Extracellular matrix

Learning objectives
-

Describe the composition, structure and function of the extracellular matrix

(ECM) and its components, including collagens, non-collagenous proteins
and proteoglycans.

Outline the sequence of steps in the biosynthesis and post-translational

modification of collagens and elastin, including the structure and synthesis
of crosslinks.

Discuss the functional roles of the ECM in tissues.

Describe the pathways of biosynthesis and turnover of proteoglycans.

Discuss the structure and function of integrins as receptors for ECM

components.

Describe paradigmatic pathologies involving ECM components.

GLYCONJUGATES
Glycoconjugates are proteins or lipids that contain covalently linked sugar
(glycan) chains.
The major classes of glycans in glycoconjugates are oligosaccharides and
glycosaminoglycans.
Glycoproteins (protein + oligosaccharides). Proteins with 1 or more
oligosaccharides covalently bound to the polypeptide backbone (co- and/or posttranslational modification).
Proteoglycans (protein + glycosaminoglycans). The basic proteoglycan unit
consists of a "core protein" linked to one or more glycosaminoglycan chain(s)
(heavily glycosylated).
Glycolipids (lipid + oligosaccharides). Membrane lipids whose polar head is
modified with oligosaccharides.

Glycoproteins
N- and O-glycosidic linkages

Monosaccharides commonly found in glycoproteins

D-Galactose (Gal)
D-Glucose (Glc)
D-Mannose (Man)
N-Acetyl-D-Glucosamine (GlcNAc)
N-Acetyl-D-Galactosamine (GalNAc)
-L-Fucose (Fuc)
Neuraminic Acid (Neu)*
N-Acetyl-Neuraminic Acid (Neu5Ac)*
* Sialic Acids

Sugar modifications
(amine substitution of alcohol yields amino sugars)

Other sugar modifications

(sialic acids)

N-linked glycoproteins
N-glycosidic bond between N-acetylglucosamine and the NH2 function in the
side chain of an Asn residue in the Asn-X-Ser/Thr consensus sequence.

Types of N-linked oligosaccharides

N-linked glycoproteins have a common core (chitobiose) made of a branched
pentasaccharide (2GlcNAc + 3Man) to which other monosaccharides are attached.

Co- and post-translational synthesis of N-linked oligosaccharides

O-linked glycoproteins
O-glycosidic bond between Xyl, Gal, GalNAc, or GlcNAc and the OH function in
the side chain of a Ser/Thr residue (no clear consensus motif).

Major types of O-linked core sugars

1. Gal-Gal-Xyl Serine
2. GalNAc Ser/Thr
3. Gal hydroxylysine

Xylosyl-serine

Galactosyl-hydroxylysine

Common glycerophospholipids

Gangliosides

Oligosaccharides are conformationally mobile

Erythrocyte glycocalyx

Proteoglycans
(central strands with numerous projections)

Proteoglycans can be regarded as a special

class of glycoproteins that are heavily
glycosylated with glycosaminoglycans (up to
95% of their mass).
They comprise a core protein with one or more
attached glycosaminoglycan chain(s).

Proteoglycans
(bottlebrush model)

Glycosaminoglycans (GAGs)
GAG chains are long, linear carbohydrate polymers made of
repeating disaccharide units.
They may contain more than 100 sugar residues in a chain.
Under physiological conditions they are negatively charged (due
to the occurrence of sulfate and uronic acid groups).
Major monosaccharide components of GAGs are:
1. Uronic acids
D-glucuronic acid (GlcUA) and L-iduronic acid (IdUA)
2. Amino sugars
N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc)

Structure of major GAG monosaccharides

In the amino sugars, the hydroxyl group at C-2 of the hexose is replaced by an amino
group, most often acetylated and sometimes sulfated (mostly in heparin and heparan
sulfate).
In the uronic acids, C-6 of the hexose is oxidized to a carboxyl group.

