activity was below 5% of that of the wild-type enzyme. These mutations most probably affect proper folding of the polypeptides, which in turn may also affect their oligomerization. These effects are likely to lead to the marked decrease in the enzyme activity. All the mutated amino acids found in LH2 in the BS2 patients, Arg594, Gly597 and Thr604, are conserved in the human and mouse LH isoenzymes (Valtavaara et al. 1998, Passoja et al. 1998a, Ruotsalainen et al. 1999). LH monomers are shown to consist of three domains. The C-terminal domain of all three isoenzymes is responsible for the LH activity and the N-terminal domain in LH3 possesses glycosyltransferase activity, but the function of the middle domain is unknown. (Rautavuoma et al. 2002). The residues Arg594 and Gly597 in LH2(long) are located in the region that separates the middle and C-terminal domains, which is known to be a protease-sensitive region. Moreover, C-terminal recombinant LH1 and LH3 fragments beginning with the residue corresponding to Lys589 in LH2(long) have been shown to be fully active enzymes (Rautavuoma et al. 2002). Thus, it is likely that the amino acids Arg594 and Gly597 are part of an unstructured domain boundary that may function as a linker. Changes in this area may affect its flexibility and the folding capability of the flanking domains.

81 Folding of the mutant T604I appears to be normal, but the activity in standard conditions was decreased to under 10% of that of the wild-type LH2(long). The change in the activity level was caused by a 10-fold increase in the Km for 2-oxoglutarate, whereas binding efficiency of the peptide substrate or the other cosubstrates of the reaction was not affected. The maximum activity of the T604I obtained under a saturating 2-oxoglutarate concentration was about 30% of that of the wild-type. The increased Km value for 2-oxoglutarate indicates that binding of this cosubstrate is abnormal in the T604I mutant. Moreover, the T604I mutant was not able to hydroxylate the N-terminal telopeptide lysine in recombinant type I procollagen chains in cellulo. LHs belong to the superfamily of 2-oxoglutarate dioxygenases, which have a catalytic site with a common structure formed by an 8-stranded β-helix core fold. A conserved basic residue in the eighth strand binds the C-5 carboxylate moiety of 2-oxoglutarate and in LH2 the residue is Arg724. (Passoja et al. 1998b, Clifton et al. 2006). This residue is located near the second Fe2+ -binding histidine in all known 2-oxoglutarate dioxygenases except the factor inhibiting HIF, an asparaginyl hydroxylase of HIF (Schofield et al. 2004). Certain serine or threonine residues near the basic residue in the eighth strand provide an additional interaction with the C-5 carboxylate of 2-oxoglutarate in many 2-oxoglutarate dioxygenases (Clifton et al. 2006, Koski et al. 2007). Mutation of this serine residue in recombinant human LH1 is shown to reduce the enzyme activity markedly, although the Km value for 2-oxoglutarate remained normal (Passoja et al. 1998b). Unfortunately the three-dimensional structure of LHs is not known and thus the exact structural role of Thr604 in the binding of 2-oxoglutarate remains unknown. The mutant enzymes were not totally inactive, which may be important for patients carrying these mutations as during this study we observed that LH2 null mice die early, during embryonic development (Article III in this thesis). On the other hand, for example in EDS VIA LH1 is known to have alternative splicing pathways to bypass or compensate mutations and to retain some enzyme activity (Yeowell & Walker 2000). However, it is not known whether the residual activity is enough to enable the survival of patients or is LH2 using similar alternative splicing system in BS2 as LH1 in EDS VIA or whether the role of LH2 in human development is not as critical as in mouse development. However, according to published reports, two affected children carrying the BS mutation T604I deceased prenatally and one child survived only for 2 years (van der Slot et al. 2003). Thus, this mutation seems to be clinically the most severe form of the reported three forms (van der Slot et al. 2003, Ha-Vinh et al. 2004). This is a bit surprising as

