Download Type XIII collagen. Structural and functional characterization of the

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. In addition, cell biological studies are needed
to test if the bindings of type XIII collagen to cell membrane and ECM proteins cause
intracellular responses. The fact that type XIII collagen can be shed necessitates studies
on the significance of membrane-bound and shed forms of this protein. Finally, the
recently recognized fact that types XIII, XXIII, and XXV collagens form a subgroup
within the conventional collagen family of proteins, implies the possibility of functional
compensation of these molecular homologous transmembrane proteins. This hypothesis
needs to be addressed in the future.
References
Adams JC & Watt FM (1993) Regulation of development and differentiation by the extracellular
matrix. Development 117: 1183-1198.
De Arcangelis A & Georges-Labouesse E (2000) Integrin and ECM functions: roles in vertebrate
development. Trends Genet 16: 389-395.
Areida SK, Reinhardt DP, Muller PK, Fietzek PP, Kowitz J, Marinkovich MP & Notbohm H
(2001) Properties of the collagen type XVII ectodomain. Evidence for n- to c-terminal triple
helix folding. J Biol Chem 276: 1594-1601.
Aumailley M & Smyth N (1998) The role of laminins in basement membrane function. J Anat 193
( Pt 1): 1-21.
Baker KN, Rendall MH, Patel A, Boyd P, Hoare M, Freedman RB & James DC (2002) Rapid
monitoring of recombinant protein products: a comparison of current technologies. Trends
Biotechnol 20: 149-156.
Banyard J, Bao L & Zetter BR (2003) Type XXIII collagen, a new transmembrane collagen
identified in metastatic tumor cells. J Biol Chem 278: 20989-20994.
Battaglia C, Mayer U, Aumailley M & Timpl R (1992) Basement-membrane heparan sulfate
proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein
core. Eur J Biochem 208: 359-366.
Bayes M, Hartung AJ, Ezer S, Pispa J, Thesleff I, Srivastava AK & Kere J (1998) The anhidrotic
ectodermal dysplasia gene (EDA) undergoes alternative splicing and encodes ectodysplasin-A
with deletion mutations in collagenous repeats. Hum Mol Genet 7: 1661-1669.
Beck K & Gruber T (1995) Structure and assembly of basement membrane and related extracellular
matrix proteins. In: Richardson D & Steiner M (eds) Principles of Cell Adhesion. CRC Press,
Boca Raton, FL, 219-252.
Bendixen C, Gangloff S & Rothstein R (1994) A yeast mating-selection scheme for detection of
protein-protein interactions. Nucleic Acids Res 22: 1778-1779.
Blakely BT, Rossi FM, Tillotson B, Palmer M, Estelles A & Blau HM (2000) Epidermal growth
factor receptor dimerization monitored in live cells. Nat Biotechnol 18: 218-222.
Boot-Handford RP, Tuckwell DS, Plumb DA, Rock CF & Poulsom R (2003) A novel and highly
conserved collagen (proα1(XXVII)) with a unique expression pattern and unusual molecular
characteristics establishes a new clade within the vertebrate fibrillar collagen family. J Biol
Chem 278: 31067-31077.
Borradori L & Sonnenberg A (1999) Structure and function of hemidesmosomes: more than simple
adhesion complexes. J Invest Dermatol 112: 411-418.
Borza DB, Bondar O, Ninomiya Y, Sado Y, Naito I, Todd P & Hudson BG (2001) The NC1
domain of collagen IV encodes a novel network composed of the α1, α2, α5, and α6 chains in
smooth muscle basement membranes. J Biol Chem 276: 28532-28540.
58
Boudko SP & Engel J (2004) Structure Formation in the C terminus of Type III Collagen Guides
Disulfide Cross-Linking. J Mol Biol 335: 1289-1297.
Boutaud A, Borza DB, Bondar O, Gunwar S, Netzer KO, Singh N, Ninomiya Y, Sado Y, Noelken
ME & Hudson BG (2000) Type IV collagen of the glomerular basement membrane. Evidence
that the chain specificity of network assembly is encoded by the noncollagenous NC1 domains.
J Biol Chem 275: 30716-30724.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
Brekken RA & Sage EH (2001) SPARC, a matricellular protein: at the crossroads of cell-matrix
communication. Matrix Biol 19: 816-827.
Briesewitz R, Epstein MR & Marcantonio EE (1993) Expression of native and truncated forms of
the human integrin α1 subunit. J Biol Chem 268: 2989-2996.
Camper L, Hellman U & Lundgren-Åkerlund E (1998) Isolation, cloning, and sequence analysis of
the integrin subunit α10, a β1-associated collagen binding integrin expressed on chondrocytes. J
Biol Chem 273: 20383-20389.
Chien CT, Bartel PL, Sternglanz R & Fields S (1991) The two-hybrid system: a method to identify
and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A
88: 9578-9582.
Chou MY & Li HC (2002) Genomic organization and characterization of the human type XXI
collagen (COL21A1) gene. Genomics 79: 395-401.
Cierniewska-Cieslak A, Cierniewski CS, Blecka K, Papierak M, Michalec L, Zhang L, Haas TA &
Plow EF (2002) Identification and characterization of two cation binding sites in the integrin β3
subunit. J Biol Chem 277: 11126-11134.
Coligan JE, Kruisbeck AM, Margulies DH, Shevach EM & Strober W (1991) Chapter 5:
Immunofluorescence and cell sorting. Current Protocols in Immunology. Greene Publishing and
Wiley-Interscience, New York.
Colognato H & Yurchenco PD (2000) Form and function: the laminin family of heterotrimers. Dev
Dyn 218: 213-234.
Colorado PC, Torre A, Kamphaus G, Maeshima Y, Hopfer H, Takahashi K, Volk R, Zamborsky
ED, Herman S, Sarkar PK, Ericksen MB, Dhanabal M, Simons M, Post M, Kufe DW,
Weichselbaum RR, Sukhatme VP & Kalluri R (2000) Anti-angiogenic cues from vascular
basement membrane collagen. Cancer Res 60: 2520-2526.
