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Type Xviii Collagen

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Type Xviii Collagen TYPE XVIII COLLAGEN HARRI Characterization of the primary structure and expression ELAMAA pattern of different variants in Xenopus laevis, characterization of the human gene structure and analysis of transgenic mice Faculty of Medicine, expressing endostatin Department of Medical Biochemistry and Molecular Biology, Biocenter Oulu, University of Oulu OULU 2004 HARRI ELAMAA TYPE XVIII COLLAGEN Characterization of the primary structure and expression pattern of different variants in Xenopus laevis, characterization of the human gene structure and analysis of transgenic mice expressing endostatin Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of the Department of Pharmacology and Toxicology, on December 3rd, 2004, at 1 p.m. OULUN YLIOPISTO, OULU 2004 Copyright © 2004 University of Oulu, 2004 Supervised by Professor Taina Pihlajaniemi Reviewed by Docent Klaus Elenius Doctor Takako Sasaki ISBN 951-42-7568-3 (nid.) ISBN 951-42-7569-1 (PDF) http://herkules.oulu.fi/isbn9514275691/ ISSN 0355-3221 http://herkules.oulu.fi/issn03553221/ OULU UNIVERSITY PRESS OULU 2004 Elamaa, Harri, Type XVIII collagen. Characterization of the primary structure and expression pattern of different variants in Xenopus laevis, characterization of the human gene structure and analysis of transgenic mice expressing endostatin Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology, and Biocenter Oulu, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland 2004 Oulu, Finland Abstract In this work the type XVIII collagen has been studied by using several approaches, such as different animal models. The primary structure of frog, Xenopus laevis, type XVIII collagen and the expression pattern of its variants during early embryogenesis have been elucidated. The gene structure of human type XVIII collagen was characterized and the localization and processing of its longest variant was studied by generated antibodies. In addition, the function of the proteolytically released C-terminal part of type XVIII collagen, endostatin, was studied by generating transgenic mice expressing endostatin. The primary structure of X. laevis type XVIII collagen is comprised of three N-terminal variants resembling their mammalian counterparts. The sizes of the polypeptides are 1285, 1581, and 1886 residues. The most conserved regions are the C-terminal endostatin region and the cysteine-rich domain in the N-terminus. Whole-mount in situ hybridization reveals different expression patterns for variants during embryogenesis. The short variant is the most abundant, whereas the two longest variants exhibit more restricted expression. The gene structure of human type XVIII collagen reveals an exon-intron organization that is conserved with mouse. The length of the human gene is about 105 kb and contains 43 exons. The third variant of type XVIII collagen has a conserved cysteine-rich domain with homology to the extracellular part of frizzled proteins. This third variant is localized to developing muscle and lung, and is also found in serum. In cell culture, the proteolytic fragments of the N-terminus, including the cysteine-rich motif, are also detected. Endostatin function was studied by generating mouse lines expressing endostatin under the keratin-14 promoter, which drives the expression mainly in the skin. Three independent transgenic mouse lines were achieved with varied expression levels. The phenotype was seen in the eye with lens opacity and abnormal morphology of epithelial cells in the lens. In the skin, a broading of the basement membrane in the epidermis dermis junction was detected. Immunoelectron microscopy analysis revealed a polarized orientation of type XVIII collagen in the basement membrane. In transgenic mice, altered localization of endogenous type XVIII collagen was seen, suggesting displacement of the endogenous type XVIII collagen with transgenic endostatin leading to disorganized basement membrane. Keywords: basement membrane, collagen type XVIII, endostatins, protein isoforms Acknowledgements This research was carried out at the Department of Medical Biochemistry and Molecular Biology. I wish to thank my supervisor, Professor Taina Pihlajaniemi, for giving me the opportunity to work in her group and introducing me to the field of matrix biology. I wish to express my appreciation and respect for Research Professor emeritus Kari Kivirikko for creating, together with Professor Taina Pihlajaniemi, excellent conditions for many scientists. I also wish to thank Professor emeritus Ilmo Hassinen, Professor Leena Ala- Kokko, Docent Johanna Myllyharju, and Professor Peppi Karppinen for their contributions to department management. I am very grateful to Docent Klaus Elenius and Dr. Takako Sasaki