82 the effects of the T604I mutation in the recombinant LH2 were not as severe as those of the Arg594, Gly597 mutations. The type and amount of collagen cross-links existing in the tissue determine in part its mechanical strength as they stabilize the collagen fibrils. Cross-links are formed via two different routes, the allysine and hydroxyallysine routes. The hydroxylation state of the telopeptide cross-link lysine residues determines the route and in the hydroxyallysine route these lysines are hydroxylated, while in the allysine route they are not. The mature pyridinoline cross-links HP and LP, which predominate in tissues requiring strength such as bone and cartilage, are formed via the hydroxyallysine route and thus require the presence of hydroxylysine in the telopeptide cross-link sites. (Knott & Bailey 1998, Eyre & Wu 2005). BS patients have a highly abnormal cross-linking profile in the bone collagen. The amount of LP and HP is dramatically decreased, while the amount of allysine- derived cross-links is increased. (Bank et al. 1999). Thus, it can be deduced that the BS2 mutations decrease the LH2 activity to an extent that results in changes in the cross-linking profile, which causes the clinical consequences observed in the patients.

6.3 Inactivation of mouse Plod2 leads to lethality during early embryonic development

Mutations in the PLOD1 gene lead to LH1 deficiency and EDS VIA, which is a severe but not lethal connective tissue disorder. The mouse model for this human syndrome shows a milder phenotype, but both have an increased risk of arterial rupture, for instance (Article I in this thesis). Mice lacking the LH3 enzyme die during embryonic development due to abnormal processing of type IV collagen leading to basement membrane fragmentation (Rautavuoma et al. 2004, Ruotsalainen et al. 2006). Recently one patient was shown to have compound heterozygote mutations in the PLOD3 gene (Salo et al. 2008). The syndrome is severe with symptoms overlapping several connective tissue disorders and the patient also has a stillborn sibling. Mutations in the gene encoding LH2 cause BS2, where patients have severe skeletal problems, but it is usually not lethal. In this study we have generated a novel LH2 null mouse line from an ES cell line, in which the Plod2 gene is disrupted by a gene-trap insertion cassette. As the human BS2 is not lethal, it was surprising that LH2 null mice died during embryonic development, after E10.5. However, our previous in vitro studies showed that recombinant LH2 enzymes carrying the mutations identified

83 in BS2 patients are not totally inactive and, thus, the residual LH2 activity may be indispensable for the patients (Article II in this thesis). LH2 null embryos developed seemingly normally until E10.5, but died after this without any obvious signs of growth retardation. However, about a fifth of the null embryos were observed to have accumulated blood inside the fetal membranes while the heart was still contracting. This could indicate problems in the cardiovascular system or the placenta. BS2 patients are not reported to have direct cardiovascular symptoms. However, in Plod2gt/gt embryos some changes were seen in the endothelium of aorta, which could affect the structural resistance of the tissue. The cause of death of all BS patients is not available, however. Two children having a BS2 mutation deceased prenatally and one child survived for 2 years (van der Slot et al. 2003). Because of the limited amount of published clinical data about these patients, the possible role of the placenta, for instance, cannot be excluded. The placenta of mouse and human are fairly different, but they also have considerable similarities (reviewed in Rossant & Cross 2001). Although the interest in the development of placenta and its defects is increasing, knowledge about it is still extremely limited and far behind the knowledge of embryonic development. As a tissue originating from a differentiating embryo, it is affected by the same genetic defects as the embryo itself (Rossant & Cross 2001). The mutant embryo may die before the phenotype would be apparent if the gene is required in the development and function of the placenta. LH2 is widely expressed in the embryo and also in the extraembryonic tissues. On the embryonic side of the placenta LH2 is expressed in the chorionic plate and mesenchymal tissue surrounding embryonic vessels, where the maternal and fetal blood are in close proximity to each other and the tissue between the compartments must be thin but firm. Moreover, the role of the placenta sets also certain other structural requirements for the tissue, such as substantial blood flow in the chorionic plate-attached umbilical cord (Inman & Downs 2007). The cause of death of the null embryos could also be a collapse in these tissue structures leading to hemorrhage. The major fibril-forming collagens in the placenta are type I, III and V collagens (Iwahashi et al. 1996, Arai & Nishiyama 2007), but there is a limited amount of data available about the collagen types in the chorionic plate at different developmental stages. Type I, IV and V collagens, but not type III collagen, were expressed in the chorionic plate of the E10.5 embryos, but clear differences were not observed between the Plod2wt/wt and Plod2gt/gt tissues. Interestingly, immunofluorescence staining of collagens III and V but not type I was slightly weaker at least in the yolk sac of

