The Extracellular Matrix

FK UNDANA
22 Oktober 2015
DR. dr. Agnes Kwenang
Departement of Biochemistry
Medical Faculty
Hasanuddin Unirversity
Learning objective :
After reading this topic you should be able to describe:
* The composition, structure, components,
including collagen, noncollagenous proteins,
proteoglycans of the ECM.
* The functional roles of the ECM in tissues
* The pathway of biosynthesis and further of
proteoglycans.
* The pathology of genetic diseases resulting from
error in the synthesis or turnover of ECM
components.
The ECM is a complex network of secreted macromolecules located
in the extracellular space.

It plays a central role in regulating basic cellular processes, including

- proliferation,
- differentiation,
- migration,
- even survival.
The macromolecular network of the ECM is made up of
- collagens,
- elastin,
- glycoproteins,
- proteoglycans
that are secreted by a variety of cell types including
- fibroblasts,
- chondrocytes,
- osteoblasts.
The ECM components are
- relatives abundance,
- distribution, and
- molecular organization
 vary enormously among tissues and
 dramatically impact
- the structural and
- functional properties of the tissue.
The component of the ECM are
intimate contact with their cells of origin
and form a three-dimensional gelatinous
bed in which the cells thrive.

Proteins in the ECM are

also bound in the cell surface,
so that they transmit mechanical signals
resulting from stretching and
compression of tissues.
Changes in these ECM characteristics are
associated with chronic diseases,
such as
- arthritis, (rheumatoid arthritis,
osteoarthritis)
- atheroslerosis,
- cancer,
- fibrosis.
 Several diseases: due to genetics disturbances;
* collagen synthesis
- osteogenesis imperfecta
- types of the Ehlers-Danlos syndrome

* proteoglycans (GAGs)
- mucopolysaccharidosis

Changes occur in the ECM during

- the aging process.
 Types of ECMs

Basement membrane (basal lamina)

– Epithelia, endothelia, muscle, fat,
nerves
Elastic fibers
– Skin, lung, large blood vessels
Stroma or interstitial matrix
Bone, tooth, and cartilage
Tendon and ligament
Most mammalian cells are located in tissues where
they are surrounded by a complex extracellular matrix
(ECM) = “connective tissues”.
The ECM contains three mayor classes of biomolecules:
1). The structural proteins, *collagen,
*elastin,
*fibrillin
2). Specialized proteins such as * fibronectin,
* laminin
3). Proteoglycans
An overview of connective tissue

Supporting the epithelial

cell layer is basal lamina,
beneath which are
collagen, elastic fibers,
and proteoglycans
I. COMPOSITION OF THE ECM.
A. Fibrous Proteins
1. Collagen: -Types of collagen
- Synthesis and secretion of collagen
2. Elastin: - Tropoelastin
- Elastic Properties of elastin
3. Proteoglycans : - Structure and function of the
proteoglycans
- Synthesis of the proteoglycans
- Degradation of proteoglycans
II. INTEGRINS
III. ADHESION PROTEINS
IV. MATRIX METALLOPROTEINASEs
COLLAGENS

