TOPIC: EXTRACELLULAR MATRIX

By

Dr.
Topic: Extracellular Matrix
Number of competencies: (02)
Number of procedures that require certification: (NIL)
Num COMPETENCY Dom Level Suggest Sugge Vert Ho
ber The student should be able to ain K/KH ed sted ical riz
K/S/ / Teachin Asses inte ont
A/C SH/P g sment grat al
Learnin meth ion Int
g od egr
method ati
on
B List the functions and K KH Lecture Writte
components of the , Small n/
I9.1
extracellular matrix (ECM). group Viva
discussi voce
on
Specific Learning
Objectives

List the functions of K K

extracellular matrix
Enumerate the different K K
components of ECM
Describe the structure of K KH
collagen
Describe the structure and K KH
functions of
mucopolysaccharides
Explain the role of K KH
mucopolysaccharides in
formation of extracellular
matrix
Enumerate the functions K K
of Proteoglycans in
extracellular matrix
Explain briefly the role of K KH
non-collagen proteins in
extracellular matrix
formation
Explain the role of Laminin, K KH
Elastin and Fibronectin
B
Discuss the involvement of ECM K KH Lecture Written Gen
I9.2
components in health and , Small / eral
disease. group Viva Med
discussi voce icine
on

Specific Learning Objectives

Enumerate the various genetic K K
disorders from abnormalities in
the synthesis of collagen
Discuss briefly the K KH
pathophysiology of Ehler Danlos
syndrome, Marfan
syndrome Alport syndrome
Explain the alterations of ECM K KH
components in osteoarthritis or
rheumatoid arthritis
 Extracellular matrix (ECM) or Ground substance or
"connective tissue."

Definition:-Extracellular matrix is a complex network of

material surrounding the mammalian cells in tissues.

Composition of Extracellular Matrix

-The ECM contains three major classes of biomolecules:

(1) structural proteins - collagen, elastin, and fibrillin
(2) specialized proteins - fibronectin and laminin
(3) proteoglycans
(4) Water-65%

Extracellular matrix is produced by the cells of connective

tissues such as fibroblasts and epithelial cells
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Biomedical Importance:
The ECM has been found to be involved in many normal and
pathologic processes—eg, it plays important role in development,
inflammatory states, and in the spread of cancer cells.

Involvement of certain components of the ECM has been

documented in both rheumatoid arthritis and osteoarthritis.

Several diseases (eg, osteogenesis imperfecta and a number of

types of the Ehlers-Danlos syndrome) are due to genetic
disturbances of the synthesis of collagen.

Specific components of proteoglycans (the glycosaminoglycans;

GAGs) are affected in the group of genetic disorders known as the
mucopolysaccharidoses.

Changes occur in the ECM during the aging process.

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Functions of the Extracellular Matrix

[Link] holds the cells together and provides a porous

pathway for diffusion of nutrients and oxygen to

individual cells.

[Link] protects the organs and also provides elasticity

where required, for example, in blood vessels, lungs, and

skin.

[Link] regulates cell processes such as growth,

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migration and differentiation.
[Link] matrix directs morphology of a tissue by

interacting with cell-surface receptors and by binding to the

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surrounding growth factors which then stimulate signaling

pathways.

[Link] proteins and glycosaminoglycans of ECM form a gel like

structure which provides a flexible mechanical support and

functions as a cushion and lubricant against mechanical shocks .

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[Link] extracellular matrix support growth and wound

healing. For instance, bone growth depends on the

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extracellular matrix since it contains the minerals needed

to harden the bone tissue.

[Link] extracellular matrix has an important role in tissue

repair which can be utilized as a therapeutic target.

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Collagen:
-constitutes approximately 25% of the total body
protein in mammals.

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--is the major component of most connective tissues,
its distribution varies in different tissues; for instance,
collagen forms 90% of organic matrix in bones, 85% of
tendons, 70% of skin and 4% of liver.

--Principle producers of collagen fibers are fibroblasts,

epithelial and smooth muscle cells

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Types of Collagen
-At least 28 distinct types of collagen made up of over
30 distinct polypeptide chains (each encoded by a
separate gene) have been identified in human tissues.

