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Connective tissue is formed by cells and an extracellular matrix. The extracellular matrix provides structure and regulates cell behavior through fibrillar proteins like collagen and elastin. Collagen is the most abundant protein in mammals and forms fibrils that give tissues strength and flexibility. Collagen synthesis involves extensive post-translational modifications within and outside cells, including hydroxylation, glycosylation, and cross-linking. These modifications are essential for collagen's signature triple helical structure and high tensile strength. Over 30 collagen types exist with diverse structures and tissue distributions that contribute to their unique functions.

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Biochemistry of connective tissue - extracellular matrix Lenka Fialová Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University, Prague 1
Connective tissue The connective tissue is formed by: CELLS and EXTRACELLULAR MATRIX (intercellular matrix) 2
Extracellular matrix Function ▫ stabilisation of tissue structure ▫ regulation cell behavior survival, development, migration, proliferation ▫ membrane filtration barrier (glomerules) ▫ exchange of different metabolites, ions and water ▫ reparation function ▫ immune processes ▫ participation in inflammation 3
Cells of connective tissue ▫ Fibroblasts ▫ Chondroblasts (cartilage) ▫ Osteoblasts (bone) ▫ Odontoblasts (tooth) These cells synthetise extracellular matrix. 4
Extracellular matrix Parts of the extracellular matrix • FIBRILLAR PROTEINS (collagen, elastin) • insoluble in water, no hydratation • GLYCOPROTEINS (e.g. fibronectin,laminin) • GLYCOSAMINOGLYCANS AND PROTEOGLYCANS • soluble in water, easily hydratated 5 Saccharide content increases
Extracellular matrix FIBRILLARY PROTEINS ▫ Structural proteins collagen firmness elastin elasticity 6
COLLAGENS The most abundant proteins in mammals. They form approximately 25 % of all body proteins. 7
Collagens Collagenum gr. kolla glue; gr. gennao constitute By boiling collagen is denatured to a colloid solution (gelatine). From the nonpurified collagen the glue arises. Incidence • main protein of the extracellular matrix • component of tendons, cartilages, bones, and teeth (dentin and cement), skin and vessels. Properties • fibrillary proteins • nonsoluble (glyco-) proteins • HIGH STRENGHT, BUT ALSO SUPPLENESS 8
Structure of collagen
Collagens Collagen has an characteristic amino acid composition and their specific sequence. Primary structure • Characteristic AA composition • Characteristic AA sequence Mature collagen contains no tryptophan and almost no cysteine − from the nutritional point of view not fully valuable protein.
Primary structure of collagen Characteristic AA composition • Fundamental amino acids ▫ Glycine 33 % (x Hb 4 %) ▫ Proline 13 % (x Hb 5 %) • Derived amino acids ▫ 4-Hydroxyproline 9 % (x Hb 0 %) ▫ 5-Hydroxylysine 0,6 % (x Hb 0 %) Origin by posttranslational modification Hb = hemoglobin Chaacteristic for collagen High content
Primary structure of collagen Fundamental AA ▫ Glycine ▫ Proline • Derived AA ▫ 4-Hydroxyproline ▫ 5-Hydroxylysine
Primary structure of collagen Characteristic AA sequence Triplet – Gly – X – Y – • Every third AA is GLYCINE • On the next position frequently PROLINE • On the third position frequently hydroxyproline, ev. hydroxylysine Proline Hydroxyproline (Hydroxylysine) Glycine
Primary structure of collagen Example of AA sequence of a part of the polypeptide chain -Gly-Pro-Hyp- Globular proteins rarely show periodicity in AA sequence.
Secondary structure of collagen Collagen helix • levorotatory helix • steeper rising • 3,3 AA/turn • intrachain hydrogen bonds not present • proline prevents formation of α-helix or β-pleated sheet α α α α-helix (the most common secondary structure in proteins) • dextrorotatory helix • gradual rising • 3,6 AA/turn • stabilization by intrachain hydrogen bonds Comparison of collagen helix to the α-helix, which represents the most common secondary structure in proteins.
Triple helix Three α α α α-chains of collagen Triple helix Relatively rigid This structure is responsible for the tensile strenght.
Triple helix The origin is dependent on the oddness of the primary structure • High presence of glycine ▫ smallest amino acid, no side chain (only –H) ▫ placed in the centre of triple helix, where no space is available ▫ close contact between the chains Triple helix is stabilized by hydrogen bonds between each peptide bond -NH group of glycine and C=O group of the peptide bond of the adjacent polypeptide chain.
Collagen chains The collagen chain is extraordinarly long and contains approximately 1000 AA. The collagen chains are called α1 − α3. ▫ They differ in AA representation ▫ Products of different genes − e.g. α1(I) or α2(V) ▫ Roman digit labels the collagen type More than 30 different types of collagen exists.
The representation of chains differs in individual types of collagens. The collagens may form homotrimers or heterotrimers. Homotrimers ▫ molecule of collagen is formed by three identical chains; ▫ e.g. collagen type III is formed by three α1(III) chains Heterotrimers ▫ molecule of collagen is formed by different chains; ▫ e.g. collagen type I is assembled of two α1(I) chains and one α2(I) chain Collagen chains
Collagen synthesis Collagen is an example of a protein, whose synthesis is connected with many posttranslational modifications (treatment of the polypeptide chain), which take part intra- and extracellularly.