* a family of proteins
* the mayor component of most connective tissues,
* constitutes: 25% -30% total protein mass in the body
* have an important role in tissue - architecture and
- integrity,
in mediating a wide variety of
- cell-cell and
- cell-matrix interactions.
* > 25 distinct types of collagens have been identified.
* all collagen types have a triple helical structure
* mature collagen type I,
- containing 1000 amino acids
- each polypeptide sub unit = α-chain into is twisted a left-handed
polyproline helix of three residues per turn.
Three of these α-chain are then wound into
a right-handed super helix forming a rodlike molecule
1.4 nm diameter
300 nm long.
* a striking of collagen is characteristic the occurrence of glycine residues
at every third position of the triple helical portion of the α-chain. This is
necessary because glycine is the only amino acid small enough to be
accomodated in helix.
The repeating structure, represented as (Gly-X-Y)n, is an absolute
requirement for the formation of the triple helix.
While X and Y can be another amino acids, about
- 100 of X positions are proline
- 100 of the positions are hydroxyproline.
Proline and hydroxyproline confered the rigidity on the collagen molecule.
Hydroxyproline is formed by the posttranslational hydroxylation of
peptide- bound proline residues catalyzed by the enzyme prolyl
hydroxylase, whose cofactors are vitamin C and α-ketoglutarate.
Lysine in the Y position may also be posttranslationally modified to
hydroxyliysine through the action of lysyl hydroxylase, an enzyme with
similar cofactors. Some of these hydroxy-lysine may be further
modified by the addition of galactose or galactosyl-glucose through an
O-glycosidic linkage, a glycosylation site that is unique to collagen.
Collagen fibers are further stabilized by the formation of covalent
cross-links, both within and between the triple helical units.
These cross-links form through the action of lysyl oxidase, a copper-
dependent enzyme that oxidatively deaminates the ε-amino groups of
certain lysine and hydroxylysine residues, yielding reactive aldehydes.
Such aldehydes can form aldol condensation products with other
lysine- or hydroxylysine-derived aldehydes or form Schiff bases with
the ε-amino groups of unoxidized lysines or hydroxylisines.
• These reactions, after further chemical rearrangement,
result in the stable covalent cross-links that are important
for the tensile strength of fibers. Histidine may also be
involved in certain cross-links.

• Type IV collagen, the best-characterized example of a

collagen with discontinous triple helix, is an important
component of basements membrane, where is forms a
meshlike network.
Hydroxylation of
proline and lysine
residues in
collagen.
Proline and lysine
residues within
the collagen
chains are
hydroxylated by
reactions that
require vitamin C.
 Formation of cross links
. in collagen
A. Lysine residues are
oxidized to allysine (an
aldehyde).
Allysine may react
with an unmodified
lysine residue to form
a Schiff base (B),
or two allysine
residues may undergo
an aldol condensation
(C).
Molecular features
of collagen from
primary sequence
up to the fibril
each individual
polypeptide chains
is twisted into a
left-handed helix of
three residues (Gly-
X-Y) per turn, and
all of this chains
are then
woundinto a right-
handed superhelix
Diseases caused by mutations in collagen genes or by deficiencies in the activities of
posttranslational enzymes involved in the byosynthesis of collagen

Gene or enzyme Disease

COl1A1, COL1A2 Osteogenesis imperfecta
Osteoporosis
Ehlers- Danlos syndrome Type VII
autosomal dominant.
COL2A1
COL3A1 Severe chondrodysplasias
COL4A3-COL 4A6 Ehlers-Danlos syndrome Type IV
Alport syndrome (autosomal and X-linkedform)
COL7A1
Epidermolysis bullosa,dystrophic
COL10A1
Schmid metaphysial chondrodysplasia

Lysyl hydroxylase Ehlers-Danlos syndromeType VI

Procollagen N-proteinase Ehlers-Danlos syndrome Type VII (autosomal

recessive)
Lysyl hydroxylase
Menkes disease
Classification of Collagens , Based primarily on the structures that they form

Class Type
Fibril forming I, II, III, V, and XI
Network-like IV, VIII, X
FACITs IX, XII, XIV, XVI, XIX
Beaded filaments VI
Anchoring fibrils VII
Transmembrane domain XIII, XVI
Others XV, XVIII

