1. Glycosaminoglycans and
Proteoglycans
Dr. M. Jawad Hassan
Compositions are combinations of carbs and protein. Lipids have a non existent role here. Combinations of polysaccharides and proteins. Today, these substances known as glycosaminoglycans, glycan is a synonym for polysaccharide. Next macromolecules that are predominantly proteins, proteoglycans, then more important in functional sense, two lectures on molecular immunology, two areas, immune system that is innate, and we will start to look at some of the molecular aspects of inflammation. Process that governs infections, interactions with microbial world.
Then process where by antibodies are made, acquired immune system is another topic, then extracellular macromolecules fibrous proteins, such as collagen.
First structures, tie them to properties.

2. Extracellular Macromolecules
macromolecule % carb.
glycosaminoglycans* (GAGs) 100
proteoglycans*
glycoproteins
fibrous proteins
Examples of functions:
mechanical support lubrication
cushioning adhesives
cell spacers selective filters
Here are the extracellular macromolecules classified in structural terms, by how much carb or protein they have. As we go down the table, start out with substances that are only carbs, polysaccharides then substances that are predominantly carbohydrates. Then third line, glycoproteins, mostly proteins that have some sugar as part of their structure. As you see fibrous proteins fall into this category, only in that they have token amount of carbohydrate, essentially protein, polypeptide. Then, short list of types of function these substances are involved in. These are here to indicate the diversity of function that these are involved in. These two terms, glycosaminoglycan, often abbreviated as GAG’s, can see that glycan, polysaccharide, and its modified by the fact that the sugar in these glycans has a nitrogen and amino group and that distinguishes them from the typical polysaccharide such as cellulose and glycogen. These go by other names, mucopolysaccharides, heard about those in histo. Then the proteoglycans also go by mucoproteins. Muco meaning that both substances were first found in mucous secretion, characterized from those. That’s why these terms are considered obsolete because they imply that they are confined to mucous secretion, where in fact they are just about everywhere.
* mucopolysaccharides, mucoproteins, respectively 1

3. GAG structure exist as:
A sugar
exist as:
independent molecules e.g., hyaluronate & heparin
parts of larger structures e.g., in proteoglycans
heteropolysaccharides repeating structure: disaccharide (AB)n ABABAB…
where A is usually 1 uronic acid (hexose with C6 as COO– )
& B is 1 glycosamine (amino sugar) derivative
unbranched
glycosidic linkage
anomeric C of 1 unit linked to hydroxyl of adjacent unit
B sugar
Chemical structures of these materials, GAGs have a structure consisting of, polysaccharides, and like all polysacc the units that oppose the polysacc are monosacc linked up in characteristic way. The GAG’s have an independent existence, the molecules are polymers on their own and the two ones we will talk about aout are hyaluronate and heparin, GAG’s of 100 percent polysacc. Also the GAGs are parts of other structures ,the proteoglycans. The GAG structure consists of these two types of sugars, recognize that hexose similar to glucose, except that there is a modified part of this structure, instead of being glucose, which has a hydroxyl group on numebr 6, it’s a carboxyl group. Known as uronic acid. We have glucuronic acid, sugar derived from glucose, and the polymer structure consists of , heterosacc, not every unit is identical. So far, glycogen cellulose and amylose, its glucose over and over, those are homopolysacc. Here its heterosacc, repetition is of disaccharide. If we have the A sugar, and the B sugar, the polymer would be some variation on repeating the A unit linked to the B unit, that’s what makes these polysaccharides. Abbreviate where AB is disaccharide repeated n times. Here is the A sugar, uronic acid, a carboxyl group, and the B sugar is an amino sugar, we have the prototype is glucose but one hydroxyl, number two is now amino group instead of hydroxyl group, glycosamine in general, or if its glucose then glucosamine. These polymers are also unbranched, linear chains, and linkage, these are the three sugars, linkage involves anomeric carbon, number 1 carbon generally, only one that has two bonds to a heteroatom, usually oxygen. And that is known as glycosidic linkage. The linkage then goes to a hydroxyl of adjacent one. 3

