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Course 4. Biomolecules and their interactions

Module 19: Structures and conformations of polysaccharide cellulose, amylase, chitin,

carbohydrate conjugates

OBJECTIVE

The main aim of this module is to introduce the students

the importance of polysaccharides

their structures and

to provide insights into how the physical properties of polysaccharides are relevant

for their biological functions

1. INTRODUCTION

Two or more monosaccharides linked to each other by glycosidic bond generate polysaccharides, also

referred to as glycans. Homopolysaccharides consist of one type of monosaccharide while

heteropolysaccharides contain more than one type of monosaccharide. Polysaccharides, as opposed to

proteins and nucleic acids, form branched and linear polymers. The reason for this is that glycosidic

linkage can form between any of the hydroxyl groups of the monosaccharide. Exoglycosidases and

endoglycosdiases are enzymes that hydrolyze monosaccharide units from a polysaccharide.

1.1 Disaccharides

A disaccharide consists of two sugars joined by an O-glycosidic bond. The hemiacetal OH of one

monosaccharide and an OH of the second monosaccharide, dehydrate to establish the bond called

a glycosidic bond. A glycosidic bond is formed between anomeric carbon and the alkoxy oxygen.

The disaccharide lactose (Fig. 1) occurs naturally in milk.

Upon hydrolysis it yields D-galactose and D- glucose.

Its abbreviated name is Gal(β1 4)Glc.

The disaccharide maltose (figure 1) contains two D-glucose residues joined by a glycosidic

linkage between C-1 (the anomeric carbon) of one glucose residue and C-4 of the other.

Figure 1

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1.1.1 Formation of maltose

Maltose is formed when two molecules of glucose are linked to each other by an O-

glycosidic bond. This glycosidic bond is formed when a hydroxyl group of one

glucose molecule (the one on the right in Fig. 2) reacts or condenses with the

intramolecular hemiacetal of the glucose molecule (on the left). A water molecule is

eliminated during this condensation process, resulting in the formation of a glycosidic

bond. On the contrary, if the glycosidic bond is attacked by a water molecule, it is

referred to as hydrolysis. Glycsodic bonds can be attacked by acids but not by bases,

hence disaccharides can be hydrolyzed to release their monosaccharide components by

boiling with dilute acid. N-glycosidic bonds link the anomeric carbon of one sugar to a

nitrogen atom in glycoproteins and nucleotides.

Sugars that can be oxidized by cupric ion (as discussed in module 18) are referred to

as reducing sugars. This reaction can only occur with the linear forms of sugars, which

though exist in equilibrium with the cyclic forms of sugars in aqueous solution. When

the anomeric carbon is involved in the formation of a glycosidic bond, that sugar

cannot exist in the linear form and is referred to as a non-reducing sugar. In the case of

maltose, as the C-1 of the glucose molecule on the left is not involved in forming the

glycosidic bond, it can undergo reactions typical of reducing sugars. Sucrose on the

other hand is defined as a non-reducing sugar.

Figure 2

2. Polysaccharides

Polysaccharides also known as glycans consists of monosaccharides linked together by glycosidic

bonds.

They generally do not have defining molecular weights like proteins. Unlike proteins,

polysaccharides do not require a template for their synthesis.

Polysaccharides can be classified as homopolysaccharides or heteropolysaccharides.

Hydrolysis

Condensation

H2O

H2O

OH

HO

H

OH

CH2OH

H

H

H

HO

OH

H

OH

CH2OH

HOH

H

H

HO

O

Hemiacetal Acetal

OH

HO

H

OH

CH2OH

HH

OH

H

HO

OH

HO

H

OH

CH2OH

HOH

H

HO

α-D-Glucose β-D-Glucose

Hemiacetal

Alcohol

Maltose

α-D-glucopyranosyl-(1 4)-D-glucopyranose

H

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Homopolysaccharide contains only a single type of monomeric unit whereas

hetropolysaccharides consists of two or more different kinds of monomeric units.

2.1 Homopolysaccharides/Storage polysaccharides

Starch and glycogen are considered as storage homopolysaccharides, while cellulose and chitin

serve structural roles in plant cell walls and the exoskeletons of animals.

2.1.1 Starch

Starch is considered the most vital storage polysaccharide in plants while glycogen

serves the same purpose in animal cells. These polysaccharides are hydrated and

stored as large granule inside the cells.

Starch is the storage form of glucose in plants, where it is predominantly stored

in cholorplasts.

It is a branched chain of D-glucose.