FACITs = fibril associated collagens

with interrupted triple helices
Type IV collagen contain a
globular carboxy-terminal
domain(A), which forms
tropocollagen dimers
(hexamers of collagen,
(B).
Four dimers associate at
the amino-terminal
domains to form a 7S
domain (C) and the
tetramers form
a lattice (D), which
provide structural
support to the basal
lamina.
 Steps in collagen biosynthesis
Location Process
Rough endoplasmic reticulum Synthesis of preprocollagen, insertion
of the procollagen molecu le into
the lumen of the ER
Hydroxylation of proline and lysine
Lumen of ER residues; glycosylation of selected
hydroxylysin residues
Lumen of ER and Golgi apparatus Self-assembly of the tropocollagen
molecule, initiated by disulfide
bond foomation in the carboxy
terminal extension; triplehelix
formation
Sectretory vesicle
Procollagen prepared for secretion
Extracellular from cell
Cleavage of the propeptides,
removing the amino- and carboxy-
terminal extensions, and self-
assembly of the collagen
molecules into fibrils, and the fiber
• Genetic Diseases result from abnormalities in the synthesis of collagen
A number of disease are due to mutations in collagen genes or in genes
encoding some of the enzymes in these posttranslational modification.
The diseases affecting bone (eg, osteogenesis imperfecta) and cartilage
(eg, chondrodysplasia).
Ehlers-Danlos syndrom.
Type IV, reflecting abnormalities in Type II.
Type VI, due to deficiency of lysyl hydroxylase
Type VII C, a deficiency of procollagen N-proteinase
Alport syndrome,
- both X-linked and autosomal ,
- affecting the structure of type IV collagen fibers.
- charasteristic abnormalities of the structure of the basement membrane
and lamina densa of the renal glomeruli.
Epidermolysis bullosa
- due to mutations in COL7A1), affecting the structure of type VII
- another variant, is due to mutations in keratin.
Scurvy
- is not a genetic disease
- due to a deficiency of vitamin C
- impaired synthesis of collagen due to deficiencies of prolyl and lysyl
hydroxylases (required vit C as a cofactor)
Menkes disease
- deficiency of copper results in defective cross-linking of collagen and
elastin by copper-dependent enzyme lysyl oxidase.
ELASTIN
- confers extensibility & elastic recoil on lung, blood vessels, ligaments.
- that appears to be only one genetic type of elastin, although variants arise
by alternative splicing of the hnRNA for elastin.
- synthetized as a soluble monomer of ~70 kDa called tropoelastin
- After secretion from the cell, certain lysyl residues of tropoelastin are
oxidatively deaminated to aldehydes by lysyl oxidase.
-The mayor cross-link formed in elastin are the desmosines, which result
from the condensation of three of these lysine-derivedaldehydes with an
unmodified lysi ne to form a tetrafunctional cross-link unique to elastin
- Elastin is highly insoluble and extremely stable and has low turnover rate
Williams Beuren syndrome
- the mutations, by affecting synthesis of elastin (supra valvular aortic
stenosis)
- a decrease of elastin is found in conditions such as pulmonary emphysema,
cutis laxa, and aging of the skin.
The cDNA structure of elastin, indicating the repeating cross-linking
and hydrophobic domains
 Mayor differences between colagen end elastin

Collagen Elastin
1. Many different genetic type One genetic type
2. Triple helix No triple helix, random coil
conformations permitting streching
3. (Gly-X-Y)n, repeating structure No (Gly-X-Y)n, repeating structure
4. Presence of hydroxylysine No hydroxylysine
5. Carbohydrate-containing No carbohydrate
6. Intramolecular aldol cross-link Intramolecular desmosine cross-link
7. Prestension of extension peptides No extension peptides present during
during biosynthesis biosynthesis
FIBRILLIN
MARFAN SYNDROME
- is due to mutations in the gene for fibrillin-1, a protein present in microfibrils
- Fibrillin-1 is a large glycoprotein (350kDa).
- It is secreted into the ECM by fibroblasts and becomes incorporated into the
insoluble microfibrils, which appear to provide a scaffold for deposition of
elastin.
- Fibrillin-2 (chromosome 5),
- mutation in this gene are linked to causation of congenital contractural
arahnodactyly, but not to Marfan syndrome
- may be important in deposition of microfibrils early in development
 Mutation in gene (on Chrom 15) for fibrillin-1, a
large glycoprotein present in elastin-associated
microfibril
↓
Abnormalities of the structure of fibrillin-1
↓
Structures of the suspensory ligament of the of
eye, the periosteum, and the media of the
aortaaffected.
Elevated levels of TGF-β may contribute to the
pathology.
↓
Ectopia lentis, arachnodactyly, and dilation of the
ascending aorta.
Probable sequence of events in the causation of the mayor signs
exhibited by patients with Marfan syndrome.
FIBRONECTIN

Schematic representation of fibronectin.