Costell M, Gustafsson E, Aszodi A, Mörgelin M, Bloch W, Hunziker E, Addicks K, Timpl R &
Fässler R (1999) Perlecan maintains the integrity of cartilage and some basement membranes. J
Cell Biol 147: 1109-1122.
Couchman JR (2003) Syndecans: proteoglycan regulators of cell-surface microdomains? Nat Rev
Mol Cell Biol 4: 926-937.
Couchman JR, Ljubimov AV, Sthanam M, Horchar T & Hassell JR (1995) Antibody mapping and
tissue localization of globular and cysteine-rich regions of perlecan domain III. J Histochem
Cytochem 43: 955-963.
Debeer P, Schönmakers EF, Twal WO, Argraves WS, de Smet L, Fryns JP & Van de Ven WJ
(2002) The fibulin-1 gene (FBLN1) is disrupted in a t(12;22) associated with a complex type of
synpolydactyly. J Med Genet 39: 98-104.
Dickeson SK, Mathis NL, Rahman M, Bergelson JM & Santoro SA (1999) Determinants of ligand
binding specificity of the α1β1 and α2β1 integrins. J Biol Chem 274: 32182-32191.
Ding B, Price RL, Goldsmith EC, Borg TK, Yan X, Douglas PS, Weinberg EO, Bartunek J,
Thielen T, Didenko VV & Lorell BH (2000) Left ventricular hypertrophy in ascending aortic
stenosis mice: anoikis and the progression to early failure. Circulation 101: 2854-2862.
Drubin DG & Nelson WJ (1996) Origins of cell polarity. Cell 84: 335-344.
Dunlevy JR & Hassell JR (2000) Heparan sulphate proteoglycans in basement membranes.
Perlecan, agrin, collagen XVIII. In: Iozzo RV (ed) Proteoglycans: Structure, Biology, and
Molecular Interactions. Marcel Dekker, Inc., New York, 275-326.
Edmondson DG & Roth SY (2001) Identification of prorein interactions by far western analysis. In:
Current Protocols in Molecular Biology. 20.6.1-20.6.10.
59
Ekblom P, Lonai P & Talts JF (2003) Expression and biological role of laminin-1. Matrix Biol 22:
35-47.
Emsley J, Knight CG, Farndale RW, Barnes MJ & Liddington RC (2000) Structural basis of
collagen recognition by integrin α2β1. Cell 101: 47-56.
Engel J & Furthmayr H (1987) Electron microscopy and other physical methods for the
characterization of extracellular matrix components: laminin, fibronectin, collagen IV, collagen
VI, and proteoglycans. Methods Enzymol 145: 3-78.
Erb EM, Tangemann K, Bohrmann B, Müller B & Engel J (1997) Integrin αIIb β3 reconstituted
into lipid bilayers is nonclustered in its activated state but clusters after fibrinogen binding.
Biochemistry 36: 7395-7402.
Erickson AC & Couchman JR (2000) Still more complexity in mammalian basement membranes. J
Histochem Cytochem 48: 1291-1306.
Ettner N, Göhring W, Sasaki T, Mann K & Timpl R (1998) The N-terminal globular domain of the
laminin α1 chain binds to α1β1 and α2β1 integrins and to the heparan sulfate-containing
domains of perlecan. FEBS Lett 430: 217-221.
Fearon ER, Finkel T, Gillison ML, Kennedy SP, Casella JF, Tomaselli GF, Morrow JS & Van
Dang C (1992) Karyoplasmic interaction selection strategy: a general strategy to detect proteinprotein interactions in mammalian cells. Proc Natl Acad Sci U S A 89: 7958-7962.
Fields S & Song O (1989) A novel genetic system to detect protein-protein interactions. Nature
340: 245-246.
van der Flier A & Sonnenberg A (2001) Function and interactions of integrins. Cell Tissue Res
305: 285-298.
Fox JW, Mayer U, Nischt R, Aumailley M, Reinhardt D, Wiedemann H, Mann K, Timpl R, Krieg
T & Engel J. (1991) Recombinant nidogen consists of three globular domains and mediates
binding of laminin to collagen type IV. EMBO J 10: 3137-3146.
Frank S, Boudko S, Mizuno K, Schulthess T, Engel J & Bachinger HP (2003) Collagen triple helix
formation can be nucleated at either end. J Biol Chem 278: 7747-7750.
Franzke CW, Tasanen K, Schäcke H, Zhou Z, Tryggvason K, Mauch C, Zigrino P, Sunnarborg S,
Lee DC, Fahrenholz F & Bruckner-Tuderman L (2002) Transmembrane collagen XVII, an
epithelial adhesion protein, is shed from the cell surface by ADAMs. EMBO J 21: 5026-5035.
Franzke CW, Tasanen K, Schumann H & Bruckner-Tuderman L (2003) Collagenous
transmembrane proteins: collagen XVII as a prototype. Matrix Biol 22: 299-309.
Friedrich MV, Göhring W, Mörgelin M, Brancaccio A, David G & Timpl R (1999) Structural basis
of glycosaminoglycan modification and of heterotypic interactions of perlecan domain V. J Mol
Biol 294: 259-270.
Galarneau A, Primeau M, Trudeau LE & Michnick SW (2002) β-lactamase protein fragment
complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat
Biotechnol 20: 619-622.
Gardner H, Kreidberg J, Koteliansky V & Jaenisch R (1996) Deletion of integrin alpha 1 by
homologous recombination permits normal murine development but gives rise to a specific
deficit in cell adhesion. Dev Biol 175: 301-313.
Garten W, Hallenberger S, Ortmann D, Schafer W, Vey M, Angliker H, Shaw E & Klenk HD
(1994) Processing of viral glycoproteins by the subtilisin-like endoprotease furin and its
inhibition by specific peptidylchloroalkylketones. Biochimie 76: 217-225.
Geiger B, Bershadsky A, Pankov R & Yamada KM (2001) Transmembrane crosstalk between the
extracellular matrix and the cytoskeleton. Nat Rev Mol Cell Biol 2: 793-805.
George EL, Baldwin HS & Hynes RO (1997) Fibronectins are essential for heart and blood vessel
morphogenesis but are dispensable for initial specification of precursor cells. Blood 90: 30733081.
