WEEK 7 (Extracellular matrix) Flashcards
What is the importance of the extracellular matrix?
- acts as a filler that lies between the tightly packed cells in a tissue
- retains a level of water and homeostatic balance
- holds the cells together
- keeps cells from moving to other locations and prevents large molecules and particles from reaching contiguous and distant cells
What are the basic components of the extracellular matrix?
- structural proteins
- proteoglycans
- adhesion proteins (link components of matrix to each other and to cells)
- collagens, elastin and laminin (fibrous proteins/principal structural proteins of connective tissue)
What are the properties of Proteoglycans?
- form gel of ECM
- found in interstitial connective tissue (synovial fluid, vitreous humor, arterial walls, bone, cartilage, cornea)
- variety of proteins in the matrix (collagen, elastin, fibronectin, laminin)
- consist of polysaccharides called GLYCOSAMINOGLYCANS (GAGs) linked to a core protein
- may contain over 100 glycosaminoglycan chains and consist of up to 95% carbohydrate by weight
- interact with a variety of proteins in the matrix (collagen, elastin, fibronectin, laminin)
What are glycosaminoglycans composed of?
repeating units of disaccharides
What are Glycosaminoglycans (GAGs)?
Long, unbranched, heteropolysaccharides composed of repeating disaccharide chains where one of the sugars is an N-acetylated amino sugar (either N-acetylglucosamine or N-acetylgalactoseamine) and the other is an acidic sugar
What glycosaminoglycan is the only exception?
Keratan Sulfate
contains galactose rather than an acidic sugar
What are the amino sugars usually found in glycosaminoglycans?
either D-glucosamine or D-galactosamine
the amino group is usually acetylated, eliminating its positive charge.
What are the acidic sugars usually found in glycosaminoglycans?
either D-glucuronic acid or L-iduronic acid
What are the characteristics of D-glucoronic acid or L-iduronic acid?
- uronic sugars
- contain carboxyl groups that are negatively charged at physiologic pH
- carboxyl groups and sulphate groups give GAGs their strongly negative nature
What gives Glycosaminoglycans their strongly negative nature?
carboxyl groups and sulphate groups
What mechanism do Proteoglycans follow?
The negatively charged carboxylate and sulfate groups on the proteoglycan bind to the positively charged ions and form hydrogen bonds with trapped water molecules, creating a hydrated gel
What are the properties of the gel?
- provides a flexible mechanical support for the ECM
- acts as a filter
- allows diffusion of ions, H2O and small molecules
- slows diffusion of proteins and movement of cell
- acts as a lubricant
What is the importance of Hyaluronan?
- the only glycosaminoglycan occurring as a single long polysaccharide chain
- only glycosaminoglycan not sulphated
- distributed widely throughout connective, epithelial and neural tissues
What is the effect of the high concentration of negative charges found in GAGs?
repeating disaccharide chains tend to be:
- extended in solution
- repel each other
- surrounded by a shell of water molecules
What happens when a solution containing GAGs is compressed and when the compression is released?
When a solution containing GAGs is compressed, the water is squeezed out and the GAGs are forced to occupy a smaller volume
When the compression is released, the GAGs spring back to their original, hydrated volume because of the REPULSION of their negative charges
What does the compression property of GAGs contribute to?
- resilience of cartilage
- synovial fluid
- vitreous humor of the eye
Describe the structure of Proteoglycans
- contain many chains of GAGs (mucopolysaccharides)
- function extracellularly
- long, negatively charged glycosaminoglycan chains repel each other (therefore occupies a very large space and acts as “molecular sieves” determining which substances enter/leave cells
- differ in monosaccharides present in their repeating disaccharide units
- GAGs are linked to proteins (except for hyaluronic acid)
Describe the structure of Proteoglycans
- contain many chains of GAGs (mucopolysaccharides)
- function extracellularly
- long, negatively charged glycosaminoglycan chains repel each other (therefore occupies a very large space and acts as “molecular sieves” determining which substances enter/leave cells
- differ in monosaccharides present in their repeating disaccharide units
- GAGs are linked to proteins (except for hyaluronic acid)
Which amino acids are GAGs usually attached covalently to?
Serine & Threonine
Which amino acid is Keratin sulfate I usually attached to?
Asparagine
What is the structure of glycosaminoglycans?
