GOOD MORNING

EXTRACELLULAR
MATRIX

Janet Myla Quizon Bonleon, MD, FPPS, FPAPP

 Extracellular Matrix
• “the substance between cells”
• “material in the intercellular space”
• All cells in solid tissue are surrounded by
ECM
• Can surround cells as fibrils that contact the
cells on all sides
• A sheet called the basement membrane that
cells can ‘sit on’
 Extracellular Matrix
• 2 groups of biochemicals that make up
the basic ECM
– Polysaccharides
• Complex chains of sugar molecules
– Glycoproteins
• Polysaccharides joined to proteins
• Proteoglycans
 Extracellular Matrix
• Embedded can be various types and
amounts of structural and insoluble collagen
fibers and flexible elastic fibers that give
resilience to the tissues
• Modified forms
– Bone
– Exoskeleton (insect)
– Shells (animals)
– Cell wall (plants)
 Where does it come from?
• All cells make ECM
• Specialist cells produce specific types
– Fibroblast cells secrete connective tissue ECM
– Osteoblast cells secrete bone-forming ECM
– Chrondroblast cells secrete cartilage-forming
ECM
– Fibroblasts & epithelial cells make basement
membrane
3 MAJOR CLASSES OF BIOMOLECULES
• STRUCTURAL PROTEINS
– Collagen, elastin, fibrillin
• SPECIALIZED PROTEINS
– Fibrillin, fibronectin, laminin
• PROTEOGLYCANS
– Long chains of repeating disaccharides
(glycosaminoglycans or mucopolysaccharides)
attached to special core proteins
 Extracellular Matrix
• Provides :
– Mechanical support
– A biochemical barrier
– A medium for :
• Extracellular communication that is assisted
by CAMs
• The stable positioning of cells in tissues
through cell matrix adhesion
• The repositioning of cell migration during
cell development & wound repair
 Extracellular Matrix
• Provides :
– Tensile strength for tendons
– Compressive strength for cartilage
– Hydraulic protection for many types
of cells
– Elasticity to the walls of blood
vessels
Cell adhesion molecules (CAMs)

• Belong mainly to glycoproteins

• Located at the cell surface and form
different types of complexes & junctions to
join :
– Cells to cells
– Cells to ECM
– ECM to the cell cytoskeleton
Cell Adhesion Molecules (CAMs)

• Assist :
– The adhesion of cells to one another to
provide organized tissue structure
– The transmission of extracellular cues
and signals across the cell membrane
– The migration of cells through the
regulation of CAM assisted adhesions
COLLAGEN
 Collagen

• Most abundant protein in the world

• Major component of most connective
tissues
• Provide extracellular framework
• In human – 19 types
• Subdivided into classes
 Collagen
• Dispersed as a gel  give support to the
structure as in ECM or vitreous humor of
the eye

• Bundled in tight, parallel fibers that provide

great strength as in tendons
 Types of Collagen
• FIBRIL-FORMING COLLAGENS

– Types I, II & III

– Rope-like structure
 Types of Collagen
• NETWORK-FORMING COLLAGENS

– Types IV & VII

– Form 3-D mesh
– Major part of basement membranes
 Types of Collagen
• FIBRIL-ASSOCIATED COLLAGENS

– Types IX & XII

– Bind to the surface of collagen
fibrils, linking these fibrils to one
another and to components in the
ECM
 Collagen
• Collagen I
– Major collagen of skin & bone

• Collagen II
– Major collagen of cartilage

ALL COLLAGEN TYPES HAVE A

TRIPLE HELICAL STRUCTURE
 Collagen Type I
• Contain ~1,000 amino acids
• Entire molecule is triple helical
• Type IV – discontinuous (globular
structure interspersed)
• Tensile strength
 Collagen Type I
• Each polypeptide sub-unit or alpha chain is
twisted into a left-handed helix of 3 residues
per turn

• Three of these alpha chains are then wound

into a right-handed superhelix  rod-like
molecule
 Collagen Type I
• Characteristic : occurrence of
GLYCINE residues at every 3rd
position of the triple helical portion of
the alpha chain

• Gly – X – Y – Gly – X – Y -
a.a. sequence
 Collagen Type I
• Necessary because glycine is the only a.a.
small enough to be accommodated in the
limited space available in the central core of
the triple helix

• X,Y = any other a.a.

