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ECM in Health

1. Main Components of the ECM

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The extracellular matrix (ECM) is
composed of various proteins that give rise to different structures and
properties of the ECM. The main components of the ECM are collagen,
proteoglycans, laminin, and fibronectin. The composition of the ECM will differ
from organ to organ, giving rise to ECM that have distinct properties that are
appropriate for the organ to function.

 

1.1 Collagen

            Collagen is one of the major
proteins of the ECM and is the most abundant in humans. It has been widely
studied with 28 different collagen types discovered.1-4 Each type is composed of
homotrimers or heterotrimers of left handed helical ? chains that are twisted
to form a right handed triple helix structure.5
There are over 40 distinct ? chains in humans as well as other proteins that contain
collagen like domains.2, 5 The collagen superfamily is
a large group of proteins that all contain the Gly-X-Y motif, where X and Y are
usually either proline or hydroxyproline, of each ? chain is characteristic of
collagens.5, 6 Despite the large amounts of
bulky proline, the right handed helical structure is stabilized by the small
glycine, interchain hydrogen bonds, and electrostatic interactions involving
lysine and aspartate.6, 7

Collagens can be categorized according
to the supramolecular structures that they form. There are fibrillar collagens
that form fibrous structures that can be found in tendons, cartilage, skin, and
cornea.2, 3 Each collagen fiber is made
up of several types of collagen, depending on the tissue they are found. Collagen
type I is the most abundant fibrillar collagen which is found in connective
tissues except the cartilage including skin, bone, tendon, and cornea.8

All fibrillar collagens are first
produced as precursors. The ? chains are assembled together in the rough
endoplasmic reticulum to form the triple helical structure. This is where
hydroxylation of proline and lysine as well as glycosylation takes place to
initiate the formation of the triple helical structure.9
The procollagen is then brought to the golgi apparatus where it is prepared for
cellular export. Processing of the procollagen happens either during or after
secretion in the ECM.10-13 The C terminal propeptide is
cleaved off by specific metalloproteinases. It has been shown that failure to
remove the C terminal of the collagen leads to high solubility of collagen that
prevents it from forming fibrils.14
For collagen types I, II, and III, the N-propeptides are cleaved off while for
type V, XI, and other fibrillar collagens, the N-propeptides remain. This modifies
the shape and diameter of the fibril without affecting fibril formation.4,
14-16 The N-propeptides of type V
and XI collagens protrude out of the gaps between collagen molecules to prevent
lateral growth via steric hindrance and charge interactions.14, 15 Type V and XI collagens are
currently believed to be responsible for nucleating and modulating fibril
formation of collagen. 14, 15 It has been shown that the
deletion of collagen V in mice leads to failure of fibril assembly despite its
low amounts in the total collagen content in most tissues.17

Once the microfibrils are formed, these
may bind with other microfibrils so that they will grow into larger fibers.
This process is mediated by other extracellular matrix proteins.18
Small leucine rich proteoglycans (SLRPs) such as decorin and biglycan have
collagen binding motifs allowing them to modulate fiber growth, size,
morphology, and content.4, 18,
19 Another su­­­­bfamily of
collagen are fibril-associated collagens with interrupted helices (FACIT) that
do not form fibrils themselves but are associated with the surface of collagen
microfibrils.2
Their primary function is to mediate the formation of higher order structure
via binding with other extracellular matrix proteins such as SLRPs and
proteoglycans.15, 20 The supramolecular assembly
of collagen is further stabilized by lysyl oxidase, which leads to overall
enhanced mechanical properties. The N terminal and C terminal ends of
individual collagen molecules are covalently cross linked by lysyl oxidase both
within and between microfibers20.
Overall, these post translational modifications of collagen contributes to the
great tensile strength of collagen.21

