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TYPE XIII COLLAGEN Structural and functional characterization of the ectodomain and identification of the binding ligands HONGMIN TU Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu OULU 2004 HONGMIN TU TYPE XIII COLLAGEN Structural and functional characterization of the ectodomain and identification of the binding ligands Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of the Main Building of the Medical Campus, on April 16th, 2004, at 12 noon. O U L U N Y L I O P I S TO, O U L U 2 0 0 4 Copyright © 2004 University of Oulu, 2004 Supervised by Professor Taina Pihlajaniemi Reviewed by Docent Nisse Kalkkinen Professor Cay Kielty ISBN 951-42-7318-4 (nid.) ISBN 951-42-7319-2 (PDF) http://herkules.oulu.fi/isbn9514273192/ ISSN 0355-3221 OULU UNIVERSITY PRESS OULU 2004 http://herkules.oulu.fi/issn03553221/ Tu, Hongmin, Type XIII collagen Structural and functional characterization of the ectodomain and identification of the binding ligands Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland 2004 Oulu, Finland Abstract Type XIII collagen is a transmembrane protein consisting of a short intracellular portion, a transmembrane anchor, and a long extracellular domain with a mainly collagenous sequence. Histochemical and cell biological studies have revealed that type XIII collagen has a wide distribution in various tissues and that it is mostly localized to cell-cell and cell-matrix contacts. In order to study type XIII collagen at the molecular level, the protein was expressed in insect cells as a homotrimer. The recombinant protein was found to reside in the plasma membrane of insect cells with its N-terminus intracellular and C-terminal part extracellular, i. e. in a type II orientation. The trimerization of type XIII collagen chains was initiated by 21 amino acid residues adjacent to the transmembrane domain on the extracellular side, and this sequence was found to be conserved in several other collagenous transmembrane proteins. In addition to the transmembrane form, the ectodomain of type XIII collagen was secreted into the cell culture medium, a result of proteolytic cleavage by furin-like proteases at the non-collagenous NC1 domain. The ectodomain was purified from the insect cell culture medium with a typical collagenous composition and conformation, and it showed as a 150 nm-long rod in rotary shadowing electron microscopy. Furthermore, the recombinant ectodomain showed high affinity binding to several extracellular matrix proteins, e. g. fibronectin, nidogen-2, and perlecan, as well as to heparin. The type XIII collagen ectodomain also showed selective recognition to collagen receptor integrins. Integrin α1 and α11 I domains bind to type XIII collagen with a high affinity, and both integrins α1β1 and α11β1 mediate cell attachment to type XIII collagen. The present results suggest that type XIII collagen shares common aspects with other collagenous transmembrane proteins in terms of chain association and ectodomain shedding. However, it is notably distinct in its structure and binding specificity compared to other types of collagen and cellsurface proteins. The data imply that type XIII collagen might participate in multiple cell-cell and cell-matrix interactions. Keywords: cell adhesion, chain association, collagen, extracellular matrix, integrin Acknowledgements This work was carried out at Biocenter Oulu and the Department of Medical Biochemistry and Molecular Biology, University of Oulu, during the years 1997-2003. I wish to express my deepest gratitude to my supervisor, Professor Taina Pihlajaniemi, M. D., Ph. D. for giving me the opportunity to work in her excellent group and for introducing me to the field of matrix biology. Her well-conceived guidance and enthusiastic support during each step of the thesis process have been of great benefit to the present work. I also wish to express my appreciation and respect for Research Professor emeritus Kari Kivirikko, M. D., Ph. D. for his never-ending enthusiasm for stimulating innovative and pioneering scientific projects. Professor Leena Ala-Kokko, M. D., Ph. D. is highly respected for her great scientific achievements and excellent leadership. Docent Johanna Myllyharju, Ph. D. deserves my sincere gratitude for her fruitful research accomplishments and her guidance, help, and caring towards me during these years. Professor emeritus Ilmo Hassinen, M. D., Ph. D. and Professor Peppi Karppinen, M. D., Ph. D. are warmly acknowledged for their contributions to department management and in creating a friendly working environment. Professor Cay Kielty, Ph. D. and Docent Nisse Kalkkinen, Ph. D. are warmly acknowledged for their valuable comments and criticisms on my thesis manuscript. I am also deeply grateful to Sandra Hänninen, M. Sc. for her fast and careful revision of the language of this book. Part of the research work in this thesis, which has been of crucial importance, was performed in several collaborating laboratories. The late Dr. Rupert Timpl and Dr. Takako Sasaki are warmly acknowledged for sharing their outstanding expertise in matrix biology and their efficient, logical, and focused ways in research, in addition to providing me the opportunity to initiate the structural and biochemical analyses of type XIII collagen in their world-class lab. I also wish to acknowledge Professor Dick Heinegård for allowing me to learn the surface plasmon resonance technique in his well-organized group and state-of-the-art facilitated lab. Particularly, I am deeply grateful to Charlotte Wiberg, Ph. D. for teaching me to operate the Biacore™ instrument step by step. Päivi Pirilä, Ph. D. deserves my appreciation for her patience and excellent knowledge of biophysical chemistry that are very important for analyzing the kinetic protein-ligand interaction data. Professor Kalervo Hiltunen, M. D., Ph. D. is acknowledged for familiarizing me with the circular dichroism instrument and for allowing me to do part of the biochemical analysis experiments in the well-equipped and pleasant lab at the Department of Biochemistry. Moreover, I am grateful to Professor Jyrki Heino, M. D., Ph. D. for attracting me to the fascinating integrin research field and for proposing the collaboration with his intelligent group members, especially Jarmo Käpylä, Ph. D. and Petri Nykvist, Ph. D. Professor Donald Gullberg, Ph. D. deserves my sincere respects for his knowledge of cell biology and for collaborating with his excellent group. My colleagues at the Department of Medical Biochemistry and Molecular Biology own my deep gratitude. Especially, I wish to thank Anne Snellman, Ph. D. for her patient guidance when I started the projects, her help and friendship during these years. Furthermore, Anne and the other pioneers of the XIII team, Pikko Huhtala, Ph. D., Pasi Hägg, M. D., Ph. D., Ari-Pekka Kvist, Ph. D., and Malin Sund, M. D., Ph. D. are warmly acknowledged for their contributions to exploring the importance of the type XIII collagen, which formed the essential basis of the work presented in this thesis. I also want to express my sincere appreciation to others working in our team for their never-fading passion and unremitting efforts towards the challenging scientific projects. This thesis could not have been completed without the excellent technical assistance of Sirkka Vilmi, and it would have taken much longer without the considerable help of Liisa Äijälä with my other tasks at the core facility of Biocenter Oulu. In addition, the extremely supportive administration, laboratory management, and technical service at the Department of Medical Biochemistry and Molecular Biology cannot be neglected. Thereby, Marja-Leena Kivelä, Auli Kinnunen, Pertti Vuokila, Marja Leena Karjalainen, Seppo Lähdesmäki, and Risto Helminen are due my special thanks for their conscientious work and genuine kindness. The present thesis benefitted enormously from the seminars and courses organized by Biocenter Oulu, as well as its financial support for my working visits to the collaborating laboratories. I also benefitted from my previous working experience in my home country. Herein, I wish to acknowledge Professors Xia Qichang, Lin Qishui, and Li Boliang at the Shanghai Institute of Biochemistry, Chinese Academy of Sciences, for providing me the envying opportunities to get training and experience in the academic field. I would not have enjoyed life in Oulu so much without my dear friends and family. Here, I wish to express my sincere admiration to Docent Raija Sormunen, Ph. D. for her optimistic attitude towards life and amazing interests in Asian culture. I also thank my Chinese friends in Oulu for sharing their experience and knowledge in many aspects. My parents, Jin Shunfang and Tu Xinyuan, are sincerely acknowledged for their encouragement and support for so many years. Above all, I want to thank my husband, Jingdong Shan, and our son, Yuanqi for their supreme love. This thesis could not have been possible were it not for their caring and understanding. This work was mainly supported by grants from the Finnish Centre of Excellence Programme (2000-2005) of the Academy of Finland (44843), the European Commission (BIO4-CT96-0537 and QLK3-2000-00084), and the Sigrid Jusélius Foundation. Oulu, March 2004 Hongmin Tu Abbreviations aa Aβ ADAMs AMY ATCC C1q amino acid amyloid β peptide a disintegrin-and-metalloproteinase Alzheimer disease amyloid-associated protein American Type Culture Collection a collagen-like subcomponent of the first component of complement, C1 CD circular dichroism C. elegans Caenorhabditis elegans CHO Chinese hamster ovary CLAC-P collagen-like Alzheimer amyloid plaque component precursor COL collagenous domain DMEM Dulbecco’s modification of Eagle’s medium ECM extracellular matrix EDA ectodysplasin-A EDTA ethylenediaminetetraacetic acid EGFP enhanced green fluorescent protein ELISA enzyme-linked immunosorbent assay FACIT fibril-associated collagens with interrupted triple helices FCS fetal calf serum GCG Genetics Computer Group GST glutathione S-transferase kDa kilo daltons KD dissociation constant MARCO macrophage receptor with collagenous structure MEFs mouse embryonic fibroblasts MIDAS metal ion-dependent adhesion site MOI multiplicity of infection MSR macrophage scavenger receptor NC noncollagenous MULTIPLEXIN multiple triple helical domains and interruptions PAGE PCR PVDF RNAi SPARC SPR SRCL SV40 TRITC Xaa Yaa polyacrylamide gel electrophoresis polymerase chain reaction polyvinylidene fluoride RNA interference secreted protein, acidic and rich in cysteine surface plasmon resonance scavenger receptor with C-type lectin Simian Virus 40 tetramethyl rhodamine isothiocyate any amino acid any amino acid List of original articles This thesis is based on the following articles, which are referred to in the text by their Roman numerals: I Snellman A, Tu H, Väisänen T, Kvist A-P, Huhtala P & Pihlajaniemi T (2000) A short sequence in the N-terminal region is required for the trimerization of type XIII collagen and is conserved in other collagenous transmembrane proteins. EMBO J 19: 5051-5059. II Tu H, Sasaki T, Snellman A, Göhring W, Pirilä P, Timpl R & Pihlajaniemi T (2002) The type XIII collagen ectodomain is a 150-nm rod and capable of binding to fibronectin, nidogen-2, perlecan, and heparin. J Biol Chem 277: 23092-23099. III Nykvist P, Tu H, Ivaska J, Käpylä J, Pihlajaniemi T & Heino J (2000) Distinct recognition of collagen subtypes by α1β1and α2β1 integrins. α1β1 mediates cell adhesion to type XIII collagen. J Biol Chem 275: 8255-8261. IV Tu H, Rodriguez-Sanchez B, Popova S, Zhang W-M, Gullberg D & Pihlajaniemi T Type XIII collagen is a high-affinity ligand for α11β1 integrin. Manuscript. Contents Abstract Acknowledgements Abbreviations List of original articles Contents 1 Introduction ...................................................................................................................15 2 Review of the literature .................................................................................................17 2.1 Extracellular matrix ................................................................................................17 2.1.1 Collagen...........................................................................................................17 2.1.2 Fibronectin.......................................................................................................19 2.1.3 Basement membrane proteins..........................................................................19 2.2 Cell receptors for extracellular matrix proteins ......................................................21 2.2.1 Integrins...........................................................................................................22 2.3 Membrane-associated proteins with collagenous domains.....................................23 2.3.1 Type XIII collagen...........................................................................................25 2.3.1.1 Gene structure and chromosomal localization of type XIII collagen........25 2.3.1.2 Cellular and tissue localization of type XIII collagen...............................26 2.3.1.3 Recombinant expression of type XIII collagen.........................................26 2.3.1.4 Physiological functions of type XIII collagen ..........................................26 2.3.1.5 Chain association and shedding................................................................27 2.3.2 Type XIII collagen-like collagens....................................................................28 2.3.2.1 Type XXIII collagen .................................................................................28 2.3.2.2 Type XXV collagen (CLAC-P).................................................................28 2.3.3 Type XVII collagen .........................................................................................29 2.3.4 Other transmembrane proteins with collagenous sequences............................29 2.4 Assays for protein-ligand interactions ....................................................................29 2.4.1 Solid phase assays ...........................................................................................30 2.4.2 Surface plasmon resonance assays ..................................................................30 2.4.3 Other assays.....................................................................................................31 3 Outlines of the present research.....................................................................................32 4 Materials and methods................................................................................................... 