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Introduction

  • The most abundant heteropolysaccharides in the body.
  • Highly negatively charged molecules,with extended conformation that imparts high viscosity to the solution.
  • GAGs are located primarily on the surface of cells or in the extracellular matrix (ECM).
  • Along with the high viscosity of GAGs comes low compressibility, which makes these molecules ideal for a lubricating fluid in the joints.
  • Their rigidity provides structural integrity to cells and provides passageways between cells, allowing for cell migration.

GAGs of physiological significance

The specific GAGs of physiological significance are:

  • Hyaluronic acid,
  • Dermatan sulfate
  • Chondroitin sulfate
  • Heparin
  • Heparan sulfate, and
  • Keratan sulfate.

Chemistry

  • These molecules are long unbranched polysaccharides containing a repeating disaccharide unit. [acidic sugar-amino sugar]n
  • Although each of these GAGs has a predominant disaccharide component, heterogeneity does exist in the sugars present in the make-up of any given class of GAG.

Nature of amino sugars (figure-1)

The disaccharide units contain either of two modified amino sugars,

  • N-acetyl galactosamine (GalNAc) or
  • N-acetylglucosamine (GlcNAc),

Amino sugars

Figure-1- Amino sugars- β-D Glucosamine and β-D Galactosamine

The amino sugar may also be sulfated on carbon 4 or 6 or on non acetylated nitrogen.

Nature of acid sugar

Uronic acid represents acid sugar in the form of:

  • Glucuronate or
  • Iduronate

The acidic sugars contain carboxyl groups that are negatively charged at physiological pH, (figure-2) and together with the sulfate groups, give glycosaminoglycans their strongly negative nature.

Acid sugars

Figure-2- The acid sugars present in Glycosaminoglycans are D- Glucuronate and L- Iduronate.

Structure- function relationship

Because of their large number of negative charges, these heteropolysaccharides chains tend to be extended in solution. They repel each other and are surrounded by a shell of water molecules. When brought together they “slip” past each other. This produces the slippery consistency of mucous secretions and synovial fluid. When a solution of GAG is compressed, the water is squeezed out and GAGs are forced to occupy a smaller volume. When the compression is released the GAGs get back to their original, hydrated volume because of the repulsion of the negative charges. This property contributes to resilience of synovial fluid and vitreous humor of eye.

THE SPECIFIC GAGs OF PHYSIOLOGICAL SIGNIFICANCE ARE:

1) Hyaluronic acid – The repeating disaccharide unit is:

Glucuronic acid and N Acetylglucosamine (figure-3)

(D-Glucuronate + GlcNAc) n

Hyaluronic acid

Figure-3- structure of Hyaluronic acid

Occurrence:  Hyaluronic acid is found in –

  • Synovial fluid,
  • ECM of loose connective tissue, umbilical cord and vitreous humor of the eye.

Function

  • It serves as a lubricant and shock absorber.
  • It is the only GAG that is not limited to animal tissue but is also found in bacteria.
  • Hyaluronic acid is unique among the GAGs because it does not contain any sulfate and is not found covalently attached to proteins.
  • It forms non-covalently linked complexes with Proteoglycans in the ECM.
  • Hyaluronic acid polymers are very large (100 – 10,000 k Da) and can displace a large volume of water.

2) Dermatan sulfate- The repeating disaccharide unit is L-Iduronic acid and N-Acetyl Galactosamine with variable amount of Glucuronic acids (figure-4).

(L-Iduronate + GalNAc sulfate) n

 Dermatan sulfate

Figure-4- structure of Dermatan Sulfate

Occurrence:  It is found in skin, blood vessels and heart valves

3) Chondroitin sulfate- The repeating disaccharide unit is Glucuronic acid and N-Acetyl galactosamine with sulfate on either C-4 or C-6. Based on presence of sulfate group, it may be labeled as Chondroitin-4-Sulfate or Chondroitin-6-Sulfate (figure-5).

(D-Glucuronate + GalNAc sulfate) n

Chondroitin sulfate

Figure-5-Structure of Chondroitin Sulfate

Occurrence:  It is found in cartilages, tendons, ligaments, heart valves and aorta.