Glycosaminoglycans
(repeating disaccharide units)

Composition and distribution of major GAGs

Proteoglycan

Characteristic
disaccharide

Sulfation

Tissue location

Hyaluronic acid (HA)

[4GlcUA1
3GlcNAc1]

None

Joint and ocular fluids

Chondroitin sulfates (CS)

[4GlcUA1
3GalNAc1]

GalNAc (4-O, 6-O)

Cartilage, tendons,
bone

Dermatan sulfate (DS)

[4IdUA1
3GalNAc1]

IdUA (2-O), GalNAc (4-O)

Skin, valves, blood

vessels

Heparan sulfate (HS)

[4IdUA1
4GlcNAc1]

GlcNAc (6-O)

Cell surfaces

Heparin (Hep)

[4IdUA1
4GlcNAc1]

GlcNH2 (2-N, 6-O), IdUA

(2-O)

Mast cells, liver

Keratan sulfates (KS)

[3Gal1
4GlcNAc1]

GlcNAc (6-O), Gal (6-O)

Cartilage, cornea

GalNAc, N-acetylgalactosamine; GlcNH2, glucosamine; GlcUA, D-glucuronic acid; IdUA, L-iduronic acid.

Linkage between GAGs and core protein

With the exception of hyaluronic acid (HA), all GAGs are attached to a Ser/Trh residue of
the core protein via the trisaccharide Gal-Gal-Xyl.
Keratan sulfate (KS) is present as both free and core protein-bound GAG. However, when
attached to a core protein, the linkage is either through an N-linked (KS I) or an O-linked
(KS II) oligosaccharide.

Biosynthesis of proteoglycans

The core protein is synthesized on ribosomes and translocated into the lumen of
the rER.

The link tetrasaccharide Gal-Gal-Xyl is attached to a Ser/Thr side chain on the

core protein while this is still in the rER, and acts as a primer for polysaccharide
growth.

Further glycosylation occurs in the Golgi apparatus in multiple enzymatic steps.

These involve position- and linkage-specific glycosyltransferases, epimerases and

sulfotransferares.

Some glycosyltransferases catalyse the transfer of monosaccharides to

Tyr/Ser/Thr (to give O-linked glycoproteins) or Asn (to give N-linked
glycoproteins) residues. Mannose groups may be transferred to Trp residues to
generate C-mannosyl-Trp.

The newly synthesized proteoglycan is exported in secretory vesicles to the

extracellular matrix.

Synthesis of chondroitin-6-sulfate

Synthesis of glucuronic acid

Degradation of proteoglycans
GAGs are degraded in the lysosome.
The protein component is hydrolyzed by proteases, while the GAG chains are degraded by
the sequential action of acid hydrolases.

Degradation of heparan sulfate

Mucopolysaccharidosis
As with degradation of glycosphingophospholipids, if one of the enzymes involved in the
stepwise pathway is missing, the entire degradation process is halted at that point and
the undegraded molecules accumulate in the lysosome, leading to some forms (more
than a dozen) of lysosomal storage diseases (mucopolysaccharidosis).
These diseases can be diagnosed by the identification of specific GAG chains in the
urine, followed by assay of the specific hydrolases in leukocytes and fibroblasts.

Syndrome

Deficient enzyme

Product accumulated in lysosomes

and secreted in urine

Hunters

Iduronate sulfatase

Heparan and dermatan sulfate

Hurlers

-Iduronidase

Heparan and dermatan sulfate

Morquios A

Galactose-6-sulfatase

Keratan sulfate

Morquios B

-Galactosidase

Keratan sulfate

Sanfilippos A

Heparan sulfamidase

Heparan sulfate

Sanfilippos B

N-Acetylglucosaminidase

Sanfilippos C

N-Acetylglucosamine-6sulfatase

Functions of proteoglycans
Occupy space between cells and collagen
Organize water molecules
- compressibility
- flexibility/elasticity
- a certain degree of rigidity (due to their highly negative charge)
- silly putty properties (bounce + resilience)

Highly viscous
- lubricating fluid in the joints

Specific binding to other macromolecules

Associate with collagen fibers

- network
- in the bone, combine with calcium salts (calcium carbonate, hydroxyapatite)

Anchoring of cells to matrix fibers

Cell migration and adhesion

- passageways between cells

Structure of aggrecan

Associations between proteoglycans and hyaluronic

acid form an aggrecan structure in the extracellular
matrix (ECM). The extension of this structure yields a
three-dimensional array of proteoglycans bound to
hyaluronic acid, which creates a stiff matrix or
bottlebrush structure in which collagen and other
ECM components are embedded.