• X 100 = proline & Y 100 = hydroxyproline
 confer rigidity
 Collagen Type I

• Hydroxylysine is also found

• “quarter staggered”  banded appearance
• STABLE  non-covalent cross-links
within & between the triple helical units
• Important enzyme : lysyl oxidase
• Process : deamination
 Biosynthesis of Collagen
• Complex
• Many posttranslational events 
hydroxylation of proline & lysine
• Cardinal principle : SELF ASSSEMBLY
• Synthesized on the ribosomes in a precursor
form  PREPROCOLLAGEN
 Biosynthesis of Collagen

• Preprocollagen (ribosomes) – contains a

lead or signal sequence that directs the
polypeptide chain into the endoplasmic
reticulum

• Leader segment is enzymatically removed –
ER

• Hydroxylation of proline & lysine residue
and glycosylation of hydroxylysine – ER

• PROCOLLAGEN – with polypeptide
extensions both amino and carboxyterminal
ends
– Not present in mature collagen
– Extensions peptide contains cysteine residues

• Amino terminal polypeptide – form
intrachain disulfide bonds
• Carboxyterminal end – form both intra- &
interchain disulfide bonds
• Disulfide bonds – assists in the registration
of the 3 collagen molecules to form the
triple helix, winding from the carboxy
terminal end

• Triple helix – no further hydroxylation of
the proline or lysine or glycosylation of the
hydroxylysine takes place

• Secretion by the Golgi apparatus

• Extracellular enzymes – PROCOLLAGEN
AMINOPROTEINASE (amino) &
PROCOLLAGEN
CARBOXYPROTEINASE (carboxyl)

• Remove extension peptides at the terminal
ends (cleavage sites : crypts of the cell
membrane)

• Propeptides are removed

• Triple helical collagen molecule
spontaneously assemble into the collagen
fibers

• Further stabilized by inter- & intrachain
cross-links

• Collagen once formed  metabolically
stable


• Increase breakdown in starvation &

inflammation
BIOMEDICAL IMPORTANCE
• GENETIC DISEASES
– Causes
• Mutation in the collagen genes
(30)
• Mutation in the genes encoding
enzymes in the
posttranslational modification
BOMEDICAL IMPORTANCE
• EHLERS-DANLOS SYNDROME
– Clinical features : hyperextensibility of the
skin, abnormal tissue fragility, increase in joint
mobility
– Type IV (11 types) – most serious because of
the tendency to rupture spontaneously, arteries
or bowel (abnormality in the Type III collagen)
BIOMEDICAL IMPORTANCE
• EHLERS-DANLOS SYNDROME
– Type VI – deficiency of lysyl hydroxylase, with
marked hypermobility of the joints & tendency
to ocular rupture
– Type VIIC – deficiency of procollagen N-
proteinase; with abnormal, thin, irregular
collagen fibrils, marked joint hypermobility and
soft skin
BIOMEDICAL IMPORTANCE
• ALPORT’S SYNDROME
– Type IV collagen affected (major
collagen found in the basement
membrane of the renal glomeruli)
– Hematuria  ESRD
BIOMEDICAL IMPORTANCE
• EPIDERMOLYSIS BULLOSA
– Minor trauma  skin breaks & blisters
– Affects type VII collagen (form delicate fibrils
that anchor the basal lamina to collagen fibrils –
dermis)
– Decrease the number of anchoring fibrils 
blistering
BIOMEDICAL IMPORTANCE
• EPIDERMOLYSIS BULLOSA
SIMPLEX