Function

It is the most abundant GAG. In cartilages it binds collagen and holds fibers in a tight, strong network.

4) Heparin sulfate – The repeating disaccharide unit is:

L-Iduronic acid and D- Glucosamine with variable amounts of Glucuronic acid. Most glucosamine residues are bound in Sulfamide linkages (figure-6). Sulfate is also found on C-3 or C-6 of Glucosamine and C-2 of uronic acid (An average of 2.5 Sulfate per disaccharide unit)

(D-Glucuronate sulfate +N-Sulfo-D-glucosamine) n

Heparin sulfate

Figure-6- structure of Heparin Sulfate

Occurrence: Heparin is a component of intracellular granules of mast cells lining the arteries of the lungs, liver and skin (contrary to other GAGs that are extra cellular compounds, it is intracellular).

Function– It serves as an anticoagulant.

5) Heparan sulfate: Heparans have less sulfate groups than heparins. The repeating disaccharide unit is same as Heparin. Some Glucosamines are acetylated

Occurrence- It is an extracellular GAG found in basement membrane and as a ubiquitous component of cell surfaces

6) Keratan sulfate –The repeating disaccharide unit is galactose and N-Acetyl glucosamine (No uronic acid). The sulfate content is variable and may be present on C-6 of either sugar (figure-7).

(Gal + GlcNAc sulfate) n

Keratan sulfate

Figure-7- Structure of Keratan sulfate

Occurrence:  cornea, bone, cartilage; Keratan sulfates are often aggregated with Chondroitin sulfates.

Proteoglycans (mucoproteins) 

Proteoglycans are formed of glycosaminoglycans (GAGs) covalently attached to the core proteins. They are found in all connective tissues, extracellular matrix (ECM) and on the surfaces of many cell types. Proteoglycans are remarkable for their diversity (different cores, different numbers of GAGs with various lengths and compositions).

Structure of Proteoglycans

All of the GAGs, except Hyaluronic acid are found covalently attached to protein forming proteoglycan monomers.

Structure of Proteoglycan monomer

A Proteoglycan monomer found in cartilage consists of a core protein to which the linear GAG chains are covalently linked. These chains which each may be composed of more than 100 monosaccharides extend out from the core protein and remain separated from each other because of charge repulsion. The resulting structure resembles a ‘Bottle brush’ (figure-8). In cartilage proteoglycans, the species of glycosaminoglycans include Chondroitin sulfate and Keratan sulfate.

 

Proteoglycan polymer

Figure-8- structure of Proteoglycan monomer (Bottle Brush)

 Linkage between the carbohydrate chain and the protein

The linkage of GAGs such as (heparan sulfates and Chondroitin sulfates) to the protein core involves a specific trisaccharide linker (Galactose-galactose-Xylose). The protein cores of Proteoglycans are rich in Serine and Threonine residues which allow multiple GAG attachments.

An O-Glycosidic bond is formed between the Xylose and the hydroxyl group of Serine. Some forms of Keratan sulfates are linked to the protein core through an N-asparaginyl bond (N-Glycosidic linkage)

Proteoglycan Aggregates- The proteoglycan monomers associate with a molecule of Hyaluronic acid to form Proteoglycan aggregates (figure-9). The association is not covalent, but occurs primarily through ionic interactions between the core protein and Hyaluronic acid. The association is stabilized by additional small proteins called Link proteins.

Proteoglycan aggregate

Figure-9- structure of proteoglycan aggregate

Functions of Proteoglycans

They perform numerous vital functions within the body.

GAG dependent functions can be divided into two classes: the biophysical and the biochemical.

1) The biophysical functions depend on the unique properties of GAGs: the ability to fill the space, bind and organize water molecules and repel negatively charged molecules. Because of high viscosity and low compressibility they are ideal for a lubricating fluid in the joints. On the other hand their rigidity provides structural integrity to the cells and allows the cell migration due to providing the passageways between cells.

2) The other, more biochemical functions of GAGs are mediated by specific binding of GAGs to other macromolecules, mostly proteins. Proteoglycans participate in cell and tissue development and physiology.