Giancotti FG & Ruoslahti E (1999) Integrin signaling. Science 285: 1028-1032.
Giancotti FG (2000) Complexity and specificity of integrin signalling. Nat Cell Biol 2: E13-E14.
Giudice GJ, Emery DJ & Diaz LA (1992) Cloning and primary structural analysis of the bullous
pemphigoid autoantigen BP180. J Invest Dermatol 99: 243-250.
60
Göhring W, Sasaki T, Heldin CH & Timpl R (1998) Mapping of the binding of platelet-derived
growth factor to distinct domains of the basement membrane proteins BM-40 and perlecan and
distinction from the BM-40 collagen-binding epitope. Eur J Biochem 255: 60-66.
Golbik R, Eble JA, Ries A & Kühn K (2000) The spatial orientation of the essential amino acid
residues arginine and aspartate within the alpha1beta1 integrin recognition site of collagen IV
has been resolved using fluorescence resonance energy transfer. J Mol Biol 297: 501-509.
Gordon MK, Gerecke DR, Hahn RA, Bhatt P, Goyal M & Koch M (2000) Cloning of a new
collagen, type XXIII, expressed in cornea. Invest Ophthalmol Vis Sci 41:
Gruenwald S & Heitz J (1993) Baculovirus Expression Vector System: Procedures & Methods
Manual. Pharmingen, San Diego, CA.
Gullberg D & Lundgren-Åkerlund E (2002) Collagen-binding I domain integrins--what do they do?
Prog Histochem Cytochem 37: 3-54.
Gullberg D, Tiger CF & Velling T (1999) Laminins during muscle development and in muscular
dystrophies. Cell Mol Life Sci 56: 442-460.
Gullberg D, Velling T, Sjöberg G & Sejersen T (1995) Up-regulation of a novel integrin α-chain (α
mt) on human fetal myotubes. Dev Dyn 204: 57-65.
Gumbiner BM (1996) Cell adhesion: the molecular basis of tissue architecture and morphogenesis.
Cell 84: 345-357.
Hägg P, Rehn M, Huhtala P, Väisänen T, Tamminen M & Pihlajaniemi T (1998) Type XIII
collagen is identified as a plasma membrane protein. J Biol Chem 273: 15590-15597.
Hägg P, Väisänen T, Tuomisto A, Rehn M, Tu H, Huhtala P, Eskelinen S & Pihlajaniemi T (2001)
Type XIII collagen: a novel cell adhesion component present in a range of cell-matrix adhesions
and in the intercalated discs between cardiac muscle cells. Matrix Biol 19: 727-742.
Hall ZW (1995) Laminin β2 (S-laminin): a new player at the synapse. Science 269: 362-363.
Hashimoto T, Wakabayashi T, Watanabe A, Kowa H, Hosoda R, Nakamura A, Kanazawa I, Arai
T, Takio K, Mann DM & Iwatsubo T (2002) CLAC: a novel Alzheimer amyloid plaque
component derived from a transmembrane precursor, CLAC-P/collagen type XXV. EMBO J
21: 1524-1534.
Henry MD & Campbell KP (1996) Dystroglycan: an extracellular matrix receptor linked to the
cytoskeleton. Curr Opin Cell Biol 8: 625-631
Hirako Y, Usukura J, Nishizawa Y & Owaribe K (1996) Demonstration of the molecular shape of
BP180, a 180-kDa bullous pemphigoid antigen and its potential for trimer formation. J Biol
Chem 271: 13739-13745.
Hogan B, Beddington R, Costantini F & Lacy E (1994) Manipulating the Mouse Embryo: A
Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY.
Hopf M, Göhring W, Kohfeldt E, Yamada Y & Timpl R (1999) Recombinant domain IV of
perlecan binds to nidogens, laminin-nidogen complex, fibronectin, fibulin-2 and heparin. Eur J
Biochem 259: 917-925.
Hopf M, Göhring W, Mann K & Timpl R (2001) Mapping of binding sites for nidogens, fibulin-2,
fibronectin and heparin to different IG modules of perlecan. J Mol Biol 311: 529-541.
Hopf M, Göhring W, Ries A, Timpl R & Hohenester E (2001) Crystal structure and mutational
analysis of a perlecan-binding fragment of nidogen-1. Nat Struct Biol 8: 634-640.
Hostikka SL & Tryggvason K (1988) The complete primary structure of the α2 chain of human
type IV collagen and comparison with the α1(IV) chain. J Biol Chem 263: 19488-19493.
Hostikka SL, Eddy RL, Byers MG, Höyhtyä M, Shows TB & Tryggvason K (1990) Identification
of a distinct type IV collagen α chain with restricted kidney distribution and assignment of its
gene to the locus of X chromosome-linked Alport syndrome. Proc Natl Acad Sci USA 87: 16061610.
Hudson BG, Reeders ST & Tryggvason K (1993) Type IV collagen: structure, gene organization,
and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse
leiomyomatosis. J Biol Chem 268: 26033-26036.
Hudson BG, Tryggvason K, Sundaramoorthy M & Neilson EG (2003) Alport's syndrome,
Goodpasture's syndrome, and type IV collagen. N Engl J Med 348: 2543-2556.
61
Humphries MJ (2002) Insights into integrin-ligand binding and activation from the first crystal
structure. Arthritis Res 4 Suppl 3: S69-S78.
Humphries MJ, McEwan PA, Barton SJ, Buckley PA, Bella J & Paul MA (2003) Integrin structure:
heady advances in ligand binding, but activation still makes the knees wobble. Trends Biochem
Sci 28: 313-320.
Hutter H, Vogel BE, Plenefisch JD, Norris CR, Proenca RB, Spieth J, Guo C, Mastwal S, Zhu X,
Scheel J & Hedgecock EM (2000) Conservation and novelty in the evolution of cell adhesion
and extracellular matrix genes. Science 287 : 989-994.
Hynes RO & Zhao Q (2000) The evolution of cell adhesion. J Cell Biol 150: F89-F96.