4. GAG structure: repeating units4
GAG A sugar B sugar hyaluronate glucuronate N-acetyl glucosamine * 2 5
Glycosidic linkage between A sugar and a B sugar where the linkages are B 1,3, sometimes alpha sometimes beta, always involve number one carbon and then number 3 or 4. this other linkage is beta 1,4. polymer, GAG is hyaluronate, the A sugar is glucuronate and the B sugar is glucosamine where amino group is acetylated.

5. GAG structure: repeating units4
GAG A sugar B sugar hyaluronate glucuronate N-acetyl glucosamine
chondroitin sulfate glucuronate N-Ac galactosamine 4-SO4
dermatan sulfate iduronate "
heparan sulfate glucuronate glucosamine N-SO3, 6-SO4
heparin iduronate 2-SO "
keratan sulfate galactose N-Ac glucosamine 6-SO4
*opposite configuration in iduronate glucuronate/iduronate: epimers at C5 glucose/galactose: epimers at C4 * 2 5
Others where GAG has these names, with chondroitin sulfate the A sugar is glucuronate, but the B sugar is an amino sugar but instead of glucosamine its galactosamine. The difference between glucose and galactose, epimers of one another, number four carbon, put a line that represents the way the hydroxyl group comes off. Minor difference. The other difference, chondroitin has instead of acetyl group, the hydroxyl group is a sulfate. This hydroxyl in number four position in a different direction and sulfated instead of being free hydroxyl group. Another variation is dermatan sulfate. Initially isolated from skin. A sugar is iduronate, difference is config around a single carbon, here its the number 6 carbon, line represents the config of carboxyl, so it comes off down instead of up. Main point is there are structural differences and they are seemingly minor. In some way these small structural diff translate into significant difference in function. Then heparin sulfate, glucuronate is the A sugar, amino sugar is sulfated in tow places, hydroxyl and n sulfate in number 2 position. Heparin is iduronate, sulfated, then keratan sulfate is where A sugar is not a uranic acid, actually galactose. This gives at least some idea of the range of structural variation among these structures. Again in this column they are GAG’s that have these variations of this repeating structure.

6. GAG structure: repeating units
GAG A sugar B sugar hyaluronate glucuronate N-acetyl glucosamine
chondroitin sulfate glucuronate N-Ac galactosamine 4-SO4
heparan sulfate " glucosamine N-SO3 6-SO4
heparin iduronate 2-SO "
dermatan sulfate iduronate N-Ac galactosamine 4-SO4
keratan sulfate galactose N-Ac glucosamine 6-SO4
*opposite configuration in iduronate * 2 5

7. Hyaluronate A B A B A B – – – 1 2 3 4 5 6
mol wt: 106 – 107 (5000 – 50,000 monosaccharide units)
very polar: 2 hydroxyls/unit 6 heteroatoms/unit COO– every other unit
binds cations: Na+, Ca++
Display of HA in motion
A B A B A B
hyastk2.gif –
Huge molecule, at least a million molecular weight. Important property, knowing the hydroxyl groups are there, that this is a polar structure, each unit has two hydroxyl groups and every other one has a charged group, carboxyl group. This will be a polar molecule, poly anion, will bind positively charged things like sodium and calcium, any cationic substance that is out in the extracellular space. As a result of the negative charges, any like charges repel one another, makes it an extended structure. – – 1 2 3 4 5 6
(glucuronate–N-acetyl glucosamine)3 (glcUA–glcNAc)3