Synthesis and posttranslational modifications of collagen Synthesis of polypeptide chain Hydroxylation of proline and some lysine residues Glycosylation of selected hydroxylysine residues Formation of –S-S- bonds in extension peptides Triple helix formation Secretion of procollagen Intracellular processes
Proteolytic removal of propeptides Assembling of collagen fibrils Formation of cross-links Extracellular processes Posttranslational modifications of collagen
Posttranslational modifications in the course of collagen synthesis INTRACELLULAR PROCESSES
Hydroxylation of proline and lysine residues Enzymatically catalyzed reaction ▫ Prolylhydroxylase ▫ Lysylhydroxylase cofactors Vitamin C !!! α α α α-ketoglutarate Reaction needs oxygen. One O atom forms -OH group of hydroxyproline, the other becomes part of the originating succinate. Dioxygenases contain Fe
Hydroxylation of the proline and lysine residues Reactions catalyzed by prolylhydroxylase Vitamin C Maintains Fe2+ in a reduced state Dioxygenase contains Fe
Hydroxylation of proline and lysine Reaction catalyzed by prolylhydroxylase • reaction highly specific only for proline attached in the polypeptide chain to the amino group of the glycine -Gly-Pro-Ser-Gly-Pro-Pro-Gly-Leu- -Gly-Pro-Ser-Gly-Pro-Hyp-Gly-Leu- N-end C-end Hydroxylation
Hydroxylation of proline and lysine Importance of proline and lysine residues hydroxylation Hydroxyproline • necessary for origin of triple helix by formation of hydrogen bonds between individual chains Hydroxylysine • glycosylation on the formed -OH group
Nonhydroxylated chain is not able to mature The stable triple helix cannot be formed Immediate degradation inside the cell Loss of collagen in the matrix Falling out of teeth Vascular fragility Poor wound healing Deficiency of vitamin C
Vitamin C deficiency Avitaminosis - scurvy Manifestation of avitaminosis in oral cavity • swollen reddish gums • falling out of the teeth
Glycosylation • Attachment of galactose or galactosylglucose to -OH group of the hydroxylysine Enzymatically catalyzed reaction ▫ Galactosyltransferase ▫ Glucosyltransferase Number of saccharide units depends on the type of collagen - e.g.: ▫ Type I (tendons) – 6 units ▫ Type II (lens envelope) – 110 units
Glycosylated residue of hydroxylysine in the molecule of collagen Mechanism of glycosylation is different than that in the glycosylation of serine or asparagine. O OH OH OH CH2OH C HN C H CH2 H2C CH H2C NH2 O O OH O OH O CH2OH OH Glycosylation Galactosyltransferase Glucosyltransferase β-glycoside bond
Formation of –S-S- bonds Disulphide bonds • in the region of C-terminal propeptides ▫ interchain and intrachain disulphide bonds • in the region of N-terminal propeptides ▫ intrachain disulphide bonds Importance • necessary for initiation of triple helix formation starts from the C-end • secretion out of the cell
Formation of –S-S- bonds I S I S I I S I S I C-end N-end interchain intrachain disulphide bonds intrachain disulphide bonds
Procollagen Function of propeptides • Start the formation of triple helix in ER intracellularly. • Prevent a premature fibril formation – extracellularly. N-terminal propeptide of the procollagen globular domain C-terminal propeptide of the procollagen globular domain TROPOCOLLAGEN mature molecule of the collagen N-end C-end
Posttranslational modifications in the process of collagen synthesis EXTRACELLULAR PROCESSES
N-terminal propeptide of procollagen (PINP) MARKERS OF BONE FORMATION (detection in serum or in plasma) Cleaving of the propeptides C-terminal propeptide of procollagen (PICP) Extracellular proteinase Aminopeptidase Extracellular proteinase Carboxypeptidase PROCOLLAGEN TROPOCOLLAGEN monomer
Tropocollagen ▫ Greek tropé - turn, induce a turn ▫ monomer of the collagen – mature molecule of collagen ▫ Mr = 300 000 N-terminal telopeptide of collagen (INTP) nonhelical area of chains TROPOCOLLAGEN monomer C-terminal telopeptide of collagen (ICTP) nonhelical area of chains MARKERS OF BONE DEGRADATION (detection in serum or in the urine)
The way of aggregation of fibrillary collagen • Regular arrangement along the row and in the adjacent row • Monomers in one row are not linked end to end (gap 40 nm) • The adjacent row is displaced by ¼ of the length • In the arrangement of monomers act the weak noncovalent bonds Formation of fibrils Polymeration Tropocollagen Collagen fibril (spontaneous)
Tropocollagen Collagen fibrils Polymerisation Collagen fibers Assembling of collagen fibrils
Formation of cross-links Collagen fibers are stabilized by formation of the covalent cross-links, which can be formed either within the tropocollagen molecule between the three chains − intramolecular cross-links and between the tropocollagen molecules − intermolecular cross-links. Intermolecular cross-links Intramolecular cross-links
Formation of cross-links Function of cross-links stabilization and strengthening of collagen fibril high breaking strength lower extensibility Cross-linking
Formation of cross-links Character of cross-links ▫ covalent bonds Examples ▫ aldol cross-link intramolecular ▫ pyridinoline a deoxypyridinoline cross-links histidine-aldol cross-link intermolecular
Aldol cross-link ▫ Cross-link on the N-end of tropocollagen is formed between the lysine residues of two chains Mechanism of formation 1. oxidative deamination of lysine, aldehyde formation ▫ by the enzyme lysyloxidase ▫ aminooxidase, containing Cu2+ ▫ prosthetic group – pyridoxalphosphate 2. Aldol condensation of aldehyde groups spontaneous reaction, two aldehydes form a cross-link
Pyridinoline and deoxypyridinoline cross-link ▫ Cross-link between N-end of one tropocollagen molecule and C-end of the adjacent tropocollagen molecule Pyridinoline ▫ of 3 hydroxylysine residues Deoxypyridinoline ▫ of 2 hydroxylysine and 1 lysine residues ▫ more specific for bone and dentine
Mechanism of origin 1. step – oxidative deamination of lysine to aldehyde ▫ catalyzed by lysyloxidase enzyme 2. step – formation of ketoamine ▫ nonenzymatic reaction of oxidized hydroxylysine with nonoxidized lysine (hydroxylysine) | OH OH OH Pyridinoline and deoxypyridinoline cross-links -CH-CH2-NH2 + O=CH-CH-CH2- -CH-CH2-NH-CH2-CO-CH2-
Pyridinoline a deoxypyridinoline cross-links 3. step – formation of the pyridine ring ▫ Interaction of ketoamine with the free aldehyde group of the hydroxylysine closes the heterocyclic pyridine ring, linking covalently three diferent collagen chains Intermolecular cross-bridge Allysin Hydroxyallysine Hydroxylysine
Pyridinoline a deoxypyridinoline cross-links ▫ In the course of bone degradation these cross bridges are separated from collagen fibers, released to blood and excreted to urine. ▫ The pyridinoline and particularly the deoxypyridinoline bridges may be determined in blood and urine. MARKERS OF BONE DEGRADATION
Fibrillar collagens – e.g. types I, II, III, V • „typical“ collagens forming fibrils Collagens associated with collagen fibrils – for example types VI, IX, XII, XIV, XVI • Triple helix is interrupted by sections making possible the bending of the molecule. • These collagens attach to the surface of collagen fibrils and join them together and connect them to other constituents of extracellular matrix Net forming collagens – types IV, VIII and X • Do not form typical fibrils • Net like arrangement • Nonhelical globular domains on the ends of the molecule Overview of collagens - classes
Anchoring collagens – type VII • forms anchoring fibers • strengthen the connection of dermis and epidermis Transmembrane collagens – types XIII and XVII • integral membrane proteins Overview of collagens-classes
Overview of collagens Some fibrilar collagens Type Molecular Occurrence structure I [α1(I)]2 [α2(I)] widely present, skin, vessels, tendons, gingiva, bone, cement, dentin, periodontal ligaments II [α1(II)]3 cartilage, vitreous body III [α1(III)]3 skin, vessels, lungs, gingiva, cement, dentin, periodontal ligaments V [α1(V)]3, [α1(V)2 α2(V)] skin, smooth muscle, bone, cement, dentin
Types of collagen Some collagens associated with fibrils Type Molecular Occurrence structure VI [α1(VI) α2(VI) α3(VI)] laterally associated with collagen type II, widely present, bone, gingiva, cement, periodontal ligaments IX [α1(IX) α2(IX) α3(IX)] laterally associated with collagen type II, cartilage, vitreous body, periodontal ligaments XII [α1(XII)]3 associated with collagen type I in soft tissues, periodontal ligaments
Oveview of collagens Some net forming collagens Type Molecular Occurrence structure IV [α1(IV)]2 [α2(IV)] basal membranes, formation of two-dimensional net gingiva, periodontal ligaments
Disorders of collagen synthesis Increased collagen synthesis • fibroses Decreased collagen synthesis • genetic disorder • acquired disorders
Disorders of collagen synthesis Increased collagen synthesis - FIBROSIS • hepatic cirrhosis • pulmonary fibrosis • atherosclerosis Tissue damage stimulates collagen synthesis by fibroblasts ▫ e.g. damaged hepatocytes are replaced by fibrous connective tissue − hepatic cirrhosis
Disorders of collagen synthesis Increased collagen synthesis ▫ bacterial infections also stimulate collagen synthesis Prevention of infection spreading − ABSCES x Some bacteria (Clostridia) produce collagenases, which degrade tropocollagen.