33 4.1 Recombinant production of human type XIII collagen in insect cells (I, II, III, IV) ......................................................................................................................................33 4.1.1 Construction of recombinant baculoviruses (I)................................................33 4.1.2 Expression of recombinant type XIII collagen in insect cells (I).....................34 4.1.3 Immunofluorescence staining of insect cells expressing type XIII collagen (I) ..................................................................................................................................34 4.1.4 N-terminal sequencing of type XIII collagen expressed in insect cells (I) ......35 4.1.5 Inhibition of type XIII collagen shedding in insect cells by a furin protease inhibitor and by heparin (I, II) ..................................................................................36 4.1.6 Purification of the recombinant human type XIII collagen ectodomain (II, III, IV) ............................................................................................................................36 4.1.7 Isolation of pepsin-resistant fragments from the recombinant type XIII collagen (II) ..............................................................................................................37 4.2 Production of integrin I domains (III, IV) ..............................................................37 4.3 Europium labelling of the recombinant integrin α1 and α2 I domains (III)...........38 4.4 Expression of recombinant integrin α1β1 and α2β1 in CHO cells (III) ................38 4.5 Binding assays (II, III, IV)......................................................................................38 4.5.1 Solid phase assays (II, III, IV) .........................................................................38 4.5.2 Surface plasmon resonance assays (II) ............................................................39 4.5.3 Heparin affinity chromatography (II) ..............................................................40 4.6 Cell spreading assays (III, IV) ................................................................................40 4.6.1 Assays for integrin α1β1- or α2β1-mediated cell attachment (III) .................40 4.6.2 Assays for integrin α11β1-mediated cell spreading (IV).................................40 4.7 Monoclonal antibody production (II) .....................................................................41 4.8 Electron microscopy (I, II) .....................................................................................41 4.8.1 Immunoelectron microscopy of insect cells expressing type XIII collagen (I)42 4.8.2 Rotary shadowing electron microscopy (II) ....................................................42 4.9 Circular dichroism spectroscopy (II) ......................................................................42 4.10 Sequence comparison of collagenous transmembrane proteins (I).......................43 5 Results ...........................................................................................................................44 5.1 Type XIII collagen is a type II transmembrane protein (I) .....................................44 5.2 A conserved plasma membrane-adjacent sequence is important for type XIII collagen chain association (I) .......................................................................................45 5.3 The ectodomain of type XIII collagen is partly shed to the cell culture medium (I, II)..................................................................................................................................45 5.4 The type XIII collagen ectodomain is a collagenous rod with two flexible hinges (II).................................................................................................................................46 5.5 The recombinant type XIII collagen ectodomain interacts with several major extracellular matrix proteins in vitro (II) ......................................................................47 5.6 Heparin is a high-affinity ligand of type XIII collagen (II) ....................................48 5.7 The type XIII collagen ectodomain selectively recognizes integrin α1 and α11 I domains (III, IV)...........................................................................................................49 5.8 Integrins α1β1 and α11β1 mediate cell attachment to type XIII collagen (III, IV)49 5.9 Localization of α11β1 integrin on cells spreading on type XIII collagen is associated with the focal adhesion molecule vinculin (IV) ..........................................50 6 Discussion ..................................................................................................................... 51 7 Future perspectives........................................................................................................56 References 1 Introduction The extracellular matrix (ECM) surrounds cells and consists of collagens, proteoglycans, and other molecules that interact with each other, as well as with the cell-surface proteins, to form complex and irregular networks. The ECM exerts mechanical forces on the cells and also supplies feedback signals to them. Most cells in connective tissues have to be attached and spread on the ECM to survive; if the contact is destroyed, the cells start a process of apoptosis caused by anoikis, the ancient Greek word for homelessness. Therefore, elucidation of structural properties of ECM proteins and identification of their ligands may shed some light on the biological roles of cell-ECM interactions. Type XIII collagen is a type II transmembrane protein composed of three collagenous domains (COL 1-3) separated and flanked by four non-collagenous domains (NC 1-4). A highly hydrophobic transmembrane segment, located at the N-terminal NC1 domain, anchors the protein to the cell membrane. Previous in situ hybridization analyses showed a wide distribution of type XIII collagen in muscle, skin, bone, and other various connective tissues. More intensive immunostaining studies revealed the location of type XIII collagen at focal adhesion sites of the cultured fibroblast cells, in myotendinous junctions in skeletal muscle, in intercalated discs of cardiac muscle, and at cell-cell contacts within eye retina, which imply that type XIII collagen has a cell adhesion-related function. However, the expression level of type XIII collagen in native tissues or cultured mammalian cells is rather low, which makes it difficult to isolate the native protein for molecular characterization of its structure and function. The successful expression of the recombinant human type XIII collagen homotrimer in insect cells has facilitated the investigation of its molecular structure and chain association. Different constructs with various deletions were expressed to study the triple helical formation and the ectodomain secretion. The critical sequences for type XIII collagen chain association were aligned with other collagenous transmembrane proteins to demonstrate their importance and conservation. The secreted type XIII collagen ectodomain was isolated from the culture medium of the insect cells infected with the virus coding for type XIII collagen lacking the cytosolic portion, which showed the highest shedding efficiency. Furthermore, the purified type XIII collagen was analyzed with rotary shadowing electron microscopy to reveal its collagenous structure, and other molecular features were studied by various biochemical assays. 16 Work has been directed towards discovering the function of this ubiquitous collagen. Although a type XIII collagen null-mouse is not yet available, defects in genetically modified mice with deletions at the NC1 or COL2 domains have led to the suggestion that type XIII collagen participates in the stabilization of the basement membrane and maintains the adherence junctions in the heart, which implies possible interactions between type XIII collagen and ECM or cell surface molecules. Thus, the putative ECM binding ligands within the major ECM components were searched using solid phase and surface plasmon resonance assays. In addition, the interactions between type XIII collagen and collagen receptor integrins were studied. These studies contribute to the clarification of the molecular properties of type XIII collagen molecules and to understanding the roles of type XIII collagen in cell-ECM and cell-cell adherence. 2 Review of the literature Cell-cell adherence and cell-extracellular matrix interactions are important for growth, development, wound healing, immune responses, and other various physiological processes in biological bodies. There are huge numbers of studies on the molecular, cellular, and pathological aspects in this field, which is impossible to be reviewed in detail in this section. Therefore, the general information regarding the biological molecules studied in this thesis and the central techniques involved in the studies were selected for discussion. 2.1 Extracellular matrix The extracellular matrix (ECM) is an intricate meshwork-like substance that underlies all epithelia and endothelia, and surrounds all connective tissue cells. It promotes cell adhesion, migration, differentiation, and proliferation and provides a supporting structure to which cells adhere. The ECM is composed of two major classes of macromolecules, i.e. fibrous proteins (e.g. collagen, elastin, fibrillin, fibronectin and laminin) and proteoglycans, usually composed of a protein core covalently linked to glycosaminoglycan chains. Due to the topic of the thesis study, this review focuses on collagens (2.1.1), fibronectin (2.1.2), and several basement membrane proteins (2.1.3). 2.1.1 Collagen The collagens are a family of proteins with multiple functions in the constitution and maintenance of the ECM of most organisms (Pihlajaniemi & Rehn 1995, Prockop & Kivirikko 1995, Myllyharju & Kivirikko 2001, Kielty & Grant 2002, Myllyharju & Kivirikko 2004). All collagen molecules are composed of three polypeptide chains, i.e. α chains, which form a triple helix. The three α chains can be identical or different. The collagenous sequences are usually composed of the repeats of Gly-Xaa-Yaa, which are important for the collagen triple helix formation. The Yaa position in the collagenous 18 sequence is frequently hydroxyproline or hydroxylysine, which increase the stability of the triple helix. To date, 27 types of collagen have been identified based on the primary structure of the collagen protein (Myllyharju & Kivirikko 2001, Kielty & Grant 2002, Myllyharju & Kivirikko 2004). The most abundant collagens, types I, II and III, are fibril-forming collagens with widespread distribution and structural roles in connective tissues that support the major organs. The microfibrillar collagen type VI, which assembles extensive filaments in the ECM, is a major collagenous component of skin. Collagen types V, VII, and XI are also involved in forming fibrils. The FACIT collagens, types IX, XII, XIV, XVI and XX, are associated with the generation of interstitial collagen fibers by decorating the surface of the fibrils and organizing them into correct three-dimensional patterns within the ECM. In addition, two types of newly identified collagens, XIX and XXI, are structurally related to FACIT collagens based on several features in the sequences of these collagen types. Although type XIX collagen is mostly located at basement membrane zones, it is not clear yet if these two collagen types are involved in interstitial collagen formation (Chou & Li 2002, Myers et al. 2003). The fibrillar collagen groups were expanded by two new members, types XXIV (Koch et al. 2003) and XXVII (Boot-Handford et al. 2003, Pace et al. 2003). Besides the interstitial collagens, there are several types of network forming collagens in the ECM. Type IV collagen is one of the major widespread components forming networks of cell basement membrane, where two members of MULTIPLEXIN collagen, types XV and XVIII, are also located (Rehn & Pihlajaniemi 1996, Erickson & Couchman 2000, Ortega & Werb 2002, Kalluri 2003). All of these collagens are conserved in Drosophila and C. elegans (Hutter et al. 2000, Hynes & Zhao 2000). Interestingly, all three basement membrane collagens are the precursors of angiogenesis inhibitors (Marneros & Olsen 2001), namely arresten from the collagen IV α1 chain (Colorado et al. 2000), canstatin from the collagen IV α2 chain (Kamphaus et al. 2000), tumstatin from the collagen IV α3 chain (Maeshima et al. 2000), restin from collagen XV (Ramchandram et al. 1999), and endostatin from collagen XVIII (O’Reilly et al. 1997). Other network-forming collagens are types VIII and X, which assemble to form hexagonal lattices surrounding the corneal endothelial cells (type VIII) or in cartilage (type X) (Myllyharju & Kivirikko 2001, Kielty & Grant 2002, Myllyharju & Kivirikko 2004). The collagen types XIII, XVII, XXIII, and XXV are grouped into transmembrane collagens composed of an N-terminal cytosolic portion, a transmembrane anchor, and an extracellular domain containing interrupted collagenous sequences (Myllyharju & Kivirikko 2001, Kielty & Grant 2002, Myllyharju & Kivirikko 2004). The structure and physiological functions of these proteins are discussed in more detail in section 2.3. Two newly identified collagen types remain to be defined in terms of their roles in ECM formation. One is type XXII collagen with its cDNA sequence published in GenBank (Acc. No. AF406780), and another is type XXVI collagen, which is specifically expressed in testis and ovary as a secreted protein (Sato et al. 2002). It is well-known that collagens are the most abundant proteins in the mammalian body and they are the major proteins comprising the ECM. However, different types of collagen showed distinct functions due to their various structures and cellular locations, which have been demonstrated by the mutations detected in human patients and 19 genetically modified animals (Vuorio 1986, Vuorio & de Crombrugghe 1990, Kivirikko 1993, Olsen 1995, Prockop & Kivirikko 1995, Myllyharju & Kivirikko 2001, Kielty & Grant 2002, Myllyharju & Kivirikko 2004). 2.1.2 Fibronectin Fibronectin is a large glycoprotein found in all vertebrates, and it exhibits structural and adhesive features in cell-associated fibrillar matrices. Fibronectin is encoded by one gene and the protein is composed of two 220-kDa polypeptides linked at their C-terminal domains by two disulfide bonds. The rest of the molecule folds to form several individual domains with specific binding affinity to collagens, cell surface receptors, and heparin sulphate, respectively. Therefore, fibronectin can act as an anchor between proteoglycan and cell receptors (Ruoslahti 1999, Pankov & Yamada 2002). The cell surface receptor binding domain of fibronectin contains a unique amino acid sequence RGD for integrin recognition (Ruoslahti & Pierschbacher 1987, Ruoslahti 2003). The fibronectin used in most studies, including the experiments for the original article II in this thesis, is isolated from human plasma, where it is in a soluble form and circulates in the blood and other body fluids. However, fibronectin can also be assembled at cell surfaces into insoluble fibronectin filaments that are released into the ECM (Morla et al. 1994, Hynes 1999b). It has been well understood that fibronectin is one of the primary cell adhesion molecules that regulates cell migration, differentiation, and growth (Hynes 1990, Geiger et al. 2001). In addition, it plays important roles in development and wound healing (Geiger et al. 2001). Lack of fibronectin resulted in fetal lethality in mice characterized by abnormalities in the formation of the extra embryonic membranes, notochord, blood vessels and heart (George et al. 1997). 2.1.3 Basement membrane proteins Typically the basement membrane is a zone about 100 nm thick consisting of a network of intrinsic macromolecular components covering epithelia and surrounding muscle cells, fat cells and peripheral nerve axons (Beck & Gruber 1995). One subdivision of basement membrane is basal lamina that is visualized under an electron microscope as an electrondense membrane 30-70 nm in thick and lamina lucida. The main constituents of the basement membrane network are type IV collagen, laminins, nidogens, and heparan sulphate proteoglycans (Yurchenco & Schittny 1990, Yurchenco & O'Rear 1994). The basement membrane network attached to the epithelial cells is crucial for maintaining the mechanical integrity of the epidermis. This has been revealed by several autoimmune blistering diseases of the skin that are caused by disruption of cell-basement membrane junctions (Stanley 1995). In addition, basement membranes provide cues for epithelial cell polarization (Drubin & Nelson 1996). In muscles the basement membrane is responsible for localization of the cell receptors and transporters (Hall 1995, Gumbiner 1996). The C. elegans genome sequence reveals that the basement membrane protein 20 components are highly conserved through metazoan evolution (Hutter et al. 2000, Hynes & Zhao 2000). Furthermore, the atomic resolution structural studies suggest that the nature and location of the protein-protein interaction surfaces in basement membranes are also highly conserved (Hopf et al. 2001). Type IV collagen is a major component in most basement membranes. In mammals six genes, COL4A1-COL4A6, encode the six α chains (α1(IV)-α6(IV)) of type IV collagen, by which three triple helical protomers, (α1)2α2(IV), α3α4α5(IV), and (α5)2α6(IV), are formed (Soininen et al. 1987, Hostikka & Tryggvason 1988, Hostikka et al. 1990, Myers et al. 1990, Leinonen et al. 1994, Mariyama et al. 1994, Oohashi et al. 1994, Zhou et al. 1994, Hudson et al. 2003). (α1)2α2(IV) exists in all basement membranes, while α3α4α5(IV) and (α5)2α6(IV) are restricted mostly to the kidney, lung, testis, cochlea, and eye (Kleppel et al. 1989, Hudson et al. 2003). The selection of the α chain for trimer formation is determined by the molecular recognition sequences in the non-collagenous domain of the C-terminus (Boutaud et al. 2000, Borza et al. 2001). Due to the frequent interruption of collagenous Gly-Xaa-Yaa sequence repeats, the triple helical structure of type IV collagen is more flexible than that of fibrillar collagens. However, type IV collagen molecules