Collagen, Proteoglycans, and Glycoproteins PDF

Peculiarities of the connective tissue structural proteins, proteoglycans, and glycoproteins doc. dr. Odeta Arandarčikaitė Department of Biochemistry Peculiarities of the connective tissue extracellular matrix Fibroblasts...

Peculiarities of the connective tissue structural proteins, proteoglycans, and glycoproteins doc. dr. Odeta Arandarčikaitė Department of Biochemistry Peculiarities of the connective tissue extracellular matrix Fibroblasts components between the cells Chondroblasts (cartilage) Osteoblasts (bone) synthesis Collagen Hyaluronic acid Proteoglycans Glycoproteins Elastin  Peculiarities of the connective tissue extracellular matrix = fibers + ground substance Collagen Hyaluronic acid Elastin Proteoglycans Glycoproteins Function: matrix is responsible for the specific structure and function of the tissues (the matrix of bone is rigid and inflexible, but that of cartilage is firm but pliable) tissue proliferation, development, shape changes serves also as a reservoir for growth factors and cytokines  Collagen Main protein of the extracellular matrix Collagens are the most abundant proteins in mammals (~30% of the total protein mass) In the human body over 90% collagen type I Replacement of collagen (liver – 90 days, bones – 1 year, skin 110-120 days) Characterized as the protein with the largest number of post-translational modifications  Translation Pre-pro-collagen form is synthesized Primary sequence of amino acid is strict: glycine as every third residue accounts for the stability of the helical structure owing to its property of being the smallest amino acid X and Y can be any amino acid but are mostly occupied by the proline residue can be any of the other amino acids – X – Y -glycine – X – Y - I H Post-translational modification - hydroxylation pre-pro-collagen is transported to the endoplasmic reticulum (ER) pre- amino acid sequence at N-terminal end is cleaved pro-collagen proline and lysine residues at 3 position (Y) are hydroxylated to hydroxyproline and hydroxylysine residues Enzymes: prolyl hydroxylase and lysyl hydroxylase α-ketoglutarate O2 glycine – X - Y Fe2+ proline or lysine (post-translational reducing agent vitamin C modification hydroxylation) Proline hydroxylation fosters and stabilizes formation of the collagen triple helix Hydroxylation of proline and lysine Lysyl hydroxylase Prolyl hydroxylase Fe2+ Fe2+ In the case of ascorbic acid deficiency, a lack of proline and lysine hydroxylation, interchain H-bond formation is impaired, as it formation of a stable triple helix and collagen fibrils cannot be cross-linked, greatly decreasing the tensile strength of the assembled fiber (disease scurvy). Post-translational modification - glycosylation hydroxylysine residues galactosyl transferase glycosyl transferase The importance of this glycosylation: fibrils formation the sugar molecules bound to collagen are arranged so that their hydrophobic face is against the hydrophobic collagen fibrils  Post-translational modification - phosphorylation -glycine – X – Y - serine or treonine phosphorylation Phosphorylation of Serine and Threonine residues X position of the Gly-X-Y Enzymes kinases recognises unfolded collagen chains Phosphotylated peptidic chains are capable to form stable triple-helixes Phosphorylation increases triple-helix stability Qiu Y1, Poppleton E1, Mekkat A2, Yu H2, Banerjee S3, Wiley SE3, Dixon JE3, Kaplan DL4, Lin YS2, Brodsky B5. Enzymatic Phosphorylation of Ser in a Type I Collagen Peptide. Biophys J. 2018 Dec 18;115(12):2327-2335. doi: 10.1016/j.bpj.2018.11.012.  Triple helix assembly Pro-α collagen might be several types with different amino acid sequences (coded by different genes) Polipeptidic chains in a triple helix can be the same or different Collagen type I (2 chains α1 and 1 chain α2) Collagen type II (3chains α1)  Triple helix assembly Triple helix formation starts with disulfide bonds formation at C-terminal between 3 pro-α chains The triple helix is stabilized by an interchain hydrogen bonding network (carbonyl group of hydroxyproline and hydroxyl group of glycine) Each procollagen is composed of a left-handed helix. 