Other than fibrillar and FACIT
collagens, there are also network forming collagens such as type IV, VIII, and
X. These are found in the basal lamina of basement membranes.2
Collagen IV forms a tetramer through their 7S N-terminal domain. Each of these
collagen IV molecules is bound to another collagen IV molecule via their
C-terminal NC1 domain of each ?-chain, forming a hexamer.2
These two domains of collagen IV allow it to form a stable collagen network
that separates the basal lamina from the interstitial stroma.22 Other extracellular matrix
proteins such as laminin, nidogen, and perlecan can be found in the basal
lamina that strengthens this barrier to effectively maintain the organization
of the cells in the body.22, 23

Although different types of collagen are
able to build various types of supramolecular structures that form the basis of
the architecture of the ECM, the contribution of other ECM proteins such as
proteoglycans, laminins, and fibronectin cannot be ignored. They largely
influence the chemical and physical properties of the extracellular matrix such
as through their growth factor binding motifs and innate chemical properties.
Furthermore, they also serve as connectors between the cells and the ECM.

 

1.2 Proteoglycans

            Proteoglycans are characterized as proteins
that have glycosaminoglycans (GAGs) covalently bonded to them. These GAGs are
long chains of negatively charged disaccharide repeats that can either be
heparin sulphate, chondroitin/dermatan sulphate, hyaluronan, and keratin
sulphate. Only hyaluronan is not sulphated. Due to the negative charge of these
GAGs, they are able to sequester water and cations, which gives them their
space filling and lubrication functions.24
Proteoglycans are classified based on their structure and the distribution and
density of their GAGs. They can be further classified based on where they are
found.25 For the purpose of this
review, only transmembrane proteoglycans and those found in the pericellular and
extracellular space will be discussed.

            There are thirteen genes that encode
for cell surface proteoglycans. Seven of these encode for transmembrane
proteoglycans, which are four syndecans, CSPG4/NG2, betaglycan, and phosphocan.
On the other hand, six of the genes encode for the GPI-anchored proteoglycans
called glypicans. For the purpose of this review, only syndecans will be
discussed. The other cell surface proteoglycans are reviewed by Iozzo & et.
al..25 Syndecans have an
intracellular domain, transmembrane domain, and ectodomain. The GAGs are found
attached to the ectodomain, which are usually heparan sulphates. This
ectodomain can be shed off through the action of MMPs. The ectodomain of
syndecans is natively disordered, which allows it to interact with a wide
variety of molecules giving it a broad range of biological functions. Some of
its functions involve binding to growth factors and morphogens, facilitating
exosome uptake, and being co-receptors of receptor tyrosine kinases.

            One of the proteoglycans found in
the pericellular area or the basement membrane is perlecan. Perlecan is a large
heparan sulfate proteoglycan (HSPG) that has multiple domains, each with
different binding sites and functions. The heparan sulfate can bind to 3 sites
on domain I and 1 site on domain V.26
These heparan sulfates can bind to a variety of molecules such as growth
factors, growth factor receptors, collagen, and other ECM proteins. In the
basement membrane, it binds to collagen IV, nidogen, and laminin, linking all
of these proteins which further strengthens the basement lamina.22, 23,
27

            Proteoglycans found in the
extracellular space are hyalectans and SLRPs. All hyalectans have the same
structure in which they have their hyaluronic acid binding N terminal and a
lectin binding C terminal with GAGs attached in between the N and C terminal
ends. Hyalectans are encoded by 4 distinct genes: aggrecan, versican, neurocan,
and brevican.25 Aggrecan is found mostly in
the bone in the cartilage and brain and neurocan and brevican are found in the
central nervous system. On the other hand, versican is found in the ECM of
almost any tissues and organs.28
They can serve as molecular bridges between the cell surface and the
extracellular matrix.25 Versican has been shown to
bind to collagen type I and fibronectin, which are both substrates of integrins.29
The binding of versican to fibronectin’s RGD motif leads to loss of cell
adhesion as it sequesters fibronectin from the cell’s integrins.28, 29

            The largest family of proteoglycans
are SLRPs with 18 distinct gene products with multiple splice variants and
processed forms. These proteins have a relatively short protein core with a
central region dominated by leucine rich repeats (LRRs). They are expressed in
all extracellular matrices even in development suggesting their involvement in
directing organ size and shape during embryonic development and homeostasis. Decorin
and biglycan are SLRPs that have collagen binding motifs and regulate collagen
fiber assembly along with other proteoglycans.