Disorders of collagen synthesis Decreased collagen synthesis • Genetically conditioned ▫ Ehlers-Danlos syndrome ▫ osteogenesis imperfecta • Acquired disturbances ▫ lathyrism ▫ copper deficiency ▫ vitamin C deficiency
Ehlers-Danlos syndrom ▫ heterogenous group of diseases caused by defects of enzymes necessary for synthesis of collagen or by abnormalities in the procollagen gene Manifestations ▫ extreme extensibility of connective tissue and skin ▫ hypermobility of joints ▫ contortionists ▫ risk of rupture of vessels or of the intestine Disorders of collagen synthesis
Disorders of collagen synthesis Osteogenesis imperfecta ▫ group of diseases caused by mutation in collagen type I ▫ exchange of Gly for an AA having larger side chain ▫ formation of triple helix is not possible ▫ degradation of polypeptide chains not forming triple helix Manifestations ▫ abnormal bone fragility ▫ bone fractures even in small injuries ▫ in more serious cases prenatal fractures Lat. imperfectus incomplete
Dentinogenesis imperfecta ▫ group of diseases caused by mutation in α1(I) ▫ associated with osteogenesis imperfecta Manifestations ▫ thin enamel ▫ discolouring of teeth (yellow, brown, grey) ▫ opalescence of the teeth ▫ lower mechanical resistance of the teeth Disorders of collagen synthesis
Disorders of collagen synthesis Disturbance of cross-link formation • Causes ▫ copper deficiency (part of lysyloxidase) ▫ animal food containing β-aminopropionitrile (contained in seeds of sweet pea − Lathyris odoratus) – blocks lysyloxidase − lathyrism Manifestations extreme fragility of connective tissue (bones, vessels)
ELASTIN Elastin is the main protein of elastic fibers, providing elasticity to the tissues. 61
Elastin Occurrence • in arteries, particularly in aorta • in skin, tendons and loose connective tissue (relatively low content) • in lungs Synthesis takes place in early development or after tissue damage Half-time is approximately 70 years (lower content in elderly people).
Elastin Properties EXTENSIBILITY AND CONTRACTILITY ▫ resembles the rubber ▫ after extension elastin is able to return to original size and original form ▫ tensile strength is lower than in collagen ▫ hydrophobic, practically insoluble in aqueous solutions
Primary structure of elastin Occurrence of amino acids ▫ 1/3 glycine ▫ high content of nonpolar AA (Ala, Val, Leu, Ileu) ▫ low hydroxyproline ▫ no hydroxylysine − elastin is not glycosylated Sequence of amino acids ▫ typical triplet as in collagen is not present Alternation of short hydrophobic and hydrophilic sections. Hydrophilic sections, which represent a minority part, are rich in lysine, which takes part in forming of cross - links.
Secondary and tertiary structure of elastin Secondary structure ▫ elastin does not form a regular secondary structure ▫ elastin has an character of random coil conformation enabling extension and contraction Tertiary structure ▫ a stable secondary structure is not expressed
Elastin synthesis Synthesis of polypeptide chain Hydroxylation of proline residues Secretion of tropoelastin Tropoelastin (globular structure, Mr = 70 000) Formation of cross-links Three-dimensional netting Intracellular processes Extracellular processes
Cross-links in elastin Cross-links ▫ there is a large number of covalent cross-links in elastin ▫ some are similar as in collagen ▫ key step is an oxidative deamination of some lysine residues by copper- containing lysyloxidase (the same enzyme as in formation of cross-links in collagen) ▫ cross-links may be formed within one polypeptide chain or between 2 – 4 chains • Desmosine ▫ cross-link completely specific for elastin ▫ arises from 4 side chains of LYSINE (3 oxidized and 1 nonoxidized) ▫ determines the high elasticity of elastin Linking of polypeptide chains of elastin by cross-links constitutes a three- dimensional netting explaining the „rubber-like“ properties of elastin.
GLYCOSAMINOGLYCANS
Glycosaminoglycans (GAG) (Mucopolysaccharides) Characteristic ▫ heteropolysaccharides (100% polysaccharides) ▫ not branched polysaccharide chains ▫ long chains (70 – 200 monosaccharides) ▫ repeating disaccharide units Muco – these substances were first detected in mucus
Glycosaminoglycans Polysaccharide chain of GAG is formed by repeating disaccharide units. [URONIC ACID – AMINO SUGAR]n OR [MONOSACCHARIDE - SULFONATED AMINO SUGAR]n
Glycosaminoglycans Uronic acids present in GAG O OH OH OH OH COO D-glucuronic acid
Glycosaminoglycans Amino sugars present in GAG
Glycosaminoglycans Modifications of amino sugars in GAG ▫ Acetylation of aminogroup − elimination of a positive charge ▫ Attachment of a sulphate on OH C-4 or C-6 (ester bond), or on nonacetylated amino group − increase of a negative charge
Glycosaminoglycans Characteristics • high number of acidic groups ▫ - COO- (uronic acids ) ▫ - OSO3 - (amino sugars with sulphate groups) • highly negative charge at physiological pH (polyanions) Na+ Na+ Na+ Na+ H20 H20 H20 H20 H20
Glycosaminoglycans Characteristic (cont.) ▫ chains repel each other and in solution tend to straighten ▫ negatively charged groups bind cations – Na+, K+ ▫ osmotically active ▫ strongly hydrophilic (1 g proteoglycans/50 g of water) occupy larger volume when compared with proteins ▫ in low concentrations form hydrated gel determine the turgor of extracellular matrix ▫ act as a filter allowing the diffusion of small molecules (e.g. ions, water) and prevents the diffusion of proteins and movement of cells
Glycosaminoglycans Types of glycosaminoglycans Seven types (groups) of GAG ▫ differ in occurrence of monosaccharides, type of glycoside bond, grade and localisation of sulphate groups
Groups of glycosaminoglycans Chondroitin-4-sulphate ▫ cartilage ▫ bone ▫ vascular wall ▫ aorta ▫ cornea ▫ dentin, cement ▫ gingiva, periodontal ligaments Repeating disaccharide GLUCURONATE + N-ACETYLGALACTOSAMINE-4- sulphate Chondroitin-6-sulphate ▫ embryonal connective tissue ▫ heart valves ▫ cartilage ▫ bone ▫ vascular wall, aorta ▫ cornea ▫ predentin, cement ▫ periodontal ligaments Repeating disaccharide GLUCURONATE + N-ACETYLGALAKTOSAMINE-6-sulphate
Groups of glycosaminoglycans Chondroitin-4-sulphate Chondroitin-6-sulphate Both are the most abundant GAG in the body Both bind collagen and firmly connect the fibrils. Depletion of chondroitinsulphate in the cartilage is the main cause of osteoartritis.