84 the Plod2gt/gt embryos than in the wild-type. The size of the mouse placenta is increasing markedly between E10–E14 (Arai & Nishiyama 2007), and thus the tissue must come up with the fast-changing mechanical requirements. Thus, mechanically inadequate collagenous structures may break down during the fast development. As LH2 is required for the formation of appropriate ECM, it may be necessary for the mechanical integrity of the placenta. Rodents have a Reichert’s membrane (RM) in the placenta, between the parietal endoderm cells and trophoblast cells, that is lacking in humans (Gardner 1983). The membrane contains high levels of type IV collagen and collagen α1(IV)2α2(IV) knock-out mice are reported to die between E10.5–E11.5 because of the failure of this membrane (Pöschl et al. 2004). Moreover, LH3 knock-out mice die at E9.5 because of abnormal collagen IV synthesis. The parietal endoderm cells producing the RM as well as the mesodermal part of the visceral yolk sac have strong LH2 expression. Thus, we thought that the LH2 null mice may also have a failure in the RM, but a clear defect was not found. Although no clear abnormalities were observed, a possible role of the membrane failure in the phenotype cannot be fully excluded. In addition, the mesodermal part of the visceral yolk sac of Plod2gt/gt embryos seemed to have sites that appeared loose. These loosened parts were partly surrounding vitelline vessels and a collapse in this site may also be the cause of vessel rupture leading to the observed bleeding between the yolk sac and the amnion. Type III collagen was found to be expressed in the endodermal part and types I and V in the mesodermal side of the yolk sac at E10.5. Interestingly, as indicated earlier, immunofluorescence staining showed that the amount of type V and III but not type I collagen may be slightly lower in the yolk sac of the Plod2gt/gt embryos. Moreover, collagen V knock-out mice are shown to have a similar hemorrhage phenotype as the LH2 null mice at the time of their death at E10 (Wenstrup et al. 2004). Generally this could indicate inadequate support in the LH2 null tissues, especially for the blood vessels. Abundant expression of type I collagen begins in mice at E12 and the Mov 13 mice that cannot synthesize the α1(I) collagen polypeptide die soon after this thorugh rupture of the major blood vessels of the embryo (Schnieke et al. 1983, Löhler et al. 1984). Type III collagen expression in mouse is even stronger than that of type I between E8.5 – E12.5 (Niederreither et al. 1994). About 10% of collagen III knock-out mice survive to adulthood, but have a much shorter lifespan relative to the wild-type (Liu et al. 1997). The major cause of death in collagen III knock-out mice is blood vessel ruptures. Interestingly, as indicated above, collagen V knock-out mice are reported to have a similar hemorrhage