3) Heparin acts as an anticoagulant and is used in the clinical practice.

 

 

 

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Glycogen, Starch and Inulin are storage polysaccharides.

1) Glycogen 

  • Glycogen is a readily mobilized storage form of glucose.
  • It is a very large, branched polymer of glucose residues (Figure-1) that can be broken down to yield glucose molecules when energy is needed.
  • Most of the glucose residues in glycogen are linked by α-1,4-glycosidic bonds.
  • Branches at about every eighth to tenth residue are created by α-1,6-glycosidic bonds.
  • It is the storage polysaccharide in animals and is sometimes called, ‘Animal starch’, but it is more branched than amylopectin present in starch.

Glycogen structure

Figure- 1- The structure of glycogen, the branch point is created by α-1,6-glycosidic linkage 

  • It is hydrolyzed by both α and β-amylases and by glycogen phosphorylase. The complete hydrolysis yields glucose.
  • Glycogen on reaction with iodine gives a reddish-brown color.
  • Glycogen is stored in muscle and liver. The concentration of glycogen is higher in the liver than in muscle (10%versus 2% by weight), but more glycogen is stored in skeletal muscle overall because of its much greater mass.
  • Glycogen is present in the cytosol in the form of granules ranging in diameter from 10 to 40 nm.
  • In the liver, glycogen synthesis and degradation are regulated to maintain blood-glucose levels as required to meet the needs of the organism as a whole. In contrast, in muscle,these processes are regulated to meet the energy needs of the muscle itself.

Glycogen is not as reduced as fatty acids are and consequently not as energy rich, but still animals store energy as glycogen?

All excess fuel is not converted to fatty acids. Glycogen is an important fuel reserve for several reasons:

  • The controlled breakdown of glycogen and release of glucose increase the amount of glucose that is available between meals. Hence, glycogen serves as a buffer to maintain blood-glucose levels.
  • Glycogen’s role in maintaining blood glucose levels is especially important because glucose is virtually the only fuel used by the brain, except during prolonged starvation.
  • Moreover, the glucose from glycogen is readily mobilized and is therefore a good source of energy for sudden,strenuous activity. Unlike fatty acids, the released glucose can provide energy in the absence of oxygen and can thus supply energy for anaerobic activity.

2) Starch

  •  It is a polymer of glucose, found in roots, rhizomes, seeds, stems, tubers and corms of plants, as microscopic granules having characteristic shapes and sizes.
  • Most animals,including humans, depend on these plant starches for nourishment.
  • The intact granules are insoluble in cold water, but grinding or swelling them in warm water causes them to burst. The released starch consists of two fractions.
  • About 20% is a water-soluble material called Amylose.
  • The majority of the starch is a much higher molecular weight substance, consisting of nearly a million glucose units, and called amylopectin.

(a) Amylose

  • It is a linear polymer of α-D-glucose, linked together by α 1→4 glycosidic linkages.
  • It is soluble in water,reacts with iodine to give a blue color and
  • The molecular weight of Amylose ranges between 50, 000 – 200, 000.

Structure of Amylose

 Figure-2- Structure of Amylose

(b) Amylopectin 

  • It is a highly branched polymer,insoluble in water, reacts with iodine to give a reddish violet color.
  • The molecular weight ranges between 70, 000 – 1 000, 000.
  • Branches are composed of 25-30 glucose units linked by α 1→4 glycosidic linkage in the chain and by α 1→6 glycosidic linkage at the branch point.

 Amylopectin

Figure-3 – Structure of Amylopectin

Hydrolysis: Hydrolysis of starch with hot dilute acids or by enzymes gives dextrins of varying complexities, maltose and finally D-glucose

3) Inulin

  • Inulin is a polysaccharide of fructose (and hence a fructosan) found in tubers and roots of dahlias, artichokes, and dandelions.
  • It is readily soluble in water and is not hydrolysed by intestinal enzymes.
  • It has a lower molecular weight than starch and colors yellow with iodine.
  • It is used to determine the glomerular filtration rate, Inulin is of particular use as it is not secreted or reabsorbed in any appreciable amount at the nephron allowing GFR to be calculated, rather than total renal filtration. However, due to clinical limitations, Inulin is rarely used for this purpose and creatinine values are the standard for determining an approximate GFR.

