Extracellular Matrix

1. Biochemistry of
Extracellular Matrix
Jana Novotná

2. Composition of Extracellular
Matrix (ECM)
• Cells (mesenchymal origin)
- fibroblasts
- smooth muscle cells
- chondroblasts
- osteoblasts and epitelial cells
• Organic fibrillar matrix
• Organic nonfibrillar matrix
• Water

3. Function of ECM
• Provides support and anchorage for cells.
• Regulates and determine cells dynamic behaviour :
- polarity of cells
- cell differentiation
- adhesion
- migration
• Provides mechanical support for tissues and organ
architecture
- growth
- regenerative and healing processes
- determination and maintenance of the structure
• Place for active exchange of different metabolites, ions,
water.

4. Structure of ECM
• collagen
– the main ECM component, forms the main fibres
• elastin
• proteoglycans
- heteropolysacharides
• structural glycoproteins
- fibronectin, laminin

5. Collagen
• The most abundant protein in the body, making 25%-35% of all the
whole-body proteins.
• Collagen contributes to the stability of tissues and organs.
• It maintains their structural integrity.
• It has great tensile strenght.
• The main component of fascia, cartilage, ligaments, tendons, bone
and skin.
• Plays an important role in cell differentiation, polarity, movement.
• Plays an important role in tissue and organ development.

6. Collagen Structure
Collagen is insoluble glycoprotein (protein + carbohydrate)
Collagen polypeptide primary structure:
- G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – G −
,
G - glycine, X - proline or hydroxyproline, Y – lysin or hydroxylysine, A – amino
acid
Proline and hydroxyproline constitute about 1/6 of the total
sequence, provide the stifness of the polypeptide chain.
Carbohydrates : glucose, galactose

7. • Three helical polypeptide units twist to form a triple-helical collagen
molecule: a molecular "rope" which has some bending stiffness and does
not undergo rotation.
• The tropocollagen molecule has a length of approximately 300 nm and a
diameter close to 1.5 nm.
• In the typical fibrillar collagens, only short terminal portions of the
polypeptides (the telopeptides) are not triple helical.

8. 1. Synthesis of a chains of pre-procollagen
on ribosomes.
2. Hydroxylation of lysine and
proline in rER/Golgi by lysyl-5-
hydroxylase and prolyl-4-
hydroxylase.
3. Glycosylation: addition of
galactose and glucose to some
hydroxylysine residues
(galactosyl transferase and
glycosyl transferase).
4. Assembly of a-chains to form
procollagen. Reaction needs the
formation of disulphide bonds
between registration peptides,
at both ends of the prepro-collagen.
Synthesis

9. HSP47 as a Specific Collagen Chaperone

10. 5. Secretion of procollagen molecules by exocytosis into the
extracellular space.
6. Cleavage of registration peptides is catalysed by procollagen
peptidases. The resulting molecule is called tropocollagen.
7. Oxidation – deamination of the hydroxylysine, the removal of (NH2)
group has a net oxidative effect and the formation of covalent cross-links.
Reaction is catalyzed by lysine oxidase (or catalase).
8. Self-assembly or polymerization of tropocollagen molecules form
collagen fibrils. Cross-linkage between adjacent tropocollagen
molecules stabilizes the fibrils.

12. Posttranslation Modification of Collagen
Molecule
Hydroxylation of some prolyl and lysyl residues
prolyl-4-hydroxylase, lysyl-5-hydroxylase
Cofactors:
O2 (or superoxid)
a-ketoglutarate
Fe2+
vitamin C (ascorbic acid)
Proline + a-ketoglutarate + O2 + Fe2+ → 4-hydroxy-proline
+ Fe3+ + CO2 + succinate
Hydroxyproline stabilizes molecule of tropocollagen.

13. Aldol Condensation

14. Amadori Product - Ketamine

15. Lysinonorleucine Cross-link

16. The typical staggered array of tropocollagen molecules in
the collagen fibril. The telopeptides participate in covalent
crosslinking.