3 such helices are coiled to form a right-handed superhelix Triple helix assembly Changes from a single helix to a triple helix: Each turn of the triple helix contains three amino acid residues, every third amino acid is in close contact with the other two strands in the center of the structure Glycine – smallest amino acid (H– as side chain) and can fit in center The hydroxyproline residues in collagen are required for stabilization of the triple helix by hydrogen bond formation Formation of this structure provide higher bending rigidity Secretion to extracellular matrix Procollagen has a central region of triple helix flanked by non- helical N- and C- terminals The procollagen molecules move through the Golgi apparatus, where they are packaged in secretory vesicles The vesicles fuse with the cell membrane, causing the release of procollagen molecules into the extracellular space  Formation of tropocollagen In extracellular matrix, the procollagen molecules are cleaved by enzymes: N- and C-procollagen peptidases Function of propeptides: initiate the formation of triple helix in ER Triple-helical tropocollagen molecules - is a monomer of mature collagen (in Greek tropé - turn) N-procollagen peptidase C-procollagen peptidase N-propeptide C-propeptide markers of bone formation detected in blood  Cross-links formation Individual tropocollagen monomers spontaneously associate to form collagen fibrils Arrangement along the row is displaced by ¼ of the length (most effective length for mechanical resistance to tensile forces)  Cross-links -covalent bonds formation 1. Deamination of some lysine or hydroxylysine residues to aldehydes (allysine or hydroxyallysine) Enzyme: lysyl oxidase 2. Aldehydes form covalent bonds with nearby lysine and hydroxylysine residue allysine hydroxylysine  Cross-links -covalent bonds formation These covalent bonds form cross-links between tropocollagens: Intramolecular cross-linking, in which two a chains within the same molecule may be covalently linked Intermolecular cross-linking that involves the formation of covalent bridges between chains in different molecules Provide to collagen strength and rigidity Extensive cross- linking make collagen insoluble Boiling: collagen is denatured to a colloid solution (gelatine)  Intermolecular cross link an intramolecular cross-link is always a type of ring-closing reaction Mature collagen are characterized by formation of cross-links Intermolecular connections of lysine or hydroxylysine side chains of collagen fibrils : Pyridinoline originates from connecting three hydroxylysine fragments Deoxypyridinoline from two hydroxylysines with one lysine If the collagen is degraded, these cross-links are released into circulation and content of pyridinolines/deoxypyridinolines in urine reflects intensity of bone resorption  Collagen fibrillogenesis Mature collagen fibrils are large cable-like bundles microfibrils The distribution of collagen and fibril thicknesses are modulators of tissue strength The tensile strength of mature fibrils collagen is remarkable: a fiber 1 mm in diameter can hold a load of 10 to 40 kg without breaking collagen fibre  Facts about tropocollagen The structural unit: tropocollagen Each subunit consists of 3 polypeptidic chains are wounded to form a triple helix (I type collagen is formed from 2 α1 and 1 α2) Each collagen helix consists of ~ 1000 amino acid residues. The helix is left-handed The helix contains 3 amino acids per turn, with a pitch of 0.94 nm Each unit of tropocollagen is about 1.5 nm wide and 300 nm long, molecular mass~285 kDa Covalent bonds form cross-links between tropocollagens – mature collagen fibrils Collagens classification All collagens have specific 3 polypeptidic chains with at least one fragment of triple helix The arrangement of the fibrils gives individual characteristics to tissues Types: about 29 Marking: Roman numerals I, II... doi: 10.1242/jcs.203950 doi: 10.1242/jcs.203950 Fibril-forming collagens (types I, II, III, V, XI, XXIV, XXVII) Fibril-associated collagens with interrupted triple helices (IX, XII, XIV, XVI, XIX, XX, XXI, XXII, XXVI) Collagens capable of forming hexagonal network (e.g., VIII, X) Basement membrane collagen (IV) Collagens that assemble into beaded filaments (e.g., type VI) Anchoring fiber-forming collagens (e.g., VII) Plasma membrane-spanning collagens (XIII, XVII, XXIII, XXV) Collagens with unique domain organization (XV, XVIII)  Collagen diseases Scurvy disease: Lack of ascorbic acid (Vitamin C) Proline and lysine hydroxylation disorders Genetic diseases Non-hydroxylated chain is not able to form stable triple helix Immediate degradation inside the cell Collagenopathies: Loss of collagen in the matrix defects of collagen fibers Falling out of teeth synthesis, or inability to Fragile blood vessels form fibers properly (have Poor wound healing been identified more than Skin lesions, bleeding gums 1000 mutations in 23 genes coding 13 types of collagen). Ehlers-Danlos syndrome: deficiency of enzyme Lysyl oxidase (crosslinking disorders). Join hypermobility, skin strechy, aneurysms. Glycation  Glycosylation ☺ non-enzymatic covalent addition of sugar Post-translational modification molecules to collagen hydroxylysine residues (Enzymes: galactosyl transferase, glycosyl transferase) the glycating sugar binding site is cross-links without glycosylation the collagen molecules do between collagen molecules not form structural fibrils sugar molecules bound to collagen are arranged so that their hydrophobic face is against the hydrophobic collagen fibrils binding collagen molecules together forces them to have a fixed orientation lower mechanical properties cells can not recognize the collagen, because sugar molecules are added accidentally diabetics, where the background level of sugar in the tissue is excessively high in areas of chronic inflammation, dying cells release a variety of sugar molecules  Dynamic equilibrium of collagen Synthesis Catabolism /Degradation: Fibrosis - increased synthesis overgrowth, scarring 1. phagocytosis of collagen into cells and degraded in lysosomes 2. collagenolysis in osteoclast-mediated *Each 1 year are replaced 3 kg of bone resorption collagen in your body. 3. extracellular enzymes proteases *Healing wound synthesis and degradation equilibrium after 3-5 weeks.  Proteolitic enzymes Specific proteases or proteolitic enzymes (break peptidic bonds) Cells synthesize an inactive form of these enzymes and activate them where they are needed Families: Cysteine proteinase, Matrix metalloproteinases  1. Cysteine proteinase Enzyme family: cysteine proteinase Cathepsin K is expressed in osteoclast lysosomes and released during bone remodeling and resorption. Cathepsin K is abile to catabolize elastin, collagen, and gelatin in bone and cartilage. Cathepsin K is degraded by Cathepsin S, called controlled Cathepsin cannibalism. Cathepsin K expression is stimulated by inflammatory cytokines, breast cancers or glioblastoma. Cathepsins B and L cleave collagen types II, IX,XI and destroy cross- linked collagen matrix at low pH  2. Matrix metalloproteinases Family of extracellular proteinases (degrading proteins) Zinc-dependent endopeptidases Isoforms arking: Arabic numerals 1, 2... Function: tissue remodeling and degradation of extracellular matrix, glycoproteins, proteoglycans “aggressive” - secreted as inactive proenzymes, requiring activation - cleavage of a propeptidic fragment Gelatinases (MMP-2, MMP-9) – cleave collagen IV, V, VII, X, elastin Collagenase – cleave collagen I, II, III Enzymes gene expression rises during wound healing, inflammation, cancer Matrix Metalloproteinases activity regulation: 1. Gene expression coordination Physiologically extracellular matrix components degradation important in cell migration, tissue remodeling during grow and differentiation. 2. Proenzyme conversion to active form Removal of propeptide is required to activate proteolytic function of newly synthesized enzymes. Activated MMP can activate other MMP (autoactivation). 3. Tissue level activation regulation by circulating protein or tissue inhibitors of metalloproteinases (TIMP). (TIMP synthesis disorders in metastasize of cancer cells, atherosclerosis). In human exist more than 20 types. Cleave all proteins in extracellular matrix (MMP-1, MMP-8, MMP-13).  Degradation of collagen by enzymes Collagenases hydrolizes the triple helix of Cathepsin cleaves collagen molecules collagen into 1/4 and 3/4 length fragments. within the native triple helix, generating fragments of various sizes.  Elastin Elastic fibers =90 % elastin (polymer of tropoelastin) + 10% fibrillin Cellular location: extracellular matrix Function: elasticity, stretch and recoil Solubility: insoluble protein Structure monomer: tropoelastin (secreted by fibroblasts and smooth muscle cells) Covalent cross-links of tropoelastins: desmosines – unique to elastin There is only one tropoelastin gene (The expression of this gene mainly occurs before birth and in the first few years of life when the cells of elastic tissues produce the elastin required for the body to develop. At young age, gene expression is turned down).  