are self-assembled by covalent interactions at the N- and C-termini to form stable networks (Langeveld et al. 1991, Sundaramoorthy et al. 2002). Type IV collagen is directly involved in two human diseases, Alport’s syndrome and Goodpasture’s syndrome (Hudson et al. 1993, Tryggvason et al. 1993, Tryggvason 1996, Hudson et al. 2003). Besides type IV collagen, laminins are also important components forming the network in basement membranes. Laminins self-assemble, providing a bridge between the cell surface and basal lamina due to their long length, which allows them to span the entire width of basement membrane, as well as their ability to bind to type IV collagen and cell surface receptors including integrins (Timpl 1996). Laminin proteins are a large family of heterotrimeric glycoproteins composed of 3 distinct polypeptide chains that form a cruciform molecule with 1 long arm and 3 short arms (Beck & Gruber 1995). To date, at least 18 different laminin isoforms have been identified (Colognato & Yurchenco 2000). More and more data have suggested that laminins play important roles in development, cell differentiation, and migration (Aumailley & Smyth 1998, Gullberg et al. 1999). One interesting finding is that laminin-1, together with type IV collagen, appears at the 2-cell stage when using an in vitro early mammalian development model, although in vivo the three chains of laminin 1 are detected later, i.e. γ1 and β1 at the 8-cell stage of the pre-implantation mouse embryo, and the α1 chain at the 16-cell stage (Hogan et al. 1994, Ekblom et al. 2003). These data suggest that laminin is possibly the earliest adhesion protein during embryonic development. In addition, recent studies, using differentiating embryoid bodies derived from mouse embryonic stem cells, revealed that embryonic basement membrane assembly and differentiation requires laminin polymerization and interaction with cell surfaces (Li et al. 2002). The supramolecular assembly of the basement membrane network requires another group of proteins, nidogens, which form stable complexes with laminin and bind to type IV collagen and perlecan (Mayer et al. 1995, Timpl 1996, Timpl & Brown 1996, Hopf et al. 1999, Erickson & Couchman 2000, Hutter et al. 2000). In mammals, two nidogen isoforms, nidogen-1 and nidogen-2, have been identified, and nidogen-2 compensates for nidogen-1 deficiency in transgenic mice (Fox et al. 1991, Kohfeldt et al. 1998, Miosge et 21 al. 2002). However, in C. elegans nidogen is not required for basement membrane assembly, but it directs the nerve position (Kim & Wadsworth 2000). In addition to nidogens, BM-40 (osteonectin or SPARC) also binds to type IV collagen and modulates cell-matrix interactions (Lane & Sage 1994, Pottgiesser et al. 1994, Göhring et al. 1998, Brekken & Sage 2001). BM-40 is a Ca2+-binding glycoprotein mostly expressed in bone, gut mucosa, healing wounds, and other remodelling tissues (Lane & Sage 1994). Moreover, the BM-40-null mice reveal that the BM-40 gene is necessary for lens transparency (Yan & Sage 1999). Similar to BM-40, a newly recognized ECM family, fibulins, also has Ca2+-binding properties and the potential to interact with various proteins including fibronectin and basement membrane proteins (Timpl et al. 2003). The in vivo functions of fibulins have been studied by transgenic mouse models and human inherited disease analyses (Kostka et al. 2001, Tartellin et al. 2001, Debeer et al. 2002). The fibulin 1-null mice show that the fibulin-1 gene is important for the development of skin, muscle and perineural tissues (Kostka et al. 2001). Proteoglycans are also important and ubiquitous constituents of basement membranes; for example, heparan sulphate proteoglycans have shown a special ability to filter solutes in blood and in the glomerulus of the kidney (Iozzo 1998, Dunlevy & Hassell 2000, Erickson & Couchman 2000). Among various basement membrane proteoglycans, perlecan is the most abundant and well characterized (Iozzo et al. 1994, Iozzo & San Antonio 2001). Perlecan is composed of an 80-nm-long core protein with 5 domains consisting of globules and short rods, and three large heparan sulphate or chondroitin sulphate chains coupled to its N-terminal portion (Paulsson et al. 1987). It is widely distributed in the basement membranes of various tissues, and also in some connective tissues lacking a classical basement membrane, e.g. cartilage and lymph nodes (Couchman et al. 1995). The perlecan heparin sulphate chains bind to laminin, type IV collagen, and fibronectin (Battaglia et al. 1992, Ettner et al. 1998), whereas the core protein interacts with nidogens, fibulins, and fibronectin (Friedrich et al. 1999, Hopf et al. 2001a). Therefore, perlecan makes the basement membrane supermolecular network more stable. Perlecan also binds to several kinds of growth factors and cell adhesion molecules, and consequently it promotes tumor cell proliferation and tumor angiogenesis (Jiang & Couchman 2003). Studies with mouse models have suggested more physiological functions of perlecan. Perlecan-null mice showed severe defects in heart, brain, and cartilage (Costel et al. 1999). Interestingly, the mice lacking the N-terminal heparan sulphate attachment sites of perlecan appeared abnormal in eye development (Rossi et al. 2003). 2.2 Cell receptors for extracellular matrix proteins The ECM interacts with the cell surface, and the dynamic communication between cells and ECM provides signals for cell adhesion, polarization, and differentiation, and controls apoptosis (Hynes 1992, Adams & Watt 1993, Ruoslahti & Reed 1994, Gumbiner 1996, Hynes 1999a, De Arcangelis & Georges-Labouesse 2000, Giancotti 2000, Geiger et al. 2001). For all of the ECM molecules, interactions with their cell receptors are 22 important for the biological function of these molecules. The most abundant ECM receptor is integrin, which is introduced in detail in section 2.2.1. Other cell surface receptors for the ECM include dystroglycan and syndecan, described in detail in a number of reviews, e.g. Henry & Campbell KP 1996, Hynes 1999a, Moiseeva 2001, and Couchman 2003. All of these ECM receptors transduce signals to and from the actin cytoskeleton (Geiger et al. 2001). 2.2.1 Integrins Integrins are a family of membrane glycoproteins that generally function as cell receptors for extracellular matrix proteins. They are heterodimers consisting of non-covalently associated α and β subunits. The two subunits collaborate to transduce signals between cells and the extracelluar matrix (Hynes 1992, Giancotti & Ruoslahti 1999, van der Flier 2001, Hynes 2002a, 2002b, Martin et al. 2002). According to the latest genome sequences of different species, 18 α and 8 β subunits, and 24 combinations have been identified in vertebrates (Gullberg & Lundgren-Åkerlund 2002, Zhang et al. 2003). Integrins have a generally conserved structure. Cryoelectron microscopy shows that they have an extracellular globular head comprising the N-terminal portions of α and β chains, a pair of membrane-spanning helices, and short C-terminal cytoplasmic tails (Erb et al. 1997). All integrins contain a region in their β subunits that contains a MIDAS-like motif for cation binding, while nine of the α subunits contain an I domain (also called an A domain) that is inserted into the head region where it plays a central role in ligand binding (Cierniewska-Cieslak et al. 2002, Humphries 2002, Humphries et al. 2003, Mould et al. 2003). Recently the crystal structure of the extracellular portion of integrin αvβ3, produced recombinantly in insect cells, was solved at a 3.1 Å resolution showing a bent conformation. Thus it is probably in an “inactive” conformation (Xiong J-P et al. 2001). Most integrins have their specific extracelluar ligands (Liddington & Ginsberg 2002, Watt 2002). Cell-fibronectin and cell-laminin interactions have been studied in more detail than cell-collagen interactions (Hynes 1992, Ruoslahti 1999). Four collagenbinding integrins-α1β1, α2β1, α10β1 and α11β1-have been identified (Emsley et al. 2000, Gullberg & Lundgren-Åkerlund 2002). All of the four collagen receptor integrins possess an I domain that mediates interaction with native collagens. Tissue-specific signals might selectively choose a particular collagen-binding integrin for migration (Gullberg & Lundgren-Åkerlund 2002). Studies of ECM or cell membrane ligands for these integrin types could help to understand more about their functions or features. α1β1 is widely distributed in various tissues, but its expression is limited to certain type of cells, i.e. most capillary endothelial cells and smooth muscle cells (Voigt et al. 1995). α1β1 specifically binds to type IV collagen, laminin-1/-2, matrilin-2, and the Cpropeptide of type I collagen, but only with low affinity to type I collagen. Type XIII collagen is also a potential ligand for α1β1 (original article III). The α1-deficient mice showed very subtle phenotypic changes and only hypercellular dermis and mild cell proliferation defects were detected (Gardner et al. 1996, Pozzi et al. 1998). On the other 23 hand, in vivo antibody inhibition studies suggested that α1β1 plays a role in inflammatory reactions (Sampson et al. 2001). α2β1 integrin has been shown to be a collagen receptor on a number of cells and it is widely expressed during embryonic development. Both α1 and α2 integrins are present in fibroblasts both in vitro and in vivo. However, the α2 subunit is not expressed in skeletal muscle. α2β1 and its I domain bind to collagen type I and the C-propeptide of collagen I, laminin-1, laminin-2, and the cartilage protein chondroadherin. Mapping of the binding site in collagen I showed that the helical GFOGER and similar sequences are important for binding (Knight et al. 1998, Xu et al. 2000). This was confirmed by the crystal structure of a complex between the I domain of integrin α2β1 and a triple helical collagen peptide containing the critical GFOGER motif (Emsley et al. 2000). The α2-knockout mice are characterized by pre-implantation lethality (De Arcangelis & GeorgesLabouesse 2000). α10β1 integrin was identified from chondrocytes (Camper et al. 1998) and it was found mostly in type II collagen-expressing tissues (Tulla et al. 2001). α10 has the highest identity with the most recently identified integrin subunit α11. However, α1, α2 and α11 integrin are also detected in chondrocytes. It is not clear if these integrins compensate for each other or if they have their own specific functions. α11β1 was identified in cartilage, intervertebral discs, and cornea. It is widely expressed in a variety of mouse tissues with the highest expression level in uterus, and it is also expressed in the heart and skeletal muscle of human tissues (Velling et al. 1999). In vivo, α11β1 is localized in fibroblasts adherent to collagen networks, which suggested that the α11β1 integrin might be involved in the ordered collagen matrix organization. However, α11 is not detected in vivo in developing human embryonic muscle, and no expression in muscle cells was observed. The distribution pattern in human embryos shows that α11 expression is restricted to mesenchymal non-muscle cells in the interstitial collagen network area (Tiger et al. 2001). The highest expression level of α11 in human embryonic tissues is in the areas adjacent to forming cartilage. In vitro assays showed that α11β1 binds more efficiently to collagen I than to collagen IV, and it is involved in cell migration, collagen deposition, and collagen reorganization (Gullberg et al. 1995, Tiger et al. 2001). 2.3 Membrane-associated proteins with collagenous domains Due to their structural similarity, four transmembrane collagens and five transmembrane proteins with collagenous domains, namely types XIII, XVII, XXIII, and XXV collagens, type I macrophage scavenger receptors (MSRs), MARCO, type I SRCL, the human complement subcomponent C1q, and the human EDA protein, are grouped as collagenous transmembrane proteins (Thiel & Reid 1989, Hirako et al. 1996, Rehn & Pihlajaniemi 1996, Resnick et al. 1996, Bayes et al. 1998, Hägg et al. 1998, Gordon et al. 2000, Ohtani et al. 2001, Hashimoto et al. 2002, Sankala et al. 2002, Banyard et al. 2003, Franzke et al. 2003, Söderberg et al. 2003). All of the collagenous transmembrane proteins have an N-terminal transmembrane anchor and one or more collagenous segments interspersed by one or more coiled-coil motifs. The schematic structures of 24 these proteins are presented in Fig. 1, and a detailed discussion of the protein structure, biosynthesis, tissue distribution, and biological function is presented in recently published reviews (Franzke et al. 2003, Snellman & Pihlajaniemi 2003). Fig. 1. Structural comparison of the collagenous transmembrane proteins. Collagenous structures are denoted by triple helices; globular domains by white circles; coiled-coil domains by grey circles or grey boxes; and transmembrane domains by black boxes. The figure is compiled from the references mentioned in the text. 25 2.3.1 Type XIII collagen 2.3.1.1 Gene structure and chromosomal localization of type XIII collagen Type XIII collagen is a transmembrane protein consisting of three collagenous domains (COL 1-3) interrupted and flanked by four non-collagenous domains (NC 1-4) as shown in Fig. 2. The human α1(XIII) chain contains an N-terminal non-collagenous domain, NC1, that consists of a 38-residue cytosolic domain, a 23-residue transmembrane domain, and a 60-residue extracellular non-collagenous segment, as well as the following extracellular domains: a 104-residue COL1, 34-residue NC2, 172-residue COL2, 22residue NC3, 236-residue COL3, and an 18-residue NC4 (Pihlajaniemi et al. 1987, Tikka et al. 1988, Pihlajaniemi & Tamminen 1990, Tikka et al. 1991, Hägg et al. 1998, Snellman et al. 2000). The mouse type XIII collagen structure is highly homologous to that of the human one. Recently two coiled-coil motifs were discovered at the NC1 and NC3 domains directing collagenous chain association (Latvanlehto et al. 2003). The human type XIII collagen consists of 41 exons and the mouse gene of 42 exons, with sizes of 140 kb and 135 kb, respectively (Tikka et al. 1991, Kvist et al. 1999). The human and mouse type XIII collagen genes are located on chromosome 10 in both species (Shows et al. 1989, Kvist et al. 2001). The overall identity between the human and mouse proteins is 90 %. The primary transcript of the type XIII collagen gene undergoes complex alternative splicing. A number of splice variants of the type XIII collagen gene have been identified in different tissues and cells, but it is not clear whether there are functional differences between these variants (Juvonen & Pihlajaniemi 1992, Juvonen et al. 1992, Juvonen et al. 1993, Peltonen et al. 1997). Fig. 2. Schematic structure of type XIII collagen. The domains are as follows: COL1-3, collagenous domains (white boxes); NC1-4, non-collagenous domains (light grey boxes); transmembrane domain (black box) and the coiled-coil domains (dark grey boxes). M represents the initiation methionine, C denotes a cysteine residue, and (R)4 is a furin proteolytic cleavage site consisting of four arginine residues. 26 2.3.1.2 Cellular and tissue localization of type XIII collagen Based on cell fractionation studies, type XIII collagen is located on the plasma membrane (Hägg et al. 1998). Moreover, biotinylation of cell surface proteins has revealed that the N-terminal portion is inside the cell and the mainly collagenous portion is outside the cell (Hägg et al. 1998). Type XIII collagen has a wide tissue distribution and has been found to be expressed in the epidermis, hair follicles, bone, muscle, lung, kidney, and various other human fetal tissues by using in situ hybridization (Sandberg et al. 1989, Juvonen et al. 1993, Sandberg-Lall et al. 2000). Immunofluorescence analysis detected type XIII collagen at the myotendinous junction in skeletal muscle, the intercalated discs of cardiac muscle both in human and mouse, and at cell-cell contacts within the retina of the adult and developing human eye (Hägg et al. 2001, Sandberg-Lall et al. 2000). In addition, type XIII collagen is also expressed in developing mouse, and it has been detected in cartilage, bone, skeletal muscle, lung, skin, and other tissues (Sund et al. 2001a). More interestingly, type XIII collagen shows significantly high expression in the central and peripheral nervous systems of the developing mouse fetus. However, the expression level of type XIII collagen is generally low. The localization of type XIII collagen in developing mouse resembles that of the integrin β1 subunit and the cell adhesion molecule vinculin (Sund et al. 2001a). In cultured fibroblasts, type XIII collagen has been found to be localized with vinculin and talin to focal adhesion sites (Hägg et al. 2001). Taken together, these data imply the role of type XIII collagen in cell-cell and cell-matrix interactions. 