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

9. 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 lysyl oxidase (or catalase). 
8. Self-assembly or polymerization of tropocollagen molecules form collagen fibrils. Cross-linkage between adjacent tropocollagen molecules stabilizes the fibrils. 

11. 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.

12. Crosslinking of tropocollagen - aldol condensation

13. Amadori product - ketamine

14. Lysinonorleucine cross-link

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

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

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

18. Collagens Classification
Fibril-forming collagens (I, II, III, V, X)
Fibril-associated collagens (FACIT)
Network-forming collagens
Anchoring fibrils collagens
Transmembrane collagens
Basement membrane collagens
Other collagens with unique function

19. Molecular composition
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

20. Basement membrane collagensIV
[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.

21. 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

22. 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.

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

24. 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)

25. 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.

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

27. Hyaluronic Acid & Keratan Sulfate

28. Chondroitin 6-sulfate & Heparan Supfate

29. Dermatan Sulfate & Heparin

30. 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.

31. 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.

32. Biosynthesis of Heparan Sulfate proteoglycan

33. Degradation of Heparan Sulfate Proteoglycan

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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.

41. 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

42. 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.

43. Integrins Groups of transmembrane protein receptors
The bridges for cell-cell and cell-ECM interactions.
Mediate the attachment between a cell and its surroundings.
Integrins perform outside-in signaling and they also operate an inside-out mode, trigger chemical pathways – signal transduction - which results in a response (activation of transcription) such as regulation of the cell cycle, cell shape, etc.)
Link cytoskeleton to ECM
Fibronectin receptor is best known

45. Tenascins
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.