Groups of glycosaminoglycans Keratansulphate ▫ intervertebral disc ▫ bone ▫ cornea ▫ predentin, cement ▫ periodontal ligaments Repeating disaccharide GALACTOSE + N-ACETYLGLUKOSAMIN-6-sulphate No uronic acid !! Dermatansulphate ▫ predominantly in skin ▫ vessels, heart valves ▫ tendons ▫ lungs ▫ gingiva, periodontal ligament ▫ cement Repeating disaccharide IDURONATE + N-ACETYLGALAcTOSAMIN-4-sulphate Differs from chondroitin-4-sulphate only by inverse configuration on C-5 in glucuronate, changed by epimeration to iduronate.
Groups of glycosaminoglycans Heparin ▫ deposited intracellularly in granules of mastocytes along arteries in the liver, lungs and skin ▫ anticoagulant effect Heparansulphate ▫ extracellularly deposited in basal membranes and cell surfaces ▫ larger than heparin ▫ gingiva, periodontal ligaments, cement Repeating disaccharides IDURONAT-2-SULPHATE + N-SULPHO-GLUCOSAMIN-6-SULPHATE
Hyaluronic acid (hyaluronate) Repeating disaccharide GLUCURONATE + N-acetyl-GLUCOSAMIN Both monosaccharide units are glucose derivatives. They do not contain any sulphate groups. • Characterized by abnormal length (up to 25 000 disaccharide units - Mr 106 – 107) • Polysaccharide chain is coiled to levorotatory helix stabilized by intramolecular hydrogen bonds. Groups of glycosaminoglycans
Hyaluronic acid (hyaluronate) Occurrence ▫ proteoglycan aggregates ▫ vitreous body ▫ synovial fluid (lubricating function) ▫ umbilical cord ▫ production increases during wound healing ▫ gingiva, periodontal ligaments ▫ cement Groups of glycosaminoglycans
Groups of glycosaminoglycans Hyaluronic acid (hyaluronate) Hyaluronate unlike other GAG is not bound covalently to any core protein. X Hyaluronate forms with other proteoglycans aggregates. Proteoglycans are attached noncovalently to hyaluronate by the N-end domain of the core protein (electrostatic interaction) with the help of link protein. Proteoglycan (glycosaminoglycans and core protein) Hyualuronate
Glycosaminoglycans Forms of GAG existence • part of larger structures (proteoglycans) • independent molecules (heparin, hyaluronate)
PROTEOGLYCANS Proteoglycans are formed by glycosaminoglycans, attached to core protein.
Proteoglycans Glycosaminoglycan (non branched saccharide chain) Proteoglycan (glycosaminoglycans and core protein) Proteoglycan aggregate (proteoglycans and hyaluronate) Hyualuronate
Proteoglycans Function of proteoglycans increase of the pressure resistance sieve for macromolecules – restriction of their diffusion lubrication effect hydratation of joint cartilages adhesion of cells and their migration involvement in the development of cells and tissues binding of signal molecules in bone tissue – binding of calcium salts
Proteoglycans Glycosaminoglycans (except for hyaluronate) are covalently bound to so called core protein. Parts of proteoglycans ▫ Glycosaminoglycans (polysaccharides) 95 % ▫ Protein 5 % Core protein Glycosaminoglycans
Proteoglycans Attachment of glycosaminoglycan chain to core protein: ▫ O-glycoside bond Through the reaction of -OH group of serine or threonine of the core protein with trisaccharide Xyl-Gal-Gal ▫ N-glycoside bond Through the reaction of amide nitrogen of asparagine
Proteoglycans • Proteoglycans are characterized by structural diversity: ▫ different core proteins ▫ different GAG chains ▫ different length of GAG chains • Proteoglycans differ also in localisation: ▫ proteoglycans attached to basal membrane ▫ interstitial proteoglycans
Function forms proteoglycan aggregates with hyaluronate • cartilage, gingiva • binds to collagen • belongs to a group of small proteoglycan rich in leucine • gingiva • present in basal membrane • long core protein • forms a barrier limiting penetration of macromolecules through the basal membrane Selected proteoglycans Proteoglycan Versican Aggrecan Decorin Perlecan Typ GAG chondroitinsulphate dermatansulphate chondroitinsulphate keratansulphate chondroitinsulphate dermatansulphate heparansulphate
Selected proteoglycans Proteoglycan Versikan Aggrecan Decorin Perlecan Occurrence in tissues of oral cavity periodontal ligaments, cement periodontal ligaments, cement, dentine development of different tissues
ADHESION GLYCOPROTEINS Ensure specific interactions between cells and molecules of extracellular matrix. 93
Adhesion glycoproteins • Functions of adhesion glycoproteins ▫ attachment of cells to extracellular matrix ▫ organization of the compounds of extracellular matrix • Long flexible molecules with several binding sites for: ▫ collagen ▫ other matrix proteins ▫ polysaccharides ▫ cell receptors (integrins – cell adhesion receptors) 94
Adhesion glycoproteins • Selected representatives of adhesion glycoproteins ▫ fibronectin ▫ laminin ▫ osteonectin ▫ chondronectin 95
Adhesion glycoproteins • Fibronectin ▫ Formed by two subunits arranged to the shape of letter V ▫ Binding sites for: collagen, heparansulphate, hyaluronate integrins ▫ Functions as a connection of cells in extracellular matrix containig fibrillary collagen 96
Adhesion glycoproteins • Laminin ▫ Formed by three different chains arranged to the shape of cross ▫ High relative molecular mass Mr = 950 000 ▫ Binding sites for: collagen of type IV heparansulphate, hyaluronate, cell adhesion receptors ▫ Adhesion glycoprotein of the basal membrane – connect collagen type IV and other compounds of the membrane 97

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biochemistry-of-connective-tissue-dentistrykopptx.pdf

  • 1.
    Biochemistry of connective tissue -extracellular matrix Lenka Fialová Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University, Prague 1
  • 2.
    Connective tissue The connectivetissue is formed by: CELLS and EXTRACELLULAR MATRIX (intercellular matrix) 2
  • 3.
    Extracellular matrix Function ▫ stabilisationof tissue structure ▫ regulation cell behavior survival, development, migration, proliferation ▫ membrane filtration barrier (glomerules) ▫ exchange of different metabolites, ions and water ▫ reparation function ▫ immune processes ▫ participation in inflammation 3
  • 4.