Hynes RO (1990) Fibronectins. In: Rich A (ed). Springer-Verlag Series in Molecular Biology.
Springer-Verlag, New York.
Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69: 11-25.
Hynes RO (1999a) Cell adhesion: old and new questions. Trends Cell Biol 9: M33-7.
Hynes RO (1999b) The dynamic dialogue between cells and matrices: implications of fibronectin's
elasticity. Proc Natl Acad Sci U S A 96: 2588-2590.
Hynes RO (2002a) A reevaluation of integrins as regulators of angiogenesis. Nat Med 8: 918-921.
Hynes RO (2002b) Integrins: bidirectional, allosteric signaling machines. Cell 110: 673-687.
Iozzo RV & San Antonio JD (2001) Heparan sulfate proteoglycans: heavy hitters in the
angiogenesis arena. J Clin Invest 108: 349-355.
Iozzo RV (1998) Matrix proteoglycans: from molecular design to cellular function. Annu Rev
Biochem 67: 609-652.
Iozzo RV, Cohen IR, Grassel S & Murdoch AD (1994) The biology of perlecan: the multifaceted
heparan sulphate proteoglycan of basement membranes and pericellular matrices. Biochem J
302 ( Pt 3): 625-639.
Ivaska J, Käpylä J, Pentikainen O, Hoffren AM, Hermonen J, Huttunen P, Johnson MS & Heino J
(1999a) A peptide inhibiting the collagen binding function of integrin α2I domain. J Biol Chem
274: 3513-3521.
Ivaska J, Reunanen H, Westermarck J, Koivisto L, Kähäri V-M & Heino J (1999b) Integrin α2β1
mediates isoform-specific activation of p38 and upregulation of collagen gene transcription by a
mechanism involving the α2 cytoplasmic tail. J Cell Biol 147: 401-416.
Jiang X & Couchman JR (2003) Perlecan and tumor angiogenesis. J Histochem Cytochem 51:
1393-1410.
Juvonen M & Pihlajaniemi T (1992) Characterization of the spectrum of alternative splicing of
α1(XIII) collagen transcripts in HT-1080 cells and calvarial tissue resulted in identification of
two previously unidentified alternatively spliced sequences, one previously unidentified exon,
and nine new mRNA variants. J Biol Chem 267: 24693-24699.
Juvonen M, Pihlajaniemi T & Autio-Harmainen H (1993) Location and alternative splicing of type
XIII collagen RNA in the early human placenta. Lab Invest 69: 541-551.
Juvonen M, Sandberg M & Pihlajaniemi T (1992) Patterns of expression of the six alternatively
spliced exons affecting the structures of the COL1 and NC2 domains of the α1(XIII) collagen
chain in human tissues and cell lines. J Biol Chem 267: 24700-24707.
Kalluri R (2003) Basement membranes: structure, assembly and role in tumour angiogenesis. Nat
Rev Cancer 3: 422-433.
Kamphaus GD, Colorado PC, Panka DJ, Hopfer H, Ramchandran R, Torre A, Maeshima Y, Mier
JW, Sukhatme VP & Kalluri R (2000) Canstatin, a novel matrix-derived inhibitor of
angiogenesis and tumor growth. J Biol Chem 275: 1209-1215.
Kielty CM & Grant ME (2002) The collagen family: structure, assembly, and organization in the
extracellular matrix. In: Royce, PM & Steinmann B (eds) Connective Tissue and Its Heritable
Disorders. Wiley-Liss, Inc., New York, 159-221.
Kim S & Wadsworth WG (2000) Positioning of longitudinal nerves in C. elegans by nidogen .
Science 288: 150-154.
Kivirikko KI (1993) Collagens and their abnormalities in a wide spectrum of diseases. Ann Med
25: 113-126.
Kleppel MM, Santi PA, Cameron JD, Wieslander J & Michael AF (1989) Human tissue
distribution of novel basement membrane collagen. Am J Pathol 134: 813-825.
62
Knight CG, Morton LF, Onley DJ, Peachey AR, Messent AJ, Smethurst PA, Tuckwell DS,
Farndale RW & Barnes MJ (1998) Identification in collagen type I of an integrin α2 β1-binding
site containing an essential GER sequence. J Biol Chem 273: 33287-33294.
Koch M, Laub F, Zhou P, Hahn RA, Tanaka S, Burgeson RE, Gerecke DR, Ramirez F & Gordon
MK (2003) Collagen XXIV, a vertebrate fibrillar collagen with structural features of
invertebrate collagens: selective expression in developing cornea and bone. J Biol Chem 278:
43236-43244.
Kohfeldt E, Sasaki T, Göhring W & Timpl R (1998) Nidogen-2: a new basement membrane protein
with diverse binding properties. J Mol Biol 282: 99-109.
Kostka G, Giltay R, Bloch W, Addicks K, Timpl R, Fässler R & Chu ML (2001) Perinatal lethality
and endothelial cell abnormalities in several vessel compartments of fibulin-1-deficient mice.
Mol Cell Biol 21: 7025-7034.
Kvist A-P, Latvanlehto A, Sund M, Eklund L, Väisänen T, Hägg P, Sormunen R, Komulainen J,
Fässler R & Pihlajaniemi T (2001) Lack of cytosolic and transmembrane domains of type XIII
collagen results in progressive myopathy. Am J Pathol 159: 1581-1592.
Kvist A-P, Latvanlehto A, Sund M, Horelli-Kuitunen N, Rehn M, Palotie A, Beier D &
Pihlajaniemi T (1999) Complete exon-intron organization and chromosomal location of the
gene for mouse type XIII collagen (col13a1) and comparison with its human homologue. Matrix
Biol 18: 261-274.
Lamberg A, Helaakoski T, Myllyharju J, Peltonen S, Notbohm H, Pihlajaniemi T, Kivirikko KI
(1996) Characterization of human type III collagen expressed in a baculovirus system.
Production of a protein with a stable triple helix requires coexpression with the two types of
recombinant prolyl 4-hydroxylase subunit. J Biol Chem 271: 11988-11995.
Landegren U (1984) Measurement of cell numbers by means of the endogenous enzyme
hexosaminidase. Applications to detection of lymphokines and cell surface antigens. J Immunol
Methods 67: 379-388.