ECM structure

ECM as reinforced concrete

The overall structure of the cartilage ECM can be likened to that of the vertical reinforced concrete
slabs poured during the construction of large buildings, in which steel rods (collagen fibers) are
embedded in an amorphous layer of cement (the proteoglycan aggregates). Collagen stabilizes the
network of proteoglycans in cartilage in much the same way that the reinforcing rods in the
concrete provide structural strength for the cement walls. The structure of earthquake-resistant
buildings, like the ECM, provides a balance between integrity and flexibility.

ECM structural glycoproteins

Bind collagens, proteoglycans and cells

anchoring collagen fibers and proteoglycans to the cell membrane
bridging molecules that mediate the interaction between cells and
ECM

Major ECM structural glycoproteins

fibronectin
laminin

Fibronectin (FN)
Fibronectin is a high molecular weight glycoprotein (~440 kDa)
It binds to integrins (membrane-spanning receptors)
It also interacts with ECM components such as collagen, fibrin,
heparin and heparan sulfate
At least 20 different tissue-specific isoforms of fibronectin have
been identified, all produced by alternative splicing of a single
precursor mRNA
Cellular fibronectin, mainly produced by fibroblasts, is assembled
into insoluble fibrils
Plasma fibronectin, mostly secreted by hepatocytes, circulates in
a soluble form

Model structure of FN
Dimers stabilized at the C-terminal end by inter-chain disulfide bonds
Each protomer subunit is organized into domains (type I, II, and III), and each of
this has several homologous repeats in the protein sequence. Therefore,
fibronectin has a string of beads appearance
Clusters of domains form binding regions for different ECM components
- collagen type I, II and III
- heparin/heparan sulfate
- fibrin

Binding to cell integrins is mediated by type III modules 9 and 10, and involves
specific primary sequence motifs
- Arg-Gly-Asp (RGD)
- Pro-X-Ser-Arg-Asn (PXSRN)

Fibronectin functions

Fibronectin is involved in cell adhesion, differentiation,

growth, migration

Anchoring basal lamina to other ECM components

Fibronectin is necessary for embryogenesis (guiding cell

attachment and migration during embryonic development)

The loss of fibronectin from the surface of many tumor cells

may contribute to their release into the circulation and
penetration through the ECM (metastasis)

Along with fibrin, plasma fibronectin is deposited at sites of

injury, forming a clot that stops bleeding and protects the
underlying tissue

Fibronectin binding to integrins

Integrins guide formation of fibronectin fibrils

A. Compact soluble FN
binds integrins.

B. FN binding induces
reorganization of actin
filaments and signaling.

C. Cell contractility leads

to changes in FN
conformation, exposing
FN interaction domains
and allowing formation
of FN fibrils.
Mao and Schwarzbauer, Matrix Biol. 2005

Laminin (LN)
LN is a high molecular weight glycoprotein (~850 kDa)
About 50 oligosaccharides are linked to the LN N-terminus, which
makes LN one of the most complicated glycoproteins ever
It is heterotrimeric (, , and chains), and folds into a crossshaped molecule. LN trimers undergo reversible self-association
to form polymers in the presence of calcium
Five , four , and three chains have been identified so far,
which can associate to form at least 15 LN variants
LN interacts with cell integrins via multiple binding sites
LN binds ECM components, including heparan sulfate (directly),
and collagen IV (mostly indirectly via nidogen/entactin)

Model structure of LN

Laminin functions

Cell adhesion
Cell differentiation
In conjunction with entactin, LN
generates a scaffold for anchoring
of cells and ECM molecules in the
basement membrane