2.3.1.3 Recombinant expression of type XIII collagen Due to the low protein expression level of type XIII collagen in native tissues and in cultured mammalian cells, a recombinant expression system has been used to produce the protein for further characterization. This was achieved using insect cells co-expressing recombinant type XIII collagen and the key enzyme for collagen synthesis, prolyl 4hydroxylase (Snellman et al. 2000). The recombinant type XIII collagen protein assembles into a stable triple helical molecule with three α-chains associated into a disulfide-bonded homotrimer. Chain association was significantly enhanced by proline hydroxylation. In addition, the type XIII collagen ectodomain was purified under denaturing conditions, and it has been used to generate a pan-collagen antibody for detecting several types of collagen in Western blotting analyses (Snellman et al. 2000). In the recombinantly expressed type XIII collagen, three pepsin and trypsin/chymotrypsin resistance peptides were identified and thought to represent the collagenous domains, COL1-3, folded into the triple helical conformation (Snellman et al. 2000). 2.3.1.4 Physiological functions of type XIII collagen The physiological function of type XIII collagen has been studied using mouse models. A gene-targeted mouse strain, Col13α1N/N, lacking the cytosolic part, transmembrane 27 domain, and coiled-coil segment of the NC1 portion of type XIII collagen, displays a mild but progressive myopathy with increasing age due to abnormalities in the basement membrane and muscle cell interaction, showing ruptures and irregularities in the basement membrane structure (Kvist et al. 2001). This phenotype suggests a function of type XIII collagen in stabilizing the basement membrane zone in skeletal muscle. Surprisingly, the N-terminally mutant molecules were found to be located correctly in focal adhesions of fibroblasts derived from the Col13α1N/N mice. In the skeletal muscle of these mice, the mutant protein was found adjacent to the sarcolemma. Thus it was considered possible that the mutant protein is partially functional, but the cytosolic and transmembrane domains were necessary for sarcolemmal-basement membrane stability (Kvist et al. 2001). To understand the molecular properties of this N-terminally altered type XIII collagen, several variants of the protein chain were expressed in insect cells (Latvanlehto et al. 2003). The mutant protein was found to be secreted into the cell culture medium, and the shedding was found to be caused by furin-like protease cleavage similar to the authentic protein. Biochemical charaterization of the N-terminally mutant type XIII collagen revealed that it was correctly folded into the triple helical conformation with respect to the COL2 and COL3 domains, but the COL1 domain was unfolded (Latvanlehto et al. 2003). The second mouse model (COL2del) was generated by overexpressing the mutant type XIII collagen chains with a 90-amino acid in-frame deletion in the conserved collagenous COL2 domain (Sund et al. 2001b). In contrast to the mild phonotype in the Col13α1N/N mice, the COL2del mice showed fetal lethality due to reduced angiogenesis and defects in the adherence junctions in the heart, which suggested important roles for type XIII collagen in cell adhesion, and consequently in mouse development (Sund et al. 2001b). The physiological functions of type XIII collagen have also been studied in vitro by using the recombinant type XIII collagen ectodomain. For example, type XIII collagen has been found to enhance neurite outgrowth, suggesting that it might be involved in nervous system development (Sund et al. 2001a). 2.3.1.5 Chain association and shedding Type XIII collagen has been found to be shed into the media in cultured keratinocytes (Peltonen et al. 1999). Moreover, the recombinant human type XIII collagen expressed in insect cells also has its ectodomain secreted (Snellman et al. 2000). More in vivo studies are needed to prove the physiological significance of the shedding of the type XIII collagen ectodomain. Twenty one amino acid residues residing on the NC1 domain have been demonstrated to be essential for collagen chain association (original article I). This restricted sequence is also conserved in type XXIII and XXV collagen (Snellman & Pihlajaniemi 2003). A second coiled-coil structure is located in the NC3 domain of type XIII collagen and in the corresponding domains of types XXIII and XXV, which is important for the association of the C-terminal part of the molecule. The absence of the NC1 coiled-coil domain results in a lack of disulfide-bonded trimers and misfolding of the membrane-proximal collagenous domain COL1 (Latvanlehto et al. 2003). 28 2.3.2 Type XIII collagen-like collagens Two newly identified transmembrane collagens, types XXIII and XXV, are structurally similar to type XIII collagen as all of them contain a short N-terminal cytosolic domain, a transmembrane domain, and three collagenous domains separated and flanked by four non-collagenous domains (Snellman & Pihlajaniemi 2003). Thus, types XIII, XXIII and XXV collagens form a subgroup of structurally similar transmembrane collagens. 2.3.2.1 Type XXIII collagen Type XXIII collagen has been identified in cornea and rat prostate carcinoma cells as a transmembrane collagen (Gordon et al. 2000, Banyard et al. 2003). It is composed of 532 amino acids in mouse and 540 amino acids in human. The protein contains an N-terminal cytosolic domain, a transmembrane region, and three collagenous domains separated and flanked by non-collagenous sequences, thereby showing structural homology to types XIII and XXV collagen. Type XXIII collagen isolated on the cell surface and can be shed to the ECM by furin proteases (Banyard et al. 2003). The ectodomains of type XXIII collagen are easily aggregated to form a multimeric complex and can bind to heparin with low affinity (Banyard et al. 2003). Two predicted chain association domains (coiled-coil motifs) shown from the primary sequence are located at the NC1 and NC3 domains, indicating its structural similarity to type XIII collagen (Latvanlehto et al. 2003). 2.3.2.2 Type XXV collagen (CLAC-P) Another newly identified transmembrane collagen is type XXV collagen, which is also named CLAC-P (collagen-like Alzheimer amyloid plaque component precursor) because of its specific expression in brain (Hashimoto et al. 2002). The human type XXV collagen is composed of 654 amino acids, with three Gly-Xaa-Yaa collagen-like repeat motifs flanked by four non-collagenous domains. The domain structure and the protein sequence of type XXV collagen are partially homologous to that of type XIII collagen (~43.0 % at the amino acid level and ~49.9% at the nucleic acid level). The CLAC-P/Col XXV gene is located on human chromosome 4q and is expressed specifically in the brain and at low levels in the heart, testis, and eye. CLAC, the extracellular portion of CLACP/Col XXV, is secreted by furin protease cleavage, and the N-terminus of the secreted CLAC is pyroglutamate-modified. CLAC is specifically bound to the fibrillized amyloid β peptide (Aβ) and becomes an integral component of amyloid deposited in Aβ brains. The blocked N-terminus and collagen-like triple helical structure is expected to hamper attacks by a number of proteases and protect the deposited Aβ from proteolysis in Aβ brains (Hashimoto et al. 2002). Recently another Alzheimer disease amyloid-associated protein (AMY) has been identified, and it shows a strong homology with CLAC-P (Söderberg et al. 2003). According to coiled-coil element analyses, type XXV collagen contains a potential 22-amino acid residue coil-coil motif adjacent to the transmembrane 29 domain, and another one in the NC3 domain that is highly conserved in types XIII and XXIII collagen (Latvanlehto et al. 2003). 2.3.3 Type XVII collagen Type XVII collagen is a rod-like, flexible, triple helical macromolecule, which also belongs to the group of collagenous type II transmembrane proteins (Franzke et al. 2003). It is composed of a globular intracellular N-terminal domain of 466 amino acids, a short transmembrane domain of 23 amino acids, and an extracellular C-terminal domain of 1008 amino acids with mainly collagenous sequences (Giudice et al. 1992, Schäcke et al. 1998). Type XVII collagen is one of the components of hemidesmosomes, where it is associated with the α6β4 integrin, mediating adhesion of epidermal keratinocytes and epithelial cells to the basement membrane (Borradori & Sonnenberg 1999). Patients with mutations in the COL17α1 gene have a blistering disorder with epidermal detachment from the basement membrane (McGrath et al. 1996). Another group of patients with autoimmune diseases have autoantibodies against both full-length and the ectodomain of type XVII collagen, and they show diminished epidermal adhesion and skin blistering (Schumann et al. 2000). The type XVII collagen is shed physiologically by ADAMs protein sheddases, and this process is activated by furin (Franzke et al. 2002). 2.3.4 Other transmembrane proteins with collagenous sequences Several other proteins also belong to group of the transmembrane collagenous proteins. They are MSRs, MARCO, type I SRCL, the A-chain of the C1q complex, and EDA (Thiel & Reid 1989, Resnick et al. 1996, Bayes et al. 1998, Ohtani et al. 2001, Sankala et al. 2002, Snellman & Pihlajaniemi 2003). These proteins are not classified as collagens because they do not possess structural roles in the extracellular matrix, although the collagenous sequences in these molecules are important for their distinctive structures and functions. Notably, both MSR and C1q bind to the fibrillized form of the Amyloid β peptide, which is similar to type XXV collagen (Hashimoto et al. 2002). 2.4 Assays for protein-ligand interactions Gene knockout and genetic mutant screening are important tools to obtain information about gene function, as well as the possible associated features of the target gene. However, to fully understand complicated biological processes, it is important to identify compounds interacting with the products of the gene of interest. The most commonly used methods for such purposes are solid phase assays, surface plasmon resonances (SPR) assays, immunoprecipitations, and two-hybrid systems. 30 2.4.1 Solid phase assays In solid phase assays, antigens or proteins are immobilized on membranes or modified microscopic slides. Specific interacting proteins or antibodies from a mixture that bind to the immobilized proteins generate a signal, which is detected either by immunoassay, fluorescence imaging, or mass spectroscopy (Macy et al. 1988, Coligan et al. 1991, Lizardi et al. 1998, Lueking et al. 1999, Wiltshire et al. 2000). Since the enzyme-linked immunosorbent assay (ELISA) does not need expensive instruments, it is still a common method for detecting protein-ligand interactions. With the advances in DNA chip technology, the conventional ELISA concept was adopted for the development of a protein chip assay (Stoica et al. 2001), i. e. the purified protein of interest is covalently bound to the chip and lysates or extracts are run over the surface. One important feature of the solid phase assay is the specificity of the antibodies or the purity of the labelled protein. However, solid phase assays cannot provide information about a dynamic process. A modification of the solid phase assay allows the identification of protein interactions by far western analysis. Proteins of interest are immobilized on PVDF or nitrocellulose membranes after being separating by SDS-PAGE, and are then probed with a radiolabelled or chemically labelled non-antibody protein. This method is used when the proteins of interest are insoluble in other assays or are not able to be expressed in cells (Edmondson & Roth 2001). 2.4.2 Surface plasmon resonance assays Surface plasmon resonance (SPR) is an in vitro technique to detect interactions between different molecules and directly measure the kinetic parameters of the interaction (Liedberg et al. 1995, Rich & Myszka 2001, Wilson 2002). The SPR technology is based on an optical phenomenon (Rich & Myszka 2000). The most popular SPR device is the Biacore® instrument (Biacore). In practice, a soluble protein is immobilized on a chip and the target molecules (analyte) are flowed over the protein surface. When a target protein associates with the ligand, the optical sensor detects the changes in the angle of minimum reflectance from the interface. Molecular interactions can be visualized directly. A clear benefit of using SPR is that it provides an accurate measurement of kinetic rate constants for a ligand-analyte binding reaction (Göhring et al. 1998, Rich & Myszka 2000, Baker et al. 2002). Surface plasmon resonance technology has also been integrated with mass spectrometry to enhance its application in biospecific interaction analyses. The SPR biosensor surface captures the analytes specifically bound to the immobilized ligands, and subsequently the interacting proteins or peptides are further identified with mass spectrometry (Williams & Addona 2000). This new technology has been used in proteomic studies (McDonnell 2001, Rich & Myszka 2003). However, SPR cannot be used for all types of protein-protein studies, especially in the case of strong binding where it is difficult to achieve dissociation, or for complicated interaction processes such as those involving ligands with multiple-binding sites and cooperative 31 network interactions. It is important to note that the interpretation of the kinetic analysis data requires good knowledge and training in biophysical chemistry. 2.4.3 Other assays Protein-protein interactions can be analyzed in vivo using so-called two-hybrid systems in yeast or in mammalian cells (Fields & Song 1989, Chien et al. 1991, Fearon et al. 1992, Bendixen et al. 1994, Yang et al. 1995). The benefits may include decreasing nonspecific binding. For example, protein-protein interactions have been monitored in mammalian cells by complementation of β-lactamase enzyme fragments (Wehrman 2002). In this system, proteins are fused to the inactive α or ω fragments of β-lactamase, and when one protein (fused to α) interacts with its ligands (fused to ω), both inactive fragments can complement each other to form a functional enzyme in mammalian cells. The activity of β-lactamase can be assayed in intact cells using a fluorescent substrate. This assay can be used in vitro as a simple colorimetric method using cephalosporin nitrocefin as the substrate, as well as be applied to clonal selection for cells expressing interacting protein partners (Galarneau et al. 2002). Another similar assay employs αcomplementation of two mutant forms (∆α and ∆ω) of β-galactosidase to identify the dimerization of the epidermal growth factor receptor (Blakely et al. 2000). However, this system is not always useful for ECM proteins since these molecules are secreted out of the cell, which results in difficulties in using yeast or mammalian cell two-hybrid methods. Co-precipitation of proteins from cell or tissue extracts is another much used approach for studying physiological interactions between proteins of interest. The most common method is co-immunoprecipitation, i.e. an antibody is used for precipitation (Phizicky & Fields 1995). In practice, the antibody is immobilized to a solid affinity support, and the associated proteins are fractionated by SDS-PAGE and detected by immunoblotting with specific antibodies that recognize the putative associated proteins. The coimmunoprecipitation assay has been widely applied in studies of the cell signalling pathway (Wary et al. 1998, Liu et al. 1999). The assay requires specific antibodies that can immunoprecipitate the proteins under non-denaturing conditions, or the proteins must be tagged and detected with antibodies for the tag. 3 Outlines of the present research The cellular transmembrane localization and wide tissue distribution of type XIII collagen is suggestive of unique characteristics compared to other known collagen types. Although the physiological functions of type XIII collagen are still not fully understood and human disorders affecting this collagen have not yet been discovered, meticulous structural and functional studies have been carried out at the molecular, cellular, and pathological levels since the cDNA of type XIII collagen was partly cloned in 1987 (Pihlajaniemi et al. 1987). The present thesis work was initiated when the type XIII collagen was recombinantly expressed in insect cells as a stable homotrimer, and with a relatively high yield compared to the low expression amount in native tissues and cultured mammalian cells. A series of deletion variants were constructed and expressed to study the biosynthesis of type XIII collagen. However, isolation of full-length type XIII collagen from the infected insect cells was difficult due to the highly hydrophobic transmembrane domain that anchors the protein to the plasma membrane. Therefore, the research goal was adjusted to focus on the purification of the shed ectodomain and the subsequent analysis of its biochemical features. The cell biological and genetically modified mouse model studies performed by my colleagues in the same research group showed the significant importance of type XIII collagen in cell adhesion, which directed the second part of this thesis, i.e. identification of the biological ligands for type XIII collagen in the ECM and on the cell surface. Therefore, the specific aims of the present research were: 1. to study the mechanisms of trimer formation and shedding of type XIII collagen α1 chains, 2. to purify the type XIII collagen ectodomain for structural and functional analyses and monoclonal antibody production, 3. to identify the ECM ligands for type XIII collagen, and 4. to identify the collagen receptor integrins for type XIII collagen recognition. 