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-Although several of these are present only in small
proportions, they may play important roles in
determining the physical properties of specific tissues.

Types of collagen are numbered by Roman numerals as

I,II,III………IX,X …….

Collagen type I to IV represent more than 95% of total

collagen.
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Structure of Collagen:
-All collagen types have a triple helical structure.
-In some collagens, the entire molecule is triple helical, whereas

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in others the triple helix may involve only a fraction of the
structure.
Collagen Type I Is Composed of a Triple Helix Structure & Forms
Fibrils:
- Type I Mature collagen, contains approximately 1,000 amino
acids in each polypeptide chain and possesses triple helical
structure throughout the molecule.
-It is composed of three similar polypeptide chains. They are also
called as alpha chain

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Right-handed collagen triple helix formed from
three left-handed α-chains.
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-each polypeptide subunit or alpha chain is twisted into
a left-handed polyproline helix of three residues per
turn.
-Three of these alpha chains are then wound into a
right-handed superhelix, forming a rodlike molecule

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1.4 nm in diameter and about 300 nm long.

-A striking characteristic of collagen is the occurrence

of glycine residues at every third position of the triple
helical portion of the alpha chain.

This is necessary because glycine is the only amino acid

small enough to be accommodated in the limited space
available down the central core of the triple helix.
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26

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This 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 any other amino acids, about 100 of the X
positions are proline and about 100 of the X positions are
hydroxyproline. Most of the time X is for proline and for

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hydroxyproline.

Collagen has an unusual amino acid composition with 33% of the

total residues being glycine (Gly), 10% proline (Pro), 10%
hydroxyproline (Hyp) and 1% hydroxylysine (Hyl).

Proline and hydroxyproline confer 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 ascorbic acid (vitamin C) and -
ketoglutarate.
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Lysines in the Y position may also be posttranslationally
modified to hydroxylysine through the action of lysyl
hydroxylase, an enzyme with similar cofactors.

Some of these hydroxylysines may be further modified

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by the addition of galactose or galactosyl-glucose
through an O-glycosidic linkage, a glycosylation site
that is unique to collagen.

The triple helical structure of collagen is stabilized by

hydrogen bonds, covalent cross links, electrostatic and
hydrophobic interactions and vander waals forces

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Lysine Hydroxylysine

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Glycosylated hydroxylysine.

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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

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Functions of Collagen:

- extracellular framework

- Strength, support and shape to the tissues and

organs

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- Great tensile strength and helps to maintain
structural integrity of cells

-Proper alignment of cells -cell proliferation and

their differentiation to different tissues and organs.

- Collagen,exposed in blood vessels contributes to

thrombus formation.
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Genetic Disorders from abnormalities in the
synthesis of Collagen:
- Genetic Diseases

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- About 30 genes encode the collagens
- Biosynthesis is complex, involving at least eight
enzyme-catalyzed posttranslational steps.
Diseases are due to mutations in collagen genes
or in genes encoding some of the enzymes
involved in these posttranslational modifications

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[Link]-Danlos Syndrome:
- is a group of inherited disorders (atleast 10 types
are known) due to mutation in gene for collagen or
due to defect in posttranslational processing of
collagen.

-principal clinical features are hyperextensibility of

the skin, increased joint mobility and abnormal
tissue fragility
Since 1997,the Villefranche classification of 6 subtypes based on their
phenotype and molecular defects have been used
Vascular subtype is the most serious because of its
tendency for spontaneous rupture of arteries or the
bowel, reflecting abnormalities in type III collagen.

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Patients with Kyphoscoliosis, exhibit progressive
curvature of the spine (scoliosis), marked joint
hypermobility and a tendency to ocular rupture due to
a deficiency of enzyme lysyl hydroxylase.

Dermatosparaxis, manifested by marked joint

hypermobility and soft skin.

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[Link] syndrome (Hereditary Nephritis):
- Type IV collagen fibers
- The major collagen found in the basement
membranes of the renal glomeruli, inner ear and
eye.