8. Hyaluronate: structure & properties6
extended structure (charge repulsion)
hydrophilic: binds 10 –100 × wt in H2O
additional, loosely associated H2O, so that volume occupied ~1000 × weight
Display of HA with glcUAs in CPK
hyacpk2.gif –
Negative charges are spaced maximally, that makes this structure very extended. This is six unit here. Extended structure is a consequence of its high density of negatively charged carboxyl groups. – – 2 3 1 4 5 6
(glcUA–glcNAc) glcUAs in space-filling form (CPK)

9. Hyaluronate solutions viscous, gel–like, compression-resistant
occurrence: EC matrix, esp. in developing tissue healing wounds synovial fluid
functions: lubricant shock absorber flexible cement attachment site path for cell migration
made by fibroblasts
degraded by hyaluronidase hyaluronidase
bacterial hyaluronidase facilitates spread of infection
Alberts et al. Fig
Thousands of these repeated units. Solutions that contain hyaluronate are viscous compared to water itself. They are more akin to a gel like structure. One property is it makes the material more resistant to compression, distortion and change. Find these in various parts of extracellular matrix, tissue that is developing where there is a lot of cell turnover, one of the ways that space is made is this hyaluronate. Wounds that are healing have high concentrations of this. Over time fluid in many joints, synovial fluid, has its properties of something similar to egg white, gelatinous. Glycogen particle, looks like this, if we look at fibrous protein. One molec of hyaluronate enormous volume, at this size or this level, does look like this structure, is not extended, at this level of magnification, this stretch would be a huge number of monomer units. Eventually it does bend slightly and with perhaps fill a relatively compact space. Here are some of the functions that have been attributed to hyaluronate, fluid containing it would act as lubricant. By resisting compression and changes of shape, property as shock absorber, at higher concentration acts as cement, adhesive material that is not a rigid cement but flexible. These molecules are places where cells will attach and by providing space that don’t contain cells, means by which cells can migrate during process of development. Synthesized by the cells known as fibroblasts. They undergo turnover, enzyme that breaks down these macromolecules known as hyaluronidase. Significant fact is that some bacteria secret such enzymes, which when they infect the ECM are important in breaking down the material that is in that space, enables the bacteria to spread. Not all bacteria have this, typically in infection that is more serious, going to because host defenses don’t thwart the tendency for bact to spread out. 7

10. Heparin mol wt ~ 104 ~ 40 monosaccharide units
made & released from mast cells in lungs & liver
heparin
cell
GAG diff because of size, small molecule. Fewer monosacc. Here is the repeating structure, each monomer has two negative charges, so this would be a extended and polyanionic material. In the resting state, heparin is on the surface of the cell. Heparin goes into action when something cleaves the heparin from cells surface, now its free to do its job. 8

11. Heparin mol wt ~ 104 ~ 40 monosaccharide units
made & released from mast cells in lungs & liver
also associated with luminal surface of endothelium
anticoagulant
forms complex with antithrombin III
this complex binds to thrombin, inactivating it
as a result, clot growth is limited
fast-acting, making it therapeutically useful
heparin
cell
Short description is anticoagulant, interrupts and slows process of blood clot. Here is the means by which it does that, something that is used therapeutically, anticoagulating that can be injected and halt clotting. Heparin has the value that it is very fast acting, anticoagulant. 8

12. Extracellular Macromolecules
macromolecule % carb.
glycosaminoglycans* (GAGs) 100
proteoglycans*
glycoproteins
fibrous proteins
Examples of functions:
mechanical support lubrication
cushioning adhesives
cell spacers selective filters
The next category are proteoglycans, that name implies that these are substances that are polysacc. Also contain protein, 5-10%.
* mucopolysaccharides, mucoproteins, respectively