Linking the ECM to the cytoskeleton: integrins (I)

Linking the ECM to the cytoskeleton: integrins (II)

Colocalization of integrins and actin cytoskeleton

General organization of integrins

Integrins are a family of adhesion molecules
involved in interactions between the cell
and the surrounding extracellular matrix
(ECM).
The heterodimerization of 19 -integrin and
8 -integrin subunits is thought to yield 25
integrin heterodimers.
The and chains span the cell membrane,
interacting with the ECM outside the cell
and the cytoskeleton and signaling
molecules inside. In this manner, integrins
can transduce signals from the ECM into
biochemical and mechanical events in the
cytoplasm that ultimately lead to
alterations in cell morphology and
function.
The ovals contain abbreviations for components of
the complex signaling cascade that conveys
information from the integrin molecule to the
nucleus of the cell.

Integrins need to be activated

Integrin
adhesiveness
can
be
dynamically regulated through a
process termed inside-out signaling.
Ligand binding transduces signals
from the cellular environment to the
interior of the cell through outside-in
signaling.
Protein structure analyses have
provided
insights
into
the
mechanisms
whereby
integrins
become activated to bind ligand and
how ligand binding translates to
changes in intracellular signaling.
Adair and Yeager, Meth. Enzymol. 2007

Inside-out signaling
(a model for integrins activation)

Involves
an
initial
switchblade-like
motion
when the headpiece extends.
Downward movement of the
7-helix leads to subunit
hybrid domain swing out,
separation of the knees, and
opening of the headpiece for
high affinity ligand binding.
Activation can occur by PKC
stimulation, GPCR activation,
or binding of proteins such as
talin to the subunit tail.
A delicate equilibrium among
the different conformation
states exists.

Inside-out signaling
(talins and kindlins)
Integrin activation, which involves a conformational reorganization of the integrinintegrin dimer
such that its affinity to the matrix ligand is radically increased, is essential for the initiation of focal
adhesions.
Two groups of proteins, the talins (talin 1 and 2) and the kindlins (kindlin 2 (also known as FERMT2
and MIG2) and kindlin 3 (also known as FERMT3)), both of which bind to cytoplasmic domains of
integrins and connect them with the actin cytoskeleton, are crucial for integrin activation.
Talins bind actin through an I/LWEQ motif at their carboxy-terminal tail domain. At the same time,
the FERM (four point one, ezrin, radixin and moesin) domain at the amino terminus of talin interacts
with an NPXY motif in the conserved cytoplasmic tail of the integrin subunit.
Compared with other proteins that link integrins to actin, talin has a special role as it binds to the
cytoplasmic domain of the integrin subunit, thereby triggering the transition of the entire integrin
integrin dimer from an inactive to an active conformation that is capable of high-affinity interactions
with ECM ligands.
The binding of talin alone, however, seems to be insufficient for complete integrin activation. It was
recently shown that kindlin 2 and kindlin 3 (which is expressed in platelets and other haematopoetic
cells), are required for maximal integrin activation. Kindlin 2 and 3 can directly bind to integrin tail
NPXY motifs that are distinct from those used by talin. Then, in cooperation with talin, kindlins
trigger integrin activation.

Geiger B. et al, Environmental sensing through focal adhesions (2009) Nature Reviews Molecular Cell Biology, 10: 21-33

Outside-in signaling
Transmembrane integrin heterodimers in their
extended and active form bind to extracellular matrix
ligands such as fibronectin, vitronectin, laminin or
collagen.
Different cations have markedly different effects on
ligand affinity: in general, Mn2+ supports ligand
binding, Mg2+ does so to a lesser extent, and Ca2+
does not support ligand binding at all.
Upon ligand binding, a conformational change occurs
that renders the cytosolic tail of integrins accessible
for intracellular interactors leading to the
recruitment of diverse proteins such as talin, vinculin
and -actinin that link integrins to the actin
cytoskeleton which in turn allows the generation of
traction forces through actomyosin contraction (focal
adhesion).
Signaling and adaptor proteins (e.g., the Src tyrosine
kinase, focal adhesion kinase [FAK], paxillin, and the
integrin-linked kinase [ILK]) are also recruited that
mediate a multiplicity of biological effects (gene
expression, differentiation, proliferation).