Lane TF & Sage EH (1994) The biology of SPARC, a protein that modulates cell-matrix
interactions. FASEB J 8: 163-173.
Langeveld JP, Noelken ME, Hard K, Todd P, Vliegenthart JF, Rouse J & Hudson BG (1991)
Bovine glomerular basement membrane. Location and structure of the asparagine-linked
oligosaccharide units and their potential role in the assembly of the 7 S collagen IV tetramer. J
Biol Chem 266: 2622-2631.
Latvanlehto A, Snellman A, Tu H & Pihlajaniemi T (2003) Type XIII collagen and some other
transmembrane collagens contain two separate coiled-coil motifs, which may function as
independent oligomerization domains. J Biol Chem 278: 37590-37599.
Lees JF, Tasab M & Bulleid NJ (1997) Identification of the molecular recognition sequence which
determines the type-specific assembly of procollagen. EMBO J 16: 908-916.
Leinonen A, Mariyama M, Mochizuki T, Tryggvason K & Reeders ST (1994) Complete primary
structure of the human type IV collagen α4(IV) chain. Comparison with structure and
expression of the other α(IV) chains. J Biol Chem 269: 26172-26177.
Li S, Harrison D, Carbonetto S, Fässler R, Smyth N, Edgar D & Yurchenco P (2002) Matrix
assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell
differentiation. J Cell Biol 157: 1279-1290.
Liddington RC & Ginsberg MH (2002) Integrin activation takes shape. J Cell Biol 158: 833-839.
Liedberg B, Nylander C & Lundström I (1995) Biosensing with surface plasmon resonance--how it
all started. Biosens Bioelectron 10: i-ix.
Liu S, Thomas SM, Woodside DG, Rose DM, Kiosses WB, Pfaff M & Ginsberg MH (1999)
Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses. Nature
402: 676-681.
Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC & Ward DC (1998) Mutation detection
and single-molecule counting using isothermal rolling-circle amplification. Nat Genet 19: 225232.
Lueking A, Horn M, Eickhoff H, Bussow K, Lehrach H & Walter G (1999) Protein microarrays for
gene expression and antibody screening. Anal Biochem 270: 103-111.
63
Macy E, Kemeny M & Saxon A (1988) Enhanced ELISA: how to measure less than 10 picograms
of a specific protein (immunoglobulin) in less than 8 hours. FASEB J 2: 3003-3009.
Maeshima Y, Colorado PC, Torre A, Holthaus KA, Grunkemeyer JA, Ericksen MB, Hopfer H,
Xiao Y, Stillman IE & Kalluri R (2000) Distinct antitumor properties of a type IV collagen
domain derived from basement membrane. J Biol Chem 275: 21340-21348.
Mariyama M, Leinonen A, Mochizuki T, Tryggvason K & Reeders ST (1994) Complete primary
structure of the human α3(IV) collagen chain. Coexpression of the α3(IV) and α4(IV) collagen
chains in human tissues. J Biol Chem 269: 23013-23017.
Marneros AG & Olsen BR (2001) The role of collagen-derived proteolytic fragments in
angiogenesis. Matrix Biol 20: 337-345.
Martin KH, Slack JK, Boerner SA, Martin CC & Parsons JT (2002) Integrin connections map: to
infinity and beyond. Science 296: 1652-1653.
Mayer U, Zimmermann K, Mann K, Reinhardt D, Timpl R & Nischt R (1995) Binding properties
and protease stability of recombinant human nidogen. Eur J Biochem 227: 681-686.
Mazzorana M, Snellman A, Kivirikko KI, van der Rest M & Pihlajaniemi T (1996) Involvement of
prolyl 4-hydroxylase in the assembly of trimeric minicollagen XII. Study in a baculovirus
expression system. J Biol Chem 271: 29003-29008.
McAlinden A, Smith TA, Sandell LJ, Ficheux D, Parry DA & Hulmes DJ (2003) Alpha-helical
coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily. J Biol
Chem 278: 42200-42207.
McDonnell JM (2001) Surface plasmon resonance: towards an understanding of the mechanisms of
biological molecular recognition. Curr Opin Chem Biol 5: 572-577.
McGrath JA, Gatalica B, Li K, Dunnill MG, McMillan JR, Christiano AM, Eady RA & Uitto J
(1996) Compound heterozygosity for a dominant glycine substitution and a recessive internal
duplication mutation in the type XVII collagen gene results in junctional epidermolysis bullosa
and abnormal dentition. Am J Pathol 148: 1787-1796.
McLaughlin SH & Bulleid NJ (1998) Molecular recognition in procollagen chain assembly. Matrix
Biol 16: 369-377.
Miosge N, Sasaki T & Timpl R (2002) Evidence of nidogen-2 compensation for nidogen-1
deficiency in transgenic mice. Matrix Biol 21: 611-621.
Moiseeva EP (2001) Adhesion receptors of vascular smooth muscle cells and their functions.
Cardiovasc Res 52: 372-386.
Morla A, Zhang Z & Ruoslahti E (1994) Superfibronectin is a functionally distinct form of
fibronectin. Nature 367: 193-196.
Mould AP, Barton SJ, Askari JA, Craig SE & Humphries MJ (2003) Role of ADMIDAS cationbinding site in ligand recognition by integrin α5β1. J Biol Chem 278: 51622-51629.
Myers JC, Jones TA, Pohjolainen ER, Kadri AS, Goddard AD, Sheer D, Solomon E &
Pihlajaniemi T (1990) Molecular cloning of α5(IV) collagen and assignment of the gene to the
region of the X chromosome containing the Alport syndrome locus. Am J Hum Genet 46: 10241033.
Myers JC, Li D, Amenta PS, Clark CC, Nagaswami C & Weisel JW (2003) Type XIX collagen
purified from human umbilical cord is characterized by multiple sharp kinks delineating
collagenous subdomains and by intermolecular aggregates via globular, disulfide-linked, and
heparin-binding amino termini. J Biol Chem 278: 32047-32057.