13. Proteoglycans (PGs) Examples
composed of as many as 200 GAG chains covalently bonded to a core protein via serine side chains
molecular weight range: 105 – 107
GAG chains: chondroitin sulfate, heparan sulfate, dermatan sulfate, keratan sulfate
Examples
decorin
many connective tissues
binds type I collagen, TGF-b
perlecan
basal laminae
structural & filtering function
aggrecan
syndecan (slide 13)
from Alberts et al. Fig
Alberts T 19-3: Dcrn GAG chndSO4 /drmSO4
GAG chains
core
protein
The proteoglycans or PG’s contain a variety structurally, always have a GAG and they always have at least one protein. Proteoglycan example of PG, protein in green, generally known as core protein, and GAG is dashed line in red. Many have more than one GAG chain but this one, decorin is the simplest example. In general, GAG chains are linked covalently to the protein and by way of serine side chain, hydroxyl side chain, and they have this wide rage of molecular weight. GAG’s mentioned, these sulfates, these are the names of the GAG parts of these structures. Decorin is found in many connective tissues, often bound to one of the collagen types. Perlecan has different arrangement of core proteins and several GAG chains attached, and then aggregan ,extended core protein, with a very large number of GAG chains attached in this fashion , bottle brush analogy. 9

14. PG in basal lamina of renal glomerulus
adapted from Alberts et al., 3 ed., Fig
network of fibrous proteins & perlecan PG forms filter
entactin
Here is a surface, made up of a few types of proteins, collagen, fibrous protein. Here is proteoglycan that forms part of this structure. Meshwork functions to act as a filter. This part of the kidney in effect filters out big things from little things, little things like water and sodium chloride get through ,but cells and proteins, that are part of blood, if this is filtering blood are held back by meshwork, and they don’t bind to this structure, delicate process. This is an example of how a complex molecular structure is composed of some of these PG’s and fibrous proteins.
GAG: heparan SO4
perlecan
laminin
type IV collagen 10

15. Proteoglycans: aggrecan
~100 GAG chains/molecule
~100 monosaccharides/GAG chain
each "bristle" = 1 GAG chain
each GAG chain is either chondroitin sulfate
or keratan sulfate
GAG chains linked to ser side chains of core protein
The aggrecan, single core protein linked up to a few GAG chains that can be either chondroitin sulfate or keratan sulfate.
based on Alberts et al. Fig
4ed
core
protein
GAG chains 11

16. An aggregate of aggrecans & hyaluronan
 1m 
major GAG–PG in cartilage
link proteins bind non-covalently
with bound H2O, disperses shocks, compressive force
~ cell size
adhesion proteins link to collagen & cells
degraded by chondroitin sulfatase, etc
core protein
link proteins
These get more complex, structure seen in cartilage commonly, consisting of several aggregans, presented by bottle brush structures that are non covalently bound by link proteins to a single large molecule of hyaluronate. That’s what the blue line represents, result of this aggregate is that the material is very good at resisting compressive forces and its huge, roughly the size of a cell. This represents the whole structure of which this picture is just a small part. A few microns in length. These things can get huge, make the point that there are enzymes that break these materials down, and some times there are bacteria that can secrete these enzymes, makes these bacteria bad guys in the sense that they will contribute to destruction of tissues over time. In gingivitis, that goes on to perio disease, this is part of the story, these materials, enzymes that contribute to break down and irreversible breakdown of tissue.
hyalur- onan
keratan sulfate
chondroitin sulfate 12
Alberts et al. Fig

17. Repeating units of some common glycosaminoglycans of extracellular matrix
linear polymers composed of
repeating disaccharide units
Glucoronic acid
N-Acetylglucosamine

18. Proteoglycans: syndecan
cell-surface PG
core protein domains
intracellular
transmembrane
extracellular
5 GAGs attached
functions
interactions
cell-cell
cell-matrix
growth factor receptor
GAG chains
outside
Even though they are primarily ECM, core protein extends inside the cell. Trans membrane domain, and outer part has the GAG chain. This syndecan and PGs like it, important in interaction between cells and cells in extracellular matrix.
inside
core
protein
Lehninger et al. Fig. 9-22 13

19. Proteoglycan structure
Proteoglycans are glycosaminoglycans-containing macromolecules
of the cell surface and extracellular matrix
Proteoglycan structure