for their accurate review and valuable comments on my thesis manuscript. Sandra Hänninen, M.Sc. is acknowledged for her careful revision of the language of the manuscript. The collaboration between different laboratories and researchers has been crucial for my thesis. I would like to thank all my co-authors for their invaluable efforts. I wish to express my deepest gratitude to Dr. Olivier Destrée and the members of his research group in the Hubrecht laboratory in Utrecht. Dr. Destrée and his group wished me warmly welcome to their group and introduced me to the frog world during my several visits. I wish to thank Docent Helena Autio-Harmainen for her contribution to this work. I also wish to thank Docent Raija Sormunen and her excellent team for sharing their expertise in ultrastructural analysis. I am grateful for the valuable comments from Docent Raija Soininen, and expert in transgenic technology. Particularly, I am very grateful to Marko Rehn for helping me to take my first steps in the scientific field, and his support and advice during these years. Especially I wish to thank Päivi Tuomaala for her excellent technical assistance, which has been extremely valuable for my thesis. I also wish to thank Outi Laurila, Aira Harju, Sirpa Kellokumpu and Anna-Liisa Oikarinen for their valuable technical assistance. I am also grateful to the people in the laboratory animal center, Liisa Asikkala, Sirpa Tausta and Päivi Moilanen. In addition, special thanks go to Marja-Leena Kivelä and Auli Kinnunen for their kind help in secretarial matters, Seppo Lähdesmäki and Pertti Vuokila their help in a variety of practical matters, and Ari-Pekka Kvist and Risto Helminen for helping me with the computers. I have been privileged to work with pleasant researchers who are willing to offer help and consultation. I would like to thank all members of Taina´s group for their support. I am grateful to Ritva Heljasvaara for her comments on my thesis manuscript. I would especially like to thank my friends Joni Mäki and Lauri Eklund. We have shared a lot during our work and leisure time. I also want to thank all of my colleagues from the old downstairs office, L023, for creating a friendly atmosphere. I owe my deepest gratitude to my parents Maire and Eero for their love and support during these years, and my warmest thanks to my brother Kari and sister Satu and their families for their friendship and many hilarious moments. Above all, I owe my greatest gratitude to my wife Sari for her love and caring, and sharing everyday life. You have encouraged me and reminded me of the existence of intriguing life outside of work. I am deeply indebted to our new family member, the sweet daughter, who came to brighten our family life and cheered up her father during the thesis process. This work was supported by grants from the Emil Altonen Foundation, the Research and Science Foundation of Farmos, and the Finnish Cultural Foundation. Oulu, October 2004 Harri Elamaa Abbreviations BM basement membrane bp basepairs C- carboxy- COL collagenous domain CRD cysteine-rich domain fz Frizzled domain GAG glycosaminoglycan HSPG heparin sulphate proteoglycan IEM immunoelectron microscopy kb kilobase kDa kilodalton K14 keratin 14 N- amino- NC non-collagenous PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction RT-PCR reverse transcription polymerase chain reaction SDS sodium dodecyl sulphate VEGF vascular endothelial growth factor List of original articles This thesis is based on the following articles, which are referred to in the text by their Roman numerals: I Elamaa H, Peterson J, Pihlajaniemi T & Destrée O (2002) Cloning of three variants of type XVIII collagen and their expression patterns during Xenopus laevis development. Mech Dev 114: 109-13. II Elamaa H, Snellman A, Rehn M, Autio-Harmainen H & Pihlajaniemi T (2003) Characterization of the human type XVIII collagen gene and proteolytic processing and tissue location of the variant containing a frizzled motif. Matrix Biol 22: 427-42. III Elamaa H, Sormunen R, Rehn M, Soininen R & Pihlajaniemi T (2004) Endostatin overexpression specifically in the lens and skin leads to cataract and ultrastructural alterations in basement membranes. Am. J. Pathol., In press. Contents Abstract Acknowledgements Abbreviations List of original articles Contents 1 Introduction ...................................................................................................................13 2 Review of the literature .................................................................................................14 2.1 Cryptic angiogenesis inhibitors ..............................................................................14 2.2 Collagen superfamily..............................................................................................15
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