Glycosaminoglycans & Proteoglycans

Question 1

Glycosaminoglycans

Answer 1

1. GAG’s (glycosaminoglycans) are long, unbranched, heteropolysaccharide chains composed of repeating disaccharide units. These units consist of 1 (N-acetylated) amino sugar (D-glucosamine or D-galactosamine) followed by 1 uronic acid (D-glucuronic acid or L-iduronic acid)
2. The length of the GAG’s varies from repeats of 15 to repeats o fup to 30,000

Question 2

Representative sugars found in GAGs

Answer 2

1. Glucosamine will be found with its amino group acetylated in GAGs
2. GAG oligosaccharide chains are polyanionic (negatively charged):
   
   1. Carboxyl groups on uronic acid
   2. Presence of sulfate groups on amino or hydroxyl groups of sugars; mostly on hydroxyl groups
   3. Elimination of positive charge on amino group by acetylation
   4. All these combined give highly negatively charged molecules

Question 3

Proteoglycans

Answer 3

1. Proteoglycans and GAGs are used interchangeably since they are essential the same thing
2. Proteoglycans are complexes of carbohydrtes (95%) and protein (5%), reaching molecular masses of 2 million Da. Proteoglycans are extremely heterogeneous with respect to such variables as function, tissue distribution, core proteins, and ssociated GAG’s.
3. Extracellular matrix consists of three major components:
   
   1. Collagen and other fibrous proteins
   2. Adhesive proteins (laminin, fibronectins)
   3. Proteoglycans
4. ECM is highly diverse functionally as well as biochemically
5. Most human cells are totally dependent on ECM. Loss of this dependence (anchorage dependence) is characteristic of cancer cells
6. Resilience of cells with GAGs:
   
   1. Presence of negative charges attracts many counter-ions, which lead to hydration
   2. Hydratory properties of GAG’s are critical for specific function of being able to be compressed and relaxed

Question 4

GAG classification

Answer 4

1. The GAG’s are classified on the basis of:
   
   1. Composition of disaccharides
   2. Glycosidic linkage
   3. Amount and location of sulfation
2. GAG’s (and proteoglycans) have been shown to play critical roles in development and homeostasis as a function of their hydratory ability, to alter protein-protein interactions, and their ability to modulate enzymatic activity.
3. These activities specifically indclude the following:
   
   1. Organization of basement membranes
   2. Formation of hydrated matrices
   3. Binding of cytokines(cell-to-cell communication in immune response), growth factors, morphogens (maintaining shape of the cells)
   4. Co-reception for growth factor receptors
   5. Promotion of cell motility and cell attachment

Question 5

Specific Examples of GAG’s

Answer 5

1. Chondroitin (cartilage) 4- and 6- sulfates
   
   1. Most abundant GAG in the body
   2. Found in cartilage, tendons, ligaments, and aorta, often a part of large aggregates
   3. Participate in compressibility of cartilage
   4. 1 sulfate group on amino sugar
2. Dermatan (skin) sulfate
   
   1. Found primarily in skin, but also in blood vessels and heart valves
   2. 1 sulfate group on amino sugar
3. Keratan sulfates
   
   1. Found primarily in cornea of eye, playing a role in corneal transparency
   2. 2 sulfate groups on amino sugar and urnoic acid each
4. Heparin
   
   1. Intracellular (previous were all extracellular)
   2. Found in secretory vesicles in mast cells, and is released upon injury
   3. Anticoagulant – it binds to Anti-Thrombin III and enhances AT III’s activity as an anticoagulant (thus enhancement of inhibition of thrombin, which is a coagulant)
   4. One of the most negative charged molecules known
   5. Commercial ones is 15,000 Da but in nature is 60-40,000 Da.
   6. 3 Total sulfate groups; 2 on amino and 1 on acid.
5. Hyaluronic Acid
   
   1. Found in high concentration in embryonic tissue; may play a role in cell migration
   2. Important shock absorber in cartilage
   3. Found in bacteria
   4. Not covalently attached to protein
   5. Not sulfated
6. Heparan sulfate
   
