1. EXTRACELLULAR MATRIX
Jana Novotná

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

3. Function of ECM Provides support anchorage and 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. structural glycoproteins
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 structure:
- G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – G – A –
– A – G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – G – 
– A – A – G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – 
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.

8. Synthesis
1.  Synthesis of a chains of pre-procollagen on ribosomes.  A signal protein directs them to the RER .
2. Cleavage of signal protein forms procollagen.

9. Hydroxylases need Fe2+ as a cofactor
3. Hydroxylation of lysine and proline to hydroxylysine and to hydroxyroline
lysyl-5- hydroxylase and prolyl-4-hydroxylase
a-ketoglutarate, O2 and ascorbic acid is necessary to activate the hydroxylases.
Hydroxylases need Fe2+  as a cofactor
Proline + a-ketoglutarate + O2 + Fe2+ → 4-hydroxyprolin + Fe3+ + CO2 + succinate

10. 4. Glycosylation: addition of galactose and glucose to some hydroxylysine residues.  The enzymes galactosyl transferase and glycosyl transferase are required for this process.

11. 5. Assembly of three a-chains to form procollagen in Golgi apparatus
5. Assembly of three a-chains to form procollagen in Golgi apparatus. This involves the formation of disulphide bonds between parts of the polypeptide chains known as registration peptides, which occur at both ends of the pre-procollagen. 
6. Secretion of procollagen molecules by exocytosis into the extra cellular space.

12. 7. Cleavage of registration peptides occurs in the extra cellular space, and is catalysed by procollagen peptidases.  The resulting molecule is called tropocollagen.
8. Oxidation – deamination of hydroxylysine. It involves the removal of an amino group (NH2), which has a net oxidative effect and the formation of covalent cross-links.  It is catalyzed by lysine oxidase (or catalase). 

14. 8. Self-assembly or polymerization of tropocollagen molecules form collagen fibrils.
Cross-linkage between adjacent tropocollagen molecules stabilizes the fibrils. 

15. Collagen – fiber formation
Collagen types I, II, III, V, IX, X
Cross striated structure in

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

17. Collagen Distribution
Type Molecule composition Tissue
Fibrilar Collagens
I [a1(I)2a2(I)] Skin, tendon,bone, ligaments, dentin
II [a1(II)]3 Cartilage, vitreus humor
III [a1(III)]3 Skin, muscle, blood vessels
V [a1(V)]3 Similar to type I, fetal tissue
Sheet-forming collagen
IV [a1(IV)2a2(IV)]
[a1(IV)a2(IV)a3(IV)] All basal laminaes

18. 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.
The most common interchain cross-link in elastins is the result of the conversion of the amine groups of lysine to reactive aldehydes by lysyl oxidase. This results in the spontaneous formation of desmosine cross-links. 

19. Proteoglycans
Proteoglycans represent a special class of glycoproteins that are heavily glycosylated (95%).
They consisit of core protein with one or more attached glycosamino glycan chain(s).

20. Glycosaminoglycans (GAG)
Glycosaminoglycan (GAG) chains are long, linear carbohydrate polymers
under physiological conditions they are negatively charged (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)

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

23. Hyaluronic acid
D-glukuronic acid + GlcNAc

24. Synthesis
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.
Then sugars are added by glycosyl transferase. The completed proteoglycan is then exported in secretory vesicles to the extracellular matrix of the cell.

25. Glycosaminoglycan Classification
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

26. Function of Proteoglycans
organize water molecules
- resistant 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

27. 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

28. Fibronectin Structure
Dimer connected at C-terminal by S-S linkage
Rigid and flexible domains
Cell binding domain RGDS
(arg, gly, asp, ser)
- binding receptor in cell membranes
Domain is binding to
- collagen type I, II and III
- heparin sulfate
- hyaluronic acid
- fibrin

29. Fibronectin Function cell adhesion cell differentiation
anchoring basal laminae to other ECM
blood clothing
- clothing process, link to fibrin

30. Laminin Structure and Function
cross-shaped glycoprotein
3 polypeptide chains
domain bind
- collagen type IV
- heparin
- heparin sulfate
cell surface receptor
cell adhesion
cell differentiation
anchoring the glycoprotein to basal laminae