            Overall, proteoglycans vary in form
and structure that confer different functions in the ECM. They are integral in
the maintenance of a healthy ECM without which would lead to

 

1.3 Laminin

            Laminins are trimeric glycoproteins
consisting of an ?, ?, and ? chains that are often found in the basal lamina or
some mesenchymal compartments.4 The twelve mammalian ?, ?,
and ? chains can theoretically create 60 unique laminins but only 16
combinations have been observed so far.23, 30 The ? chains vary in size
from 200 and 400 kDa while ? and ? chains have sizes from 120 to 200 kDa. A
trimer can then have a size varying from 400 to 800 kDa.30
With a rotary shadowing electron microscopy, laminins look like cross-shaped
molecules.23, 31,
32 The three chains form an
?-helical coiled coil structure that forms the long arm of the cross while the
three short arms are composed of one chain each.23
At the end of the long arm are 5 laminin G-like (LG) domains from the ? chain
that serve as attachment sites for the cell. Integrins, dystroglycan, Lutheran
glycoprotein, or sulfated glycolipids bind to these LG domains.30
At the end of each short arm are laminin N-terminal (LN) domains that are
important for laminin polymerization and basement membrane assembly.23

Laminins have cell type specific
functions such as adhesion, differentiation, migration, phenotype maintenance,
and apoptotic resistance.30
Through binding of integrins, laminins are able to create a dynamic link
between the cell and the ECM.30
Different heterotrimeric laminins will have different integrin heterodimers
binding partners.30
This allows the induction of signaling pathways and organization of
intracellular cystoskeleton.30, 33 Although the binding of
laminins to integrin is primarily mediated by the LG domains on the ? chain,
the ? and ? chains can also influence binding. It was found that ?2 containing
laminins have higher affinity to integrins ?3?1 and ?7X2?1 when compared to ?2
containing laminins in vitro. The difference in binding affinity was attributed
to the 20 amino acid residues on the coiled coil domain of the long arm. 34
Furthermore, it was found that the glutamic acid residues found on the C
terminal end of ?1 and ?2 chains are necessary for integrin binding.35

The binding of laminin to the cell
surface is essential in laminin polymerization.23
Laminin polymerization is mediated by the LN domains found at the end of each
short arm. The short arms form a ternary node with the short arms of other
laminin molecules. However, it is still unknown whether the laminin-type
epidermal growth factor-like (LE) domains found next to the LN domains play a
role in the formation of the ternary node since LN domains cannot be produced
without the LE domains.23
Laminins have a unique role in basement membrane assembly and they initiate
this through laminin polymerization. Genetic ablation of ?1 or  ?1 chains proved to be embryonically lethal
due to the resultant failure of heterotrimeric formation and basement membrane
assembly.36, 37 On the other hand, genetic
ablation of other basement membrane components led to defects later in
development and did not prevent basement membrane formation.38-42

Laminins interact with other components
of the basement membrane such as nidogen, perlecan, and collagen IV.23, 30 Collagen IV in the basement
membrane is seen as the maturation of the basement membrane that is essential
for structural stability later in development.23, 40 The exact mechanism how
laminins bind to collagen IV is still debated upon. Initial studies indicated
that nidogen binds to laminin through the LE domains of the ?1 chain and
collagen IV, thus serving as an intermediary between the two networks found in
the basement membrane. However, studies have shown that nidogen might not be
the major bridge in connecting laminins and collagen IV.23
It was then found that the interaction between laminins and collagen IV was
mediated by heparan sulfates.43
Perlecan was thought to mediate this function. However, it was found that
genetic ablation of perlecan in mice still had the collagen IV intact.23, 40 It is then postulated that
agrin, another pericellular HSPG, serves as a compensating candidate. In this
model, both perlecan and agrin would bind to the nidogen containing laminin
network and to the collagen IV’s 7S and NC1 domains.44, 45