4 Materials and methods Detailed descriptions of the materials and methods applied in the studies can be found in the original articles I-IV. 4.1 Recombinant production of human type XIII collagen in insect cells (I, II, III, IV) 4.1.1 Construction of recombinant baculoviruses (I) In order to produce type XIII collagen in insect cells, human collagen XIII full-length cDNA was constructed by linking a PCR-generated 5’ fragment covering nucleotides 1272 of the human type XIII collagen gene (Tikka et al. 1991) to the previously described cDNA clone E-26 (Pihlajaniemi & Tamminen 1990). The construct was named huXIII and included the whole coding sequence of the human type XIII collagen α1 chain except the alternatively spliced exons 4B, 13, 29, and 33, which were not present in the original cDNA clone E-26 (Pihlajaniemi & Tamminen 1990, Hägg et al. 1998). The huXIII cDNA was ligated to the transfer vector pVL1392 (Invitrogen) to form the construct pVLwthumanXIII. The construct lacking the cytosolic portion of human type XIII collagen, del1-38, was generated by PCR using huXIII cDNA as the template. It lacked the first 124 nucleotides and introduced a new initiation methionine by replacing the original residue leucine at position 39 adjacent to the transmembrane domain. The cDNA del1-38 was also ligated to pVL1392 resulting in the construct pVLdel1-38. Construct pVLdel1-83 was produced by directly inserting the cDNA E-26 into pVL1392. The cDNA del63-83 lacking nucleotides 197-259, and the cDNA del442-668 lacking the nucleotides proceeding nucleotide 1196 from the human type XIII collagen cDNA were both generated by PCR and ligated to pVL1392 to form pVLdel63-83 and pVLdel442668, respectively. In the next step, the recombinant viruses wthumanXIII, del1-38, del183, del63-83, and del442-668 were produced by transfecting Spodoptera frugiperda Sf 9 34 insect cells with each of the pVL constructs and the modified Autographa californica nuclear polyhedrosis virus DNA (AcNPV, Pharmingen) using the Baculo Gold transfection kit (Pharmingen). The recombinant viruses were collected, amplified, plaque-purified, and re-amplified (Gruenwald & Heitz 1993). Another recombinant virus containing the mouse type XIII collagen gene was generated in a slightly different way. The cDNA moXIII(689) encoding the mouse fulllength type XIII collagen gene was ligated to the vector pFastBac1 (GibcoBRL, Life Technologies) resulting in the construction of pBacmoXIII(689)HIS, which contained an extra sequence coding for six histidine residues just before the stop codon. The recombinant bacmid DNA was used to trnasfect the Sf9 cells according to the BAC-TOBAC Baculovirus Expression System Instruction Manual (GibcoBRL, Life Technologies). The recombinant virus moXIII(689)HIS was collected and amplified as described above. 4.1.2 Expression of recombinant type XIII collagen in insect cells (I) The insect cells High Five or Sf9 (Invitrogen) were cultured as monolayers at 28°C in the TNM-FH medium (Sigma) supplemented with 10 % fetal bovine serum (Bioclear) or in the serum-free HyQ CCM3 medium (Hyclone). For the recombinant protein production, cells were infected at a density of 6×105 cells/ml with a virus coding for the collagen XIII variant at MOI=5, and coinfected with the virus coding for both the α and β subunits of human prolyl 4-hydroxylase (4PHαβ; Nokelainen et al. 1998) at MOI=1. The cosubstrate of the enzyme, ascorbate, was added to the culture medium daily at 80 µg/ml. The cells and culture medium were harvested 48 h post-infection, and separated by centrifuging at 340×g for 10 min. The cells were washed with PBS once and then homogenized in 70 mM Tris, 300 mM NaCl, 0.2% Triton, pH 7.4, containing CompleteTM protease inhibitor (Boehringer Mannheim). The homogenate was kept in the same solution for 30 min before centrifuging at 8000 ×g for 10 min to separate the supernatant and pellet. All of the steps above were performed at 4ºC or in ice. The pellet was further extracted with 1% SDS at room temperature for 10 min and centrifuged at 8000 ×g for 10 min to remove the insoluble materials. The samples from media, supernatants, and pellets were analyzed by SDS-PAGE under reducing or non-reducing conditions, and detected by Western blotting with antibodies against type XIII collagen (Hägg et al. 1998, Snellman et al. 2000, original article III). 4.1.3 Immunofluorescence staining of insect cells expressing type XIII collagen (I) High Five insect cells were attached to coverslips (Menzel-Gläser, Germany) coated with poly-L-lysine (or poly-D-lysine, GIBCO) and infected with viruses coding for human type XIII collagen variants and 4PHαβ as described in section 4.1.2. After a 48-h infection, the coverslips were rinsed with PBS and the cells were fixed, but not 35 permeabilized, with 3 % paraformaldehyde for 5 min at room temperature. Subsequently the coverslips were incubated in 0.2 % BSA-PBS for 1 h to block the unoccupied surface, and then in the first antibody XIII/NC3-1 (Hägg et al. 1998) solution at a dilution of 1:200 for 1 h followed by an incubation with the secondary swine anti-rabbit antibody conjugated to TRITC (Dako) at a 1:100 dilution for 1 h. All of the immunostaining steps were performed at room temperature and the samples were washed extensively before changing to the next incubation solution. Finally, the coverslips were mounted onto SuperFrost glass slides (Menzel-Gläser, Germany) using Immu-mount medium (Shandon), and the immunostaining was examined and photographed under a Leitz Aristoplan microscope. 4.1.4 N-terminal sequencing of type XIII collagen expressed in insect cells (I) As described in section 4.1.2, High Five insect cells were co-infected with the viruses del1-38 and 4PHαβ for 48 h. The cells and media were separated and the media were centrifuged further at 40 000 ×g for 45 min to remove the viruses. The supernatant was adjusted to pH 7.0 and then mixed for 2 h at 4 °C with P11 cation exchange resins (Whatman) pre-equilibrated with 50 mM sodium phosphate buffer, pH 7.0. The beads were collected by centrifuging at 340 ×g for 5 min and washed three times with the equilibration buffer, and finally the adsorbed proteins were step-eluted by the same buffer added with increased concentrations of NaCl. The fractions containing type XIII collagen were concentrated using the Ultrafree protein concentrator (Millipore) for further analyses. Meanwhile, the infected cells were homogenized in PBS containing CompleteTM Protease Inhibitor (Boehringer Mannheim). After centrifugation at 280 ×g for 10 min, the pellet was further treated with 500 mM Na2CO3 on ice for 2 h to partly destroy the plasma membrane structure. The membrane debris was collected by centrifuging at 40 000 ×g for 1.5 h. The proteins bound to the plasma membrane were extracted with PBS containing 0.1% Triton, 10% glycerol, and 2 M urea, followed by centrifugation at 12 000 ×g for 20 min. The supernatant was concentrated by filter centrifugation as mentioned above or by precipitation with 75% ethanol at –20 °C. The samples isolated from media and cell membrane were analyzed with SDS-PAGE under reducing conditions, electroblotted onto a ProBlott membrane (Applied Biosystems), and detected by Coomassie Blue staining and the antibody XIII/NC3-1, which recognized the NC3 domain of type XIII collagen (Hägg et al. 1998). The putative bands stained with Coomassie Blue were cut out and loaded into a 477A protein sequencer (Applied Biosystems) for automatic N-terminal sequencing. 36 4.1.5 Inhibition of type XIII collagen shedding in insect cells by a furin protease inhibitor and by heparin (I, II) The High Five cells were infected with del1-38 and 4PHαβ viruses in HyQ CCM3 medium as in section 4.1.2. The furin protease inhibitor decanoyl-RVKR-chloromethyl ketone (Bachem, Basel, Switzerland), an antagonist to the furin recognition site R105RRR at the N-terminal part of type XIII collagen, in 100 µl methanol with a concentration of 100 µM was added to the culture medium 24 h post-infection, and an equal volume of methanol was added to another culture plate as a control. After a 48-h infection, the cell lysates and media were prepared and analyzed as described in section 4.1.2 using the antibody XIII/NC3-1. For the heparin treatment, heparin (Sigma) solution was added before infection to monolayer or suspension cultures of the High Five cells (Invitrogen) at a final concentration of 57 µg/ml culture media (10 units/ml). The virus infection of the cells and the analyses of collagen XIII expression were performed as described in section 4.1.2. 4.1.6 Purification of the recombinant human type XIII collagen ectodomain (II, III, IV) High Five insect cells were infected in the serum-free medium HyQ CCM3 (Hyclone) or Express Five (Invitrogen) with the viruses del1-38 (MOI=5) and 4PHαβ (MOI=1), as monolayers with 7.6×104 cells/cm2 or in suspension with 1×106 cells/ml at 27°C. Ascorbate phosphate (80 µg/ml) was added to the culture medium daily. The medium was harvested 48 h post-infection by centrifuging at 340 ×g for 10 min to remove the cells, and filtered through a 0.22-µm membrane to remove the debris. To purify the collagen XIII ectodomain, the medium was loaded into a HiTrap Q 5-ml column (Amersham Biosciences) pre-equilibrated with 20 mM MES-0.15M NaCl-2 mM EDTA, pH 6.0. The ectodomain was eluted in a flow-through fraction and was isolated further by a HiTrap SP 5-ml column (Amersham Biosciences) using a gradient elution program on a liquid chromatography instrument ÄKTA explorer 10 (Amersham Biosciences). The fractions containing type XIII collagen were pooled and concentrated to 1 ml for the final gel filtration step, in which the sample was injected into a Sephacryl S-500 column (1.6×100 cm, Amersham Biosciences) pre-equilibrated with 20 mM HEPES-0.15M NaCl, pH 7.0 and eluted with the same buffer. The fractions corresponding to the elution volume for proteins with a molecular mass of 150-200 kDa were analyzed by SDS-PAGE under nonreducing conditions with both Coomassie blue staining and immunoblotting detection methods. The purity of the protein was further confirmed by acidic native PAGE (Reisfeld et al. 1962.). The purified sample was stored at -20 ºC or adapted to 50 mM acetic acid using an Ultrafree Centrifugal Filter Device with a molecular mass cut-off of 10 kDa (Millipore) and then lyophilized. The production yield was determined by a Micro BCA assay (Pierce) according to the supplier's instruction or by amino acid analysis performed on a 421A amino acid analyzer (Applied Biosystems). The later method was also applied for the analyses of prolyl- and lysyl- hydroxylation. The 37 glycosylation of the recombinant type XIII collagen was analyzed by an Immuno-Blot kit combined with an enzymatic deglycosylation kit (Bio-Rad). 4.1.7 Isolation of pepsin-resistant fragments from the recombinant type XIII collagen (II) The purified human type XIII collagen ectodomain was first dissolved in 70 mM Tris-30 mM NaCl, pH7.5, and then the solution was adjusted to pH 2 with 0.3 M HCl for pepsin digestion. The protein was digested with pepsin at a ratio of 100:1 w/w for 10-30 min at 22 °C, and after that, the reaction was terminated by neutralizing the buffer to pH 7.0. The digested mixture was analyzed by SDS-PAGE or applied to the final isolation, at which the pepsin-resistant fragments were separated in a 50 mM sodium phosphate-0.15 M NaCl buffer, pH 7.0 using a Superdex 200 gel filtration column (1.6 × 60 cm, Amersham Biosciences) on an ÄKTA Explorer 10 (Amersham Bioscieces). Following an SDS-PAGE analysis detected with both Coomassie blue staining and Western blotting, the fractions containing the same sizes of peptides were pooled, concentrated, and stored at -20 °C. 4.2 Production of integrin I domains (III, IV) The integrin α1 I domain cDNA was generated by PCR using human integrin α1 cDNA (Briesewitz et al. 1993.) as a template. The forward primer was 5'CACAGGGATCCGTCAGCCCCACATTT-3' and the reverse primer was 5'GTGGCTGTCGACAGCTGTGGCTTCCAG-3'. The target PCR product was ligated to a pGEX-4T-3 vector (Amersham Biosciences), and subsequently the α1 I domain was expressed as a GST fusion protein in E. coli BL21 cells according to the protocols supplied by the manufactures of the expression vector. Integrin α2 I domain cDNA was also produced by PCR using human integrin α2 cDNA (Takada & Hemler 1989) as a template, and ligated to pGEX-2T vector (Amersham Biosciences). The GST-α2I fusion protein was expressed in E. coli BL21 cells (Ivaska et al. 1999a). Integrin α11 I domain cDNA was generated by PCR using human integrin α11 cDNA (Velling et al. 1999) as a template and ligated to the pGEX-KT vector (Amersham Biosciences). The I domain was also expressed as a GST fusion protein in E. coli (Zhang et al. 2003). All of the recombinant proteins-GST-α1I, GST-α2I, and GST-α11I were purified using glutathione-sepharose (Amersham Biosciences) affinity chromatography (Ivaska et al. 1999a, Zhang et al. 2003). To produce the α1 and α2 I domains, GST was removed by proteolytic cleavage with thrombin (Amersham Biosciences) after the fusion protein had bound to the glutathione-sepharose. The purity of the recombinant I domains was tested by native PAGE and SDS-PAGE. 38 4.3 Europium labelling of the recombinant integrin α1 and α2 I domains (III) The integrin α1 and α2 I domains were labelled with europium for binding analyses. To effectively label the proteins, the pH of the sample solution was adjusted to 9.0-9.3 by 1M Na2CO3 and then the europium-labelling reagent was added according to the manufacturer’s instructions (Wallac). After incubation of the reaction mixture at 4 °C overnight, the excess reagents were removed by a Sephadex G-50 gel filtration column (Amersham Biosciences). The europium-labelled I domains were verified by timeresolved fluorometry using a Victor2 multilabel counter (Wallac), and the protein concentration was determined using Bradford’s assay (Bradford 1976, Stoscheck 1990). 4.4 Expression of recombinant integrin α1β1 and α2β1 in CHO cells (III) To express exogenous α1 integrin, CHO cells (ATCC) were transfected by electroporation with an α1 cDNA (Briesewitz et al. 1993) ligated to a pLEN-expressing vector (Dickeson et al. 1999), and co-transfected with a pAWneo2 plasmid (Ohashi et al. 1985) for selection. For α2β1 expression, the α2 cDNA was ligated to a pAW vector (Ohashi et al. 1985) and the plasmid was transfected into the CHO cells by the method described above. The cell clones stably transfected with α1 or α2 were selected by a neomycin analogue G418 (Life Technologies), and integrin expression on the cell surface was analyzed by flow cytometry using the antibodies against α1 or α2 integrin (Ivaska et al. 1999b). 