EXTRACELLULAR MATRIX(ECM) Dr. Eman Khairy Lecturers of Medical biochemistry & Molecular Biology

By the end of this topic you will be able to: Recognize the importance of ECM and its component in the health and diseases. Describe the structural and functional properties of collagen and elastin, the major proteins in ECM. Describe the general properties of proteoglycans , fibronectin, and laminin.

ECM contains 3 major classes of biomolecules The space between cells that bind cells and tissue together ECM contains 3 major classes of biomolecules Fibrous (structural protein) Collagen Elastin Fibrillin Proteoglycan Chondrotin sulphate Dermatan sulphate Hyalouronic acid Adhesion proteins Fibronectin Laminin Integrin

ADHESION MOLECULES Link component of matrix protein to each other and to cells Cell A Direct cell to cell contact adhesion proteins Cell B ECM cell to ECM contact

Specific domain bind to specific cellular receptor Adhesion proteins Cytoskeletal proteins Integrin Adhesion proteins Specific domain bind to specific cellular receptor spans cell membrane and interact with intracellular protein , Actin filamints

Collagen Collagen is glycoprotein 28-30 types in human Provides Extracellular framework of animal body The main component of loose connective tissue ,blood vessels ligaments, tendons, bone and skin. The most abundant protein in the body, making 25%-35% of all the whole-body proteins. Collagen is glycoprotein

Collagen structure Collagen fibers Collagen fibrils Tropocollagen (triple helix) Single chain of amino acids

Triple helical collagen supercoils Collagen structure (Gly –X-Y )n Y proline Glycine X any amino acid Triple helical collagen supercoils (right handed)

Collagen structure Glycine Proline Lysine ,hydroxylysine Third amino acid of collagen 33% Proline Y is frequently proline or hydroxyproline 21% of collagen Responsible for left handed alpha helix Lysine ,hydroxylysine Some Y position Form interchain covalent cross link. Form O linked glycosidic bond

Hydroxyproline and hydroxylysine Result from hydroxylase enzyme. Hydroxylation need vitamin C TYPE OF POST-TRANSLATIONAL MODIFICATION Hydroxylysine is site for attachment of disaccharide.

Absent amino acids in collagen Tryptophan Cysteine

Biosynthesis 1. Formation of pro-α chains 2. Hydroxylation 3. Glycosylation 4. Assembly and secretion 5. Extracellular cleavage of procollagen molecules 6. Formation of collagen fibrils 7. Cross-link formation Extended Modular Program

Where is collagen synthesized? Osteoblast Chondroblast Fibroblast

1. Formation of pro-α chains The newly formed polypeptide (prepro-α chains) contain a special signal sequence at their n-terminal ends Directs the passage of the prepro-α chain into the lumen of the RER Then it is cleaved giving a precursor of collagen called a pro-α chain Extended Modular Program

By prolyl hydroxylase and lysyl hydroxylase enzymes 2. Hydroxylation Proline and lysine residues are hydroxylated to hydroxyproline and hydroxylysine By prolyl hydroxylase and lysyl hydroxylase enzymes Extended Modular Program

Some hydroxylysine residues are glycosylated with 3. Glycosylation Some hydroxylysine residues are glycosylated with glucose or galactose

Extended Modular Program

4. Assembly and secretion tropocollagen tropocollagen 5. Extracellular cleavage of procollagen molecules Propeptides Extended Modular Program

6. Formation of collagen fibrils Extended Modular Program

Oxidative deamination allysine + hydroxyallysine 7. Cross-link formation Oxidative deamination Cu dependent allysine + hydroxyallysine Covalent crosslink Extended Modular Program

Extended Modular Program Summary of biosynthesis Extended Modular Program

Degradation of collagen Normal collagen is highly stable Long half life up to several months collagenase

Covalent crosslink between triple helical units Lysyl oxidase Cu dependent Oxidatively deaminate some lysyine and hydroxylysine Covalent crosslink Form allysine + hydroxyallysine.

Difference between collagen helix and alpha helix Backbone of polypeptide chain twisted about alpha carbon forming a helix Superhelix of 3 chains wrapped around each other in RT haded RT handed twist Each single chain is LT handed Intrachain hydrogen bond interchain hydrogen bond 3.6 aa /turn 3 aa /turn No specific aa sequence. (Glycine X-Y)n

Collagen structure function relationship Tight parallel fibers Provide great tensile strength Collagen tendon Arranged at angle to each other. Resist mechanical shear Collagen of bone and teeth Loosely woven ,flexible Collagen of skin Transparent with minimal scattering of light Collagen of cornea of eye Form gel. Viterous humor of eye

Mention 2 diseases that are related to collage? 1- Scurvey 2- Osteogenesis imperfecta

Scurvey (Vit. C deficiency) Defect in hydroxylation of proline and lysine Weak and fragile collagen Collagen become unstable and easily degraded Bleeding per gum, swollen joint, poor wound healing, fragile bone and easily fracture

Osteogensis imperfecta Single base mutation Replacement of glycine with bulky amino acid Weak collagen Fragile bone with easily bone fracture from minor trauma

Elastin Major protein component of elastic tissues such as arteries, lungs, skin Highly cross linked ,insoluble, amorphous Rubber like consistency

Elastin has no regular 2ry structure ,only random coils Microfibrils Elastic fibers

glycine , valine , alanine and proline Little hydroxyproline Tropoelastin contain glycine , valine , alanine and proline Rich in lysine Little hydroxyproline No hydroxylysine

Elastic fibers synthesis Tropoelastin molecules Desmosine crosslink (3 allysyl residues+ unaltered lysine ) lysyl oxidase

Elastin is highly stable up to 70 years. Elastin degradation Elastin is highly stable up to 70 years. Elasin is degraded by elastase enzyme releases by neutrophils.