17. Collagen – Fiber Formation
Collagen types I, II, III, V, IX, X
Cross striated structure of collagen fiber reflect periodic composition of
individual tropocollagen molecules.
Collagen fibrils of 1 mm diameter support the weight of 10 kg.

18. Collagen Interactions
Fiber forming collagen and nonfibrous collagen
Tendon Cartilagous matrix

19. Collagens Classification
1. Fibril-forming collagens (I, II, III, V, X)
2. Fibril-associated collagens (FACIT)
3. Network-forming collagens
4. Anchoring fibrils collagens
5. Transmembrane collagens
6. Basement membrane collagens
7. Other collagens with unique function

21. Major Collagen Types
Fibril forming collagens
(Most abundant collagen family - 90 % of the total collagens)
Type Molecular composition Tissue distribution
I [a1(I)2 2a(I)] Bone, dermis, tendon, ligaments cornea
II [a1(II)]3 Cartilage, vitreous body, nucleus
pulposus
III [a1(III)]3 Skin, vessel wall, reticular fibres of most
tissues (lung, liver, spleen, etc)
V
XI
a1(V)a2(V)a3(V)
a1(XI)a2(XI)a3(XI)
Lung, cornea, bone, fetal membranes,
together with type I collagen
Cartilage, vitreous body

22. Basement membrane collagens
IV [a1(IV)2a2(IV)]; a1 – a6 Basement membrane
Short non-helical amino-terminal domain, a long Gly-X-Y repeat domain with
numerous small interruptions, and a highly conserved carboxyl-terminal globular
NC1 domain.
It polymerizes into a disulfide-bonded polygonal network via tetramerization
between amino-terminal domains and dimerization between NC1 domains.

23. Elastin
• Elastin is a major protein component
of tissues that require elasticity such
as arteries, lungs, bladder, skin and
elastic ligaments and cartilage.
• It is composed of soluble tropoelastin
protein containing primarily glycine
and valine and modified alanine and
proline residues.
• Tropoelastin is a ~65kDa protein that
is highly cross-linked to form an
insoluble complex.
• Polypeptide chains are cross-linked
together to form rubberlike, elastic
fibers. Each elastin molecule uncoils
into a more extended conformation
when the fiber is stretched and will
recoil spontaneously as soon as the
stretching force is relaxed.
Jaime Moore , Susan Thibeault
Journal of Voice Volume 26, Issue 3 2012 269 - 275

24. Elastin
• Desmosine (isodesmosine)
- the most common interchain
cross-link
• conversion of NH3 groups of
lysine (hydroxylisine) to
reactive aldehydes by lysyl
oxidase.
• desmosine cross-link is
spontaneously formed.

25. Proteoglycans
Special class of glycoproteins heavily glycosylated (95%).
Core protein with one or more
attached glycosamino glycan
chain(s).

26. Glycosaminoglycans (GAG)
• Long chain, linear carbohydrate polymers
• Negatively charged under physiological
conditions (due to the occurrence of sulfate and
uronic acid groups).
Disaccharide subunits are:
1. uronic acid
D-glucuronic acid or L-iduronic acid
2. aminosugar
N-acetyl glucosamin (GlcNAc) or
N-acetyl galactosamin (GalNAc)

27. Amino sugars and uronic acids are the most common building blocks of
the glycosaminoglycans.
• amino sugars  -OH at C-2 is replaced by an amino group. This amino
group is most often acetylated and sometimes sulfated.
• uronic acids  C-6 of the hexose is oxidized to a carboxyl group.

28. Linkage of GAGs to protein core by specific
trisaccharide linker

29. Hyaluronic Acid & Keratan Sulfate

30. Chondroitin 6-sulfate & Heparan
Supfate

31. Dermatan Sulfate & Heparin

32. Biosynthesis
• The protein component is synthesized by ribosomes and
transocated into the lumen of the RER.
• Glycosylation of the proteoglycan occurs in the Golgi
apparatus in multiple enzymatic steps.
• First a special link tetrasaccharide is attached to a
serine side chain on the core protein to serve as a
primer for polysaccharide growth.