Seven functional domains of fibronectin are represented; two
different types of domain for heparin, cell-binding, fibrin are
shown. The domain are composed of various combinations of
three structural motifs (I, II, III) , not depicted in the figure.
Also not shown is the fact that fibronectin is a dimer joined by
disulfide bridges near the carboxy terminals of the monomers.
The approximate location of RGD sequence of fibronectin, which
interacts with a variety of fibronectin integrin receptors on cell
surfaces, is indicated by the arrow.
LAMININ
- is a majorprotein component of renal glomerular
& other basal laminas.
- 850 kDa, 70 nm long consists of three distinct elongated polypeptide
chains (A, B1, dan B2), linked together to form an elongated cruciform
shape.
- It has potential binding sites for type IV collagen, heparin, and integrin
on cell surfaces.
- The collagen interacts with laminin (rather than directly with the cell
surface), which in turn interacts with integrins or other laminin receptor
proteins, thus anchoring the lamina of the cells.
Entactin = nidogen
- glycoprotein containing an RGD sequence; it binds to laminin and is a
major cell attachment factor
Schematic representation of a cell interacting through various
integrin receptors with collagen, fibronectin, and laminin
present in the ECM. (Speific subunits are not indicated)
Schematic representation of fibronectin interacting with an integrin
fibronect receptor situated in the exterior of the plasma membrane
of a cell of theECM and of various attachment proteins interacting
indirectly or directly with an actin microfilament in the cytosol. For
simplicityy, the attachment proteins are represened as a complex.
The structure of laminin
B. PROTEOGLYCANS
The fibrous structural proteins of the ECM are embedded formed from
proteoglycans.
Proteoglycans consist of polysaccharides called glycosaminoglycans
(GAGs).
Linked to a core protein.
The GAGs are composed of repeating units of disaccharides.
A proteoglycan may contain > 100 GAG chains and consist of up to 95%
carbohydrate by weight.
1. Structure and function.
2. Synthesis
3. Degradation
Repeating
disaccharides of
some
glycosaminoglycan
s. These repeating
disaccharides
usually contain an
N-acetylated sugar
and a uronic acid,
which usually is
glucuronic acid
and iduronic acid.
Sulfate groups are
often present but
are not included in
the sugar names in
this figure.
 Some specific functions of the glycosaminoglycans (GAGs) and
Proteoglycans
Glycosaminoglycans Proteoglycans
Hyaluronic acids Cell migration in
Embryogenesis
Morphogenesis
Wound healing
Chondroitin sulfate proteoglycans Formation of bone, cartilage, cornea
Keratan sulfate proteoglycans Transparency of cornea
Dermatan sulfate Transparency of cornea
Binds LDL to plasma walls
Heparin Anticoagulant (binds antithrombin III)
Causes release of lipoprotein lipase from
capillary walls
Heparan sulfate (syndecan) Component of skin fibroblast and aortic
wall; commonly fond in cells surfaces
Interactions between the cell membrane and
the components of the extracellular matrix
• The functional
properties of a normal
joint depend, in part, on
the presence of a soft,
well-lubricated,
deformable, and
compressible layer of
cartilaginous tissue
covering the ends of the
long bones that
constitute the joint.
• Systemic lupus
erythematosus (SLE)
was causing in the joint
tissues.
• Attachment of glycosaminoglycans to proteins. The sugar are
linked to a serine or threonine residue of the protein. A and B
represent the sugars of the repeating disaccharide.
Summary of structure of GAG and their attachments to core proteins.
The presence of link trisaccharides (Gal-Gal-Xyl) in the chondroitin
sulfates, heparin, and heparan and dermatan sulfates is shown.
Structure of heparin.
The polymer section
illustrated structural
features typical of
heparin; however the
sequence of variously
substituted repeating
disaccharide units
has been arbitrarily
selected. In addition,
non-O-sulfated or 3-
O-sulfated
glucosamine residues
may also occur.
Synthesis of chondroitin sulfate.
Sugars are added to the protein
one at atime., with UDP-sugars
serving as the precursors.
Initially a xylose residue is
added toa serine in the protein.
Then two galactose residues
are added, followed by a
glucuronic acid (GlcUA) and an
N-acetylglucosamine (Gal NAc).
Subsequent additions occur the
alternating action of two
enzymes that produce the
repeating disaccharide units.
One enzym (6) adds GlycUA
residues, and the other (7) adds
GalNAc. As the chain grows,
sulfate groups are added by
phosphoadenosine
phosphosulfate(PAPS)
“Bottle brush” structure of a proteoglycan, with a
magnified segment
Schematic representation of the proteoglycan aggrecan.
Synthesis and degradation of proteoglycans by chondrocytes.
 Defective enzymes in the Mucopolysaccharidoses