Myllyharju J & Kivirikko KI (2001) Collagens and collagen-related diseases. Ann Med 33: 7-21.
Myllyharju J & Kivirikko KI (2004) Collagens and their mutations: from man to Drosophila and
Caenorhabditis elegans. Trends Genet 20: 33-43.
Myllyharju J, Lamberg A, Notbohm H, Fietzek PP, Pihlajaniemi T & Kivirikko KI (1997)
Expression of wild-type and modified proα chains of human type I procollagen in insect cells
leads to the formation of stable [α1(I)]2α2(I) collagen heterotrimers and [α1(I)]3 homotrimers
but not [α2(I)]3 homotrimers. J Biol Chem 272: 21824-21830.
Nakayama K (1997) Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in
processing of a wide variety of precursor proteins. Biochem J 327: 625-635.
Nermut MV, Green NM, Eason P, Yamada SS & Yamada KM (1988) Electron microscopy and
structural model of human fibronectin receptor. EMBO J 7: 4093-4099.
64
Nokelainen M, Helaakoski T, Myllyharju J, Notbohm H, Pihlajaniemi T, Fietzek PP & Kivirikko
KI (1998) Expression and characterization of recombinant human type II collagens with low and
high contents of hydroxylysine and its glycosylated forms. Matrix Biol 16: 329-338.
Ohashi PS, Mak TW, Van den Elsen P, Yanagi Y, Yoshikai Y, Calman AF, Terhorst C, Stobo JD
& Weiss A (1985) Reconstitution of an active surface T3/T-cell antigen receptor by DNA
transfer. Nature 316: 606-609.
Ohtani K, Suzuki Y, Eda S, Kawai T, Kase T, Keshi H, Sakai Y, Fukuoh A, Sakamoto T, Itabe H,
Suzutani T, Ogasawara M, Yoshida I & Wakamiya N (2001) The membrane-type collectin CLP1 is a scavenger receptor on vascular endothelial cells. J Biol Chem 276: 44222-44228.
Olsen BR (1995) New insights into the function of collagens from genetic analysis. Curr Opin Cell
Biol 7: 720-727.
Oohashi T, Sugimoto M, Mattei M-G & Ninomiya Y (1994) Identification of a new collagen chain,
α6(IV), by cDNA isolation and assignment of the gene to chromosome Xq22, which is the same
locus for COL4A5. J Biol Chem 269: 7520-7526.
O'Reilly MS, Böhm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR &
Folkman J (1997) Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell
88: 277-285.
Ortega N & Werb Z (2002) New functional roles for non-collagenous domains of basement
membrane collagens. J Cell Sci 115 : 4201-4214.
Pace JM, Corrado M, Missero C & Byers PH (2003) Identification, characterization and expression
analysis of a new fibrillar collagen gene, COL27A1. Matrix Biol 22: 3-14.
Pankov R & Yamada KM (2002) Fibronectin at a glance. J Cell Sci 115: 3861-3863.
Paulsson M, Yurchenco PD, Ruben GC, Engel J & Timpl R (1987) Structure of low density
heparan sulfate proteoglycan isolated from a mouse tumor basement membrane. J Mol Biol 197:
297-313.
Peltonen S, Hentula M, Hägg P, Ylä-Outinen H, Tuukkanen J, Lakkakorpi J, Rehn M, Pihlajaniemi
T & Peltonen J (1999) A novel component of epidermal cell-matrix and cell-cell contacts:
transmembrane protein type XIII collagen. J Invest Dermatol 113: 635-642.
Peltonen S, Rehn M & Pihlajaniemi T (1997) Alternative splicing of mouse α1(XIII) collagen
RNAs results in at least 17 different transcripts, predicting α1(XIII) collagen chains with length
varying between 651 and 710 amino acid residues. DNA Cell Biol 16: 227-234.
Phizicky EM & Fields S (1995) Protein-protein interactions: methods for detection and analysis.
Microbiol Rev 59: 94-123.
Pihlajaniemi T & Rehn M (1995) Two new collagen subgroups: Membrane associated collagens
and types XV and XVIII. Prog Nucleic Acid Res Mol Biol 50: 225-262.
Pihlajaniemi T & Tamminen M (1990) The α1 chain of type XIII collagen consists of three
collagenous and four noncollagenous domains, and its primary transcript undergoes complex
alternative splicing. J Biol Chem 265: 16922-16928.
Pihlajaniemi T, Myllylä R, Seyer J, Kurkinen M & Prockop DJ (1987) Partial characterization of a
low molecular weight human collagen that undergoes alternative splicing. Proc Natl Acad Sci
84: 940-944.
Pottgiesser J, Maurer P, Mayer U, Nischt R, Mann K, Timpl R, Krieg T & Engel J (1994) Changes
in calcium and collagen IV binding caused by mutations in the EF hand and other domains of
extracellular matrix protein BM-40 (SPARC, osteonectin). J Mol Biol 238: 563-574.
Pozzi A, Wary KK, Giancotti FG & Gardner HA (1998) Integrin alpha1beta1 mediates a unique
collagen-dependent proliferation pathway in vivo. J Cell Biol 142: 587-594.
Prockop DJ & Kivirikko KI (1995) Collagens: molecular biology, diseases, and potentials for
therapy. Annu Rev Biochem 64: 403-434.
Ramchandran R, Dhanabal M, Volk R, Waterman MJF, Segal M, Lu H, Knebelmann B &
Sukhatme VP (1999) Antiangiogenic activity of restin, NC10 domain of human collagen XV:
Comparison to endostatin. Biochem Biophys Res Commun 255: 735-739.
Rehn M & Pihlajaniemi T (1996) Type XV and type XVIII collagens, a new subgroup within the
family of collagens. Cell Dev Biol 7: 673-679.
Reisfeld RA, Lewis UJ & Williams DE (1962) Disk electrophoresis of basic proteins and peptides
on polyacrylamide gels. Nature 195: 281-283.
65
Resnick D, Chatterton JE, Schwartz K, Slayter H & Krieger M (1996) Structures of class A
macrophage scavenger receptors. Electron microscopic study of flexible, multidomain, fibrous
proteins and determination of the disulfide bond pattern of the scavenger receptor cysteine-rich
domain. J Biol Chem 271: 26924-26930.