   1. Ubiquitous component of cell surfaces

Question 6

Proteoglycans continued

Answer 6

1. Proteoglycans are proteins that are covalently linked to GAG’s
2. There are at least 30 different proteoglycans
3. Representative proteoglycans include the following:
   
   1. Versican
   2. Syndecan, CD 44, Thrombomodulin
   3. Neurocan, brevican, cerebrocan, phosphocan

Question 7

Proteoglycans structure

Answer 7

1. The most common linkage between the core protein and the GAG is an O-glycosidic bond between the xylose on the trihexoside (Gal-Gal-Xyl) and a serine residue in the protein.
2. The core proteins of the proteoglycans are synthesized in the ER. Some of the formation of linkages of sugars to proteins takes place in the ER.
3. The later steps (chain elongation, chain termination, and other modifications [introduction of sulphate groups and epimerization of sugars]) occur in the Golgi.

Question 8

Proteoglycan Aggregates

Answer 8

1. A higher level of organization can occur, in which the proteoglycan (core proteins linked to GAG’s) units interact through ionic association to form proteoglycan aggregates. The proteoglycan units are associated through ionic interaction with hyaluronic acid (between GAG’s and core proteins is O-glycosidic bond, a type of covalent bond)

Question 9

Degradation of GAG’s

Answer 9

1. The degradation of GAG’s occurs in the lysosomes. The GAG’s:
   
   1. Are engulfed by the cell membrane (phagocytosis), forming a vesicle (phagosome)
   2. The vesicle fuses with the lysosome
   3. The GAG’s are hydrolyzed inside the lysosome
2. Digestion of GAG’s requires the participation of both endoglycosidases and exoglycosidases
3. The order of degradation is the reverse of the order of synthesis; “last joined, first rebalanced”
4. Lysosomes are organelles that contain acid hydrolases that degrade macromolecules introduced through phagocytosis or endocytosis; they also engage in autophagy and autolysis.

Question 10

Mutation with lysosomes

Answer 10

1. Mutations associated with the lysosomal hydrolases are known as mucopolysaccharidoses (“mucopolysaccharide” is an old name for GAG)
2. Diagnosis of specific mucopolysaccharidoses is based on analysis of oligosaccharides in the urine; if certain enzyme doesn’t work, you would expect to find the buildup of the substrate of that enzyme since it failed to be degraded
3. The identification of the terminal sugar can indicate which specific enzyme is deficient or absent
4. Diagnosis can be confirmed by DNA testing and the measurement of the activity of specific lysosomal hydrolases
5. Enzyme therapy via bone marrow and cord blood transplants have been used for the treatment of certain symptoms of these diseases.

Question 11

Clinical Correlations

Answer 11

1. Chondroitin sulfate
   
   1. It has been hypothesized that abnormal immune response to GAG’s could be involved in the development of arthritis
   2. It has been suggested that the administration of chondroitin sulfate together with glucosamine might possibly be effective in pain reduction among a subgroup of patients with moderate-to-severe knee pain
   3. It is used in alternative medicine as a treatment for osteoarthritis
2. There are commercially available injectable fillers derived from hyaluronic acid. They are used to reduce naso-labial folds (smile lines) and to augment lips
   
   1. One of these products is thought to function by stretching the dermal fibroblasts in a manner that could stimulate the skin to create more collagen.
3. Hyaluronidase (the enzyme that degrades hyaluronic aicd) is secreted by some bacteria
   
   1. This enzyme helps the bacteria spread by enzymatically loosening the connective tissue matrix and thus increase bacteria’s virulence
4. Hyaluronidase (prepared from animal or recombinant sources) has been used clinically as an adjuvant (“advantageous”) to promote the absorption and dispersion of injected drugs.
5. One of the mucopolysaccharidoses is characterized by the genetic deficiency of alpha-L-iduronidase, a lysosomal hydrolase
   
   1. A reduction of this enzyme activity can result in widespread cellular, tissue, and organ dysfunction.
   2. A synthetic version of this enzyme, laronidase, is presently in use in the treatment of Hurler Syndrome.
6. Hyaluronic acid and hyaluronidase have the potential of possibly serving as markers for detecting head and neck tumors.

Question 12

Summary

Answer 12