4.5 Binding assays (II, III, IV) 4.5.1 Solid phase assays (II, III, IV) In order to investigate the interaction between collagens and integrins, the I domains of α1 and α2 integrin subunits were labelled with europium (section 4.3) and the signal was detected by a time-resolved fluorometry, whereas the binding of the type XIII collagen ectodomain to ECM molecules was tested with an ELISA method. For the analyses of the selective collagen binding to integrin, a 96-well microtiter plate (Nunc) was coated with 100 µl of different types of collagens diluted to 15 µg/ml in PBS at 4 °C for 12 h. The collagens tested were type I collagen from rat (Sigma), type II from human (Chemicon), type II from bovine (a kind gift from Dr. M. van der Rest, Lyon, France), type II from chicken (Genzyme), type III from human (Chemicon), type IV from mouse (Sigma), type V from human (Chemicon), and recombinant human type XIII 39 (section 4. 1. 6). The wells for a blank control were coated with 100 µl of DelfiaTM Diluent II (Wallac). After coating, all of the sample wells were blocked with 100 µl of the same DelfiaTM Diluent II at 37 °C for 1 h. Europium-labelled α1I and α2I (section 4.3) were diluted to 1 µg/ml with PBS containing 2 mM MgCl2 and dispensed into the testing wells. The plate was incubated at 37 °C for 3 h, followed by extensive washing with PBS containing 2 mM MgCl2. After the interaction reaction, a DelfiaTM enhancement solution (Wallac) was added to each testing well and the amount of the bound I domain was detected by measuring the europium signal using a Victor2 multilabel counter (Wallac) for the time-resolved fluorometry. All of the experiments were performed in three parallels. To study the interaction between type XIII collagen and the ECM components, the selected ECM proteins were coated onto 96-well plates and incubated with the purified ectodomain after blocking the uncoated plastic surface. The samples were subsequently incubated with the first antibody XIII/NC3-1 and then with the secondary anti-rabbit antibody conjugated to horseradish peroxidase. Finally a substrate for the linked enzyme, 5-aminosalicylic acid (Sigma), was dispensed to the reaction and the signal was detected at 490 nm. The studies of heparin binding to the collagen XIII ectodomain were performed in a similar way except that heparin-BSA (Sigma) was coated onto the sample wells and the first antibody for detecting the bound ectodomain was the monoclonal antibody against the recombinant human type XIII collagen (section 4.7). The binding signal was monitored at 450 nm using another substrate for the horseradish peroxidase, 3, 3’,5, 5’-tetramethylbenzidine (Sigma). The ELISA method was also used to test the interaction between the integrin α11 I domain and type XIII collagen, in which the recombinant GST-α11I (section 4.2) was immobilized onto the 96-well microtiter and the collagen XIII ectodomain was dispensed as a soluble analyte in PBS containing 2 mM MgCl2 and 20 µM CaCl2. The binding was detected in the same way as that for heparin binding. 4.5.2 Surface plasmon resonance assays (II) In addition to the preliminary screening of the ECM ligands for the type XIII collagen ectodomain by ELISA (section 4.5.1), the interactions between the ectodomain and several ECM proteins were further analyzed using a surface plasmon resonance assay on a Biacore® 3000 instrument (Biacore AB, Uppsala, Sweden). The proteins were immobilized on CM5 sensor chips (research grade, Biacore AB) using a standard amine coupling method according to the manufacturer’s instructions. For each ligand, two immobilization levels were reached for different purposes, i.e. affinity analyses or binding kinetics tests. After immobilization of the ligands, the binding capacities of the selected analytes were studied by detecting the association and dissociation phases, and subsequently the chips were regenerated by a pulsed liquid treatment with 2.5 M NaCl, or in some cases with a solution of 2.5 M NaCl-5 mM NaOH. All of the proteins were tested as an immobilized ligand and vice versa as a soluble analyte. The kinetics constants were measured by fitting the association and dissociation data to the available models with 40 BIAevaluation software (version 3.1, Biacore AB) according to the manufacturer’s protocols. 4.5.3 Heparin affinity chromatography (II) To test the binding of the transmembrane type XIII collagen to the heparin molecule, the insect cells expressing recombinant membrane-bound type XIII collagen (del1-38, see section 4.1.2) were homogenized and the proteins were extracted with a Tris buffer, pH 7.4 containing 0.2 % Triton X-100. After removing the insoluble pellet, the lysate was manually loaded into a HiTrap heparin column (Amersham Biosciences) and then eluted in steps with the same extraction buffer containing 0.5M and 1M of NaCl. The same column was also applied to study the heparin binding of the purified ectodomain of type XIII collagen using a gradient elution method on an ÄKTA Explorer 10 (Amersham Biosciences). 4.6 Cell spreading assays (III, IV) 4.6.1 Assays for integrin α1β1- or α2β1-mediated cell attachment (III) The 96-well microtiter plates (Nunc) were coated at 4 °C for 12 h with different types of collagen at 1 µg/cm2 or a mixture of bovine dermal collagens (Cellon, Luxembourg) at 5 µg/cm2, and blocked with 0.1% heat-inactivated BSA at 37 °C for 1 h. CHO cells, lacking the endogenous collagen-binding integrin subunits, were stably transfected with the cDNAs of integrin α1 or α2 subunits (section 4.4), and were tested for the cell attachment. The cells were suspended in serum-free DMEM (Life Technologies) containing 0.1% glycine, and allowed to attach for 40, 80, and 120 min. After removing the unattached cells, the adherent cells were fixed with 4 % formaldehyde-5 % sucrose at 4 °C for 30 min, and then the sample wells were filled with water. The cells were subsequently examined and photographed under a phase-contrast microscope. 4.6.2 Assays for integrin α11β1-mediated cell spreading (IV) The 24-well culture plates were coated respectively with 20 µg/ml of fibronectin from human plasma (Invitrogen), type I collagen from rat tail (Collaborative Biomedical Products), recombinant human type XIII collagen (section 4.1.6), and 0.2% BSA (Serva) for a blank control, followed by blocking the uncoated surface with the same BSA solution. The wild-type C2C12 cells and the C2C12 stably expressing α11β1 integrin (Tiger et al. 2001) were added to the wells and allowed to adhere for 45 min at 37 °C in PUCK’s saline supplemented with 5 % CO2. After washing away the unattached cells, the 41 attached ones were visualized and photographed under a Leitz DMII light microscope and then lysed for quantitative analyses with a β-hexosaminidase release assay (Landegren 1984). Cell spreading was also monitored by immunofluorescence staining of SV40immortalized mouse embryonic fibroblasts (MEFs) derived from wild-type or α-null mice. The ligands were coated on glass coverslips and the cell attachment was performed as described above. After the incubation, the cells were fixed with acetone at -20 °C for 8 min or with 4 % paraformaldehyde for 15 min and then permeabilized with 0.1 % Triton/PBS for 10 min. The acetone fixation method has been optimized for α11 integrin staining, whereas the paraformaldehyde fixation has been optimized for vinculin staining. However, in double-staining of α11 integrin and vinculin, the acetone fixation was also used. The cells were then stained with a rabbit polyclonal antibody against the cytosolic tail of the subunit (Tiger et al. 2001) or a mouse monoclonal antibody against vinculin (Sigma). A Cy3-conjugated goat anti-rabbit IgG or a Cy2-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) were used for the detection. The stained cells were mounted onto the glass slides and examined under a Leitz Aristoplan microscope (Leitz Wetzlar, Germany). To visualize the focal adhesion staining in higher magnification, the primary wild-type MEFs with higher cell density were allowed to adhere to the coated coverslips in DMEM containing 10 % FCS for 2 h at 37 ºC. The stained cells were examined under Zeiss Axiophot microscope (Carl Zeiss, Germany). 4.7 Monoclonal antibody production (II) A monoclonal antibody against the type XIII collagen ectodomain was generated and prescreened by a commercial service (VTT Biotechnology, Espoo, Finland). The purified ectodomain was used to immunize mice, and the ELISA-positive sera of the immunized mice were analyzed by Western blotting using the polyclonal antibody XIII/NC3-1 (Hägg et al. 1998). Subsequently, one mouse was chosen for a final boost and only one of the resulting media of the fused cells showed positive signals in ELISA and immunoblotting. The fusion was cloned further and the IgG produced by one clone was affinity-purified. 4.8 Electron microscopy (I, II) Electron microscopy was used to detect the expression of type XIII collagen in insect cells using an immunolabelling method and to visualize the structure of the type XIII collagen ectodomain with rotary shadowing technology. 42 4.8.1 Immunoelectron microscopy of insect cells expressing type XIII collagen (I) The High Five insect cells were infected with viruses moXIII(689)HIS and 4PHαβ or only with 4PHαβ as described in section 4.1.2. After infection the cells were washed with PBS and detached from the culture plates with 8% paraformaldehyde in 0.2 M HEPES buffer, pH 7.4. The samples were then centrifuged at 6000 ×g for 4 min, and the cell pellets were kept in the same solution and fixed at room temperature for 30 min. After fixation, the pellets were freeze-protected in 2.3 M sucrose-PBS solution for 30 min, and then divided into 1 mm2 specimens and frozen in liquid nitrogen. Subsequently, the cell specimens were cut into 60 nm sections using a Leitz Ultracut cryomicrotome and placed on Formvar-coated nickel grids. The sections were blocked with 10% FCS in a 20 mM glycine-PBS solution for 10 min on ice and incubated at room temperature for 45 min with the antibody XIII/NC3-1 diluted 1:200 with 5 % FCS in 20 mM glycine-PBS. Following extensive washing with 20 mM glycine-PBS, the sections were incubated for 20 min with an AuroProbe protein A conjugated with gold particles of 10 nm in diameter (Amersham). The beads were diluted in 5 % FCS-20 mM glycine-PBS according to the manufacturer’s instructions. The sections were again washed with the buffer and postfixed in 2.5 % glutaraldehyde-PBS, pH 7.4 for 2 min. The specimens were then rinsed with distilled water followed by counterstaining and dry-protection with 0.3 % uranyl acetate-2 % methylcellulose on ice for 10 min, and finally they were visualized and photographed with a Philips 410LS electron microscope using an acceleration voltage of 60 or 80 kV. 4.8.2 Rotary shadowing electron microscopy (II) The purified and lyophilized type XIII collagen ectodomain was dissolved in 50 mM acetic acid and further diluted to a final concentration of 50 µg/ml with 40 % glycerol. The samples were sprayed onto mica discs, dried in a vacuum, and rotary shadowed with platinum/carbon. The detailed protocols were described by Engel & Furthmayr (1987). 4.9 Circular dichroism spectroscopy (II) In order to analyze the collagenous conformation of the recombinant type XIII collagen ectodomain, CD spectroscopy was utilized at 15 °C using a Jasco J710 spectropolarimeter (Jasco Inc.) according to the instrument manual. In addition, the farUV spectra of proteins were measured from 195 to 250 nm in 50 mM acetic acid, and the melting curve was obtained at 222 nm by increasing the temperature linearly at a rate of 20 °C/h. 43 4.10 Sequence comparison of collagenous transmembrane proteins (I) The protein sequences of collagen types XIII and XVII, MARCO, EDA, the macrophage scavenger receptor, and the A, B and C chains of C1q were aligned with the multiple sequence analysis program PileUp using default values (version 10.0, 1999, GCG) and the multiple alignment results were compiled using the program BOXSHADE (version 3.2, K. Hofmann and MD. Baron). Further homology analyses were performed using ANTHEPROT (version 4.9, G. Deléage). All of the programs were accessed from CSC, the Finnish IT center for Science. 5 Results 5.1 Type XIII collagen is a type II transmembrane protein (I) The type XIII collagen protein sequence reveals a transmembrane domain located in the N-terminal non-collagenous region, which suggests that the short N-terminal portion is intracellular and the long C-terminal portion with three collagenous domains is extracellular, see Fig. 2 in section 2.3.1.1. To verify this, type XIII collagen was recombinantly expressed in insect cells to study its location and orientation. High Five cells were infected with the virus wthumanXIII coding for the full-length human type XIII collagen together with the virus 4PHαβ encoding the α- and β-subunits of human prolyl 4-hydroxylase, which is a key enzyme for collagen synthesis (Lamberg et al. 1996). After infection, the cells were fixed but not permeabilized for immunofluorescence staining using the antibody XIII/NC3-1 against the NC3 domain of type XIII collagen. This resulted in a significant punctuate staining of collagen XIII on the cell surface, which indicated that the protein was localized on the plasma membrane with its C-terminus toward the extracellular environment. The results were confirmed by analyzing the expression of mouse full-length type XIII collagen containing a C-terminal histidine tag using the same method except that the cells were stained with a monoclonal anti-histidine tag antibody. In contrast, cells infected with another virus, del1-83 encoding an N-terminally truncated type XIII collagen lacking the predicted cytosolic and transmembrane domains, were also analyzed and no collagen XIII staining was observed on the cell surfaces. The High Five insect cells expressing mouse full-length type XIII collagen were further studied with immunoelectron microscopy using the XIII/NC3-1 antibody. The staining image demonstrated that the NC3 domain is located on the extracellular side of the plasma membrane, which confirms the type II orientation of type XIII collagen. 45 5.2 A conserved plasma membrane-adjacent sequence is important for type XIII collagen chain association (I) The distinguishing orientation and location of type XIII collagen imply different structural features and physiological roles compared to the classical fibrillar collagens and raised several questions, one of which is how the collagenous triple helices are formed in the transmembrane collagen XIII. To study the trimerization of type XIII collagen, the full-length α1 chains and four deletion variants were expressed in insect cells, and the recombinant products were analyzed by SDS-PAGE/Western blotting under reducing and non-reducing conditions and detected with the antibody XIII/NC3-1. The protein bands in reducing gels demonstrated the actual molecular masses of the monomers, whereas the corresponding samples analyzed in non-reducing gels revealed the capacity of disulfide-bonded trimer formation. Notably, the full-length variant and that lacking the cytosolic part (del1-38) or lacking the COL3 and NC4 domains (del442668) were associated into disulfide-bonded trimeric molecules, while the recombinant protein lacking the N-terminal 83 residues including the cytosolic and transmembrane parts (del1-83) or that lacking 21 residues in the ectodomain (del63-83) adjacent to the transmembrane domain failed to form the disulfide-linked trimer. This indicated that the 21 residues are important for chain association. Further data suggested that triple helix formation driven by the 21 residue sequence corresponds to the assembly of COL1 but not to COL2 and COL3 domains (Latvanlehto et al. 2003). Interestingly, a similar sequence was also identified in several other collagenous transmembrane proteins, namely type XVII collagen, MARCO, and EDA. Furthermore, the conserved sequence in all of these proteins is located immediately adjacent to the transmembrane anchor. 