Extracellular matrix (ECM)

Transcription

1 Medical Biochemistry & Molecular Biology Department Extracellular matrix (ECM) By the end of this topic you will be able to: Recognise 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. What is Extracellular space? Is the space that is present outside the cell. ECM is a connective tissue that contains a matrix of macromolecules. These macromolecules are synthesized and secreted by cells that live within the tissue. Proteins and polysaccharides are the biomolecules of the ECM are secreted by cells of the tissue and then assembled into an organized meshwork. ECM has different functions in different tissues (e.g ECM adds strength to bone). ECM contains 3 classes of biomolecules Fibrous (structural proteins) collagen Elastin Fibrillin Proteoglycans Chondrotin sulphate Dermatan sulphate Hyalouronic acid Adhesion proteins fibronectin Laminin Integrin 1

2 1- Collagen - Collagen is Glycoprotein - It is the most abundant protein in human. - It represents 25-30% of total protein. - It is the major component of ECM in bone, cornea, skin and blood vessels. - There are about 28 types of collagen. - Collagen is formed of three polypeptide chains (α-chains) that form a triple-helix structure. Each α chain forms a left- handed helix, while the triple helix has a right handed direction. Why Glycine is present in every third position in collagen? Glycine is in every third position because it is the smallest amino acid that can occupy the limited space in the center of the triple helix. Collagen synthesis Collagen is synthesized in : - Fibroblasts - Osteoblasts of bone - Chondroblasts of cartilage Collagen synthesis involves reactions occurring inside and out side the cell. Collagen is secreted into the extracellular matrix (ECM). 2

3 Steps of collagen synthesis Intra cellular Synthesis of pre pro-α chain which contains a specific amino acid sequence at N-terminal. This specific sequence directs the Pre- pro α chain to the lumen of *RER Removal of this specific sequence Pro-α chain Intra cellular Hydroxylation of selected prolines and lysines Glycosylation of selected hydroxy-lysines Intra cellular Assembly of 3 pro-α chains. Intrachain and interchain disulfide bonds of pro-peptide extension. *RER rough endoplasmic reticulum Procollagen molecule is secreted into ECM 3

4 N-terminal and C- terminal of Procollagen are cleaved by peptidases and Tropocollagen is formed Self assembly of Tropocollagen molecules into collagen Fibrils Extracellular Extracellular Extracellular Cross- link formation in collagen fibrils to form collagen fibers Collagen synthesis 4

5 Hydroxylation reactions and Cross-link formation Hydroxylation Cross- link formation reaction in collagen Substrates Proline or Lysine Lysine & hydroxlysine Enzyme (s) Prolyl hydroxylase or lysyl hydroxylase Lysyl oxidae Products hydroxy proline or hydroxy lysine *Allysine & hydroxyallysine Cofactor vitamin c (ascorbic Copper acid) Site intracellular extracellular *The reactive aldehydes that result (allysine and hydroxyallysine) can condense with lysine or hydroxylysine residues in neighboring collagen molecules to form covalent cross-links and, thus, mature collagen fibers - A defect in cross- link formation. Affects collagen stability - Hydroxylation reactions is an example of post-translational modification. The reaction catalyzed by lysyl oxidase is an example of oxidative deamination. Cross- link formation in collagen 5

6 Diseases due to defect in collagen synthesis 1- Scurvy (vitamin C deficiency) 2- Osteogenesis Imperfecta (O.I) Scurvy (vitamin C deficiency) Defect in hydroxy lysine Defect in hydroxylation reactions Defect in hydroxyproline/ hydroxylysine Defect in cross link formation Weak collagen (bruises on limbs due to extravasation of blood due to capillary fragilty) Defect in inter chain hydrogen bonds Scurvy with lower limb bruises 6

7 Osteogenesis Imperfecta (OI) 80% of OI due to dominant mutation in gene coding chain in type I collagen The most common mutation due to replacement of glycine by amino acid with bulky side chain Abnormal α chain which prevent formation of triple helical structure Weak collagen fibers OI is called brittle bone disease as bone is easily fracture with minor or no trauma Mild form: bone fragility, hearing loss, and blue sclera Severe form: in utero fractures, perinatal death due to pulmonary complicatons 7

8 Collagen stability: 1- The triple helix and three polypeptides are coiled in opposite directions 2-Only inter- chain hydrogen bonds between 3 polypeptide chains 3-Covalent cross links between and within triple helix units. A comparison between collagen helix and α helix α-helix The single polypeptide chain is twisted around α carbon forming α helix Collagen helix Superhelix of 3 chains warpped around each other in right handed direction Right handed direction Each single chain has left handed direction (why?) Stabilized by intra-chain hydrogen chain No intra- chain hydrogen bond Only inter-chain hydrogen bonds between 3 chains 3.6 amino acid (residue) / turn 3 amino acid/ turn No specific sequence No proline Cysteine is present Tryptophan is present Specific amino acid (Gly- X-Y) Proline is present No cysteine in mature collagen No tryptophan 8

9 Collagen have diverse structure function relationship according to the organs in which they present..how? Tendons Bone Skin Collagen of tendons is present in tight parallel fibers to provide tensile strength Collagen of bone is arranged at an angle to each other to resist mechanical shear. Collagen of skin forms loosely flexible fiber. Cornea Vitreous humor Collagen of cornea is transparent. Collagen of vitreous humor of the eye is dispersed as gel that stiffens the structure Collagen degradation: Collagen have long half life, so it is stable Degraded by collagenases It is degraded into smaller parts which phagocytosed by the cells to be degraded by lysosomal enzymes 9