- core protein
- serine side chain
- linkage region (galactose-galactose-xylose)
- repeating disaccharide units (acidic sugar-amino sugar)
Describe the linkages found in glycosaminoglycans
GAGs attached to core protein via covalent linkage are most commonly through a trihexoside (galactose-galactose-xylose) and a serine residue in the protein. An Glycosidic bond is formed between the xylose and the hydroxyl group of the serine.
What are the properties of the association between proteoglycan monomers and one molecule of hyaluronic acid?
- form proteoglycan aggregates
- association is not covalent
- occurs primarily through ionic interaction between the core protein and the hyaluronic acid
- association is stabilised by additional small proteins called LINK PROTEINS
Describe the structure and properties of Chondroitin 4 sulphates and Chrondroitin 6 sulphates
- Disaccharide unit consists of N-ACETYLGALACTOSAMINE (with Sulphate on either Carbon 4 or Carbon 6) and GLUCURONIC ACID
- most abundant GAG in the body
- found in cartilage, tendons, ligaments and aorta
- form proteoglycan aggregates, through non-covalent association with hyaluronic acid
- in cartilage, binds collagen and holds fibers in a tight, strong network
- formation of bone, cartilage, cornea
Describe the structure and properties of Dermatan Sulfate
- Disaccharide unit consists of N-acetylgalactoseamine and L-iduronic acid (with variable amounts of glucoronic acid)
- Found in skin, blood vessels and heart valves
- function: transparency of cornea
Describe the structure and properties of Keratan Sulfates I and II
- disaccharide units consists of N-acetylglucosamine and Galactose (no uronic acid); sulfate may be present on carbon 6 of either sugar
- most heterogenous GAG since they contain additional monosaccharides such as L-fucose, N-acetyl-neuraminic acid and mannose
- KS I is found in corneas
- KS II is found in loose connective tissue proteoglycan aggregates with chondroitin sulfate
- function: transparency of cornea
Describe the structure and properties of Hyaluronic acid
- Disaccharide units consist of N-acetylglucosamine and Glucoronic acid
- Different from other GAGs since not sulphated, not covalently attached to protein and not limited to animal tissue but also found in bacteria
- serves as a LUBRICANT and SHOCK ABSORBER
- Mediates CELL MIGRATION in EMBRYOGENESIS, MORPHOGENESIS & WOUND HEALING
- found in synovial fluid of joints, vitreous humor of the eye, the umbilical cord, loose connective tissue and cartilage
Describe the structure and properties of Heparin
- Disaccharide units consist of Glucosamine and Glucuronic or Iduronic acid
- Alpha-linkage joins the sugars
- Unlike other GAGs which are extracellular, Heparin is an intracellular component of mast cells that line arteries, especially in liver, lungs and skin
- Serves as an anticoagulant
- Causes release of lipoprotein lipase from capillary walls
Describe the structure and properties of Heparan Sulfate
- Disaccharide units consist of Glucosamine and Glucuronic acid or Iduronic acid
- Same structure as Heparin except some glucosamines are acetylated and there are fewer sulfates
- Extracellular GAG found in basement membrane and as a ubiquitous component of cell surfaces
- Function: component of skin fibroblasts and aortic wall & commonly found on cell surfaces
What are the functions of Proteoglycans?
- determine which substances enter/leave cells
- give resilience and flexibility to substances (permits compression and re-expansion of molecule)
What are the main types of Glycosaminoglycans found in cartilage proteoglycans?
Chondroitin sulfate and Keratin sulfate
How do infections spread in regards to the ECM?
Infections spread because the infectious agent alters the “containing” capacity of the ECM
How do cancer cells metastasise?
By altering the integrity of the matrix
Which diseases are caused due to the damage of the functional capacity of the matrix?
Rheumatoid arthritis and Osteoarthritis
What do alterations in the matrix of the renal glomerulus allow?
proteins to be excreted into urine
Which connective tissue disorders are caused by a structurally and functionally abnormal matrix?
Ehlers-Danlos syndrome & Marfan syndrome
What do functional properties of a normal joint depend on?
The presence of a soft, well-lubricated, deformable and compressible layer of cartilaginous tissue
Describe the synthesis of Proteoglycans
Protein component of proteoglycans are synthesised on the Endoplasmic Reticulum which enters the lumen of ER where initial glycosylations occur. Proteoglycans are synthesised in the ER and Golgi complex.
1) A sugar is attached to a serine, threonine or asparagine residue of the protein. Additional sugars, donated by UDP-sugar precursors add sequentially to the non-reducing end of the molecule.