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- Hematuria, ocular lesions and hearing loss
- end-stage renal disease

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[Link] bullosa:

- type VII collagen

- skin breaks,
- blisters will be formed even for a minor trauma

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-
This collagen forms delicate fibrils that anchor the
basal lamina to collagen fibrils in the dermis.
These anchoring fibrils have been shown to be
markedly reduced in this form of the disease,
probably resulting in the blistering.

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4. Osteogenesis Imperfecta(Brittle –bone disease):

- Type-1 Collagen

- Abnormal fragility of bones - mutiple fractures in infancy

and childhood.

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- The scleras are often abnormally thin and translucent
and may appear blue owing to a deficiency of connective
tissue.

Four types (mild, extensive, severe, and variable) have

been recognized, of which the extensive type occurring in
the newborn is the most ominous.

Affected infants may be born with multiple fractures and

not survive.
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- RNA splicing
- Most frequent type results in the replacement of
glycine by another bulkier amino acid, affecting
formation of the triple helix.

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- abnormal fibrils - weakening the overall structure of
bone.

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AQUIRED DISORDERS

[Link]:

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In Vit.C deficiency collagen synthesis is adversly affected,
leading to bleeding of gums, poor wound healing and
subcutaneous hemorrhage.

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[Link]:
- Due to regular intake the pulse Kesari dal( Lathyrus
sativa or sweet pea).
- β-oxalyl aminoalanine (BOAA).
- BOAA interferes with the crosslinking of lysine amino

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acid in collagen structure by inhibiting the enzyme lysyl
oxidase

7. Menkes disease:

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Role of non-collagen proteins in
Extracellular matrix formation

-Role of -Elastin
-Fibrillin
-Fibronectin
-Laminin
 ELASTIN
- found in
- elastin fibre uncoils when streched and recoils
when relaxed
- 90% amino acids are nonpolar – hydrophobic
-
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- Tropoelastin -800 amino acid residues.
- prolines of tropoelastin are hydroxylated to
hydroxyproline by prolyl hydroxylase.
- Hydroxylysine and glycosylated hydroxylysine are
not present.

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- Elastin does not contain repeat Gly-X-Y sequences,
triple helical structure, or carbohydrate moieties.

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- lysyl oxidase
- However, the major cross-links formed in elastin are
the desmosines, which result from the condensation
of three of these lysine-derived aldehydes with an
unmodified lysine to form a tetrafunctional cross-link
unique to elastin.

Once cross-linked - elastin is highly insoluble and

extremely stable and has a very low turnover rate.

Elastin exhibits a variety of random coil conformations

that permit the protein to stretch and subsequently recoil
during the performance of its physiologic functions.
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Clinical Aspects:
Williams syndrome (Williams-Beuren syndrome)
-connective tissue and CNS are affected
-mental retardation and aortic stenosis

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SCLERODERMA:
-increased accumulation of elastin

- PULMONARY EMPHYSEMA - Increased degradation of

Elastin

-Decreased synthesis of elastin is seen in Aging of the

Skin

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3. Fibrillin
-Fibrillin is a structural component of
microfibrils.

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-Found commonly in zonular fibres of the
lens, periosteum and and associated with
elastin fibres in the aorta.
-Four types of fibrillin are known and are
named as Fibrillin1,2,3 and 4

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Clinical Aspects:

Marfan Syndrome:
- Fibrillin-1
- is a relatively prevalent inherited disease affecting

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connective tissue
- eyes (ectopia lentis)
- skeletal system ( arachnodactyly, hyperextensibility) -
- cardiovascular system (dilation of the ascending aorta).

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4. Fibronectin
-major glycoprotein
- cell adhesion, growth, cell migration
-Fibronectins guide immune cells to wounded areas
and thus help in wound healing.

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-Tumor cells are deficient in fibronectin which results in
lack of adhesion among the tumor cells that may often
lead to metastasis

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Schematic representation showing interaction of
collagen, fibronectin, and laminin present in the ECM
with a typical cell (ex: fibroblast) present in the matrix.