Rich RL & Myszka DG (2000) Advances in surface plasmon resonance biosensor analysis. Curr
Opin Biotechnol 11: 54-61.
Rich RL & Myszka DG (2001) BIACORE: a new platform for routine biomolecular interaction
analysis. J Mol Recognit 14: 223-228.
Rich RL & Myszka DG (2003) A survey of the year 2002 commercial optical biosensor literature. J
Mol Recognit 16: 351-382.
Rossi M, Morita H, Sormunen R, Airenne S, Kreivi M, Wang L, Fukai N, Olsen BR, Tryggvason K
& Soininen R (2003) Heparan sulfate chains of perlecan are indispensable in the lens capsule
but not in the kidney. EMBO J 22: 236-245.
Ruoslahti E & Pierschbacher MD (1987) New perspectives in cell adhesion: RGD and integrins.
Science 238: 491-497.
Ruoslahti E & Reed JC (1994) Anchorage dependence, integrins, and apoptosis. Cell 77: 477-478.
Ruoslahti E (1999) Fibronectin and its integrin receptors in cancer. Adv Cancer Res 76: 1-20.
Ruoslahti E (2003) The RGD story: a personal account. Matrix Biol 22: 459-465.
Sampson NS, Ryan ST, Enke DA, Cosgrove D, Koteliansky V & Gotwals P (2001) Global gene
expression analysis reveals a role for the α1 integrin in renal pathogenesis. J Biol Chem 276:
34182-34188.
Sandberg M, Tamminen M, Hirvonen H, Vuorio E & Pihlajaniemi T (1989) Expression of mRNAs
coding for the α1 chain of type XIII collagen in human fetal tissues: comparison with expression
of mRNAs for collagen types I, II, and III. J Cell Biol 109: 1371-1379.
Sandberg-Lall M, Hägg PO, Wahlström I & Pihlajaniemi T (2000) Type XIII collagen is widely
expressed in the adult and developing human eye and accentuated in the ciliary muscle, the
optic nerve and the neural retina. Exp Eye Res 70: 401-410.
Sankala M, Brännström A, Schulthess T, Bergmann U, Morgunova E, Engel J, Tryggvason K &
Pikkarainen T (2002) Characterization of recombinant soluble macrophage scavenger receptor
MARCO. J Biol Chem 277: 33378-33385.
Sato K, Yomogida K, Wada T, Yorihuzi T, Nishimune Y, Hosokawa N & Nagata K (2002) Type
XXVI collagen, a new member of the collagen family, is specifically expressed in the testis and
ovary. J Biol Chem. 277: 37678-37684
Schäcke H, Schumann H, Hammami-Hauasli N, Raghunath M & Bruckner-Tuderman L (1998)
Two forms of collagen XVII in keratinocytes. A full-length transmembrane protein and a
soluble ectodomain. J Biol Chem 273: 25937-25943.
Schumann H, Baetge J, Tasanen K, Wojnarowska F, Schäcke H, Zillikens D & BrucknerTuderman L (2000) The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies
in different blistering skin diseases. Am J Pathol 156: 685-695.
Seidah NG & Chretien M (1997) Eukaryotic protein processing: endoproteolysis of precursor
proteins. Curr Opin Biotechnol 8: 602-607.
Shows TB, Tikka L, Byers MG, Eddy RL, Haley LL, Henry WM, Prockop DJ & Tryggvason K
(1989) Assignment of the human collagen α1(XIII) chain gene (COL13A1) to the q22 region of
chromosome 10. Genomics 5: 128-133.
Snellman A & Pihlajaniemi T (2003) Collagenous transmembrane proteins. Recent Res Devel Biol
Chem 2: 1-22.
Snellman A, Keränen MR, Hägg PO, Lamberg A, Hiltunen JK, Kivirikko KI & Pihlajaniemi T
(2000) Type XIII collagen forms homotrimers with three triple helical collagenous domains and
its association into disulfide-bonded trimers is enhanced by prolyl 4-hydroxylase. J Biol Chem
275: 8936-8944.
Söderberg L, Zhukareva V, Bogdanovic N, Hashimoto T, Winblad B, Iwatsubo T, Lee VM,
Trojanowski JQ & Näslund J (2003) Molecular identification of AMY, an Alzheimer disease
amyloid-associated protein. J Neuropathol Exp Neurol 62: 1108-1117.
Soininen R, Haka-Risku T, Prockop DJ & Tryggvason K (1987) Complete primary structure of the
α1 chain of human basement membrane (type IV) collagen. FEBS Lett 225: 188-194.
66
Stanley JR (1995) Autoantibodies against adhesion molecules and structures in blistering skin
diseases. J Exp Med 181: 1-4.
Stoica GE, Kuo A, Aigner A, Sunitha I, Souttou B, Malerczyk C, Caughey DJ, Wen D, Karavanov
A, Riegel AT & Wellstein A (2001) Identification of anaplastic lymphoma kinase as a receptor
for the growth factor pleiotrophin. J Biol Chem 276: 16772-16779.
Stoscheck CM (1990) Quantitation of Protein. Methods Enzymol 182: 50-68.
Sund M, Väisänen T, Kaukinen S, Ilves M, Tu H, Autio-Harmainen H, Rauvala H & Pihlajaniemi
T (2001a) Distinct expression of type XIII collagen in neuronal structures and other tissues
during mouse development. Matrix Biol 20: 215-231.
Sund M, Ylönen R, Tuomisto A, Sormunen R, Tahkola J, Kvist A-P, Kontusaari S, AutioHarmainen H, Pihlajaniemi T (2001b) Abnormal adherence junctions in the heart and reduced
angiogenesis in transgenic mice overexpressing mutant type XIII collagen. EMBO J 20:51535164
Sundaramoorthy M, Meiyappan M, Todd P & Hudson BG (2002) Crystal structure of NC1
domains. Structural basis for type IV collagen assembly in basement membranes. J Biol Chem
277: 31142-31153.