• Another variant
• Due to mutations in keratin 5
BIOMEDICAL IMPORTANCE
• SCURVY
– Deficiency of ascorbic acid
– Not genetic
– Bleeding gums, subcutaneous hemorrhage &
poor wound healing
– Impaired synthesis of collagen due to the
deficiencies of prolyl & lysyl hydroxylase
(require ascorbic acids as co-factor)
 ELASTIN
•
 ELASTIN
• Responsible for extensibility & elastic
recoil in tissues
• Not as widespread as collagen
• Present in the lungs, arterial blood vessel &
some elastin ligaments
• Smaller quantity in the skin, ear cartilage
• Only 1 genetic type
 Biosynthesis of Elastin
• Tropoelastin ( not synthesized in a preform
with extension peptides)

• Some prolines are hydroxylated to
hydroxyproline by prolyl hydroxylase
(OH-lys & glycosylated OH-lys not present)

• Secretion from the cells lysyl residues of
tropoelastin are oxidatively deaminated to
aldehydes by lysyl oxidase (same as
collagen)

• Major cross-links formed are
DESMOSINES result from condensation of
3 of the lysine-derived aldehydes with an
unmodified lysine to form a tetrafunctional
cross-link unique to elastin)

• Mature, extracellular form (elastin is highly
soluble & extremely stable with very low
turnover rate)
• Presence of a variety of random recoil
conformation – permit elastin to stretch &
recoil
 Collagen Elastin
Many different genetic One genetic type
types
Triple helix Random coil
conformations
permit stretching
(Gly-X-Y)n repeating None
structure
 Collagen Elastin
Hydroxylysine None
Carbohydrate-containing None
Intramolecular aldol Intramolecular
cross-links desmosine
cross-links
Extension peptides None
during biosynthesis
 Muscular artery (Elastin stain)
•
 Biomedical Importance
• WILLIAM’S SYNDROME
– Deletion in elastin gene (7q11.23)
– Affect connective tissue and central
nervous system
– Supravalvular aortic stenosis
 Biomedical Importance
• SKIN DISORDERS
– Accumulation of elastin (scleroderma)

• PULMONARY EMPHYSEMA. CUTIS

LAXA & AGING
– Decrease/ fragmentation of elastin
 Biomedical Importance
• -1 ANTITRYPSIN DEFICIENCY
– Normal lung - alveoli are chronically exposed
to low levels of neutrophil elastase
– Proteolytic activity can destroy the elastin in
the alveolar walls if unopposed by the
inhibitory action of -1-AT
– Deficiency sec to mutations in the -1 gene 
emphysema
– Especially smokers
 FIBRILLLIN
•
 Fibrillin
• Large glycoprotein
• Structural component of microfibrils (10-
12nm filaments)
• Secreted into the ECM by fibroblasts 
incorporated into insoluble microfibrils
• Provide a scaffold for the deposition of
elastin
 Fibrillin

• Found in the zonula fibers of lens,

periosteum
• Associated with elastin fiber in the
aorta
 Biomedical Importance
• MARFAN’S SYNDROME
– Frequently inherited connective tissue
disorder
– Autosomal dominant trait
– Ectopia lentis
– Skeletal system : tall, long digits
(arachnodactyly), hyperextensibility of
joints
– CVS dilated ascending aorta secondary to
weakness of aortic media
 Biomedical Importance
• MARFAN’S SYNDROME

– Mutation in Chromosome 15 (genes for

fibrillin)
– Long, contractural arachnodactyly –
Chromosome 5
FIBRONECTIN
 Fibronectin
• Major glycoprotein of ECM & found in
soluble form in the plasma
• 2 identical sub-units joined by 2 disulfide
bridges into functional carboxyterminals
• Has 3 types of repeating motifs (I, II & III)
organized into functional domains
 Fibronectin
• Function on domains including heparin &
fibrin, collagen, DNA & cell surfaces
• Contain Arg-Gly-Asp (RGD)sequence that
binds to the receptor
• Sequence is shared by other proteins in the
ECM that bind to integrins (a type of
transmembrane proteins present in cell
surfaces)
 Fibronectin
• Fibronectin receptor interacts indirectly
with actin (microfilaments)
• Attachment of proteins
• Attachment & interaction – important
because it affects cell behaviour
• Adhesion molecules, cell migration
(provide binding site)
 LAMININ