[Glycosalation occurs initially in the lumen of the ER and subsequently in the Golgi complex via Glycosyltransferases]
2) Once initial sugars are attached to the protein, alternating action of glycosyltransferases adds the sugars of repeating disaccharide to the GAG chain
3) Sulfation occurs after addition of sugar. 3’-phosphoadenosine 5’-phosphosulfate (PAPS) activates sulfate and provides sulfate groups
4) Epimerase converts glucuronic acid residues to iduronic acid residues
What are Glycosyltransferases?
Enzymes that add sugars to the chain
What happens to proteoglycans after synthesis?
Proteoglycans are secreted from the cell
What does the final structure of a proteoglycan resemble?
A bottlebrush with many GAG chains extending from the core protein
What are the properties of proteoglycan aggregates?
- proteoglycan monomers can associate with one molecule of hyaluronic acid to form proteoglycan aggregates
- association is not covalent and occurs primarily through ionic interactions between the core protein and hyaluronic acid
- association is stabilised by additional small proteins called LINK PROTEINS
What do proteoglycans interact with?
Adhesion proteins and fibronectin which is attached to the cell membrane protein integrin
Cross-linked fibers of collagen also associate with this complex
What are the properties of proteoglycans in cartilage?
- long polysaccharide side chains of proteoglycans in cartilage contain many anionic groups
- high concentration of -ve charges attracts cations that create a high osmotic pressure within cartilage, drawing water into this specialised connective tissue and placing collagen network under tension
- at equilibrium, tension balances the swelling pressure caused by proteoglycans
- complementary roles of this macromolecular organisation give cartilage its resilience
- cartilage can withstand the compressive load of weight bearing and then re-expand to its previous dimensions when that load is relieved
Describe the synthesis of Chondroitin Sulfate
Sugars are added to the protein one at a time with UDP-sugars serving as precursors
1) A Xylose residue is added to a serine in the protein
2) 2 Galactose residues are then added, followed by glucuronic acid (GlcUA) and N-acetylglucosamine (GalNac)
3) Subsequent additions occur by the alternating action of 2 enzymes that produce the repeating disaccharide units
Describe what happens in Proteoglycan degradation
Lysosomal enzymes degrade proteoglycans, glycoproteins and glycolipids brought into the cell by ENDOCYTOSIS. Lysosomes fuse with the endocytotic vesicles and Lysosomal proteases digest the protein component.
Carbohydrate component is degraded by lysosomal glycosidases.
ENDOGLYCOSIDASES = cleave chains into shorter oligosaccharides
EXOGLYCOSIDASES = specific for each type of linkage and removes the sugar residues, one at a time, from reducing ends
What are Mucopolysaccharidoses?
Hereditary diseases caused by a deficiency of any one of the lysosomal degradative enzymes that break down glycosaminoglycans (e.g heparan sulfate, dermatan sulfate and/or keratin sulfate)
What are deficiencies of lysosomal glycosidases?
Occur when partially degraded carbohydrates from proteoglycans, glycoproteins and glycolipids accumulate within membrane-enclosed vesicles inside cells causing an enlargement of organ with impairment of its function
What are the properties of Mucopolysaccharidoses?
- progressive disorders
- characterised by lysosomal accumulation of GAGs in various tissues causing a range of symptoms such as skeletal & ECM deformities and intellectual disability
- all autosomal-recessive disorders (except Hunter Syndrome which has X-linked inheritance)
DIAGNOSIS: measuring the patient’s cellular level of the lysosomal hydrolase
TREATMENT:
- bone marrow and cord blood transplants (transplanted macrophages produce the enzymes that degrade GAGs; have been used to treat Hurler & Hunter syndromes)
- enzyme replacement therapy (does not prevent neurologic damage)
Describe the symptoms and causes of Hurler syndrome
DEFICIENT ENZYME: alpha-L-iduronidase
ACCUMULATED SUBSTRATE: heparan sulfate & dermatan sulfate
INHERTANCE: Autosomal Recessive
SYMPTOMS:
- development delay
- skeletal abnormalities
- airway obstruction
- corneal clouding
- hepatosplenomegaly
Describe the symptoms and causes of Hunter syndrome
DEFICIENT ENZYME: iduronate-2-sulfatase
ACCUMULATED SUBSTRATE: heparan sulfate & dermatan sulfate
INHERITANCE: X-linked recessive
SYMPTOMS:
- physical deformity
- development delay
- aggressive behaviour
- no corneal clouding
Describe the causes and symptoms of SLY Syndrome
CAUSES:
- Beta-Glucuronidase deficiency
- Degradation of dermatan sulfate and heparan sulfate affected
SYMPTOMS:
- hepatosplenomegaly
- skeletal deformity
- short stature
- corneal clouding
- development disability
Describe the causes and symptoms of Sanflippo Syndrome types A-D
CAUSES:
There are four enzymatic steps necessary for the removal of N-sulfated or N-acetylated glucosamine residues from heparan sulfate
TYPE A: Heparan sulfatase deficiency
TYPE B: N-acetylglucosaminidase deficiency
TYPE C: Acetyl CoA: B-glucosaminide acetyltransferase deficiency
TYPE D: N-acetylglucosamine 6-Sulfatase deficiency
SYMPTOMS:
- severe nervous system disorders
- development disability
What is I-Cell disease?