Biochemical pathways of focal adhesions

http://www.genome.jp/kegg/pathway/hsa/hsa04510.html

The adhesome

Geiger B. et al, Environmental sensing through focal adhesions (2009) Nature Reviews Molecular Cell Biology, 10: 21-33

The adhesome
Integrin-mediated adhesions are multiprotein complexes that link the extracellular matrix to the
actin cytoskeleton. Molecular analyses of these adhesion sites indicate that the integrin
adhesome consists of 160 distinct components (see the figure).
Most of these components are intrinsic constituents of the adhesion sites (boxes surrounded by
a black frame), whereas others are transiently associated with the adhesion site and affect its
structure or signalling activity (surrounded by a dashed frame).
Examination of the molecular interactions that take place between the different constituents of
the adhesome points to an extraordinary connectivity. The entire network contains nearly 700
links, most of which (55%) are binding interactions and the rest are modification interactions,
whereby one component affects (for example, activates or inhibits) the activity of another
component.
The biological activities of the adhesome components are diverse and include several actin
regulators that affect the organization of the attached cytoskeleton, many of the adaptor
proteins that link actin to integrins either directly or indirectly, and a wide range of signalling
molecules, such as kinases, phosphatases and G proteins and their regulators.

Geiger B. et al, Environmental sensing through focal adhesions (2009) Nature Reviews Molecular Cell Biology, 10: 21-33

Focal adhesions

Focal complexes

Focal contacts

Biogenesis of focal adhesions

Legate KR et al. (2006) ILK, PINCH and parvin: the tIPP of integrin signalling Nat. Rev. Mol. Cell Biol. 7: 112

Molecular components of focal complexes

How focal complexes are assembled into focal contacts

?
?

Dynamics of focal adhesion formation

Geiger B. et al, Environmental sensing through focal adhesions (2009) Nature Reviews Molecular Cell Biology, 10: 21-33

Dynamics of focal adhesion formation

a | Selected frames from a time-lapse sequence that show the formation of new focal
adhesions (FAs), and the associated dynamics of the boundary between the
lamellipodium and the lamella. Time is indicated in minutes. Nascent FAs (a paxillinpositive spot is indicated by an arrowhead) form inside the lamellipodia. Disturbance
of the flow is seen in the phase-contrast image as a dark zone in front of the adhesion
site. Formation of FAs is followed by the advance of the lamellipodiumlamella
boundary. The newly formed contact undergoes maturation and elongates in the
direction of flow.
b | A diagram that summarizes the stages of FA formation and maturation, and the
simultaneous advancement of the boundary between fast (lamellipodium) and slow
(lamella) actin flow zones. Nascent and mature FAs are shown as red ellipses of
different sizes; and stress fibres are shown as purple lines of different thicknesses.
Note that the process by which the boundary advances between the flows in
lamellipodium (fast) and lamella (slow), and that of FA maturation, are presented in
different panels, for clarity. In fact, these two processes proceed simultaneously.

Geiger B. et al, Environmental sensing through focal adhesions (2009) Nature Reviews Molecular Cell Biology, 10: 21-33

Feedback loops in the adhesome

Geiger B. et al, Environmental sensing through focal adhesions (2009) Nature Reviews Molecular Cell Biology, 10: 21-33