    Cells of connectivetissue ▫ Fibroblasts ▫ Chondroblasts (cartilage) ▫ Osteoblasts (bone) ▫ Odontoblasts (tooth) These cells synthetise extracellular matrix. 4
  • 5.
    Extracellular matrix Parts ofthe extracellular matrix • FIBRILLAR PROTEINS (collagen, elastin) • insoluble in water, no hydratation • GLYCOPROTEINS (e.g. fibronectin,laminin) • GLYCOSAMINOGLYCANS AND PROTEOGLYCANS • soluble in water, easily hydratated 5 Saccharide content increases
  • 6.
    Extracellular matrix FIBRILLARY PROTEINS ▫Structural proteins collagen firmness elastin elasticity 6
  • 7.
    COLLAGENS The most abundantproteins in mammals. They form approximately 25 % of all body proteins. 7
  • 8.
    Collagens Collagenum gr. kolla glue; gr.gennao constitute By boiling collagen is denatured to a colloid solution (gelatine). From the nonpurified collagen the glue arises. Incidence • main protein of the extracellular matrix • component of tendons, cartilages, bones, and teeth (dentin and cement), skin and vessels. Properties • fibrillary proteins • nonsoluble (glyco-) proteins • HIGH STRENGHT, BUT ALSO SUPPLENESS 8
  • 9.
  • 10.
    Collagens Collagen has ancharacteristic amino acid composition and their specific sequence. Primary structure • Characteristic AA composition • Characteristic AA sequence Mature collagen contains no tryptophan and almost no cysteine − from the nutritional point of view not fully valuable protein.
  • 11.
    Primary structure ofcollagen Characteristic AA composition • Fundamental amino acids ▫ Glycine 33 % (x Hb 4 %) ▫ Proline 13 % (x Hb 5 %) • Derived amino acids ▫ 4-Hydroxyproline 9 % (x Hb 0 %) ▫ 5-Hydroxylysine 0,6 % (x Hb 0 %) Origin by posttranslational modification Hb = hemoglobin Chaacteristic for collagen High content
  • 12.
    Primary structure ofcollagen Fundamental AA ▫ Glycine ▫ Proline • Derived AA ▫ 4-Hydroxyproline ▫ 5-Hydroxylysine
  • 13.
    Primary structure ofcollagen Characteristic AA sequence Triplet – Gly – X – Y – • Every third AA is GLYCINE • On the next position frequently PROLINE • On the third position frequently hydroxyproline, ev. hydroxylysine Proline Hydroxyproline (Hydroxylysine) Glycine
  • 14.
    Primary structure ofcollagen Example of AA sequence of a part of the polypeptide chain -Gly-Pro-Hyp- Globular proteins rarely show periodicity in AA sequence.
  • 15.
    Secondary structure ofcollagen Collagen helix • levorotatory helix • steeper rising • 3,3 AA/turn • intrachain hydrogen bonds not present • proline prevents formation of α-helix or β-pleated sheet α α α α-helix (the most common secondary structure in proteins) • dextrorotatory helix • gradual rising • 3,6 AA/turn • stabilization by intrachain hydrogen bonds Comparison of collagen helix to the α-helix, which represents the most common secondary structure in proteins.
  • 16.
    Triple helix Three α α α α-chainsof collagen Triple helix Relatively rigid This structure is responsible for the tensile strenght.
  • 17.
    Triple helix The originis dependent on the oddness of the primary structure • High presence of glycine ▫ smallest amino acid, no side chain (only –H) ▫ placed in the centre of triple helix, where no space is available ▫ close contact between the chains Triple helix is stabilized by hydrogen bonds between each peptide bond -NH group of glycine and C=O group of the peptide bond of the adjacent polypeptide chain.
  • 18.
    Collagen chains The collagenchain is extraordinarly long and contains approximately 1000 AA. The collagen chains are called α1 − α3. ▫ They differ in AA representation ▫ Products of different genes − e.g. α1(I) or α2(V) ▫ Roman digit labels the collagen type More than 30 different types of collagen exists.
  • 19.
    The representation ofchains differs in individual types of collagens. The collagens may form homotrimers or heterotrimers. Homotrimers ▫ molecule of collagen is formed by three identical chains; ▫ e.g. collagen type III is formed by three α1(III) chains Heterotrimers ▫ molecule of collagen is formed by different chains; ▫ e.g. collagen type I is assembled of two α1(I) chains and one α2(I) chain Collagen chains
  • 20.
    Collagen synthesis Collagen isan example of a protein, whose synthesis is connected with many posttranslational modifications (treatment of the polypeptide chain), which take part intra- and extracellularly.
  • 21.
    Synthesis and posttranslationalmodifications of collagen Synthesis of polypeptide chain Hydroxylation of proline and some lysine residues Glycosylation of selected hydroxylysine residues Formation of –S-S- bonds in extension peptides Triple helix formation Secretion of procollagen Intracellular processes
  • 22.
    Proteolytic removal ofpropeptides Assembling of collagen fibrils Formation of cross-links Extracellular processes Posttranslational modifications of collagen
  • 23.
    Posttranslational modifications inthe course of collagen synthesis INTRACELLULAR PROCESSES
  • 24.
    Hydroxylation of prolineand lysine residues Enzymatically catalyzed reaction ▫ Prolylhydroxylase ▫ Lysylhydroxylase cofactors Vitamin C !!! α α α α-ketoglutarate Reaction needs oxygen. One O atom forms -OH group of hydroxyproline, the other becomes part of the originating succinate. Dioxygenases contain Fe
  • 25.
    Hydroxylation of theproline and lysine residues Reactions catalyzed by prolylhydroxylase Vitamin C Maintains Fe2+ in a reduced state Dioxygenase contains Fe
  • 26.
    Hydroxylation of prolineand lysine Reaction catalyzed by prolylhydroxylase • reaction highly specific only for proline attached in the polypeptide chain to the amino group of the glycine -Gly-Pro-Ser-Gly-Pro-Pro-Gly-Leu- -Gly-Pro-Ser-Gly-Pro-Hyp-Gly-Leu- N-end C-end Hydroxylation
  • 27.
    Hydroxylation of prolineand lysine Importance of proline and lysine residues hydroxylation Hydroxyproline • necessary for origin of triple helix by formation of hydrogen bonds between individual chains Hydroxylysine • glycosylation on the formed -OH group
  • 28.
    Nonhydroxylated chain isnot able to mature The stable triple helix cannot be formed Immediate degradation inside the cell Loss of collagen in the matrix Falling out of teeth Vascular fragility Poor wound healing Deficiency of vitamin C
  • 29.
    Vitamin C deficiency Avitaminosis- scurvy Manifestation of avitaminosis in oral cavity • swollen reddish gums • falling out of the teeth
  • 30.