5.3 The ectodomain of type XIII collagen is partly shed to the cell culture medium (I, II) Shedding of the ectodomain has been found for many transmembrane proteins (Ding et al. 2000, Franzke et al. 2002), and thus the secretion of the recombinant type XIII collagen expressed in insect cells was also tested. The cells were infected respectively with viruses directing the synthesis of the full-length collagen XIII (wthumanXIII), the protein lacking the intracellular portion (del1-38), and the C-terminally truncated one (del442-668), together with virus 4PHαβ encoding prolyl 4-hydroxylase. After infection, the cell lysates and culture media were analyzed by SDS-PAGE/Western blotting and detected with the antibody XIII/NC3-1. The results showed that approximately 50% of the del1-38 α-chains were secreted, whereas less than 10% of full-length and Cterminally truncated type XIII collagen were found in a shed form. Next, the membrane-bound and secreted del1-38 proteins were partially purified and subsequently separated by SDS-PAGE and electroblotted onto a PVDF membrane for Nterminal sequencing. Sequencing revealed that the membrane-bound form maintained its correct N-terminus starting from a methionine at residue 39 followed by the predicted 46 transmembrane domain, while the protein collected from the medium had its transmembrane domain removed. The N-terminal sequence of the secreted type XIII collagen, E109APKTSPGCN 109 (E =glutamic acid at residue 109 of human type XIII collagen), suggested that the protein was proteolytically cleaved at the site linked to the amino acid sequence R105RRR, which implied that certain furin-like protease(es) were involved in the cleavage (Nakayama et al. 1997, Seidah & Chretien 1997). In order to test this, the furin-specific inhibitor decanol-RVKR-chloromethylketone (Garten et al. 1994) was selected to test the inhibition of type XIII collagen secretion. The inhibitor (100 µM) was added to the insect cell culture medium 24 h post-infection, and the cells and media were collected 48 h postinfection for SDS-PAGE and Western blotting analysis. The data indicated that the furin protease inhibitor significantly reduced the amount of the secreted type XIII collagen ectodomain. The shedding was also inhibited when 10 units/ml of heparin was added to the cell culture medium before infection. A possible hypothesis is that the negatively charged heparin molecule interacted with the positively charged arginine clusters and consequently prevented the proteolytic cleavage. 5.4 The type XIII collagen ectodomain is a collagenous rod with two flexible hinges (II) The effective secretion of the type XIII collagen variant del1-38 made it convenient to purify the ectodomain from the insect cell culture medium. A purification method was developed involving the sequential use of anion exchange, cation exchange, and gel filtration chromatographies. This resulted in a purified ectodomain with a molecular mass of about 240 kDa shown on non-reducing SDS-PAGE. The N-terminus of this purified ectodomain was EAPKTSPGCN, which is the same as the sequence described in section 5.3. The amino acid composition analysis of the purified type XIII collagen ectodomain showed that 71.7% of the available prolines (at the Y position of the collagenous GlyXaa-Yaa amino acid triplets) and 30 % of the available lysines (at the Y position of GlyXaa-Yaa) were hydroxylated. The collagenous structure was analyzed further by limited pepsin digestion of the purified ectodomain. Four pepsin-resistant peptides were acquired with molecular masses of 20, 29, 34, and 39 kDa shown on reducing SDS-PAGE. The N-terminal sequencing of these fragments revealed that the 20-kDa fragment represented the collagen XIII COL1 domain, the 29-kDa fragment corresponded to COL2, and both the 34- and 39-kDa fragments represented the COL3 domain. Interestingly, the COL1 fragment, but not the other ones, showed an apparent molecular mass of 46 kDa in non-reducing SDS-PAGE indicating the presence of an intrachain disulfide bond. To define the 34- and 39-kDa peptides, the pepsin-digested mixture was separated by gel filtration chromatography and the fractions corresponding to the 34- and 39-kDa peptides were subjected to amino acid analysis. The 34-kDa fragment showed an identical composition to that of 39-kDa, except that the proline and lysine hydroxylation 47 levels were lower. We thus suggest that the lower molecular mass peptide has a lower glycosylation level, possibly due to a shortage of hydroxyl lysine residues. The structure of the type XIII collagen ectodomain was also studied with CD spectroscopy, which revealed a triple helical structure typical for a collagenous conformation. However, the ectodomain showed a broad melting profile from 27 to 42 °C, suggestive of a variable level of hydroxylation among the recombinant collagen XIII molecules. In fact, this was already observed when comparing the 34- and 39-kDa pepsin-resistant fragments. It is most likely that the highest melting point, 42 °C, represents the Tm of the molecules with the highest hydroxylation level and molecules with lower Tm values are under-hydroxylated. This is probably due to insufficient lysyl hydroxylase activity in insect cells. 5.5 The recombinant type XIII collagen ectodomain interacts with several major extracellular matrix proteins in vitro (II) Type XIII collagen has been found to be widely located in cell-matrix contact regions such as the basement membrane zones of normal human skin, in cultured keratinocytes, fibroblast cells, and several other human mesenchymal cell lines (Peltonen et al. 1999, Hägg et al. 2001). This suggests that type XIII collagen has the opportunity to associate with various ECM components. Therefore, the purified collagen XIII ectodomain was tested for binding in vitro with widely occurring ECM components using a surface plasmon resonance (SPR) assay on a Biacore® 3000 (Biacore AB, Uppsala, Sweden) and an ELISA solid phase assay. Using SPR, the recombinant ectodomain was found to interact with immobilized fibronectin, nidogen-2, and perlecan at apparent KD values of 2.4 nM, 5.4 nM, and 2.5 nM, respectively. The binding of type XIII collagen to fibronectin, nidogen-2, and perlecan was also confirmed by ELISA tests in which the ECM and basement membrane proteins were immobilized as ligands, and the type XIII collagen ectodomain was tested as a soluble analyte detectable by the antibody XIII/NC31. On the other hand, the fibril-forming collagen types I and III did not bind to the type XIII collagen ectodomain in any of the tests. However, some of the tested proteins showed complicated binding profiles with collagen XIII. Vitronectin, nidogen-1, fibulin-2, and types IV and VI collagen interacted with type XIII collagen moderately in ELISA tests and in the SPR assay when these proteins were analyzed as soluble analytes, but the binding was not observed when these proteins were immobilized on the CM-5 chip tested with the Biacore® 3000. Two other components, fibulin-1 and the laminin-1-nidogen-1 complex, did not show significant binding neither in ELISA as a soluble analyte nor in SPR as an immobilized ligand. However, a slow binding curve was monitored in SPR when collagen XIII was immobilized onto the CM-5 sensor chip. In addition, BM-40 did not bind to type XIII collagen in ELISA, but it interacted with collagen XIII in SPR so strongly that it was very difficult to dissociate these two molecules, which suggested that the different faces of the bound ligand are presented in these assays. Subsequently all of the isolated pepsin-digested fragments of the ectodomain were tested for binding to the immobilized fibronectin, nidogen-2, and perlecan using SPR. 48 Interestingly, all of the peptides bound to fibronectin, but none of them interacted with nidogen-2 or perlecan, which indicated that the binding sites on collagen XIII for these ECM molecules may be different and that some of these sites reside on the short noncollagenous domains. The interaction of fibronectin with the collagenous domains reflects a general interaction with triple-helical molecules. The yeast 2-hybrid system was not considered suitable in this work since it was considered possible that the interactions of type XIII collagen to other molecules rely on the collagenous triple helix conformation and glycosylation, or other post-translational modifications that are not correctly performed in the yeast cells. The recently developed mammalian cell in vivo interaction systems could shed some light on these properties, but it is uncertain if the environment inside the cells is suitable for ECM molecule interactions. Another possible way to detect ligand binding is to use affinity chromatography with the purified type XIII collagen coupled to the supporting materials, such as resins or beads. The tissue or cell extracts are loaded into the column under nearly physiological conditions and the bound molecules, i.e. the putative binding candidates, are eluted for clarification. This method has been successfully used for enzyme-substrate and growth factor-receptor interactions. However, it is not commonly used for isolating the ligands from ECM. Most components in the ECM already form networks or complexes, and several of them are in fibril forms, which make it difficult to identify each individual protein for the targeting ligand. Therefore, up to now solid phase and SPR assays have been considered the most useful methods for in vitro analysis of ECM molecule interactions. In any case, to obtain reliable results it is important to combine data from different binding assays, together with tissue or cell staining and immunoprecipitation results. 5.6 Heparin is a high-affinity ligand of type XIII collagen (II) The inhibition of type XIII collagen ectodomain cleavage by heparin (section 5.3) suggested that heparin molecules interacted with the arginine residue cluster at the cleavage site adjacent to the transmembrane domain, masking the cleavage sites for the furin-like protease. This presumption was studied further by heparin affinity chromatography, in which the membrane-bound form of the type XIII collagen variant del1-38 bound to the column so tightly that 1M NaCl was needed for its elution. The purified collagen XIII ectodomain was analyzed using heparin affinity chromatography. Surprisingly, although the four arginine residues supposed to be involved in the heparin binding were eliminated in the purified ectodomain, the ectodomain bound to the column at a low concentration of NaCl and was eluted with 0.7 M NaCl. This indicated that there are several heparin-binding sites on the type XIII collagen molecule. The interaction between the type XIII collagen ectodomain and heparin was further confirmed by an ELISA test using heparin-BSA as an immobilized ligand and the ectodomain as a soluble analyte. The estimated KD was close to 2 nM based on the determination of the type XIII collagen concentration for half-maximal saturation. These 49 in vitro data indicated that heparin and heparin-linked proteins are high affinity ligands for type XIII collagen. 5.7 The type XIII collagen ectodomain selectively recognizes integrin α1 and α11 I domains (III, IV) Although type XIII collagen is membrane-bound and thus distinct from the secreted collagens, its extensive ectodomain is nevertheless largely collagenous. This prompted us to study the interaction between the collagen XIII ectodomain and collagen receptor integrins using a solid phase assay, following the identification of the ECM ligands for the ectodomain. Firstly, collagen type XIII, together with types I-V, was coated onto microtiter wells to test the binding of the europium-labelled recombinant integrin α1 and α2I domains. In the case of binding to α1I, type XIII collagen showed the highest signal amongst all of the tested collagens, while in the case of α2I, only marginal binding of the α2I domain to collagen XIII was detected. Collagen types I-V did not show significant differences in the two binding tests, except that the interaction between human collagen V and α2I was about 50% weaker than that between collagen V and α1I. The interaction between the integrin α11 I domain and type XIII collagen ectodomain was analyzed by a solid phase assay using α11I as a solid ligand and the type XIII collagen ectodomain as a soluble analyte in a PBS buffer containing 2 mM MgCl2 and 20 µM CaCl2. The interacting proteins were detected with the monoclonal antibody against the recombinant type XIII collagen ectodomain. The binding of these two proteins was dose-dependent showing the apparent KD of 45 ± 4.4 nM, which is lower than the KD value (750 ± 50 nM) measured for the interaction between the integrin α11I domain and the type I collagen (Zhang et al. 2003). The result is suggestive of high affinity binding between collagen XIII and α11β1 integrin. 5.8 Integrins α1β1 and α11β1 mediate cell attachment to type XIII collagen (III, IV) In addition to the biochemical analyses of the binding of integrin I domains to collagens, the involvement of integrins in cell-collagen interactions was studied by expression of integrins α1β1, α2β1, and α11β1 in mammalian cells lacking the collagen receptor integrins, followed by examination of the spreading of these cells on collagen-coated surfaces. In the case of α1β1 and α2β1 expression, the CHO cells were stably transfected with cDNAs coding for integrin α1 and for α2, respectively, and the recombinantly expressed integrins were detected on the cell surface using flow cytometry. CHO-α2β1 cells allowed to attach to collagens I-V for 120 min showed the highest affinity to types I and IV and moderate adhesion to types II, III, and V. Most of the CHO-α2β1 cells attached to type IV collagen showed spreading morphology, whereas the cells attached to type I collagen had a circular shape. The spreading of CHO-α1β1 cells on collagens was 50 more selective. The CHO-α1β1 cells spread on collagen types I, III, and IV, but not on type II collagen, with the highest affinity to collagen IV. To achieve spreading of CHOα1β1 cells on type V collagen, a higher concentration of the ligand was needed compared to that used in the CHO-α2β1 spreading. Interestingly, the CHO cells expressing α1β1 integrin attached to type XIII collagen with almost the same affinity as observed for type IV collagen, but the attached cells showed a circular shape, which was morphologically different from the fibroblast-like appearance of the cells spreading on type IV collagen. On the other hand, no significant attachment of the CHO-α2β1 cells on type XIII collagen was observed. The interaction of integrin α11β1 and type XIII collagen was analyzed using C2C12 cells stably transfected with an α11 cDNA. No other collagen receptors were expressed in these cells. The recombinant integrin α11 was visualized on the plasma membrane by cell immunostaining (Tiger et al. 2001) and it was detected by Western blotting analyses of cell lysates extracted with a Triton-containing buffer, indicating that α11β1 was located on the cell membrane. Like other fibroblastic cells, C2C12 cells contain the α5β1 integrin, and thereby both wild-type and α11-transfected C2C12 cells spread on fibronectin in the same manner, which was considered saturated binding (100%) of cells to the matrix proteins in a quantitative β-hexosaminidase release assay. The C2C12-α11+ cells attached to type I collagen, confirming that the α11β1 integrin recognizes interstitial collagens (Zhang et al. 2003). Interestingly, a higher number of C2C12-α11+ cells attached to the type XIII collagen ectodomain-coated surface, and the difference of the attachment between these two collagen types were statistically significant, i.e. 80 % on collagen XIII versus 40 % on collagen I (P value =0.0035). The attached C2C12-α11+ cells started to spread on both collagens XIII and I, as well as to fibronectin after a 45 min incubation at 37 °C in a basic physiological buffer (PUCK’s saline) equilibrated with 5% CO2. 5.9 Localization of α11β1 integrin on cells spreading on type XIII collagen is associated with the focal adhesion molecule vinculin (IV) The location of integrin α11β1 in primary wild-type MEFs was studied by immunostaining using the antibody against the cytosolic tail of mouse α11 subunit. The cells spread nearly equally on types I and XIII collagens, and the integrin α11β1 was detected at the typical focal adhesion sites. The cells were further double-stained with an antibody against one of the focal adhesion markers, vinculin, together with that against α11. α11β1 was visualized at focal contacts, where vinculin was also detected. In addition, on immortalized MEFs spreading on the type XIII collagen ectodomain, α11 and vinculin also co-localized at focal adhesion sites. Interestingly, in MEFs derived from α11-null mice (Popva et al. a manuscript under preparation) vinculin was not detected at the focal adhesion sites, although the cells attached and spread on type XIII collagen similar to the wild-type MEFs. A difference in the immunostaining pattern of vinculin was not observed either for cells from wild-type or α11-null mice spreading on type I collagen. 