85 phenotype as the LH2 null mice (Wenstrup et al. 2004). Moreover, collagen V expression increases markedly at E10.5 in mice (Roulet et al. 2007). Although the site of vessel rupture or the exact cause for that remained unknown in the collagen V null mice, type V collagen was shown to be required for proper collagen fibril formation. Type V collagen participates as a quantitatively minor component to the formation of type I collagen fibrils and the lack of it prevents the fibril formation in the mesenchyme almost completely and the formed fibrils look abnormal (Wenstrup et al. 2004). The phenotype of the collagen V null mouse line is surprisingly severe, as the phenotype of the heterozygous mice resembles the classical type of Ehlers-Danlos syndrome (Wenstrup et al. 2004). The Plod2gt/gt embryos were nevertheless able to synthesize collagen fibrils. As a telopeptide LH, LH2 has a role in the synthesis of several collagen types. Lack of the enzyme may not prevent the synthesis of collagen molecules as they were seen in the extracellular space, but it may either affect the processing time, quality and/or the strength of the assembled structures as indicated earlier. Moreover, hydroxylysine residues at the triple helical regions of collagens serve as attachment sites for carbohydrates and the exact contribution of LH2 to the hydroxylation of these lysines is currently unknown. Thus, it is possible that lack of LH2 may affect the glycosylation status of collagens. It is probable that the lack of LH2 not only affects a specific collagen target but rather decreases the strength of ECM structures in general by changing the cross-link type. Underhydroxylation of the telopeptide lysines prevents the adequate formation of trivalent, mature, pyridinoline cross-links LP and HP, but the telopeptide lysyl hydroxylation also determines the type of immature divalent cross-link. At E10.5 mature collagen cross-links are not formed yet. Thus, divalent forms most probably predominate at this developmental stage, suggesting the importance of the immature forms of cross-links in developing tissues. Furthermore, our results show that the compensatory effect of the other two LH isoforms is not significant when LH2 activity is lacking. Thus, the embryonic lethality of LH2 null mice is most probably caused by the absence of the telopeptide lysyl hydroxylase activity. The dramatic consequence of the lack of one collagen-modifying enzyme is interesting as, for instance, the type I collagen null Mov 13 mice and collagen V knock-out mice die only a moment earlier or slightly later and the type III collagen knock-out mice may survive even after birth (Schnieke et al. 1983, Liu et al. 1997, Wenstrup et al. 2004). BS patients have skeletal problems such as osteoporosis, fragile bones, congenital joint contractures, progressive kyphoscoliosis and short stature. As

86 indicated earlier, unfortunately the E10.5 embryos are too young for the cross-link analysis as maturation is still ongoing, and due to the early embryonic lethality the skeletal consequences also remain unknown. However, it is interesting that the LH2 heterozygous mice also have significant changes in the cross-link pattern, particularly in bone, indicating that the amount of the LH2 needed in the process is rather high. Moreover, reduction in the amount of pyridinoline cross-links was more marked in the heterozygous calvaria than the femur. These bone types differ from each other in their origin and mechanism of ossification, which in turn affect their composition (Chung et al. 2004, van den Bos et al. 2008). The flat bone calvaria originates from the ectodermal neural crest, while the femur, representing a long bone, is of mesodermal origin (Chung et al. 2004). Flat bones are formed by straight intramembranous ossification, where the cells differentiate directly into bone-forming osteoblasts that produce a matrix that is particularly rich in type I collagen. Long bones, in turn, are ossified via a cartilage model, by endochondral ossification, which begins by a condensation of mesenchymal cells to chondrocytes. (Kronenberg 2003). The function of the long bones is to provide support for the body, whereas the role of calvaria, for instance, is more protective than supportive. Our results indicate that a decrease in LH2 activity may be less harmful in long bones. They are ossified via a cartilage model and LH2 may have more time to hydroxylate the telopeptide lysines when cartilage is replaced slowly by bone. The straight ossification process in the calvaria may in turn be too fast for the reduced amount of LH2 to keep up with and thus the collagen cross- linking is more affected. However, bone tissue is renewing constantly and an interplay between the contributing components is important. The protein composition in different bone types differs considerably, and for example the collagen amount in flat bones is higher than in long bones and the collagens are also more soluble (van den Bos et al. 2008). Interestingly, the levels of certain growth factors such as TGF-β are higher in flat bones than in long bones (Finkelman et al. 1994) and, furthermore, TGF-β is known to increase the expression of LH2(long) and the formation of pyridinoline cross-links (van der Slot et al. 2005). Thus, in addition to the time available for lysyl hydroxylation in different ossification processes, changes in the interplay of the contributing components of bone development may affect the cross-linking state of bones with reduced amounts of LH2. Moreover, both pyridinoline cross-links require telopeptide hydroxylysine, i.e. LH2(long), for their formation, and the difference between the LP and HP cross-link is only in the hydroxylation state of the helical lysine. Thus, it was interesting that the amount of HP was more severely affected