    Glycosylation • Attachment ofgalactose or galactosylglucose to -OH group of the hydroxylysine Enzymatically catalyzed reaction ▫ Galactosyltransferase ▫ Glucosyltransferase Number of saccharide units depends on the type of collagen - e.g.: ▫ Type I (tendons) – 6 units ▫ Type II (lens envelope) – 110 units
  • 31.
    Glycosylated residue ofhydroxylysine in the molecule of collagen Mechanism of glycosylation is different than that in the glycosylation of serine or asparagine. O OH OH OH CH2OH C HN C H CH2 H2C CH H2C NH2 O O OH O OH O CH2OH OH Glycosylation Galactosyltransferase Glucosyltransferase β-glycoside bond
  • 32.
    Formation of –S-S-bonds Disulphide bonds • in the region of C-terminal propeptides ▫ interchain and intrachain disulphide bonds • in the region of N-terminal propeptides ▫ intrachain disulphide bonds Importance • necessary for initiation of triple helix formation starts from the C-end • secretion out of the cell
  • 33.
    Formation of –S-S-bonds I S I S I I S I S I C-end N-end interchain intrachain disulphide bonds intrachain disulphide bonds
  • 34.
    Procollagen Function of propeptides •Start the formation of triple helix in ER intracellularly. • Prevent a premature fibril formation – extracellularly. N-terminal propeptide of the procollagen globular domain C-terminal propeptide of the procollagen globular domain TROPOCOLLAGEN mature molecule of the collagen N-end C-end
  • 35.
    Posttranslational modifications in theprocess of collagen synthesis EXTRACELLULAR PROCESSES
  • 36.
    N-terminal propeptide of procollagen(PINP) MARKERS OF BONE FORMATION (detection in serum or in plasma) Cleaving of the propeptides C-terminal propeptide of procollagen (PICP) Extracellular proteinase Aminopeptidase Extracellular proteinase Carboxypeptidase PROCOLLAGEN TROPOCOLLAGEN monomer
  • 37.
    Tropocollagen ▫ Greek tropé- turn, induce a turn ▫ monomer of the collagen – mature molecule of collagen ▫ Mr = 300 000 N-terminal telopeptide of collagen (INTP) nonhelical area of chains TROPOCOLLAGEN monomer C-terminal telopeptide of collagen (ICTP) nonhelical area of chains MARKERS OF BONE DEGRADATION (detection in serum or in the urine)
  • 38.
    The way ofaggregation of fibrillary collagen • Regular arrangement along the row and in the adjacent row • Monomers in one row are not linked end to end (gap 40 nm) • The adjacent row is displaced by ¼ of the length • In the arrangement of monomers act the weak noncovalent bonds Formation of fibrils Polymeration Tropocollagen Collagen fibril (spontaneous)
  • 39.
  • 40.
    Formation of cross-links Collagenfibers are stabilized by formation of the covalent cross-links, which can be formed either within the tropocollagen molecule between the three chains − intramolecular cross-links and between the tropocollagen molecules − intermolecular cross-links. Intermolecular cross-links Intramolecular cross-links
  • 41.
    Formation of cross-links Functionof cross-links stabilization and strengthening of collagen fibril high breaking strength lower extensibility Cross-linking
  • 42.
    Formation of cross-links Characterof cross-links ▫ covalent bonds Examples ▫ aldol cross-link intramolecular ▫ pyridinoline a deoxypyridinoline cross-links histidine-aldol cross-link intermolecular
  • 43.
    Aldol cross-link ▫ Cross-linkon the N-end of tropocollagen is formed between the lysine residues of two chains Mechanism of formation 1. oxidative deamination of lysine, aldehyde formation ▫ by the enzyme lysyloxidase ▫ aminooxidase, containing Cu2+ ▫ prosthetic group – pyridoxalphosphate 2. Aldol condensation of aldehyde groups spontaneous reaction, two aldehydes form a cross-link
  • 44.
    Pyridinoline and deoxypyridinoline cross-link ▫Cross-link between N-end of one tropocollagen molecule and C-end of the adjacent tropocollagen molecule Pyridinoline ▫ of 3 hydroxylysine residues Deoxypyridinoline ▫ of 2 hydroxylysine and 1 lysine residues ▫ more specific for bone and dentine
  • 45.
    Mechanism of origin 1.step – oxidative deamination of lysine to aldehyde ▫ catalyzed by lysyloxidase enzyme 2. step – formation of ketoamine ▫ nonenzymatic reaction of oxidized hydroxylysine with nonoxidized lysine (hydroxylysine) | OH OH OH Pyridinoline and deoxypyridinoline cross-links -CH-CH2-NH2 + O=CH-CH-CH2- -CH-CH2-NH-CH2-CO-CH2-
  • 46.
    Pyridinoline a deoxypyridinoline cross-links 3.step – formation of the pyridine ring ▫ Interaction of ketoamine with the free aldehyde group of the hydroxylysine closes the heterocyclic pyridine ring, linking covalently three diferent collagen chains Intermolecular cross-bridge Allysin Hydroxyallysine Hydroxylysine
  • 47.
    Pyridinoline a deoxypyridinoline cross-links ▫In the course of bone degradation these cross bridges are separated from collagen fibers, released to blood and excreted to urine. ▫ The pyridinoline and particularly the deoxypyridinoline bridges may be determined in blood and urine. MARKERS OF BONE DEGRADATION
  • 48.
    Fibrillar collagens –e.g. types I, II, III, V • „typical“ collagens forming fibrils Collagens associated with collagen fibrils – for example types VI, IX, XII, XIV, XVI • Triple helix is interrupted by sections making possible the bending of the molecule. • These collagens attach to the surface of collagen fibrils and join them together and connect them to other constituents of extracellular matrix Net forming collagens – types IV, VIII and X • Do not form typical fibrils • Net like arrangement • Nonhelical globular domains on the ends of the molecule Overview of collagens - classes
  • 49.
    Anchoring collagens –type VII • forms anchoring fibers • strengthen the connection of dermis and epidermis Transmembrane collagens – types XIII and XVII • integral membrane proteins Overview of collagens-classes
  • 50.
    Overview of collagens Somefibrilar collagens Type Molecular Occurrence structure I [α1(I)]2 [α2(I)] widely present, skin, vessels, tendons, gingiva, bone, cement, dentin, periodontal ligaments II [α1(II)]3 cartilage, vitreous body III [α1(III)]3 skin, vessels, lungs, gingiva, cement, dentin, periodontal ligaments V [α1(V)]3, [α1(V)2 α2(V)] skin, smooth muscle, bone, cement, dentin
  • 51.
    Types of collagen Somecollagens associated with fibrils Type Molecular Occurrence structure VI [α1(VI) α2(VI) α3(VI)] laterally associated with collagen type II, widely present, bone, gingiva, cement, periodontal ligaments IX [α1(IX) α2(IX) α3(IX)] laterally associated with collagen type II, cartilage, vitreous body, periodontal ligaments XII [α1(XII)]3 associated with collagen type I in soft tissues, periodontal ligaments
  • 52.