a rare lysosomal disease named for large inclusion bodies seen in cells of patients with the disease. GlcNAc phosphotransferase is deficient and mannose 6-phosphate is not generated on proteins destined for lysosomes.
What does the lack of M6P (mannose-6-phosphate) on amino acid cause?
It causes precursor acid hydrolases to traffic to the plasma membrane and be secreted constitutively instead of trafficking to lysosomes
What are the causes of the absent acid hydrolases from lysosomes?
macromolecule substrates for these digestive enzymes accumulate within the lysosomes which generates the inclusion bodies that define I-Cell disease
What do high concentrations of lysosomal enzymes found in the patient’s plasma and urine indicate?
Indicates that the targeting process to lysosomes is deficient
What are the symptoms and treatment for I-cell disease?
SYMPTOMS:
- skeletal abnormalities
- restricted joint movement
- coarse (dysmorphic) facial features
- severe psychomotor impairment
TREATMENT: there is no cure and death from cardiopulmonary complications usually occurs in early childhood
Why is I-Cell disease termed a mucolipidosis?
Because it has features in common with the mucopolysaccharidoses and sphingolipidoses
What is Pseudo-Hurler Polydystrophy (ML III)?
a less severe mucolipidosis form of I-cell disease in which the phosphotransferase maintains some residual enzymatic activity and it symptomatically resembled a mild form of Hurler syndrome
What is Pseudo-Hurler Polydystrophy (ML III)?
a less severe mucolipidosis form of I-cell disease in which the phosphotransferase maintains some residual enzymatic activity and it symptomatically resembled a mild form of Hurler syndrome
Describe the stages of the causation of I-Cell disease
1) Mutations in gene cause Mutant GlcNAc phosphotransferase
2) Lack of normal transfer of GlcNAc 1-P to specific mannose residues of certain enzymes destined for lysosomes
3) These enzymes lack M6P and are secreted from cells rather than targeted to lysosomes
4) Lysosomes are deficient in certain hydrolases, do not function properly and accumulate partly digested cellular material manifesting as inclusion bodies
What are Integrins?
Major cellular receptors for ECM proteins
What are the properties of Integrins?
- provide a link between the internal cytoskeleton of cells (primarily actin) and extracellular proteins
- involved in signalling options
- consist of alpha and beta subunit
- can be activated by “inside-out” or “outside-in” mechanisms
- certain integrins, such as those associated with WBCs are normally inactive and get activated during infection
How many alpha/beta dimers have been discovered?
24
What does integrin activation allow them to do?
Bind to vascular endothelial cells (leukocyte adhesion) at the site of infection
What is Leukocyte Adhesion deficiency?
A genetic disorder that results from mutations in the beta 2 integrin in which leukocytes cannot be recruited to the sites of infection
What is the mechanism behind the drugs developed to treat inflammatory and autoimmune disorders?
These drugs block either beta 2 or alpha 4 integrins on lymphocytes to treat autoimmune and inflammatory disorders by interfering with the normal WBC response to cytokines
What are the properties of adhesion proteins?
- Fibronectin & Laminin
- Extracellular glycoproteins
- Link integrins to ECM components
- Contains separate distinct binding domains for proteoglycans, collagen and fibrin allowing binding to various components of the ECM
- Contain specific binding domains for cell surface receptors (Integrins)
- Provide a mechanism for signalling between cells via both internal and external signals
What is the function of Integrins?
Integrins bind to FIBRONECTIN on the external surface, span the plasma membrane and adhere to proteins which bind to the INTRACELLULAR ACTIN filaments of the cytoskeleton
What are Matrix metalloproteinases?
The only proteases that can degrade ECM components and are carefully regulated by the tissue inhibitors of matrix metalloproteinases (TIMPS)