Takada Y & Hemler ME (1989) The primary structure of the VLA-2/collagen receptor α2 subunit
(platelet GPIa): homology to other integrins and the presence of a possible collagen-binding
domain. J Cell Biol 109: 397-407.
Tarttelin EE, Gregory-Evans CY, Bird AC, Weleber RG, Klein ML, Blackburn J & Gregory-Evans
K (2001) Molecular genetic heterogeneity in autosomal dominant drusen. J Med Genet 38: 381384.
Thiel S & Reid KB (1989) Structures and functions associated with the group of mammalian lectins
containing collagen-like sequences. FEBS Lett 250: 78-84.
Tiger CF, Fougerousse F, Grundström G, Velling T & Gullberg D (2001) α11β1 integrin is a
receptor for interstitial collagens involved in cell migration and collagen reorganization on
mesenchymal nonmuscle cells. Dev Biol 237: 116-129.
Tikka L, Elomaa O, Pihlajaniemi T & Tryggvason K (1991) Human α1(XIII) collagen gene.
Multiple forms of the gene transcripts are generated through complex alternative splicing of
several short exons. J Biol Chem 266: 17713-17719.
Tikka L, Pihlajaniemi T, Henttu P, Prockop DJ & Tryggvason K (1988) Gene structure for the α1
chain of a human short-chain collagen (type XIII) with alternatively spliced transcripts and
translation termination codon at the 5' end of the last exon. Proc Natl Acad Sci USA 85: 74917495.
Timpl R & Brown J (1996) Supramolecular assembly of basement membranes. BioEssays 18: 123132.
Timpl R (1996) Macromolecular organization of basement membranes. Curr Opin Cell Biol 8: 618624.
Timpl R, Sasaki T, Kostka G & Chu ML (2003) Fibulins: a versatile family of extracellular matrix
proteins. Nat Rev Mol Cell Biol 4: 479-489.
Tryggvason K (1996) Mutations in type IV collagen genes and Alport phenotypes. Contrib Nephrol
117: 154-171.
Tryggvason K, Zhou J, Hostikka SL & Shows TB (1993) Molecular genetics of Alport syndrome.
Kidney Int 43: 38-44.
Tulla M, Pentikainen OT, Viitasalo T, Käpylä J, Impola U, Nykvist P, Nissinen L, Johnson MS &
Heino J (2001) Selective binding of collagen subtypes by integrin α1I, α2I, and α10I domains. J
Biol Chem 276: 48206-48212.
Velling T, Kusche-Gullberg M, Sejersen T & Gullberg D (1999) cDNA cloning and chromosomal
localization of human α11 integrin. A collagen-binding, I domain-containing, β1-associated
integrin α-chain present in muscle tissues. J Biol Chem 274: 25735-25742.
Voigt S, Gossrau R, Baum O, Loster K, Hofmann W & Reutter W (1995) Distribution and
quantification of alpha 1-integrin subunit in rat organs. Histochem J 27: 123-132.
Vuorio E & de Crombrugghe B (1990) The family of collagen genes. Annu Rev Biochem 59: 837872.
67
Vuorio E (1986) Connective tissue diseases: mutations of collagen genes. Ann Clin Res 18: 234241.
Wary KK, Mariotti A, Zurzolo C & Giancotti FG (1998) A requirement for caveolin-1 and
associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell 94: 625634.
Watt FM (2002) Role of integrins in regulating epidermal adhesion, growth and differentiation.
EMBO J 21: 3919-3926.
Wehrman T, Kleaveland B, Her JH, Balint RF & Blau HM (2002) Protein-protein interactions
monitored in mammalian cells via complementation of β-lactamase enzyme fragments. Proc
Natl Acad Sci 99: 3469-3474.
Weisel JW, Nagaswami C, Vilaire G & Bennett JS (1992) Examination of the platelet membrane
glycoprotein IIb-IIIa complex and its interaction with fibrinogen and other ligands by electron
microscopy . J Biol Chem 267: 16637-16643.
Williams C & Addona TA (2000) The integration of SPR biosensors with mass spectrometry:
possible applications for proteome analysis. Trends Biotechnol 18: 45-48.
Wilson WD (2002) Analyzing biomolecular interaction. Science 295: 2103-2105.
Wiltshire S, O'Malley S, Lambert J, Kukanskis K, Edgar D, Kingsmore SF & Schweitzer B (2000)
Detection of multiple allergen-specific IgEs on microarrays by immunoassay with rolling circle
amplification. Clin Chem 46: 1990-1993.
Xiong JP, Stehle T, Diefenbach B, Zhang R, Dunker R, Scott DL, Joachimiak A, Goodman SL &
Arnaout MA (2001) Crystal structure of the extracellular segment of integrin αVβ3. Science
294: 339-345.
Xu Y, Gurusiddappa S, Rich RL, Owens RT, Keene DR, Mayne R, Hook A & Hook M (2000)
Multiple binding sites in collagen type I for the integrins α1β1 and α2β1. J Biol Chem 275:
38981-38989.
Yan Q & Sage EH (1999) SPARC, a matricellular glycoprotein with important biological
functions. J Histochem Cytochem 47: 1495-1506.
Yang M, Wu Z & Fields S (1995) Protein-peptide interactions analyzed with the yeast two-hybrid
system. Nucleic Acids Res 23: 1152-1156.
Yurchenco PD & O'Rear JJ (1994) Basal lamina assembly. Curr Opin Cell Biol 6: 674-681.
Yurchenco PD & Schittny JC (1990) Molecular architecture of basement membranes. FASEB J 4:
1577-1590.
Zhang W-M, Käpylä J, Puranen JS, Knight CG, Tiger CF, Pentikainen OT, Johnson MS, Farndale
RW, Heino J & Gullberg D (2003) α11β1 integrin recognizes the GFOGER sequence in
interstitial collagens. J Biol Chem 278: 7270-7277.
Zhou J, Ding M, Zhao Z & Reeders ST (1994) Complete primary structure of the sixth chain of
human basement membrane collagen, α6(IV). Isolation of the cDNAs for α6(IV) and
comparison with five other type IV collagen chains. J Biol Chem 269: 13193-13199.