Tropoelastin structure Polypeptidic chain ~ 700 amino acids Amino acids composition: rich (> 80%) in glycine, proline, valine, lysine and alanine residues some hydroxyproline Tropoelastin is an asymmetric molecule: N - terminus with a long narrow region ∼11 nm C - terminus branches to give a larger more open appearance “foot” like molecule is not compact but is relatively extended tropoelastin is secreted as a monomer into the extracellular matrix  Elastin structure formation tropoelastin is secreted as a monomer into the extracellular matrix the monomers spontaneously self-associate at the cell surface association is driven by hydrophobic interactions that dominate in length of the molecule The random orientation of individual tropoelastin monomers: head-to-tail (N─C), tail-to-tail (C─C), head-to-head (N─N), and lateral interactions that give rise to a randomized cross-linking structure. Elastin cross-link formation (desmosine) 1. Enzyme: lysyl oxidase oxidates lysine residue to aldehyde 2. reactions to form demosine for cross-linking: 1 lysine residue 3 allysines (aldehydes) formed by oxidative deamination of lysine residues Meaning: covalently binding 2 tropoelastin polypeptydes Interconnected network giving connective tissue its elasticity, stretch and bend in any direction.  Formation of elastin fibers During the cross-linking formation elastin mixes with fibrillin (glycoprotein) The elastic fiber consisting of an amorphous elastin core (yellow) and fibrillin-containing microfibrils (blue) Each elastin molecule in the network has multiple random-coil domains which expand and contract The highly stable cross-linking of elastin allows the entire network to stretch and recoil like a rubber band IUBMB Life, Volume: 72, Issue: 5, Pages: 842-854, First published: 13 December 2019, DOI: (10.1002/iub.2213) Fibrous networks in extracellular space Collagen and elastic fibers form fibrous networks to provide tissues integrity and mechanical stability with composite strength and elasticity Fibers interacts with many of matrix macromolecules (limited physical interactions between collagen and elastic fibers) Confer mechanical resistance through separate mechanisms  Proteoglycans Proteoglycans = glycosaminoglycan +core proteins Structure: proteins 5-15%, carbohydrates 85-95% Repeating units disaccharide (linear structure carbohydrates) Properties: - negative charge (repel negative molecules and attracts positive ions) - binds water and form hydrated matrices Function: make space and allow compressibility act as filter of ions (Ca2+) regulates cell migration and adhesion  Proteoglycans structure glycosaminoglycan (GAG) -a long polysaccharide chain with a repeating disaccharide motif glycosaminoglycan covalently linked (O-glycosidic bound) to core protein proteoglycan monomers typically are bound non-covalently to a hyaluronic acid molecule hyaluronic acid molecule occurs as single long not sulfated polysaccharidic chain “a bottlebrush” shaped molecule  Glycosaminoglycan disaccharides amino sugar acidic sugar N-acetylglucosamine Glucuronic acid N-acetylgalactosamine Iduronic acid Amino group – NH2 binding usually C2 Glucuronic acid is with C6 oxidized to a position enzyme: aminotrasferase) carboxylic acid Acetylation (acetyl-CoA binding to NH2 Epimerization of D-glucuronic acid to group enzyme: N-acetyltrasferase) L-Iduronic acid, enzymes: emirases Sulfanated, enzymes: sulfotransferases Sulfanated, enzymes: sulfotransferases  Glycosaminoglycan disaccharides Hyaluronic Hyaluronic acid acid Not Not sulfated. sulfated. Synovial Synovial fluid, fluid, Glucuronic Glucuronic acid acid connective connective tissue, tissue, N-acetylgalactosamine N-acetylgalactosamine tendon, tendon, eye, eye, skin. skin. Chondroitin Chondroitin sulfate sulfate Galactosamine Galactosamine C4 C4 or or Most Most abudant. abudant. Glucuronic Glucuronic acid acid C6 sulfate. C6 sulfate. Cartilage, Cartilage, tendons tendons N-acetylgalactosamine N-acetylgalactosamine ligaments, ligaments, aorta aorta Heparin Heparin Glucosamine Glucosamine sulfated sulfated Anticoagulant. Anticoagulant. Glucuronic Glucuronic acid acid C2 C2 and and C6 C6 Glucuronic Glucuronic Mast Mast cells, cells, lung, lung, N-acetylglucosamine N-acetylglucosamine acid acid sulfated sulfated C2. C2. skin. skin. Dermatan Dermatan sulfate sulfate Galactosamine Galactosamine C4 C4 