Connective tissue - extracelular matrix Connective tissue • The main component is extracelular matrix ® determines mechanic properties of tissue • Proportion of cells is low. Cells in the connestive tissue • fibroblasts and fibrocytes (chondroblasts and chondrocytes, osteoblasts and osteocytes) produce the major components of extracellular matrix, • histiocytes (fixed and free macrophages) scavenge foreign particles and tissue detritus, • mast cells (mastocytes) releasing heparin and histamine, • adipose cells (adipocytes), • leukocytes, namely lymphocytes, plasmocytes, and monocytes (circulating macrophages, leaving vessels through diapedesis Etracellular matrix • Fibrilar components – fibrils of collagen and reticulin,in some connective tissue types elastin fibres, and • Interfibrillar component (the amorphous ground substance) proteoglycans and hyaluronic acid, fibronectin, laminin and other cell adhesion glycoproteins, in bones, voluminous insoluble mineral component. Function of extracelular matrix Supporting function for cells Regulation: of polarity of the cells cell division adhesion motion Grow and regeneration of tissues Determining the shape of tissue Architecture of tissue and organs Membrane filtration barrier Exchange of metabolites, ions and water Collagen • The most abundant protein in the body (25% of proteins in adults) • Present in all connective tissues • Responsible for strength and flexibility • 27 (?) types of collagen known in human – they differ by structure and function • Only some types are fibilar (I, II, III, V,VI, XI ) • In all collagen types is present (at least partially) typical triplhelix Approximate collagen content in various tissues Amino acids in collagen • Fibrilar collagens (a-chains)  polymer of repeats Gly-X-Y where: in position X is very often proline, 3-hydroxyproline, Glu, His, Leu, Phe, in position Y 4-hydroxyproline, Thr, Lys, Arg. • At each third position is glycin, in average each forth amino acid is proline or hydroxyproline • The content of glycine ~30%, hydroxyproline and hydroxylysine 25% Secondary structure of chains Tropocollagen structure • The basic structural unit of collagen is triple helix 300 x 1,5 nm • in collagen type I two chains a[1](I) and one chain a[2](I) (structural formula [a[1](I)][2]a[2](I)) Glycosylation of collagen • Hydroxy groups of hydroxylysine are modified by posttranslational (cotranslational) glycosylation • Most often glucose a galactose Chain types in collagen • Amino acid composition of polypeptide chains is variable • About 30 separarate genes are known • Best described and mostl wide-spread are a[1] and [2] chains Collagen types • Fibril-forming collagens - type I,II,III, V, XI (90% of all collagens) • Network forming collagens – type IV, VIII, X • FACIT*s– type IX,XII, XIV, XVI, XIX • Transmembrane collagens – XIII, XVIII • Others….. Synthesis of collagen Fibroblasts, chondroblasts and osteoblasts • First phase has intracellular location: The synthesis of separate procollagen chains (pro-a1, pro-2, etc.) Cotranslational hydroxylation of Pro a Lys and glycosylation of hydroxylysine (Hyl) in ER Formation of procollagen from three chains Secretion of procollagen by exocytosis • Second phase is located extracellulary Removal of N- and C-terminal propeptides – formation of tropocollagen Agregation of tropocollagen units – formation of protofibrils Interaction of protofibril with proteoglycans – formation of microfibrils Collagen ripening – oxidation of lysine side chains to allysine, formation of intermolecular links Hydroxylation of proline and lysine residues Iniciation of triple helix formation Procollagen secretion • Secretory vesicles fuses with the membrane and releases procollagen into the extracellular space • Only triple helices can be secreted • Improperly coiled molecules are degraded Conversion of procollagen to tropocollagen • Specifické procollagenpeptidases catalyze the removal of non-helical pro-peptides on both ends • (In clinical biochemistry, the abbreviations PINP and PICP). Collagen degradation • Molecules of collagen are metabolically very stable, half-life may be several years. • Their breakdown is increased during starvation or inflammatory diseases. • Degradation by collagenases* Collagen and diseases of connective tissue • Fibroses - overproduction of collagen - lung fibrosis - liver cirhosis - atherosclerosis • Inherited abnormalities in collagen structure Osteogenesis imperfecta (brittle bones) mutation in the gene for a[1] and [2] chains of collagen I – impaired formation of triple helices of collagen® collagen fall prey to intracellular proteases Ehlers-Danlos syndrom group of diseses, at least 10 types characterized by stretchy skin and loose joints („india rubber“ man) impairment of collagen synthesis due to mutation of genes hyperexensibility of the skin, abnormal tissue fragility, increased joint mobility, „cigarette paper“ scars Elastin • Produced by smooth muscle cells, fibroblasts and chondrocytes • Probably only one genetic type of elastin, variants arise by alternative splicing • Amorphous structure with large amount of crosslinks, non soluble. • The main amino acids are glycine (~31%), proline (11%), valine, alanine (22%) and other hydrophobic AA • Only small portion of hydroxyproline (~1%) • No glycosylation • Chains don't have a regular secondary structure • Precursor in the synthesis is tropoelastin Proteoglycans • The main component of amorphous ground substance • glycosaminoglycans connected with core protein Function of proteoglycans and glycosaminoglycans • Formation of cell-free ground substance – Pressure resistance – Shape recovery – Lubrication of joints – Hydration of cartilages Occurence of GAG Various types of proteoglycans Agrecan – tha main proteoglycan in cartilage Versican – in many tissues, mainly vessels and skin Dekorin – small proteoglycan of many tissues Biglycan – small proteoglycan of cartilage Fibronectin • Cellular glue (cellular adhesive) • Most abundant multiadhesive glycoprotein in connective tissue • released by fibrocytes, endothelial cells, and other cell types • the liver cells secrete fibronectin into the blood plasma (concentration 150 - 700 mg / l). • large protein ~ 5000 AK • mediates the communication between collagen, proteoglycans and cell surface • it interacts with receptors on the cell surface - in this way enables the adhesion of cells to the extracellular matrix - enables interaction between the exterior and the interior of the cell • it is involved in migration of the cells Structure of fibronectin • Two polypeptide chains connected near the carboxy ends by disulfidic bond. • Different parts of fibronectin bind to cell surface receptors, proteoglycans, collagen and fibrin Integrins • Receptors for fibronectin • Transmembrane proteins • Provide a link between the internal cytoskeleton of cells and extracellular proteins such as fibronectin, collagen, laminin. • Are involved in a wide variety of cell signaling option Laminin • In addition to collagen IV is the main protein of basement membranes. • Large heterotrimer (Mr 950 000) • High affinity to ither components of basement membranes (e.g. collagen, proteoglycans, sulfatated lipids ) • Laminin aggregates reversibly into a flat polymeric network at high concentrations of Ca^2+ ions. Those polymers bind through another protein (either nidogen or entactin) type IV collagen and core proteins of proteoglycans. These interactions are the cause of anchoring epithelial cells onto components of basement membranes. These interactions are the cause of anchoring epithelial cells onto components of basement membranes. Structure of laminin • Heterotrimer cross-shaped protein. • Polypeptide subunits a, b a g are joined through disulfide bridges • Main secondary structure of all three subunits is a-helix • All three chains have extension at the amino-terminal end with globular domains.These domains allow binding of laminin to other components of extracellular matrix.