(a & b indicate α and β polypeptide chains of integrins)

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5. Laminin:
is the most abundant glycoprotein in basement
membrane.
-renal glomerular and other basal laminas

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-has got binding domains for both ECM and cell surface
receptors

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 Structure of Basal Lamina
laminin is attached to type IV collagen and the epithelial
cell layer via integrins and dystroglycans
- The relatively thick basal lamina of the renal
glomerulus has an important role in glomerular
filtration, regulate the passage of large molecules
(most plasma proteins) across the glomerulus into
the renal tubule.

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-
The glomerular membrane allows small molecules,
such as inulin (5.2 kDa), to pass through as easily as
water. On the other hand, only a small amount of
the protein albumin (69 kDa), the major plasma
protein, passes through the normal glomerulus.

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This is explained by two sets of facts:
(1) The pores in the glomerular membrane are large
enough to allow molecules up to about 8 nm to pass
through.

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(2) Albumin is smaller than this pore size, but it is
prevented from passing through easily by the negative
charges of heparan sulfate and of certain sialic acid-
containing glycoproteins present in the lamina. These
negative charges repel albumin and most plasma
proteins, which are negatively charged at the pH of
blood.

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- The normal structure of the glomerulus may be
severely damaged in certain types of
glomerulonephritis (eg, caused by antibodies directed
against various components of the glomerular
membrane) - severe albuminuria.

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Presence of small amounts of albumin in urine(
microalbuminuria-less than 300mg/day) is considered a
good and earliest marker of renal glomerular damage.

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Proteoglycans (Mucoproteins or
Mucoids).
Proteoglycans are proteins that contain covalently linked
glycosaminoglycans (GAGs).

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-The amount of carbohydrate in a proteoglycan is usually
much greater than is found in a glycoprotein
- 95% carboyhydrate and 5% protein.

- they are found in the ECM of connective tissue, synovial

fluid of joints, cartilage, mucous secretions and vitreos
humour of the eye

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Schematic representation of the proteoglycan “aggrecan”

It is very large with its overall structure resembling that of a

bottle brush.

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Degradation of GAGs :
is carried out by a battery of lysosomal hydrolases. These
include certain endoglycosidases, various
exoglycosidases, and sulfatases, generally acting in
sequence to degrade the various GAGs.
Mucopolysaccharidoses :
Deficiencies of Enzymes that Degrade
Glycosaminoglycans Result in Mucopolysaccharidoses.

They are a group of lysosomal disorders characterized by

accumulation of glycosaminoglycans in tissues and their
increased excretion.
None is common
 Biochemical Defects in Mucopolysaccharidoses (MPS)
Name Enzymatic Defect Urinary Metabolites
Mucopolysaccharidoses
Hurler’s syndrome (MPS I ) L-Iduronidase Dermatan sulfate, heparan sulfate

Hunters Syndrome (MPS II) Iduronate sulfatase Dermatan sulfate, heparan sulfate
Sanfilippo A (MPS IIIA ) Heparan sulfate N- Heparan sulfate
sulfatase (sulfamidase)
Sanfilippo B (MPS IIIB ) N-Acetylglucosaminidase Heparan sulfate
Sanfilippo C (MPS IIIC ) Glucosaminide N- Heparan sulfate
acetyltransferase
Sanfilippo D ( MPS IIID ) N-Acetylglucosamine 6- Heparan sulfate
sulfatase
Morquio A (MPS IVA) Galactosamine 6-sulfatase Keratan sulfate, chondroitin 6-
sulfate
Morquio B (MPS IVB) β-Galactosidase Keratan sulfate
Maroteaux-Lamy (MPS VI) N-Acetylgalactosamine 4- Dermatan sulfate
sulfatase (arylsulfatase B)
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Name Enzymatic Defect Urinary Metabolites

Mucolipidoses
Sly (MPS VII) β-Glucuronidase Dermatan sulfate, heparan
sulfate, chondroitin 4-

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sulfate, chondroitin 6-
sulfate
Sialidosis(ML I ) Sialidase (neuraminidase) Glycoprotein fragments
I-cell disease (ML II ) N-acetylglucosamine-1- Glycoprotein fragments
phosphotransferase
Pseudo-Hurler As for ML II but deficiency is Glycoprotein fragments
polydystrophy (ML III) incomplete

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Major Features of the Mucopolysaccharidoses

- chronic progressive course.