Skin, Skin, vessels. vessels. Iduronic Iduronic acid acid sulfate. sulfate. N-acetylgalactosamine N-acetylgalactosamine Keratan Keratan sulfate sulfate C6 C6 sulfate sulfate either Galactosamine eitherC6 Cornea. Cornea. Galactose Galactose N- N- sugars. sulfate. sugars. acetylgalactosamine N-acetylgalactosamine acetylgalactosamine  Hyalectan (lectican) The core protein of lecticans binds numerous glycosaminoglycan chains and attaches to hyaluronan with the help of link protein Some of the proteoglycans have distinct core protein structures, and others display similarities and thus may be grouped into families Many lecticans assemble along a hyaluronan backbone forming a massive, carbohydrate-rich extracellular matrix macromolecule Proteoglycans regulate the hydration of tissue extracellular matrix, which is a major determinant of overall tissue viscoelasticity Positions of potential glycosaminoglycan (GAG) substitution of aggrecan, versicans, brevicans, and neurocan are deduced from the primary structures Wei, J.; Hu, M.; Huang, K.; Lin, S.; Du, H. Roles of Proteoglycans and Glycosaminoglycans in Cancer Development and Progression. Int. J. Mol. Sci. 2020, 21, 5983. https://doi.org/10.3390/ijms21175983  Proteoglycans of the connective tissue Hyaluronan is a major component of synovial fluid, which absorbs shock and lubricates joints Hyaluronan also serves as a “backbone” to bind many aggrecan molecules, the dominant proteoglycan in cartilage. Aggrecan is proteoglycan found in cartilage and chondrocytes About 90% of aggrecan mass consists of bound glycosaminoglycans keratin sulfate and chondroitin sulfate type (100-150 GAG chains) Aggrecan functions to provide hydration in the extracellular matrix of cartilage tissues Aggrecan also plays an important role in chondrocyte differentiation and facilitates chondrocyte-extracellular matrix interactions Wei, J.; Hu, M.; Huang, K.; Lin, S.; Du, H. Roles of Proteoglycans and Glycosaminoglycans in Cancer Development and Progression. Int. J. Mol. Sci. 2020, 21, 5983. https://doi.org/10.3390/ijms21175983 Glycosaminoglycan- polysaccharide chain binding to core protein aggrecan Glycosaminoglycan- polysaccharide chain is composed of repeating glycosaminoglycan covalently (O-glycosidic disaccharides bound) linked to core protein via serine residue on the protein through a sequence serine-xylose- galactose-galactose  Proteoglycans negative charge The negative charge groups: — carboxyl groups (D-glucuronic acid or L-iduronic acid) — sulfate groups Each sugar has 1 or 2 negative charges: — binds water (extremely hidrofilic) — attract positive ions (K+ and Na+) — repel negative molecules Properties of proteoglycans: — high viscosity — resistance to compression —return to original shape —voluminous Proteoglycan of cartilage containing ~100 of proteoglycan monomers (consist of ~100 of chondroitin sulfate and keratan sulfate) via link protein attached to hyaluronic acid.  Synthesis of proteoglycans 1. Core protein synthesis in ribosomes and transported to the lumen of rough endoplasmic reticulum 2. After synthesis of O-linked glycoproteins direct addition of saccaride chain 3. Saccharide chains elongation (Golgi bodies) 4. Empirisation of glucuronic acid to iduronic acid 5. Sulfation of amino sugars 6. Proteoglycan monomers typically are bound non-covalently to a hyaluronic acid 7. The final proteoglycan is eventually transported to secretory vesicles, which may be released into the extracellular Aggrecan synthesis and degradation chondroitin sulfate rich domains keratan sulfate-rich domain link proteins hyaluronan There is a constant level of aggrecan breakdown and new synthesis Aggrecan structure is not constant throughout life, but changes due to both synthetic and degradative events Changes due to synthesis alter the structure of the chondroitin sulfate and keratan sulfate chains resulting predisposing to cartilage erosion Roughley, P.J. & Mort, J.S. J EXP ORTOP (2014) 1: 8. https://doi.org/10.1186/s40634-014-0008-7  proteases classes: serine, cysteine, aspartic and metalloproteases glucose or ribose with lysine residues in the core protein Roughley, P.J. & Mort, J.S. J EXP ORTOP (2014) 1: 8. https://doi.org/10.1186/s40634-014-0008-7 Degradation of proteoglycans 1. Phagocytosytosis to cell (enzymes: endosulfatase, heparanase) 2. The saccharide chains degradation in lysosomes by enzymes lysosomal glycosidases (defeciency of these enzymes causes