 

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Representation of ring structure of monosaccharides

The ring structures of monosaccharides can be represented as follows :

1) Fischer projection

The acyclic structure of a sugar is commonly shown by using a Fischer projection. A Fischer projection is sometimes used to illustrate the cyclic hemiacetal form of a sugar. The presence of an aldehyde group and the hydroxyl groups in an aldose make it possible for these compounds to undergo intramolecular reactions to form cyclic hemiacetal. These cyclic hemiacetals, are often more stable than is the open-chain form of the sugar. The ketoses (keto group containing sugars) form hemiketal rings

 Hemiacetal

Figure-1- In Fischer projection, α anomer has the orientation of OH towards the right side, whereas, in beta anomer, it is towards the left side.

2) Haworth projection

Rules for drawing Haworth projections

Either a six or 5-membered ring is drawn including oxygen as one atom.
Most aldohexoses are six-membered resembling Pyran- an organic compound having a 6 membered ring (Figure-2).
Aldotetroses, aldopentoses, ketohexoses are 5-membered resembling Furan- A five membered organic compound (Figure-2)

 Furan and Pyran

Figure-2- Structure of Furan and Pyran.

Numbering the rings

The numbering is done clockwise starting next to the oxygen (Figure-3)

 Numbering the rings

Figure-3- Numbering the rings

Pyranose and Furanose forms of Glucose (Figure-4)

 Pyranose and furanose forms

Figure-4- Glucopyranose and Glucofuranose forms

In Haworth configuration all groups to the right of carbon backbone in Fischer projection are oriented down (down -right) while all groups to the left of carbon backbone are oriented up, except those around C5,the reverse orientation occurs (Figure-5).

 Alpha and beta anomers (Figure-5)

 Anomers

Figure-5- When drawn in the Haworth projection, the α configuration places the hydroxyl downward, while the β is the reverse.

Mutarotation

Carbohydrates can change spontaneously between α and β configurations through intermediate open chain formation, this leads to a process known as Mutarotation. There is gradual change in optical rotation of the solution. The initial optical rotation is changed to a constant optical rotation characteristic of that sugar.

This can be explained in reference to two experiments

1) When D Glucose is crystallized at room temperature and a fresh solution is prepared, its specific rotation of polarized light is +112ο, but after 12-18 hours it changes to +52.5 ο

2) If the initial crystallization takes place at 98 ο and then solubilized, the specific rotation is found to be +19 ο, which also changes to +52.5 ο within a few hours.

This change in rotation with time is called Mutarotation.

Explanation

At room temperature the alpha form predominates and the specific rotation is+112ο, there is transient ring opening and change in configuration. In the second condition when the crystallization takes place at 98 ο, the Beta form predominates and the specific rotation is+19 ο. Both undergo Mutarotation and at equilibrium one-third molecules are α type and two third are β variety to get the specific rotation of+52.5 ο (Figure-6) . 

 Mutarotation

 Figure-6- Mutarotation of Glucose

 The constant specific rotation is due to the resultant net optical activity of both alpha and beta anomers.

 

 

 

 

 

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Isomerism in monosaccharides

The monosaccharides having asymmetric carbon atoms exhibit isomerism.

Asymmetric carbon atom- It is the carbon atom that is attached to four different groups (Figure-1)

 asymmetric carbon

Figure-1- An asymmetric carbon atom with 4 different attachments

All monosaccharides except- Dihydroxy acetone, have asymmetric carbon atoms (Figure-2)

 Glyceraldehyde and DHA

Figure-2- Glyceraldehyde has an asymmetric carbon atom whereas dihydroxyacetone lacks, thus it does not have isomers.

Based on the presence of asymmetric carbon atoms the following types of isomerism of monosaccharides are observed in the human system-

1) D and L isomerism- The orientation of the —H and —OH groups around the carbon atom adjacent to the terminal primary alcohol carbon (carbon 5 in glucose) determines whether the sugar belongs to the D or L series. When the —OH group on this carbon is on the right (as seen in figure-3), the sugar is the D isomer; when it is on the left, it is the L isomer (figure-3)

 D-L-glucose

Figure-3- D and L isomers of Glucose

Glyceraldehyde has a single asymmetric carbon and, thus, there are two stereoisomers of this sugar.