87 than the LP in the LH2 heterozygous mouse tissues. This indicates that LH2 is also involved in the hydroxylation of the helical lysine residue participating in cross-link formation.

88 7 Conclusions and future prospects

Today ECM is known to be a highly dynamic part of the human body and not just a scaffold for cells and tissues. Functional characteristics of collagens, the most common protein component of the ECM, are determined during collagen biosynthesis. In addition to collagen gene mutations, failures in the enzymes required for collagen biosynthesis and an imbalance of regulating factors, for instance, can lead to clinical consequences. Besides rare hereditary connective tissue disorders the role of ECM and its changes are now also emphasized in common disease states such as fibrosis and cancer that cause a large economic burden on the health care system. In this study we generated an LH1 null mouse line, which is the first animal model for human EDS VIA. Although the mice had generally milder symptoms, the LH1 null mice and EDS VIA patients share the same life-threatening phenotype i.e. risk of arterial ruptures. Structural weakness of arterial walls can be explained by inadequate helical lysine hydroxylation and the accompanied changes in the collagen cross-link pattern. Interestingly, in LH1 null mice the correlation between the hydroxylysine content and the amount of HP cross-links, which requires both telopeptide and helical hydroxylysine, was inconsistent. Thus, in vivo the two other isoforms, LH2 and LH3, most probably can compensate for the lack of LH1 activity, but the exact mechanism remains to be established. Interestingly, in LH2 null mice we observed that LH1 and LH3 are not able to compensate for at least the telopeptide LH activity. LH2 null mice died early during development, which was surprising as the human disorder BS2 is generally not lethal. However, our in vitro studies revealed that although the known LH2 mutations in BS2 patients are destructive and able to explain the clinical symptoms, they do not lead to complete inactivation of the enzyme. The known mutations affect the folding of the enzyme or interfere with the binding of a cosubstrate. Embyonic lethality of LH2 null mice raises the question whether a total lack of LH2 would be lethal also in humans. The exact cause of death of LH2 null mice remains unknown, but the results suggest that inadequate telopeptide lysyl hydroxylation and thus formation of pyridinolin cross-links may lead to a general weakness and collapse of tissue structures, particularly in the vessel walls. Unfortunately, analysis of collagen cross-links from LH2 null mice was impossible due to the early lethality, but the remarkable role of LH2 in cross- link formation was seen with heterozygous mice that had surprisingly severe changes in the cross-links, especially in the bone. Both mouse lines generated and

89 analyzed here emphasize the importance of LH1 and LH2 in proper cross-link formation. Although the LH2 gene-trap mouse line generated here has provided new data about LH2 and emphasized its role in mouse development, the embryonic lethal phenotype restricts studies of the contribution of LH2 to common pathological situations such as fibrosis. It would be important also to know the contribution of LH2 in tumorigenesis as the role of some other 2-oxoglutarate dioxygenases in cancer is remarkable and because increased expression levels of LH2 is connected to hypoxia and cancer. Thus, a conditional LH2 knock-out mouse line could provide answers to some of these questions.