Laminins are essential in connecting the
cell to the ECM through its interactions with both cell surface receptors and
other components of the ECM. While collagen, proteoglycans, and hyaluronic acid
comprise the major structural component of the ECM, laminins are one of the
molecules that bridge the interaction gap between the cells and the ECM.4

 

1.4 Fibronectin

            Fibronectin is a multidomain protein
that interacts with the various ECM components described previously.46, 47 Similar to laminin, it
connects the cell to the ECM.4 It is encoded by a single gene,
but it has 20 isoforms in humans as a result of alternative splicing of the
mRNA.46, 47 Its amino acid sequence
consists of repetitive domains that are categorized either as type I, II, or
III. Alternative splicing occurs at EIIIA, EIIIB, and variable regions of the
gene.46

            Similar to collagen, it forms a
fibrillar network in the extracellular matrix.46
Fibronectin naturally exists as a dimer outside the cell, mediated by the two cysteine
disulfide bonds. This dimerization is crucial for fibrillar assembly of
fibronectin. Fibronectins lacking these cysteines resulted in the secretion of
monomers that did not form fibrils.46, 48 Fibronectin matrix assembly
is mediated by selective binding to ?5?1 integrins through an RGD binding motif
and a synergy site on the fibronectin molecule.46
Both of these sites are required for the initiation of fibril assembly.46, 49 Through these integrins, the
compact and soluble secreted fibronectin is unfolded revealing cryptic binding
sites for other fibronectin molecules.46, 50,
51 Using anti integrin or anti
fibronectin antibodies have been shown to prevent fibronectin fibril formation.46, 52,
53 Fibronectin binding induces
integrin clustering that provides local high concentrations of fibronectin at
the cell surface. This phenomenon promotes fibronectin-fibronectin interactions
through the N terminal assembly domains of each molecule.46

            Once it is tethered on the cell surface by integrins, the
actin cytoskeleton can pull onto fibronectin molecules to change its
conformation.46, 47 This will affect the C
terminal regions of fibronectin, revealing cryptic binding sites for
fibronectin, heparan sulfates, heparin, collagen, and other ECM proteins.46, 47 This allows it to interact
with proteoglycans such as syndecans and perlecan that aid in matrix assembly
as well.54-58 It is through strong
noncovalent protein-protein interactions that the fibronectin network matures
and becomes insoluble. Although it is mainly through the
fibronectin-fibronectin interactions, other ECM proteins may mediate lateral
interactions between fibrils.46
This stabilizes the relatively weak binding sites at individual sites. However,
the turnover of the fibronectin matrix is still largely unexplored.46

            Due to fibronectin’s multiple binding sites for other ECM
proteins, it has been implicated in various functions including a role in
collagen type I assembly. It is postulated that fibronectin provides direction
for collagen I assembly.46
It has also been shown that without fibronectin, collagen fibrils do not
accumulate.59, 60 Furthermore, in developing
tissues, fibronectin has been shown to be present before collagen.46
The interactions between fibronectin and collagen are regulated by mechanical
forces.61 Moreover, structural
analysis revealed cooperative binding between collagen and fibronectin.62 Additionally, studies have
shown that collagen can also enhance fibronectin assembly.46, 63,
64

           

2. Function of ECM

            The
complex ECM has several functions and can influence biochemical and biophysical
processes in the cell simultaneously. While the ECM has been largely regarded
as just a scaffold that provides structure for the cells, it has several other
functions that largely impact the cell phenotype. The ECM can serve as binding
sites, controlling the adhesion and movement of cells. This is emphasized in
the complex structure and composition of the basement membrane that serves as a
barrier between epithelial cells and the interstitial stroma. In addition to
structural integrity and anchorage, the ECM components have several binding
sites for growth factors, controlling their release and presentation to target
cells. This is especially important in morphogenesis as it establishes
morphogen gradients. Finally, the ECM transmits mechanical signals to the
cells, which activates several intracellular signalling pathways and
cytoskeletal machinery. Indeed, the ECM serves several functions and here we
review the function of the ECM in the context of development and maintenance of
the stem cell niche.

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