10 Elastin Introduction - Elastin is a fibrous protein Amorphous protein (random coil) to stretch then recoil. Has rubber like properties Present in the lungs, wall of big arteries, elastic ligaments Structure: - Insoluble protein polymer (i.e non polar amino acid as glycine, valine, alanine) - Rich in proline. Hydroxyproline is present - No hydroxylysine or glycosylated hydroxy lysine Synthesis: - Elastin is an insoluble protein polymer synthesized from a precursor, tropoelastin, which is a linear polypeptide composed of about 700 amino acids that are primarily small and nonpolar (for example: glycine, alanine, and valine). - After secretion of tropoelastin, it will be deposited into scaffold (microfibrills). - Cross link formation (desmosine cross link) by lysyl oxidase, and lysyl residue is the substrate. - Once cross links formed.mature insoluble elastin is formed - Cross link in elastin produces rubbery network, so elastin can stretched in any direction. 10

11 What are the differences between Cross link formation in collagen and Elastin? Collagen s cross-link Elastin s cross-link Substrate(s) lysine& OH lysine Lysine only Product(s) Allysine/OH allysine Allysine Reaction Condensation reaction between Allysine/hydroxyallysine and lysine/ OH lysine of neighbouring molecule Condensation reaction between 3 allysine and lysine residue of other molecule Name aldo cross link Desmosine cross link Importance Gives collagen strength Gives elastin insolubility and rubbery network (stretch and recoil) N.B Elastin stability: Extremely stable with low rate of turnover Elastin degradation: By elastase enzyme secreted from neutrophils 11

12 Role of α1 antitrypsin in protection of elastin Elastin presents in the lung Elastin is protected from degradation of *elastase by α1 anti-trypsin which inactivate elastase *The main source of elastase is alveolar neutrophiles If there is α1 anti-trypsin defficiency Increase elastase activity Increase degradation of elastin *Lung emphysema * Destruction in connective tissue of alveolar wall which can t be regenerated. - α1- antitrypsin is synthesized in the liver, alveolar monocytes and macrophages. 12

13 α1- antitrypsin deficiency Homozygous genetic disorder There is a deficiency in α1- antitrypsin due to genetic defect. Lung emphysema occurs Treated by intravenous administration of α1- antitrypsin every week. N.B - Smoking causes oxidation and subsequent inactivation of methionine in α1- antitrypsin (Methionine 358 in α1- antitrypsin is required for the binding of α1- antitrypsin to its target elastase). So, α1- antitrypsin can t bind to elastase. This leads to lung emphysema. A comparison between collagen and Elastin Collagen have ~ 28 types Elastin Only one type formed of triple helix Gly- X-Y repeats Presence of hydroxy lysine Attached to oligosaccharides Intra molecular aldo cross links Provides high tensile strength without stretch No triple helix. formed of random coils.why? To permit stretching properties No specific repeats No hydroxy lysine Not attached Intramolecular desmosine cross links Permits stretch and elastic recoil 13

14 Proteoglycans Characteristics - Protein constitutes about 5-10%. While carbohydrate forms about 95% (GAG formerly called Mucopolysaccharides). - GAG have long un branched chains, formed of repeating disaccharides (aminosugaruronic acid). Amino sugar is acetylated and sulfated except in Hyalouronic acid (non sulfated) Proteoglycan monomer is polyanion due to the presence of COO,SO3 The chains of repeating disaccharide units remain separated by repulsion between negative charges The chains bind cations as Na and K and attract water by osmotic pressure into ECM Bottle brush appearance Gel like consistency of ECM 14

15 Proteoglycans structure Glycosaminoglycans + core proteins (covalent link) Proteoglycan monomer Proteoglycan monomer +hyalouronic acid +linker protein(non covalent) Proteoglycan aggregates Types of GAG: Hyaluronic acid (only free GAG, No core protein, non sulfated ) chondroitin sulfate dermatan sulfate Heparan sulfate keratan sulfate Heparin Secreted in blood by mast cell, Not present in ECM 15

16 Fibronectin It is a glycoprotein with large molecular weight It is formed of two identical subunits joined by disulfide bridge ( unlike mature collagen doesn t contain disulfide bonds) It had binding sites for fibrin, collagen, and cell surface receptor. It communicates interior of the cell with exterior If there is tissue injury, it binds with platelets to allow blood clot formation It allows movement of the cell to affected area during wound healing. Many tumor cells secret less amount of adhesion protein to allow more movement of the tumor cells. This facilitates metastasis. 16

17 Laminin Is the most abundant protein in basal lamina after type IV collagen It is heterotrimeric protein, has α,β,γ subunits It forms networks of web like structure to resist tensile force in basal lamina. It provides additional support for tissues through its ability to bind with type IV collagen What is the medical importance of ECM? The ECM is not simply a glue that holds cells together ECM serves to keep the cells from moving to other site. ECM prevent large molecule and particles from reaching the cells ECM and diseases Infection spread: Infectious agents secrets hyluronidase enzyme which degrade hyalouronic acid Cancer cell metastasize: through alternation of integrity of ECM (i.e secret less amount of fibronectin). Rheumatoid disease and osteoarthitis may occur due to damage in ECM Osteogenesis imperfecta Alternation in renal glomeruli will lead to protein excretion in urine. Mucopolysaccharidosis: due to defect in lysosomal enzyme which degrade mucopolysaccharides 17