• Laminin 1 is present on capillaries and in the rim of

muscle fibers
 Laminin
• Major protein component of renal glomerular &
other basal laminae  surround epithelial cells
• Glomerular membrane – epithelium = basal
lamina = endothelium
• Primary components of basal lamina : laminin,
entactin & type IV collagen & GAGs heparin or
heparin sulfate
• Synthesized by the underlying cells
 Laminin
• Consists of 3 distinct elongated polypeptide
chains (A, B1 and B2) linked together to
form an elongated cruciform shape
• Presence of binding cells – cell shape
• Collagen interacts with laminin  interacts
with integrin or other laminin receptor
proteins
• Anchor lamina to the cells
 Laminin
• ENTACTIN
– “nidogen”
– Glycoprotein
– Contains RGD sequence
– Binds to laminin
– Major cell attachment factor
 Biomedical Importance of
Laminin
• KIDNEY – basal lamina is thick
• Important role in glomerular filtration
• Regulates passage of large molecules (most
plasma proteins) across glomerulus 
renal tubule
• Allows small molecules to pass like water
 Laminin
•

• Laminin 1 chain in the normal human kidney

 Biomedical Importance of
Laminin
• ALBUMIN
– Pores in the glomerular membrane are large
enough to allow ~8nm molecule to pass
through
– Albumin is smaller but because of the negative
charges of heparin sulfate & some sialic acid-
containing glycoproteins in the basal lamina 
albumin is repelled
 Biomedical Importance of
Laminin

• GLOMERULONEPHRITIS
– Antibodies destroy GM
– Allows disposition of macromolecules
– Albuminuria
 PROTEOGLYCANS
and
GLYCOSAMINOGLYCANS
 Proteoglycans
• Proteins with covalently linked GAGs
• Vary in tissue distribution
• Vary in nature of core proteins, attached
GAGs & function
• Core proteins are proteins bound covalently
to GAGs
 Proteoglycans

• Amount of carbohydrate is greater than in glycoproteins

(95% of its weight)
• E.g. aggrecan = major type in cartilage, contains long
strand of hyaluronic acid (GAG) to which link proteins are
attached non-covalently
7 GLYCOSAMINOGLYCANS
1. Hyaluronic acid
2. Chondroitin sulfate
3. Keratan sulfate I
4. Keratan sulfate II
5. Heparin
6. Heparan sulfate
7. Dermatan sulfate
 Glycosaminoglycans
• Differ in
– number of amino sugar composition
– uronic acid composition
– linkages between components
– chain length of disaccharides
– presence of sulfate groups
– position of attachment to constituent sugars
 Glycosaminoglycans
• Unbranched polysaccharides made up of
repeating disaccharides
• One component is always an aminosugar
– D-glucosamine
– D-galactosamine
 Glycosaminoglycans
• Other component of the repeating
disaccharides (except keratan sulfate) is
uronic acid
• All GAGs contain a sulfate group (except
hyaluronic acid)
– L-glucoronic acid
– 5’ epimer, L-iduronic acid
• O-esters in heparin
• N-sulfate in heparan sulfate
 Harper
• Table 48-6
• Major properties of the GAG
 Biosynthesis of GAGs