33. Biosynthesis
• Then sugars are added by glycosyltransferase.
• Some glycosyltransferases catalyse sugar transfer to
tyrosine, serine and threonine to give O-linked
glycoproteins, or to asparagine to give N-linked
glycoproteins.
• Mannosyl groups may be transferred to tryptophan to
generate C-manosyl tryptophan
• The completed proteoglycan is then exported in
secretory vesicles to the extracellular matrix of the cell.

34. Biosynthesis of Heparan Sulfate
proteoglycan

35. Degradation of Heparan Sulfate
Proteoglycan

36. Glycosaminoglycan Occurence
Proteoglycans can be categorised depending upon the
nature of their glycosaminoglycan chains.
• Hyaluronic acid (does not contain any sulfate)
– non-covalent link complex with proteoglycans
• Chondroitin sulfate
– cartilage, bone
• Dermatan sulfate
– skin, blood vessels
• Heparan sulfate
– basement membrane, component of cells surface
• Keratan sulfate
– cornea, bone, cartilage, often aggregated with chondroitin
sulfate

37. Function of Proteoglycans
• organize water molecules
- resistent to compression
- return to original shape
- repel negative molecules
• occupy space between cells and collagen
• high viscosity
- lubricating fluid in the joints
• specific binding to other macromolecules
• link to collagen fibers
- form network
- in bone combine with calcium salts (calcium carbonate,
hydroxyapatite)
• cell migration and adhesion
- passageways between cells
• anchoring cells to matrix fibers

38. Structural Glycoproteins
• Direct linkage to collagen or proteoglycans
– anchoring collagen fibers to cell membrane
– covalent attachment to membrane lipid
• Major adhesive structural glycoproteins
– fibronectin
– laminin

39. Fibronectin
• High-molecular weight (~440kDa) glycoprotein
• Attached to cell membrane by membrane-spanning
receptor – integrin.
• Crosslinks and stabilizes other components of ECM
• Enhances cell addhesion to extracellular matrix
components (collagen, fibrin and heparansulfate
proteoglycans).
• Related to blood clotting - soluble FN crosslinks
platelets together using membrane bound heparin

40. Fibronectin Structure
• protein dimer connected at C-terminal by S-S linkage
• rigid and flexible domains
• cell binding domain RGD
(Arg, Gly, Asp)
- binding receptor in cell membranes
• RGD domain binds to
- collagen type I, II and III
- heparin sulfate
- hyaluronic acid
- fibrin

41. Fibronectin Function
• related to cell adhesion, differentiation, growth,
migration;
• anchoring basal laminae to other ECM;
• plasma fibronectin forms a blood cloth, along with fibrin;
• related to cell movement - groups of embryonic cells
follow a FN pathway - FN guides macrophages into
wound areas.

42. Laminin structure
and function
• cross-shaped glycoprotein
• 3 polypeptide chains
• domain bind
- collagen type IV
- heparin
- heparin sulfate
• cell surface receptor RGD
• cell adhesion
• role in cell differentiation
• anchoring the glycoprotein to
basal laminae

44. Fibrillin
• Glycoprotein, essential for
formation of elastic fibers (a
sheath surrounding the amorphous
elastin)
• Produced by fibroblasts.
• A group of three proteins, fibrillin-
1, -2 and -3.
• The main role - maintaining the
structural integrity of tissues, the
regulation of cytokines – TGF-b
• In humans, defects in the fibrillin-1
and fibrillin-2 genes have been
linked to diseases that affect the
cardiovascular, skeletal and ocular
systems, including Marfan
syndrome.

45. Integrins
• Groups of
transmembrane protein
receptors
• mediate the attachment
between a cell and its
surroundings
• integrins perform
outside-in signaling and
they also operate an
inside-out mode.
• Link cytoskeleton to ECM
• Fibronectin receptor is
best known

46. Tanescins
• Abundant in the extracellular matrix of developing
vertebrate embryo.
• Tenascin-C contains an RGD motif and is recognized by
diverse integrins. Mainly synthesized by the nervous
system and connective tissues.
• Tenascin-R is found in the nervous system
• Tenascin-X and tenascin-Y are found primarily in muscle
connective tissues.
• Tenascin-W is found in kidney and developing bone.