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- multisystem disorders
- organomegalay
- dysostosis multiplex
- mental retardation
- abnormal facies

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Some Laboratory Tests Used in the Diagnosis of a
Mucopolysaccharidosis

Urinalysis for presence of increased amounts of GAGs (

screening tests such as ( toluidine blue test and thin

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layer chromatography} are carried out.

Assays of suspected enzymes in white blood cells,

fibroblasts or possibly serum. Tissue biopsy with
subsequent analysis of GAGs by electrophoresis. Use of
specific gene tests.
Prenatal diagnosis can now be performed in at least
certain cases using amniotic fluid cells or chorionic
villus biopsy
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 Some Functions of Glycosaminoglycans and Proteoglycans
•Act as structural components of the ECM

•Have specific interactions with collagen, elastin, fibronectin,

laminin, and other proteins such as growth factors

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•As polyanions, bind polycations and cations. The GAGs present in the
proteoglycans are polyanions and hence bind polycations and cations
such as Na+ and K+. This latter ability attracts water by osmotic
pressure into the extracellular matrix and contributes to its turgor.
•Contribute to the characteristic turgor of various tissues
•Act as sieves in the ECM. GAGs also gel at relatively low
concentrations. Because of the long extended nature of the
polysaccharide chains of GAGs and their ability to gel, the
proteoglycans can act as sieves, restricting the passage of large
macromolecules into the ECM but allowing relatively free diffusion of
small molecules 84
•Facilitate cell migration (HA)

•Have role in compressibility of cartilage in weight-bearing

(HA, CS)
•Play role in corneal transparency (KS I and DS)

•Have structural role in sclera (DS)

•Act as anticoagulant (heparin)

•Are components of plasma membranes, where they may

act as receptors and participate in cell adhesion and cell-cell
interactions (eg, HS)
•Determine charge-selectiveness of renal glomerulus (HS)

•Are components of synaptic and other vesicles (eg, HS)

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Proteoglycans are Associated with Major Diseases &
with Aging:

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Hyaluronic acid may be important in permitting tumor
cells to migrate through the ECM. Tumor cells can
induce fibroblasts to synthesize greatly increased
amounts of this GAG, thereby perhaps facilitating their
own spread. Some tumor cells have less heparan
sulfate at their surfaces, and this may play a role in the
lack of adhesiveness that these cells display.

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The intima of the arterial wall contains hyaluronic acid
and chondroitin sulfate, dermatan sulfate, and heparan
sulfate proteoglycans.
Of these proteoglycans, dermatan sulfate binds plasma
low-density lipoproteins. In addition, dermatan sulfate

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appears to be the major GAG synthesized by arterial
smooth muscle cells.
Because it is these cells that proliferate in
atherosclerotic lesions in arteries, dermatan sulfate
may play an important role in development of the
atherosclerotic plaque.

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In various types of arthritis, proteoglycans may act as
autoantigens, thus contributing to the pathologic
features of these conditions. The amount of chondroitin
sulfate in cartilage diminishes with age, whereas the
amounts of keratan sulfate and hyaluronic acid increase.
These changes may contribute to the development of

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osteoarthritis, as may increased activity of the enzyme
aggrecanase, which acts to degrade aggrecan. Changes
in the amounts of certain GAGs in the skin are also
observed with aging and help to account for the
characteristic changes noted in this organ in the elderly.

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Osteoarthritis (OA) is a degenerative disease that
affects various tissues surrounding joints such as
articular cartilage, subchondral bone, synovial
membrane, and ligaments.
Progression of OA can be characterized by changes in ECM
composition and structure. Natural, healthy cartilage matrix is
mainly composed of collagen type II which provides tensile
support for the tissue. Aggrecan, a negatively charged
proteoglycan that attracts water molecules, provides the
compressive resistant and shock absorbing capability of
cartilage under loading . It has been shown that during OA,
there are sequential events that affect the integrity of
homeostatic ECM; aggrecan content is decreased, while
collagen content is increased. This change in ECM composition
predisposes the tissue for mechanical fault resulting in
significantly altered mechanical environments of the cells
within the cartilage matrix.