dissease oligosacharidosis)  Proteoglycan Function Proteoglycans are multifaceted molecules that serve several biological functions: Proteoglycans are part of the extracellular matrix of a cell and are involved in the extracellular matrix function of swelling and hydration that helps the cell to withstand the compression forces: In cartilage forms the structural component of the tissues that help in hydration, lubrication, and protection Proteoglycan serves to promote growth factor sequestering Proteoglycans are also involved in inhibiting or inducing angiogenesis or even enhanced angiogenesis Proteoglycans are also involved in moderating cell growth, cell proliferation, adhesion and regulation Cell surface proteoglycan is also involved in intercellular cell signaling pathways Proteoglycans are also involved in the release of inflammatory cytokines and chemokines from inflammatory cells  Extracellular matrix Cartilage Cartilage Collagen type Collagen type II, II, up up to to 25 25 %% of of dry dry weight, weight, Responsible for Responsible for the the tensile tensile strength strength collagens IX collagens IX and and XI XI are are in in lower lower proportion proportion Proteoglycans (mainly Proteoglycans (mainly aggrecan aggrecan and and hyaluronan) hyaluronan) Dampen mechanical Dampen mechanical pressures pressures Elastic cartilage Elastic cartilage contains contains abundant abundant elastic elastic fibers fibers The elasticity The elasticity of of structures structures like like pharynx, epiglottis, pharynx, epiglottis, and and pinna pinna Bone Bone Mainly collagen Mainly collagen type type I I Elasticity to Elasticity to avoid avoid bone bone fragility fragility Calcium phosphate Calcium phosphate crystal crystal (70% (70% of of dry dry weight) weight) Stiffness and Stiffness and hardness hardness Proteoglycans (chondroitin Proteoglycans (chondroitin sulfate sulfate 67-97 67-97 %) %) Organization of Organization of collagen collagen fibers fibers  Aging of cartilages Extracellular matrix proten syntesis in chondrocytes↓ Less voluminous, cartilage Extracellular matrix decreased water content resistance to compression Proteoglycan molecules shorter compressive stiffness Proteoglycans—smaller aggrecan molecules with a Lower mechanical properties higher keratin/chondroitin sulphate ratio Hyluronic acid is shorter Glycation of collagen ↑  Osteoarthritis Most effected are cartilages and main cells chondrocytes. downregulation of proteoglycans gene expression The reduced proteoglycan content low proteoglycan synthesis decreases compressive modulus of collagen synthesis increases cartilage and consequently exposes the tissue to greater strains when change from collagen type II to type I exposed to mechanical stress. matrix metalloproteinases expression↑ interleukin 1β (IL-1β, IL-12, IL-15), chemokines, nitric oxide (NO)↑ The changes in extracellular matrix composition and structure late phase apoptosis of chondrocytes inhibits mesenchymal stem cells chondrogenic differentiation. Maricela Maldonado, Jin Nam , The Role of Changes in Extracellular Matrix of Cartilage in the Presence of Inflammation on the Pathology of Osteoarthritis, Biomed Res Int. 2013: 284873.  Glycoproteins Structure: proteins 85-90%, carbohydrates 10-15% Glycosylation - post-translational modification by enzymes covalently attaching saccharide chains to protein chain Functions of saccharide chains: Modulation of protein molecule: viscosity, charge, conformation, increase solubility Prevent degradation of protein by proteinases  Glycosylation The sugars found in glycoproteins: glucose galactose Sacharide chains: mannose Short fucose Branched N-acetylgalactosamine Have negative charge/ N-acetylglucosamine no charge N-acetylneuraminic acid No repeating Xylose disaccharide units Types of glycoproteins structure: 1. N-linked glycoproteins synthesized and modified in the cell, the rough endoplasmic reticulum and the Golgi apparatus N-linked glycoproteins have carbohydrates (14 or more sugar residues) attached to the side chain of asparagine residues part of the cell membrane or are secreted 2. O-linked glycoproteins synthesized in the Golgi apparatus the addition of of a single sugar residue to the hydroxyl side chain of serine or threonine residues part of the extracellular matrix Types of glycoproteins structure: While O-linked and N-linked glycoproteins are the most common forms, other connections are