Disease Enzyme Accumulated products

Hunter Iduronate sulfatase HS, DS
Hurler+Scheie α-iduronidase HS, DS
Maroteaux-Lamy N-acetylgalactosamine sulfatase DS
Mucolipidosis VII β-glucuronidase HS, DS
Sanfilippo A heparan sulfamidase HS
Sanfilippo B N-acetylglucosaminidase HS
Sanfilippo D N-acetyl glucosamine 6-sulfatase HS

HS = Heparan sulfate; DS = Dermatan sulfate

Simplified scheme of
causation of a
mucopolysaccharidosis such
as Hurler syndrome in which
the affected enzyme isα-L-
iduronidase.Marked
accumulation of the GAGs in
thetissues mentioned in the
figur could cause,
hepatomegaly, splenomegaly,
disturbances of growth,
coarse facies, and mental
retardation
 Summary of the major features of the
mucopolysaccharidosis.
They exhibit a chronic progressive course.
They affect a number of organ systems (ie, they are
multisystem disorders)
Many patients exhibit organomegaly (eg, hepatomegaly, and
splenomegaly may be present)
Patients often exhibit dysostosis multiple (characterized by
severe abnormalities in the development of cartilage and
bone, and also mental retardation).
Patients often exhibit abnormal facies (facial appearnce)
Other signs sometimes found are abnormalities of hearing, of
vision, of the cardiovascular system, and of mental
development.
• BONE
• - is mineralized connective tissue.
• -is affected by many metabolic & genetic disorder
Schematic illustration of the
major cells present in
membranous bone.
Osteoblast (lighter color)
are synthesizing type I
collagen, which form the
matrix that traps cells, As
this occurs, osteoblasts
gradually differentiate to
become osteocytes
Schematic illustration of some aspects
of the role of the osteoclast in bone
resorption. Lysosomal enzymes and
hidrogen ions are released into the
confined microenvironment created by
the attachment between bone matrix
and the peripheral clear zone of the
osteoclast. The acidification of this
confined space facilitates the
dissolution of calcium phosphate from
bone and is the optimal pH for the
activity of lysosomal hydrolases. Bone
matrix is thus removed, and the
products of bone resorption are taken
up into the cytoplasm of the osteoclast,
probably digested further and
transferred into capillaries. The
chemical equation shown in the figure
refers to the action of carbonic
anhydrase Ii, described in the text.
• CARTILAGE
• The major components of cartilage are
• type II collagen and
• certain proteoglycan
• Schematic representation of
the molecular organization in
cartilage matrix. Link proteins
non covalently bind the core
protein (lighter color) of
proteoglycans to the linear
hyaluronic acid molecules
(darker color) The chondroitin
sulfate side chains of the
proteoglycan electrostatiscally
bind to the collagen fibrils,
forming a cross-linked matrix.
The oval outlines the area
enlarged in the lower part of
the figure.
• II. INTEGRINS
-are major cellular receptors for ECM proteins and provide
a link between the internal cytoskeleton of cells (primarily the
actin microfilament system) and extracellular proteins, such as
fibronectin, collagen, and laminin.
-providing a stable environment in which the cell
-involved in a wide variety of cell signaling options
-associated with leucocytes
-Leucocyte adhesion deficiency (LAD) , result from mutation in
the β2 integrin, leucocyte cannot be recruited to the sites of
infection.
• III. ADHESION PROTEINS
- are found in the ECM and link integrins to ECM
component.
- of which fibronectin is a prime example, are large