    Oveview of collagens Somenet forming collagens Type Molecular Occurrence structure IV [α1(IV)]2 [α2(IV)] basal membranes, formation of two-dimensional net gingiva, periodontal ligaments
  • 53.
    Disorders of collagensynthesis Increased collagen synthesis • fibroses Decreased collagen synthesis • genetic disorder • acquired disorders
  • 54.
    Disorders of collagensynthesis Increased collagen synthesis - FIBROSIS • hepatic cirrhosis • pulmonary fibrosis • atherosclerosis Tissue damage stimulates collagen synthesis by fibroblasts ▫ e.g. damaged hepatocytes are replaced by fibrous connective tissue − hepatic cirrhosis
  • 55.
    Disorders of collagensynthesis Increased collagen synthesis ▫ bacterial infections also stimulate collagen synthesis Prevention of infection spreading − ABSCES x Some bacteria (Clostridia) produce collagenases, which degrade tropocollagen.
  • 56.
    Disorders of collagensynthesis Decreased collagen synthesis • Genetically conditioned ▫ Ehlers-Danlos syndrome ▫ osteogenesis imperfecta • Acquired disturbances ▫ lathyrism ▫ copper deficiency ▫ vitamin C deficiency
  • 57.
    Ehlers-Danlos syndrom ▫ heterogenousgroup of diseases caused by defects of enzymes necessary for synthesis of collagen or by abnormalities in the procollagen gene Manifestations ▫ extreme extensibility of connective tissue and skin ▫ hypermobility of joints ▫ contortionists ▫ risk of rupture of vessels or of the intestine Disorders of collagen synthesis
  • 58.
    Disorders of collagensynthesis Osteogenesis imperfecta ▫ group of diseases caused by mutation in collagen type I ▫ exchange of Gly for an AA having larger side chain ▫ formation of triple helix is not possible ▫ degradation of polypeptide chains not forming triple helix Manifestations ▫ abnormal bone fragility ▫ bone fractures even in small injuries ▫ in more serious cases prenatal fractures Lat. imperfectus incomplete
  • 59.
    Dentinogenesis imperfecta ▫ groupof diseases caused by mutation in α1(I) ▫ associated with osteogenesis imperfecta Manifestations ▫ thin enamel ▫ discolouring of teeth (yellow, brown, grey) ▫ opalescence of the teeth ▫ lower mechanical resistance of the teeth Disorders of collagen synthesis
  • 60.
    Disorders of collagensynthesis Disturbance of cross-link formation • Causes ▫ copper deficiency (part of lysyloxidase) ▫ animal food containing β-aminopropionitrile (contained in seeds of sweet pea − Lathyris odoratus) – blocks lysyloxidase − lathyrism Manifestations extreme fragility of connective tissue (bones, vessels)
  • 61.
    ELASTIN Elastin is themain protein of elastic fibers, providing elasticity to the tissues. 61
  • 62.
    Elastin Occurrence • in arteries,particularly in aorta • in skin, tendons and loose connective tissue (relatively low content) • in lungs Synthesis takes place in early development or after tissue damage Half-time is approximately 70 years (lower content in elderly people).
  • 63.
    Elastin Properties EXTENSIBILITY AND CONTRACTILITY ▫resembles the rubber ▫ after extension elastin is able to return to original size and original form ▫ tensile strength is lower than in collagen ▫ hydrophobic, practically insoluble in aqueous solutions
  • 64.
    Primary structure ofelastin Occurrence of amino acids ▫ 1/3 glycine ▫ high content of nonpolar AA (Ala, Val, Leu, Ileu) ▫ low hydroxyproline ▫ no hydroxylysine − elastin is not glycosylated Sequence of amino acids ▫ typical triplet as in collagen is not present Alternation of short hydrophobic and hydrophilic sections. Hydrophilic sections, which represent a minority part, are rich in lysine, which takes part in forming of cross - links.
  • 65.
    Secondary and tertiarystructure of elastin Secondary structure ▫ elastin does not form a regular secondary structure ▫ elastin has an character of random coil conformation enabling extension and contraction Tertiary structure ▫ a stable secondary structure is not expressed
  • 66.
    Elastin synthesis Synthesis ofpolypeptide chain Hydroxylation of proline residues Secretion of tropoelastin Tropoelastin (globular structure, Mr = 70 000) Formation of cross-links Three-dimensional netting Intracellular processes Extracellular processes
  • 67.
    Cross-links in elastin Cross-links ▫there is a large number of covalent cross-links in elastin ▫ some are similar as in collagen ▫ key step is an oxidative deamination of some lysine residues by copper- containing lysyloxidase (the same enzyme as in formation of cross-links in collagen) ▫ cross-links may be formed within one polypeptide chain or between 2 – 4 chains • Desmosine ▫ cross-link completely specific for elastin ▫ arises from 4 side chains of LYSINE (3 oxidized and 1 nonoxidized) ▫ determines the high elasticity of elastin Linking of polypeptide chains of elastin by cross-links constitutes a three- dimensional netting explaining the „rubber-like“ properties of elastin.
  • 68.
  • 69.
    Glycosaminoglycans (GAG) (Mucopolysaccharides) Characteristic ▫ heteropolysaccharides(100% polysaccharides) ▫ not branched polysaccharide chains ▫ long chains (70 – 200 monosaccharides) ▫ repeating disaccharide units Muco – these substances were first detected in mucus
  • 70.
    Glycosaminoglycans Polysaccharide chain ofGAG is formed by repeating disaccharide units. [URONIC ACID – AMINO SUGAR]n OR [MONOSACCHARIDE - SULFONATED AMINO SUGAR]n
  • 71.
    Glycosaminoglycans Uronic acids presentin GAG O OH OH OH OH COO D-glucuronic acid
  • 72.
  • 73.
    Glycosaminoglycans Modifications of aminosugars in GAG ▫ Acetylation of aminogroup − elimination of a positive charge ▫ Attachment of a sulphate on OH C-4 or C-6 (ester bond), or on nonacetylated amino group − increase of a negative charge
  • 74.
    Glycosaminoglycans Characteristics • high numberof acidic groups ▫ - COO- (uronic acids ) ▫ - OSO3 - (amino sugars with sulphate groups) • highly negative charge at physiological pH (polyanions) Na+ Na+ Na+ Na+ H20 H20 H20 H20 H20
  • 75.
    Glycosaminoglycans Characteristic (cont.) ▫ chainsrepel each other and in solution tend to straighten ▫ negatively charged groups bind cations – Na+, K+ ▫ osmotically active ▫ strongly hydrophilic (1 g proteoglycans/50 g of water) occupy larger volume when compared with proteins ▫ in low concentrations form hydrated gel determine the turgor of extracellular matrix ▫ act as a filter allowing the diffusion of small molecules (e.g. ions, water) and prevents the diffusion of proteins and movement of cells
  • 76.
    Glycosaminoglycans Types of glycosaminoglycans Seventypes (groups) of GAG ▫ differ in occurrence of monosaccharides, type of glycoside bond, grade and localisation of sulphate groups
  • 77.