It contains a mixture of amylose and amylopectin (Fig. 3).

Amylose is a linear unbranched polymer of α-D-glucose units in a repeating

sequence of α1 4 glycosidic linkages.

Amylose is an isomer of cellulose but these differ from each other in their

structural properties.

Cellulose is also a glucose polymer where glucose residues are linked via β

glycosidic linkages causing it to attain a fully extended conformation that can be

tightly packed.

Amlyose on the other hand, due to the α glycosidic linkages attains a helical

coiled structure that can aggregate irregularly (Fig. 3B).

Amylopectin is a branched polymer of α1 4 glycosidic linkages and with α1

6 branching points that occur at intervals of approximately 25-30 α-D-glucose

residues.

Amylopectin is considered as one of the largest molecules that exist in nature due

to their extensive branching.

It is a reducing sugar and is present abundantly in potatoes and in seed.

If starch were to be stored as monomers, it would increase the intracellular

osmotic pressure. Hence, storing glucose as starch keeps the osmotic pressure inside

the cell under check, preventing the cells from lysis.

Figure 3A

(α1 6) branch point

Reducing endNonreducing end

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Fig. 3B depicts the arrangement of amylose and amylopectin in starch

granules. A double helical structure is formed when amylopectin (red) coils

with itself or with amylose strands Amylose (blue). Starch is mobilized for

energy production when glucose residues are enzymatically cleaved from the

nonreducing ends.

Figure 3B

2.1.2 Glycogen

It is the major storage form of carbohydrate in animals, stored as granules largely

in the liver and in muscle.

The granules consist of several clusters of smaller granules. Each granule is

comprised of one branched glycogen molecule.

It is a highly branched form similar to amylopectin but is more extensively

branched than amylopectin as the α1 6 branching occurs every 8 to 14 D-glucose

residues (Fig.4).

Glycogen has many non-reducing ends, on which glycogen phophorylase can act

to mobilize the breakdown of glycogen to generate free glucose molecules.

Glycogen debranching enzyme acts on the branches.

Both these enzymes are associated, in a tightly bound form with the glycogen

granules making the mobilization of glycogen more efficient.

Glycogen granules are more tightly packed and have more branches than starch.

Figure 4

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2.2 Homopolysaccharides/Structural polysaccharides

2.2.1 Cellulose

It is a linear, unbranched homopolysaccharide of D- glucose units joined

by β1 4 glycosidic linkages (Fig. 5).

It is a structural polysaccharide of plant cell.

Although cellulose forms a part of the human diet, it is not hydrolyzed by

human enzyme system.

Herbivores contain symbiotic microorganisms that secrete cellulases that

hydrolyzes this β1 4 linkage.

Figure 5

2.2.1 Chitin

It is a linear homopolysaccharide composed of N-acetylglucosamine residues joined

by β1 4 linkages (Fig. 6).

It is the principal component of the exoskeleton of insect and crustaceans.

It is the second most abundant polysaccharide in nature.

Figure 6

2.3 Homopolysaccharide folding

The three dimensional structure of polysaccharides are stabilized by hydrogen bonds,

hydrophobic and van der Waals interactions and electrostatic interactions. Hydrogen

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bonding has an important influence on polysaccharide folding due to the presence of

multiple OH in its structure.

The three-dimensional structures of these molecules can be described in terms of the

dihedral angles, ϕ and ψ, about the glycosidic bond. The bulkiness of the pyranose ring

and its substituents, and electronic effects at the anomeric carbon, place constraints on

the angles ϕ and ψ. Thus certain conformations are much more stable than others.

The tightly coiled helix is the most stable structure for starch and glycogen stabilized

by interchain hydrogen bonds (Fig. 7).

Figure 7

2.2 Hetropolysaccharides

Glycosaminoglycans (GAGs)

The extracellular matrix is a gel like material present in the extracellular space of the

tissues. It holds the cells together and provides a porous pathway for the diffusion of

nutrients and oxygen to individual cells. Glycosaminoglycans form an important

constituent of the extacellular matrix (ECM).

They are unique to bacteria and animals and are not present in plants.

These are negatively charged linear polymers with repeating disaccharide units.

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The repeating disaccharide unit consists of acidic sugar (uronic acid) and an

amino sugar (N-acetylglucosamine or N-acetylgalactosamine).

Some glycosaminoglycans also contain esterified sulphate groups.

They attain a extended conformation in solution forming a rodlike helix that

provides maximum separation between the negatively charged sulfate groups.