multidomain proteins that allow binding to many
different components simultaneously.
- In addition to integrin binding sites, fibronectin contains
binding sites for collagen and GAGs.
- important to allow cell migration and tissue remodeling
during growth and differentiation.
• - as the integrin molecule is bound to intracellular
cytoskeletal proteins, the adhesion proteins provide a
bridge between the actin cytoskeleton of the cell and the cells’
position within the ECM.
- Loss of adhesion protein capability can lead to either
physiologic or abnormal cell movement.
- Alternative splicing of fibronectin allows many different forms
of this adhesion protein to be expressed, including a soluble
form (versus cell-associated forms), which is found in the
plasma. The metabolic significance of these products remains to
be determined
- Fibronectin was first discovered as a large, external
transformation-sensitive protein (LETS) which was lost when
fibroblast were transformed into tumor cells
- Many tumor cells secrete less than normal amounts of
adhesion protein material, which allows for more movement
within the extracelluler milieu. This, in turn, increases the
potential for the tumor cells to leave their original location
and take root at another location within the body
(metastasis)
• IV. MATRIX METALLOPROTEINASES
-The ECM contains a series of proteases, known as the
matrix metalloproteinases, or MMPs.
- These are zinc containing proteases that use the zinc to
appropriately position water to participate in the
peoteolytic reaction. At least 23 different types of human
MMPs exist, and they cleave all proteins found in ECM,
including collagen and laminin.
- Because MMPs degrade ECM components, their expression
is important to allow cell migration and tissue remodeling
during growth and differentiation. In, addition many growth
factors bind to ECM components and, as bound components,
do not exhibit their normal growth-promoting activity.
- Destruction of the ECM by the MMPs releases these growth
factors, allowing them to bind to cell surface receptors to
initiate growth of tissues. Thus, coordinated expression of
the MMPs is required for appropriate cell movement and
growth.
 - Cancer cells that metastasize require extensive ECM
remodeling and ussualy use MMP activity to spread
throughout the body.
- A propeptide is present in newly synthetized MMPs that
contain a critical cysteine residue.
The cysteine residue in the propeptide binds to the zinc
atom the active site of the protease and prevents the
propeptide from exhibiting proteolytic activity. Removal of
the propeptide is required to activate the MMPs. Once, they
are activated, certain MMPs can activate other forms of
MMP.
- Regulation of MMP activity is quite complex.
These regulatory processes include transcriptional
regulation, proteolytic activation, inhibition by the
circulating protein α 2-macrogobulin, and regulation by a
class of inhibitors known as tissue inhibitors of
metalloporoteinase, or TIMP. It is important that the
synthesis of TMPs and MMPs be coordinately regulated,
because dissociation of their expression can facilitate
various clinical disorders, such as certain forms of cancer
and atherosclerosis.
• References
1. Baynes JW, Dominiczak: Medical Biochemistry, 2009:87.
2. Lieberman M, Marks AD: Basic medical biochemistry.
A clinical approach, 3rd ed, 2009:936-961.
3. Murray RK, et al: Harper’s Illustrated Biochemistry, 28th ed,
2009: 527-544.