1. Attachment to core proteins

2. Chain elongation
3. Chain termination
4. Further modifications
 Biosynthesis of GAGs
• ATTACHMENT TO CORE PROTEINS
– Occurs in the ER
– 3 types of linkages (GAG – core CHON)
• O-glycosidic bond between xylose & serine
• O-glycosidic bond between N-
acetylgalactosamine & serine (or Thr)
• N-glycosylamine between N-
acetylglucosamine & the amide nitrogen of
Asn, as found in N-linked glycoproteins
 Biosynthesis of GAGs
• ATTACHMENT TO CORE PROTEINS
• O-glycosidic bond between xylose & serine
• Bond unique to proteoglycans
• Formed by transfer of Xyl residue to Ser
from UDP-xylose  2 resideues of Gal +
Xyl residue  link trisaccharide
• Gal-Gal-Xyl-Ser
• Further chain growth on the terminal Gal
 Biosynthesis of GAGs

• CHAIN ELONGATION
– Nucleotide sugars & highly specific Golgi-
located glycotransferases are employed to
synthesize the oligosaccharide chains of GAGs
– “ONE ENZYME, ONE LINKAGE”
relationship
– Occurs in the golgi apparatus
 Biosynthesis of GAGs
• CHAIN TERMINATION
– Result from
• Sulfation
• Progression of the growing GAG chain
away from membrane site where
catalysis occurs
 Biosynthesis of GAGs

• FURTHER MODIFICATIONS
– Introduction of the sulfate group
– Occurs in the golgi apparatus
– Highly specific
 Functions of Proteoglycans
• Some bind to collagen or elastin – important
structural organization of ECM
• Some interact with adhesive proteins- fibronectin
& laminin
• GAGs polyanions – bind to Na & K  attract
water by osmotic pressure  cell turgor
• Act as sieves – restrict passage of large
macromolecules  able to gel
• Occupy large area of the ECM
Specific Functions
 Hyaluronic Acid
• Unbranched chain of repeating
disaccharide units containing GluUA
& GlcNAc
• During embryogenesis, permit cell
migration during morphogenesis &
wound repair
• In cartilage, provide compressibility
 Chondroitin Sulfate

• Repeating disaccharide contains

GlcUA & GalNAc
• At sites of calcification in the
endochondral bone & cartilage
• Provide endoskeletal structure
• Neurons – maintain shape
 Keratan Sulfates I & II

• Repeating Gal-GlcNAc disaccharide

units containing sulfate attached to 6’
position of GlcNAc or occasionally of
Gal
• Cornea – critical role in corneal
transparency
 Dermatan Sulfate
• Structure similar to chondroitin sulfate,
except that in place of GlcUA in -1,3
linkage to GalNac, it contains IdUA

• Sclera – maintains the overall shape of

the eye
 Heparin
• Repeating disaccharides contain
glucosamine & either of the two uronic
acids (~90% are IdUA)
• Important anticoagulant
• Binds to
– Factor IX
– Factor XI
– Plasma antithrombin III
 Heparan Sulfate