20. GAG synthesis & breakdown
activated precursors: UDP–monosaccharide derivatives e.g., UDP–glucuronate
residues added one at a time in Golgi complex
sulfate moieties
degradation
lysosomes
specific glycosidases & sulfatases
mucopolysaccharidoses
genetic disorders
accumulation of GAG due to absence of a specific glycosidase or sulfatase
–UDP –
adenine –
There is the way that they are synthesized, activated precursors are the story as with all polymers. Activated precursor for sugar is sugar linked up to UDP. UDP glucose is activated precursor for glycogen. Sulfate comes from sulfate donor known as PAPs and its often called active sulfate because its is the activated precursor, the yellow part is donated to some acceptor group. All of these are typically physiologically degraded in lysosomes once they are taken into appropriate cell, enzymes that break down the polymers, glycosidase, some more or less unusual disorders, mucopolysaccharidoses that one or more of these enzymes is missing. – – 14

21. GAG synthesis & breakdown
Synthesis of amino sugars
GlcNAc and GalNac (fructose 6 phosphate)
NANA (N-acetyle mannosamin and phophoenol pyruvate)
CMP-NANA synthetase for activation.

22. Synthesis of acidic sugars
Glucoronic acid and L-Iduronic acid
Diet, lysosomal degradation via uronic acid pathway
Active form is UDP-glucoronic acid
Synthesis of carbohydrate chain and addition of sulphates
Xylosyltransferase
sulphotransferases
PAPS is sulphar donor

23. Degradation Acid hydrolases Phagocytosis
Lysosomal degradation (endoglycosidases)

24. Properties of proteoglycans
Glycosylated proteins which have covalently attached highly anionic glycosaminoglycans (GAGs)
Highly hydrated gels (due to charged sugars). Resist compression
-Sulfated glycosaminoglycans (disaccharides) are negatively charged: bind cations and water
Core proteins link to hyaluronic acid (MW: 3 x 106)
Number of disaccharides typically found in each glycosaminoglycan chain
Heparin/Heparan sulfate (n = 15-30)
Keratan sulfate (n = 20-40)
Chondroitin sulfate (n < 250)
Hyaluron (n < 50,000)
Can be in the ECM and on the surface of cells
E.g. Syndecan (integral membrane protein with Heparan sulfate) is present on the surface of epithelial cells

25. MUCOPOLYSACCHARIDOSES (MPS)
Rare inborn errors in the degradation of glycosaminoglycans result in a series of diseases called mucopolysaccharidoses. They are characterized by mental retardation and/or structural defects.
MPS Type I
Hurler’s syndrome results from a deficiency of alpha-L-iduronidase. Heparan sulfate and dermatan sulfate accumulate. There is growth and mental retardation with characteristic facial changes.
MPS Type II
Hunters syndrome is similar to Hurler’s syndrome but the enzyme deficiency is for iduronate sulfatase and the inheritance is X-linked.
MPS Type III
Sanfilipo’s syndrome is caused by a deficiency of one of four enzymes of which three are hydrolases and one is an N-acetyltransferase. There is severe mental retardation but only mild structural features.
Other MPS Types are
IV, VI and VII. There is no MPS Type V.

26. MPS I (Hurler Syndrome)
A deficiency of L-iduronidase leads to mental retardation and structural changes due to accumulation of dermatan sulfate and heparan sulfate 26

27. MPS II (Hunter Syndrome)
X-linked disease due to a deficiency of iduronate sulfatase 27

28. MPS III (Sanfilippo Syndrome)
Deficiency in one of four degradative enzymes leads to severe mental retardation but little structural change 28

29. MPS IV (Morquio Syndrome)
Deficiency of a galactose-6-sulfatase or a beta-galactosidase leads to accumulation of keratan sulfate with normal intelligence but severe deformity 29

30. THANK YOU