6 Discussion Our aim was to study the molecular properties and biosynthetic features of type XIII collagen, a type II transmembrane protein. For this purpose, type XIII collagen was recombinantly produced in insect cells using a baculovirus expression system. The key enzyme for collagen biosysthesis, prolyl 4-hydroxylase, was co-expressed in the same cells to ensure sufficient hydroxylation of proline residues and thus formation of stable triple helices. Although the recombinant protein expression level in the insect cell expression model is not as high as that in E. coli or in yeast models, it is more useful when post-translational modification is essential for the expressed protein. Moreover, the cells can be maintained in serum-free media, which is convenient for purification of the protein from the medium. The insect cell expression system has been proven to be useful for production of the fibrillar collagen types I, II, and III, and type XII collagen with properties nearly identical to the authentic respective collagens (Lamberg et al. 1996, Mazzorana et al. 1996, Myllyharju et al. 1997, Nokelainen et al. 1998). In terms of expressing recombinant type XIII collagen, the method minimizes the problems of contaminating endogenous type XIII collagen that is not detected in the insect cells. Therefore, various constructs can be transfected into the cells for biosynthesis studies without the interference of endogenous type combined XIII collagen. Type XIII collagen has not previously been isolated as a purified triple helical protein. Here we developed a purification method for type XIII collagen shed into the culture medium. The biosynthesis of type XIII collagen was studied by expressing various deletion variants in insect cells. The data presented in paper I showed that residues 63-83 of human type XIII collagen, adjacent to the transmembrane domain at the extracellular side, are important for the formation of disulfide-bonded trimers. We also observed that 13 residues within this 21-residue motif are also conserved in type XVII collagen, MARCO, and EDA. Thus our proposal was that this 21-residue motif is essential for the correct association of the three α1(XIII) chains, and that this sequence is also important for chain association in the case of several other collagenous transmembrane proteins. (See section 5.2). Subsequently, further data on this sequence has revealed that this chain association sequence is necessary but not sufficient for formation of the disulfide-bonded trimer, as the transmembane domain also plays a role in the proper association of the three α1(XIII) chains (Latvanlehto et al. 2003). Detailed characterization of the nature of 52 the transmembrane-proximal association domain revealed it to be a short coiled-coil motif (Latvanlehto et al. 2003). In addition, another coiled-coil motif has been identified in the NC3 domain of type XIII collagen, and similar structures have been predicted in the homologous types XXIII and XXV collagens (Latvanlehto et al. 2003). The presence of coiled-coil sequences has subsequently been observed for a number of other collagens (McAlinden et al. 2003). Thus the coiled-coil motif has emerged as a previously unrecognized factor in collagen chain association and shedding. In the classical collagen types, the collagen triple helices fold from the C-terminus (Lees et al. 1997, McLaughlin & Bulleid 1998, Boutaud et al. 2000, Borza et al. 2001, Boudko & Engel 2004). On the other hand, in the collagenous transmembrane proteins, the coiled-coil structures occur at the N-terminal part and in the middle of the molecules (Snellman & Pihlajaniemi 2003). This and previous data with recombinantly expressed deletion constructs suggest that the transmembrane collagens start their chain association from the N-terminus, contrary to the folding direction of fibrillar collagens (Snellman et al. 2000, Areida et al. 2001). The fact that α-helical coiled-coil oligomerization domains have now been identified in most types of collagens as well as their location before, after, or between collagenous regions suggest that chain association can start from the both sides of the collagenous domains (Frank et al. 2003, McAlinden et al. 2003). The general roles of these recently identified coiled-coil motifs in collagen triple-helical assembly remain to be revealed. Type XIII collagen has been found to be shed in insect cells (Snellman et al. 2000) and also in keratinocytes (Peltonen et al. 1999). The present studies show that the proteolytic cleavage site resides at R105RRR↓, and the cleavage is induced by furin protease (original article I). In our studies, the variant del1-38 lacking the cytosolic portion has shown higher expression and secretion levels than full-length α1(XIII) chains. We do not know whether the cytosolic part inhibits the shedding or whether the high expression of the protein induces more shedding. The physiological function of type XIII collagen shedding remains to be studied. It is possible that full-length type XIII collagen is important for cell-matrix adhesion. This is suggested by the deficiency observed in the skeletal muscle-basement membrane interphase in Col13aN/N mice lacking the cytosolic and transmembrane domain (Kvist et al. 2001). Our studies show that the shed form is triple-helical, and thus likely to be functional. It is possible that the shed ectodomain interacts with ECM components and integrins and could thereby it could regulate matrix network formation and cell receptor stimulation. Due to the hydrophobic and easily aggregating features of the transmembrane domain, the purification of membrane-bound type XIII collagen was not successful. Instead, the ectodomain of type XIII collagen was isolated from the insect cell culture medium. The cells were transfected with viruses encoding the type XIII collagen del1-38 variant and prolyl 4-hydroxylase. In the purified type XIII collagen ectodomain, 72% of the proline in the collagenous triplet Gly-Xaa-Pro and 20 % of lysines in Gly-Xaa-Lys were hydroxylated. Although native type XIII collagen from tissues is not available to evaluate the hydroxylation data, the modification levels of the recombinant protein are close to that of other recombinant collagens expressed in insect cells (Lamberg et al. 1996, Myllyharju et al. 1997). Previously the type XIII collagen variant del1-83 was purified from insect cell lysates in a denatured form, and a monoclonal antibody was raised against it, but it turned out to 53 be a pan-collagen antibody that recognizes several types of collagens in Western blotting analysis (Snellman et al. 2000). In original article II, a new monoclonal antibody was produced from the purified type XIII collagen ectodomain with a triple helical conformation. This antibody shows specificity for recognizing type XIII collagen in Western blotting as well as in immunofluorescent staining of cells expressing type XIII collagen (Tu et al. a manuscript in preparation). We performed rotary shadowing electron microscopy to obtain information about the molecular dimensions of type XIII collagen. The type XIII collagen ectodomain was detected in the rotary shadowing electron microscope as a 150-nm rod indicative of collagenous triple helical folding. However, in contrast to the fibrillar collagen rod structure, type XIII collagen is more flexible since it has two kinks which coincided in location with the non-collagenous domains separating the three collagenous segments. The structure is markedly distinct from other cell adhesion molecules such as integrin (Nermut et al. 1985, Weisel et al. 1992) and the identified structures of other collagenous transmembrane proteins such as type XVII collagen (Hirako et al. 1996) and MSRs (Resnick et al. 1996). The elongated and flexible type XIII collagen molecule could possibly cross the basement membrane under epithelial cells and penetrate into the surrounding stroma. This highly elongated structure is suggestive of multiple interactions for type XIII collagen with proteins on the cell surface as well as in the basement membrane and in other parts of the ECM. In the ECM, numerous molecules interact with each other to form complex molecular networks. The interactions between these constituents have been defined mostly by in vitro tests between individual molecules. Hence we have chosen the most abundant ECM molecules to test their binding to the recombinant type XIII collagen ectodomain. Both solid phase and SPR assays were applied for the analyses, the former one for the preliminary screening and the latter one for the kinetic analysis. Due to the multiple binding sites of the tested ECM proteins and the type XIII ectodomain itself, the rate constants derived from the data evaluations are not unequivocal. Nevertheless, the data suggest high affinity binding to fibronectin, nidogen, and perlecan and moderate interactions to several other ECM molecules. Fibronectin is one of the major components in the ECM and is also localized to the cell surface (Geiger et al. 2001, Pankov & Yamada 2002). Although occurring at low levels, type XIII collagen can be found widely in tissues, and the observed high affinity binding in vitro raises the possibilities that type XIII collagen may affect fibronectin matrix formation or fibronectin-cell crosstalk. This might be tested by adding the recombinant type XIII collagen ectodomain to cultured cells or by overexpressing type XIII collagen in cells to study its effect on fibronectin function. Type XIII collagen has been observed in many tissues and cell-basement membrane interphases (Sandberg et al. 1989, Juvonen et al. 1993, Sandberg-Lall et al. 2000, Hägg et al. 2001, Kvist et al. 2001, Sund et al. 2001a). Thus the high affinity of the type XIII collagen ectodomain to the basement membrane components nidogen-2 and perlecan and its moderate binding to type IV collagen is intriguing. Based on the studies with isolated COL1, COL2, and COL3 domains, the interactions with the basement membrane compounds occur at the non-collagenous domains. This is in contrast to the observed interaction with fibronectin, which most possibly involved the collagenous domains of type XIII collagen. The specific interaction between type XIII collagen and basement 54 membrane molecules has also been implied in the studies of the Col13a1N/N mice (Kvist et al. 2001), as the fibroblasts derived from Col13a1N/N mice showed decreased adhesion to type IV collagen and the sarcolemma-basement membrane interphase appeared abnormal in the muscle tissues of these mice. The type XIII collagen ectodomain showed significant binding to perlecan, a heparan sulphate proteoglycan, but no further tests have been performed to detect if the binding occurred at the protein core or at the glycosaminoglycan. However, the heparin binding data supports the possibility of an interaction between type XIII collagen and heparin sulphate proteoglycans. Moreover, heparin also inhibits the secretion of the type XIII collagen ectodomain into the cell culture medium, suggesting a possible role for heparin or its related molecules in regulating the shedding of transmembrane type XIII collagen. Integrin-collagen interactions are considered important for cell-ECM communication. A number of studies have shown that collagen receptor integrins recognize specific types of collagen. One hypothesis for this phenomenon is tissue specificity. This is easily understandable in the case of skin or other connective tissues where thick basement membranes (>50nm) surround the epithelial cells. The ectodomain of an integrin molecule is only 20-30 nm long (Nermut et al. 1985, Weisel et al. 1992). In such locations, the integrin collagenous ligand is apparently type IV collagen occurring as the major component of basement membrane. On the other hand, in fibroblastic cells and most embryonic tissues in which basement membranes are rather thin or not formed, the fibrillar collagens are also accessible for integrin binding. Although there are several publications showing the specific residues or sequences that are critical for recognition, it is unclear whether the surrounding collagens give feedback to the cells to selectively express specific integrins (Knight et al. 1998, Golbic et al. 2000, Xu et al. 2000). Type XIII collagen might be a good candidate for this purpose because it resides on the plasma membrane and not only occurs on cells adjacent to a thick basal lamina, but also on fibroblasts and other cells with nearly no basement membrane. In the present studies (original article III), type XIII collagen shows affinity for α1β1 but not α2β1 integrin, which is different from other types of collagen (I-V) tested. In addition, type XIII collagen showed high affinity to α11β1 integrin (original article IV). The in vitro cell attachment tests revealed that integrin α11β1 mediates more cells attached to type XIII collagen than to type I collagen. However, CHO cells expressing α1β1 as the only active collagen receptor integrin attached to type XIII collagen but retained a round shape, whereas C2C12 cells expressing α11β1 integrin as the only collagen receptor integrin spread on the type XIII collagen ectodomain-coated surface. This cell morphological difference might be due to the different cell types used in these studies or a difference in the mechanisms of interaction between the type XIII collagen ectodomain and different integrins. The transmembrane location and possiblities to interaction with integrins raise a question that if type XIII collagen may synergise with or influence adjacent integrins. More studies are expected to be performed to answer this question. The sequence motif GFOGER in the collagenous triple helical structure has been identified as a recognition site for α1β1, α2β1, and α11β1 integrins (Knight et al. 1998, Xu et al. 2000, Gullberg & Lundgren-Åkerlund 2002, Zhang et al. 2003). Type XIII collagen does not contain the exact sequence of GFOGER, but instead it contains several GER sequences located in COL2 and COL3 domains. Two of the GER motifs have been identified within a highly conserved region at the C-terminal part of type XIII collagen, 55 which is also distant from the transmembrane domain. Future work could be directed at identifying the critical residues in type XIII collagen for integrin binding. Knowledge of the mechanism of interactions between collagen receptor integrins and type XIII collagen might help to demonstrate if specific sequences or residues on the collagen triple helix decide the selections, or if the recognition is based on some other factors that remain to be defined. All in all, type XIII collagen has been studied for over ten years with the aim to elucidate the structure and function of this unique protein. Here the molecular structure and ligand binding data support the proposal that type XIII collagen provides mechanical strength in the cell-ECM and in cell-cell adherence junctions and it may also participate in ECM network formation. 7 Future perspectives Type XIII collagen was partly cloned in 1987 and it was reported as a type II transmembrane collagen in 1998 following meticulous investigations at the molecular and cellular levels. The recombinant expression of type XIII collagen in insect cells has provided important new possibilities for studying the protein in more detail. Several ligands for type XIII collagen have been identified in the present work, and in further work it will be important to identify the specific binding sites for the different interactions. The monoclonal antibody raised against the recombinant type XIII collagen ectodomain can be a useful tool to isolate native type XIII collagen from animal tissues or cultured mammalian cells in order to compare the properties of native and recombinant protein. Furthermore, cells transfected with different variants of type XIII collagen tagged by the enhanced green fluorescent protein (EGFP) can be applied to cell biological analyses to study the contribution of each type XIII collagen domain to cell-matrix adherence. On the other hand, inactivation of the type XIII collagen gene in vivo in mice or by siRNA techniques in cells will help to understand more about the physiological function of this protein. No data exists yet regarding the role of the cytosolic portion of this molecule. Thus studies should be directed at analyzing the possible ligands of the intracellular domain of type XIII collagen. 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