• Contains glucosamine with fewer N-

sulfates than heparin
• Predominant uronic acid is GlcUA
• Acts as receptors
• Participate in mediation of cell growth
& cell-cell communication
 Aging/ Disease
• Hyaluronic acid- important in permitting
migration of tumor cells through the ECM
• Dermatan sulfate – important role in the
development of atherosclerotic plaques
• In arthritis – act as autoantigens
• Chondroitin sulfate diminishes with age in
cartilage
• Osteoarthritis – keratan sulfate & hyaluronic acid
increases in cartilage
 Mucopolysaccharidoses
• Adult tissues – GAG have a slower turnover
• Half-lives : days to weeks
• Specific enzyme deficiencies result in
certain inborn errors of metabolism
• Degradation of GAG carried out by
lysosomal hydrolysis
– Endoglycosidases
– Exoglycosidases
– Sulfatases
 Mucopolysaccharidoses
• Common : autosomal recessive
• Most studied : Hurler’s & Hunter’s
Syndromes
• None are common
• Dx : urine assay, tissue autopsy
 Muculipidoses
• Combined features common to both muco- &
sphingolipidoses
• Signs & symptoms :
– Cloudy corneas
– Mental retardation
– Stiff joints
– Cardiac abnormalities
– Hepatosplenomegaly
– Short stature – depend on specific disease & severity
 Muculipidoses
• PATHOPHYSIOLOGY
– Mutations in a gene encoding a lysosomal
hydrolase involved in the degradation of 1 or
more GAGs 
– Defective lysosomal hydrolase 
– Accumulation of substrate in various tissues –
liver, spleen, bone, skin & CNS
 BONE
&
CARTILAGE
 BONE & CARTILAGE
• Specialized forms of ECM
• Major constituents of bone
– Collagen I
– Hydroxyapatite
• Major constituents of cartilage
– Collagen II
– Certain proteoglycans
 BONE
• Inorganic or mineral component – mainly
crystalline hydroxyapatite with Na, Mg,
CO3 & Fl
• Contains ~99% of the body’s Ca
• Hydroxyapatite
– Bone strength
– Resilience
 BONE
• Dynamic
• Continuous cycles of remodeling
– Resorption  deposition of new
bone tissues
• Major cell types
– Osteoclasts  resorption
– Osteoblasts  deposition
 BONE
• Osteoblasts : synthesize most of
proteins found in bone
• Control mineralization
• Many factors are involved in the
regulation of bone metabolism
• Stimulation/ inhibition – clasts/ blasts
 BONE

• Black areas – POLYMER (collagen fibers)

• White areas – mineralized mineral (hydroxyapatite crystals)
 Bone

• Microstructure of collagen fibers in a

hydroxyapatite matrix
 BONE
Metabolic & Genetic Disorders
• OSTEOGENESIS IMPERFECTA
– Brittle bones
– Abnormal fragility of the bones
– sclerae thin & transluscent & appears blue
secondary to deficiency of connective tissue
– Mutations result in decreased expression of
collagen or abnormal pro-alpha chains 
weakening of the overall structure of the bone
 BONE
Metabolic & Genetic Disorders
• OSTEOPETROSIS (marble bone disease)
– Increased bone density
– Due to inability to resorb bone
– May occur with RTA & cerebral calcification
– Due to mutations in gene encoding carbonic
anhydrase II
 BONE
Metabolic & Genetic Diseases
• OSTEOPOROSIS
– Generalized progressive reduction in
bone tissue mass per unit volume
causing skeletal weakness
– Fractures very easily
– Possible causes : estrogens &
interleukin-1 & -6
 CARTILAGE
• Avascular tissue
• Obtains most nutrients from synovial fluid
• Exhibits slow but continuous turnover
• Principal protein -Type II collagen
• Proteoglycan – important role in its
compressibility
• Aggrecan – major proteoglycan
 Cartilage

• FPch – fibrous perichrondrium

• CHl – chondrogenic cells
• ChB – outer layer – active matrix producing chrondroblasts
• Lac – lacuna – surrounded by matrix
• CN – “cell nests” - less metabolic activity
 CARTILAGE
Metabolic & Genetic Disorders
• CHONDRODYSPLASIA
– Mixed group of hereditary disorders affecting
cartilage
– Manifestations : short-limbed dwarfism &
numerous skeletal deformities
– Best known : ACHONDROPLASIA
 CARTILAGE
Metabolic & Genetic Disorders
• ACHONDROPLASIA
– Most common cause of dwarfism
– Short limbs
– Normal trunk size
– Macrocephaly
– Other skeletal deformities
– Autosomal dominant trait (mutations)
– Very specific mutations in the gene encoding
FGFR3 (fibroblast growth factor receptor 3)
•
THANK YOU
 References
• Chapter 48 Harper
• Lipincott
quiz
1. What is the most abundant protein in the
world and a major component in most
connective tissues?

2. What component of you ECM consists of 3

distinct elongated polypeptide chains (A,
B1 and B2) linked together to form an
elongated cruciform shape?
3. What is the major constituent of bone
(collagen type I or II)?

4 & 5. Give 2 functions of your ECM.