90 References

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108 Yeowell HN, Walker LC, Murad S & Pinnell SR (1997) A common duplication in the lysyl hydroxylase gene of patients with Ehlers Danlos syndrome type VI results in preferential stimulation of lysyl hydroxylase activity and mRNA by hydralazine. Arch Biochem Biophys 347(1): 126–131. Zeng B, MacDonald JR, Bann JG, Beck K, Gambee JE, Boswell BA & Bächinger HP (1998) Chicken FK506-binding protein, FKBP65, a member of the FKBP family of peptidylprolyl cis-trans isomerases, is only partially inhibited by FK506. Biochem J 330(Pt 1): 109–114. Zhu Y, Oganesian A, Keene DR & Sandell LJ (1999) Type IIA procollagen containing the cysteine-rich amino propeptide is deposited in the extracellular matrix of prechondrogenic tissue and binds to TGF-beta1 and BMP-2. J Cell Biol 144(5): 1069– 1080. Zuurmond AM, van der Slot-Verhoeven AJ, van Dura EA, De Groot J & Bank RA (2005) Minoxidil exerts different inhibitory effects on gene expression of lysyl hydroxylase 1, 2, and 3: implications for collagen cross-linking and treatment of fibrosis. Matrix Biol 24(4): 261–270.

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110 Original articles

I Takaluoma K, Hyry M, Lantto J, Sormunen R, Bank RA, Kivirikko KI, Myllyharju J & Soininen R (2007) Tissue-specific changes in the hydroxylysine content and cross- links of collagens and alterations in fibril morphology in lysyl hydroxylase 1 knock- out mice. J Biol Chem 282(9):6588–6596. II Hyry M, Lantto J & Myllyharju J (2009) Missense mutations that cause Bruck syndrome affect enzymatic activity, folding, and oligomerization of lysyl hydroxylase 2. J Biol Chem 284(45):30917–30924. III Hyry M, Soininen R, Bank RA, Sormunen R, Miinalainen I, Talsta HL, Lantto J & Myllyharju J (2012) Lack of lysyl hydroxylase 2 in mouse leads to early lethality during embryonic development. Manuscript. Reprinted with permission from American Society for Biochemistry and Molecular Biology.

Original publications are not included in the electrical version of the dissertation.

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112 ACTA UNIVERSITATIS OULUENSIS SERIES D MEDICA

1143. Karjalainen, Minna (2011) Genetic predisposition to spontaneous preterm birth : approaches to identify susceptibility genes 1144. Saaristo, Timo (2011) Assessment of risk and prevention of type 2 diabetes in primary health care 1145. Vuononvirta, Tiina (2011) Etäterveydenhuollon käyttöönotto terveydenhuollon verkostoissa 1146. Vanhala, Marja (2012) Lapsen ylipaino – riskitekijät, tunnistaminen ja elintavat 1147. Katisko, Jani (2012) Intraoperative imaging guided delineation and localization of regions of surgical interest : Feasibility study 1148. Holmström, Anneli (2012) Etnografinen tutkimus natiivitutkimusten oppimisesta röntgenhoitajaopiskelijoiden opinnoissa 1149. Ronkainen, Hanna-Leena (2012) Novel prognostic biomarkers for renal cell carcinoma 1150. Murtomaa-Hautala, Mari (2012) Species-specific effects of dioxin exposure on xenobiotic metabolism and hard tissue in voles 1151. Jauhola, Outi (2012) Henoch-Schönlein purpura in children 1152. Toljamo, Kari (2012) Gastric erosions – clinical significance and pathology : a long-term follow-up study 1153. Meriläinen, Merja (2012) Tehohoitopotilaan hoitoympäristö : psyykkinen elämänlaatu ja toipuminen 1154. Liisanantti, Janne (2012) Acute drug poisoning: outcome and factors affecting outcome 1155. Markkanen, Piia (2012) Human δ opioid receptor : The effect of Phe27Cys polymorphism, N-linked glycosylation and SERCA2b interaction on receptor processing and trafficking 1156. Hannula, Annukka (2012) Imaging studies of the urinary tract in children with acute urinary tract infection 1157. Korvala, Johanna (2012) In quest of genetic susceptibility to disorders manifesting in fractures : Assessing the significance of genetic factors in femoral neck stress fractures and childhood non-OI primary osteoporosis 1158. Takala, Heikki (2012) Biomarkers in esophageal cancer

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