In lung, elastin is present Elastin is protected from degradation of elastase by α1 anti-trypsin which inactivate elastase If there is α1 anti-trypsin defficiency Increase elastase activity Increase degradation of elastin Lung emphysema destruction in connective tissue of alveolar wall which cannot be regenerated

α1- antitrypsin deficiency Homozygous genetic disease There is deficiency in α1- antitrypsin (α1 antiprotease) Lung emphysema occurs Treated by IV α1- antitrypsin every week.

Smoking cause oxidation and subsequent inactivation of methionine in α1- antitrypsin. So, α1- antitrypsin can’t bind to elastase . This will be lead to lung emphysema.

Differences of elastin from collagen Many different genetic types Single genetic type Triple helix No (random coils) (Gly-x-y)n No Hydroxylysine is present Contain carbohydrate Intramolecular aldol cross-links Intramolecular desmosine cross-links Widely distributed Large blood vessels,skin ,elastic ligaments High tensile strength + no stretch Stretch+ recoil

Carbohydrate chain (95%) Proteoglycans Core protein (5%) Carbohydrate chain (95%) Proteoglycan

Glycosaminoglycans (GAG) Core protein Unbranched Long Linear Repeated disaccharide units Amino sugar is sulphated or acetylated Covalent crosslink Disaccharides Amino sugar+ uronic acid

Proteoglycan monomer is polyanion due to the presence of COO ,SO3 1-The chains of repeating disaccharide units remain separated by repulsion between negative charges- Bottle brush appearance of proteoglycan

Proteoglycan monomer is polyanion due to the presence of COO ,SO3 2-The chains bind cations as Na and K and attract water by osmotic pressure into ECM- Gel like consistency of ECM

PROTEOGLYCAN AGGREGATES Hyalouronic acid PROTEOGLYCAN AGGREGATES Linker protein

Proteoglycans Proteoglycan monomer Proteoglycan aggregates Proteoglycan monomer +hyalouronic acid +linker protein(non covalent) Proteoglycan monomer Glycosaminoglycans + core proteins (covalent link)

Classification of proteoglycan is based on Disaccharide unit Type of glycosidic bond Degree and location of sulfate group

Classification of proteoglycan into six groups Hyaluronic acid (only free GAG, No core protein, non sulfated ) chondroitin sulfate dermatan sulfate heparan sulfate keratan sulfate Heparin (secreted in blood by mast cells,not present in ECM)

Proteoglycan functions All the proteoglycan classes determine structural organization of the matrix Each class has also special function

LAMININ Hetero trimeric The second most abundant protein in basal laminae after type IV collagen Hetero trimeric Alpha subunit Beta subunit Gamma subunit

Laminin Functions It can form networks of web like structure to resist tensile force in basal lamina. Bind to type IV collagen ad other components in ECM and integrin ,so provide additional support.

Fibronectin Glycoprotein of large MW Formed of 2 identical subunits joined by 2 disulfide bridges.

Fibronectin Fibronectin has binding sites for : Fibrin Collagen Heparin DNA Cell surface receptor

Fibronectin provide a bridge between actin of the cell and ECM Fibronectin interact with fibronectin receptors (integrin) Integrin interact with actin microfilaments

Fibronectin Sites ECM Soluble form in plasma

Fibronectin Functions Cell migration Communicates exterior of the cell with its interior Bind to the platelets during blood clotting

How the tumor cells metastasize? Tumor cells secrete less than normal amounts of adhesion protein Allow more free movements within ECM Increase the potential for the tumor cells to leave their original location

Medical importance of ECM Infection spread Metastasis Rheumatoid arthritis Alteration of renal glomeruli Genetic defect Osteogenesis imperfecta Mucopolysaccharidosis

The link between ECM and disease 1- Infection spread: Infectious agents secrets hyluronidase enzyme which degrade hyalouronic acid. 2- Cancer cell metastasize: Through alternation of integrity of ECM (i.e secret less amount of fibronectin).

3- Rheumatoid disease and osteoarthitis may occur due to damage in ECM 3- Rheumatoid disease and osteoarthitis may occur due to damage in ECM. 4- Alternation in renal glomeruli will lead to protein excretion in urine. 5- osteogenesis imperfecta 6- Mucopolysaccharidosis: due to defect in lysosomal enzyme which degrade mucopolysaccharides ( old name of GAG)

Mucopolysaccharidosis Gradual deterioration of health and death in childhood. Accumulation of large quantities of GAGs in various tissues Defect in normal degradation of molecules of ECM Defeciencies of lysosomal enzymes