    Groups of glycosaminoglycans Chondroitin-4-sulphate ▫cartilage ▫ bone ▫ vascular wall ▫ aorta ▫ cornea ▫ dentin, cement ▫ gingiva, periodontal ligaments Repeating disaccharide GLUCURONATE + N-ACETYLGALACTOSAMINE-4- sulphate Chondroitin-6-sulphate ▫ embryonal connective tissue ▫ heart valves ▫ cartilage ▫ bone ▫ vascular wall, aorta ▫ cornea ▫ predentin, cement ▫ periodontal ligaments Repeating disaccharide GLUCURONATE + N-ACETYLGALAKTOSAMINE-6-sulphate
  • 78.
    Groups of glycosaminoglycans Chondroitin-4-sulphateChondroitin-6-sulphate Both are the most abundant GAG in the body Both bind collagen and firmly connect the fibrils. Depletion of chondroitinsulphate in the cartilage is the main cause of osteoartritis.
  • 79.
    Groups of glycosaminoglycans Keratansulphate ▫intervertebral disc ▫ bone ▫ cornea ▫ predentin, cement ▫ periodontal ligaments Repeating disaccharide GALACTOSE + N-ACETYLGLUKOSAMIN-6-sulphate No uronic acid !! Dermatansulphate ▫ predominantly in skin ▫ vessels, heart valves ▫ tendons ▫ lungs ▫ gingiva, periodontal ligament ▫ cement Repeating disaccharide IDURONATE + N-ACETYLGALAcTOSAMIN-4-sulphate Differs from chondroitin-4-sulphate only by inverse configuration on C-5 in glucuronate, changed by epimeration to iduronate.
  • 80.
    Groups of glycosaminoglycans Heparin ▫deposited intracellularly in granules of mastocytes along arteries in the liver, lungs and skin ▫ anticoagulant effect Heparansulphate ▫ extracellularly deposited in basal membranes and cell surfaces ▫ larger than heparin ▫ gingiva, periodontal ligaments, cement Repeating disaccharides IDURONAT-2-SULPHATE + N-SULPHO-GLUCOSAMIN-6-SULPHATE
  • 81.
    Hyaluronic acid (hyaluronate) Repeatingdisaccharide GLUCURONATE + N-acetyl-GLUCOSAMIN Both monosaccharide units are glucose derivatives. They do not contain any sulphate groups. • Characterized by abnormal length (up to 25 000 disaccharide units - Mr 106 – 107) • Polysaccharide chain is coiled to levorotatory helix stabilized by intramolecular hydrogen bonds. Groups of glycosaminoglycans
  • 82.
    Hyaluronic acid (hyaluronate) Occurrence ▫proteoglycan aggregates ▫ vitreous body ▫ synovial fluid (lubricating function) ▫ umbilical cord ▫ production increases during wound healing ▫ gingiva, periodontal ligaments ▫ cement Groups of glycosaminoglycans
  • 83.
    Groups of glycosaminoglycans Hyaluronicacid (hyaluronate) Hyaluronate unlike other GAG is not bound covalently to any core protein. X Hyaluronate forms with other proteoglycans aggregates. Proteoglycans are attached noncovalently to hyaluronate by the N-end domain of the core protein (electrostatic interaction) with the help of link protein. Proteoglycan (glycosaminoglycans and core protein) Hyualuronate
  • 84.
    Glycosaminoglycans Forms of GAGexistence • part of larger structures (proteoglycans) • independent molecules (heparin, hyaluronate)
  • 85.
    PROTEOGLYCANS Proteoglycans are formedby glycosaminoglycans, attached to core protein.
  • 86.
    Proteoglycans Glycosaminoglycan (non branched saccharide chain) Proteoglycan (glycosaminoglycans andcore protein) Proteoglycan aggregate (proteoglycans and hyaluronate) Hyualuronate
  • 87.
    Proteoglycans Function of proteoglycans increase of the pressure resistance sieve for macromolecules – restriction of their diffusion lubrication effect hydratation of joint cartilages adhesion of cells and their migration involvement in the development of cells and tissues binding of signal molecules in bone tissue – binding of calcium salts
  • 88.
    Proteoglycans Glycosaminoglycans (except forhyaluronate) are covalently bound to so called core protein. Parts of proteoglycans ▫ Glycosaminoglycans (polysaccharides) 95 % ▫ Protein 5 % Core protein Glycosaminoglycans
  • 89.
    Proteoglycans Attachment of glycosaminoglycanchain to core protein: ▫ O-glycoside bond Through the reaction of -OH group of serine or threonine of the core protein with trisaccharide Xyl-Gal-Gal ▫ N-glycoside bond Through the reaction of amide nitrogen of asparagine
  • 90.
    Proteoglycans • Proteoglycans arecharacterized by structural diversity: ▫ different core proteins ▫ different GAG chains ▫ different length of GAG chains • Proteoglycans differ also in localisation: ▫ proteoglycans attached to basal membrane ▫ interstitial proteoglycans
  • 91.
    Function forms proteoglycan aggregates withhyaluronate • cartilage, gingiva • binds to collagen • belongs to a group of small proteoglycan rich in leucine • gingiva • present in basal membrane • long core protein • forms a barrier limiting penetration of macromolecules through the basal membrane Selected proteoglycans Proteoglycan Versican Aggrecan Decorin Perlecan Typ GAG chondroitinsulphate dermatansulphate chondroitinsulphate keratansulphate chondroitinsulphate dermatansulphate heparansulphate
  • 92.
    Selected proteoglycans Proteoglycan Versikan Aggrecan Decorin Perlecan Occurrence intissues of oral cavity periodontal ligaments, cement periodontal ligaments, cement, dentine development of different tissues
  • 93.
    ADHESION GLYCOPROTEINS Ensure specificinteractions between cells and molecules of extracellular matrix. 93
  • 94.
    Adhesion glycoproteins • Functionsof adhesion glycoproteins ▫ attachment of cells to extracellular matrix ▫ organization of the compounds of extracellular matrix • Long flexible molecules with several binding sites for: ▫ collagen ▫ other matrix proteins ▫ polysaccharides ▫ cell receptors (integrins – cell adhesion receptors) 94
  • 95.
    Adhesion glycoproteins • Selectedrepresentatives of adhesion glycoproteins ▫ fibronectin ▫ laminin ▫ osteonectin ▫ chondronectin 95
  • 96.
    Adhesion glycoproteins • Fibronectin ▫Formed by two subunits arranged to the shape of letter V ▫ Binding sites for: collagen, heparansulphate, hyaluronate integrins ▫ Functions as a connection of cells in extracellular matrix containig fibrillary collagen 96
  • 97.
    Adhesion glycoproteins • Laminin ▫Formed by three different chains arranged to the shape of cross ▫ High relative molecular mass Mr = 950 000 ▫ Binding sites for: collagen of type IV heparansulphate, hyaluronate, cell adhesion receptors ▫ Adhesion glycoprotein of the basal membrane – connect collagen type IV and other compounds of the membrane 97