also possible: P-glycosylation occurs when the sugar attaches to the phosphorus of phosphoserine C-glycosylation is when the sugar attaches to the carbon atom of an amino acid. An example is when the sugar mannose bonds to the carbon in tryptophan Glypiation is when a glycophosphatidylinositol (GPI) glycolipid attaches to the carbon terminus of a polypeptide  How are glycoproteins formed in the cell? 1. Protein synthesis in rogue endoplasmic reticulum 2. In Golgi oligosacharide chains are synthesized by highly specific enzymes glycosyltransferases from nucleotide sugars (UDP N- galactosamine) 3. Glycosylation is the enzymatic attachment of sugar to protein Glycosylation can occur as a type of post-translational modification Glycosylation happen co-translationally (N-glycosylation)  Glycoproteins secretion from the cell incorporation into to extracellular matrix the cell membrane Glycoproteins are incredibly diverse and have myriad functions within organisms, including roles in development, growth, homeostasis, and survival They are crucial for cellular interactions; Secreted glycoproteins can act as signaling molecules Membrane-bound glycoproteins can function as the surface receptors to which those signaling molecules bind Binding cell and extracellular matrix components together (collagen or proteoglycans)  Glycoproteins secretion from the cell incorporation into to extracellular matrix the cell membrane Glycoproteins are not constitutive but temporary proteins in the extracellular matrix: the embryonary period, in normal tissue development, tissue repairing after pathological damage tissue under remodeling processes  Laminin Structure: - α-proteins (α1...α5) - β-subunit (β1...β3) - γ-forms (γ1...γ3) Thus, there is a potential for the formation of as many as 45 different combinations of these three subunits. However, only 18 have been discovered (typical laminin 111, composed of α1β1γ1) doi: 10.4161/cam.22826  Fibrillin Large glycoprotein (350kDa) Types: fibrilin-1, fibrilin-2, firilin-3 Structure: microfibril (10–12 nm) proline-rich region high content of disulphide bonds Ca2+ binding sites Main function: tissue integrity form elastic fibers in connective tissue non-elastic tissue provide tensile strength responsible for adhesion of different extracellular components Mutation in fibrilin-1 protein impair structural integrity in the skeleton, the eye and cardiovascular system (Marfan syndrome)  Fibronectin Types: soluble plasma fibronectin, insoluble fibronectin in cell and extracellular matrix Structure: protein dimmer at C terminal connected by disulphide bonds 4 binding domains: Collagen type I, II and III Heparin sulphate Hyaliuronic acid Fibrin Functions: Maintain cytoskelet and extracellular matrix shape, cell adhesion, growth, migration, differentiation, wound heeling  Osteocalcin The most abundant non-collagenous protein in bone matrix Produced by osteoblasts as pre-pro-osteocalcin, active form 49 amino acid glutamate-rich polypeptide Contains γ-carboxyglutamic acid Osteocalcin is in parallel to collagen fibrils, which run longitudinally to bone, and it was required for optimal bone strength Osteocalcin as a biochemical marker of bone formation (Paget’s disease, renal osteodystrophy, primary hyperparathyroidism) Osteocalcin increases during periods of rapid growth, such as in children during the first year of life and during puberty  Biomarkers of osteoporosis Although biochemical markers of bone turnover cannot be used to diagnose osteoporosis, they can be measured in serum and/or urine, allowing a dynamic assessment of bone activity and health Bone formation markers: bone alkaline phosphatase (bALP), osteocalcin (OC), procollagen type I aminotellarpropeptide (P1NP), Bone resorption markers: deoxypyridoline (DPD), pyridinoline, N- and C-terminal telopeptides of type I collagen (NTx and CTx, respectively), and which have some prognostic significance for fracture Biochemical tests: vitamin D status parathyroid hormone (PTH) fibroblast growth factor 23 (FGF23) Vitamin D deficiency results in a reduced absorption of calcium and phosphate from the gut, which then increases the absorption of these ions by the bones, increasing PTH production. An inverse association of circulating PTH levels with calcium intake is generally observed, implying that PTH decreases with increasing dietary calcium intake. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9944083/