Feedback loops in the adhesome

A schematic depicting the feedback loops that interconnect the actin machinery and
integrin-mediated adhesions. Forces that are generated by actin polymerization and
myosin II-dependent contractility (step 1) affect specific mechanosensitive proteins
in the actin-linking module (perhaps talin and vinculin), the receptor module
(represented by integrins) and co-receptors (such as syndecan 4)), the associated
actin-polymerizing module (for example, zyxin and formins) and the signalling
module (represented by, for example, focal adhesion kinase and p130CAS). Acting in
concert, these interacting modules, with their particular mechanosensitive
components, form a mechanoresponsive network.
The effect on the actin cytoskeleton (step 2) depends on the integrated response of
the entire system to interactions with the matrix and to applied mechanical forces.
Stimulation of the signalling module eventually leads to the activation of guanine
nucleotide-exchange factors and GTPase-activating proteins, leading to activation or
inactivation of small G proteins, such as Rho and Rac (step 3).
These G proteins affect actin polymerization and actomyosin contractility through
cytoskeleton-regulating proteins (step 4), thus modulating the force-generating
machinery (step 5).
Geiger B. et al, Environmental sensing through focal adhesions (2009) Nature Reviews Molecular Cell Biology, 10: 21-33

Rac dependent formation of focal complex

Myosin light chain (MLC) phosphorylation is modulated by Rho

Feedback loops controlling formation and growth

of focal contacts

FAK
Src

Basic rules that regulate the fate of a focal

contact
1. Initial binding of integrins to their ECM partners takes place at
the leading edge of lamellipodia.
2. Rac is activated and drives formation of focal complexes by
promoting the assembly of a dynamic actin network in the
lamellipodium.
3. Rho drives the maturation of focal contacts via the activation
of both Dia and ROCK.
4. Pulling of focal contacts promotes tension-dependent
incorporation of new components into the nascent adhesion
sites, leading to its growth.

Effects of ECM on cell morphology

Geiger B. et al, Environmental sensing through focal adhesions (2009) Nature Reviews Molecular Cell Biology, 10: 21-33

Effects of ECM on cell morphology

An 'axis' that consists of coloured cylindrical segments depicts the biochemical diversity of the extracellular
matrix (ECM). Fibronectin is shown in green, vitronectin in blue, the Arg-Gly-Glu (RGD) peptide in red and a cellderived natural composite matrix (CDM) in yellow. Each of these matrices can be arranged into structures that
differ in their physical and geometrical properties. For example, the matrices can vary according to rigidity
(substrate compliance can be rigid or soft), ligand spacing (58 nm or 73 nm) and dimensionality (two-dimensional
(2D) or three-dimensional (3D) network). The axes highlight this diversity by showing the values of corresponding
parameters. Several possible cellular responses are shown.
A | Cells that are attached to 3D matrices assume an elongated morphology (Aa) that is similar to the shapes of
mesenchymal cells in vivo, whereas cells on 2D substrates tend to radially spread onto the substrate (Ab). The
integrins (red) localize to focal adhesions in cells on 2D substrates that are coated with fibronectin (green),
whereas they are organized into thin, elongated adhesions in the 3D matrix.
B | The response of human fibroblasts to rigid (Ba; Young's modulus (E) = 100 kPa) or soft (Bb; E = 10 kPa)
fibronectin-coated polydimethylsiloxane substrates. The organization of green fluorescent protein (GFP)paxillinlabelled focal adhesions (green) and phalloidin-labelled filamentous actin (red), as well as overall cell shape,
strongly differ in cells that are plated onto the two substrates.
C | The organization of focal adhesions differs in cells on 2D, rigid matrices of which the biochemical nature
varies. Human fibroblast cells were plated on coverslips that are coated with fibronectin (Ca) or vitronectin (Cb),
and the cells were immunostained for paxillin. Note that paxillin in vitronectin-attached cells is organized into
elongated peripheral structures, whereas classical focal adhesions are observed in cells that are attached to
fibronectin.
D | B16 melanoma cells attached to nanopatterned surfaces, the adhesive nanodots of which are spaced at
varying distances. Confocal micrographs of cells expressing GFP3 integrin (green) and stained for focal adhesion
kinase (red) indicate the successful spreading and formation of focal adhesions on the 58 nm surface (Da) and
the failure to do so on the 73 nm surface (Db).

Geiger B. et al, Environmental sensing through focal adhesions (2009) Nature Reviews Molecular Cell Biology, 10: 21-33