D-Glyceraldehyde and L-glyceraldehyde . The D and L isomers of monosaccharides are called enantiomers, as they are mirror images of each other (Figure-4)

 D and L isomers of glyceraldehyde

Figure-4- D and L Isomers of Glyceraldehyde

Biological significance

  • Most of the monosaccharides occurring in mammals are D sugars, and the enzymes responsible for their metabolism are specific for this configuration.
  • D-Ribose, the carbohydrate component of RNA, is a five-carbon aldose.
  • D-Glucose, D-mannose, and D -galactose are abundant six-carbon aldoses.
  • Some sugars naturally occur in the L form e.g. L-Arabinose and L-Fucose are found in glycoproteins.

2) Optical Isomerism- The presence of asymmetric carbon atoms also confers optical activity on the compound. When a beam of plane-polarized light is passed through a solution of an optical isomer, it rotates either to the right, dextrorotatory (+), or to the left, levorotatory (–). The direction of rotation of polarized light is independent of the stereochemistry of the sugar, so it may be designated D (–), D (+), L (–), or L (+) – figure-5

For example, the naturally occurring form of fructose is the D (–) isomer. In solution, glucose is dextrorotatory, and glucose solutions are sometimes known as dextrose.

Measurement of optical activity in chiral or asymmetric molecules using plane polarized light is called Polarimetry. The measurement of optical activity is done by an instrument called Polarimeter.

 Optical activity

Figure-5- Rotation of polarized light by an optically active solution

3) Epimers

The compounds with the same molecular formula, but differing in spatial configuration of the attached groups around a single carbon atomonly are called epimers. 

In hexoses, isomers differing as a result of variations in configuration of the —OH and —H on carbon atoms 2, 3, and 4 are known as epimers. Biologically, the most important epimers of glucose are mannose and galactose, formed by epimerization at carbons 2(figure-6) and 4(figure-7), respectively.  

 C2 epimers

Figure-6- Glucose and Mannose are C2 epimers.

 C4 epimers

Figure-7- Glucose and galactose are C4 epimers

Mannose and Galactose are not epimers of each other as they differ in configuration around 2 carbon atoms (figure-8).

 C2 and C4 epimers

Figure-8- Relationship of glucose, galactose and mannose

 4) Aldose-ketose isomerism

Compounds with the same molecular formula but differing in nature of functional group (aldehyde or keto) are aldose ketose isomers.

 Examples- Fructose and Glucose are aldose ketose isomers.

Aldose keotse isomers

Figure-9- Aldose ketose isomers (D-Glucose and D-Fructose)

Fructose has the same molecular formula as glucose but it differs in its structural formula, since there is a potential keto group in position 2, the anomeric carbon of fructose (Figure-9), whereas there is a potential aldehyde group in position 1, the anomeric carbon of glucose.

Glyceraldehyde and Dihydroxyacetone, Ribose and Ribulose are other examples of aldose -ketose isomers.

5) Anomers

In biological system the monosaccharides tend to exist in a ring form. The ring structure of an aldose is a hemiacetal, since it is formed by combination of an aldehyde (C1) and an alcohol group (Mostly C5). Similarly, the ring structure of a ketose is a hemiketal.  The ring can open and reclose allowing the rotation to occur around the carbon bearing the reactive carbonyl group yielding two possible configurations- α and β of the hemiacetal and hemiketal. The carbon about which this rotation occurs is called Anomeric carbon and the two stereoisomers are called Anomers. In alpha anomer the orientation of the OH group is towards the right side whereas in the beta anomer, it is towards the left side. (Figure-10).

 

 alpha and beta anomers

Figure-10- Alpha and beta anomers of glucose

When drawn in the Haworth projection, the α configuration places the hydroxyl downward. While the β is the reverse (figure-11).

 Haworth projections

Figure-11- Alpha and beta anomers of glucose drawn in Haworth projection

 

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