Specific pattern of sulfated and nonsulfated sugar residues are recognition sites

for variety of protein ligands.

GAGs provide high viscosity and elasticity that is the basis of their providing

strength to the ECM.

Hyaluronic acid

It is an important component of connective tissue, synovial fluid and vitreous

humor of the eye.

Is composed of about 50,000 repeating disaccharide units of D-glucuronic acid

and N-acetyl-D-glucosamine linked by β1 3 bond (Fig. 9).

Forms clear, viscous solution that enables it to act as shock absorber and lubricant

in the synovial fluid of the joints.

Hyaluronidase is an enzyme secreted by certain pathogenic bacteria that

hydrolyzes the hyaluronic acid, making the tissue more susceptible to bacterial

invasion.

All the glycosaminoglycans except hyaluronic acid are found covalently attached

to protein forming proteoglycan.

Figure 9

Because of a large number of anionic groups within hyaluronic acid molecule,

it assumes a extended rod like structure to reduce the repulsive forces amongst

the anionic groups.

The anionic groups bind to cations and water molecules.

Other examples of glycosaminoglycans include chondroitin sulfate, keratan

sulfate, dermatan sulfate, heparin sulfate etc.

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Chondroitin sulfate: consist of N-acetyl-D-galactosamine with sulphate

group, D-glucuronic acid (Figure 10). Provides tensile strength to cartilage,

tendons etc.

Dermatan sulfate: provides elasticity to skin and is present in blood vessels

and heart valves. L-iduronate substitutes the glucuronate residues that

constitute chondroitin sufate. The epimerization of the C5 of glucuronate

residue generates iduronate.

Keratan sulfate: present in cornea, cartilage, horns, hair, nails etc. Acidic

sugar (uronic acid) is not present in keratan sulfate and their sulfate content is

also variable. Repeating disaccharide unit consists of D-galactose and N-

acetyl-D-glucosamine.

The other GAGs differ from hyaluronan in the following aspects:

Other GAGs are shorter polymers.

They are linked to specific proteins resulting in the formation of proteoglycans.

One or more monomeric units differ from that of hyaluronan.

Heparin

Heparin is not a constituent of connective tissue but occurs exclusively in the

intracellular granules of the mast cells that occur in the arterial walls. It has the highest

negative charge density amongst all known biological macromolecule.

It is vital for healing wounds.

Complex formed between heparin sulfate, growth factor and growth factor receptors

initiates the proliferation and differentiation of cells.

The sulfate groups impart a certain pattern to heparin sulfate and this pattern is specific

for specific growth factors.

It is used as an anticoagulant agent. It inhibits coagulation by binding to antithrombin- a

protease inhibitor (Fig. 10).

Heparin is a protease mandatory for clotting of blood and by binding to antithrombin, its

activity is inhibited, thereby preventing blood clotting.

Heparin is added in blood samples obtained for clinical analysis and during blood

transfusion to prevent clotting of blood.

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Figure 10

(a) Chondroitin sulfate (b) Dermatan sulfate

(c) Keratan sulfate (d) Heparin

3. Glycoconjugates

Apart from storage and structural functions, polysaccharides also acts as information carriers.

Carbohydrate covalently joined to a protein or a lipid is called a glycoconjugate.

Important roles of glycoconjugates:

Communication between cells and extracellular mileu.

Label proteins for transport and localization to organelles.

Recognition sites for extracellular signaling molecules such as growth factors or

parasites.

Central players in cell-cell recognition, wound healing, blood clotting etc.

3.1 Proteoglycans

Covalent and noncovalent aggregates between proteins and glycosaminoglycans, in extracellular

matrix, form macromolecules known as proteoglycans. Various types of sulfated

glycosaminoglycan chains are linked covalently to membrane proteins or to secreted proteins to

form proteoglycans. The GAG part of the proteoglycans have the ability to interact to

extracellular proteins through electrostatic interactions with the polysaccharides. Proteoglycans

are involved in various activities such as growth factor activation and adhesion, they also act as

tissue organizers.

The basic proteoglycan unit consists of a “core protein” to which glycosaminoglycan is attached

covalently through a tetrasaccharide bridge (Fig. 11A). The GAGs are attached to a serine residue

to the core protein through the tetrasaccharide bridge. The serine residue is the part of the

consensus sequence –Ser-Gly-X-Gly, where X is any amino acid residue.

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Electron microscope shows that they have a bottlebrush-like molecular architecture (Fig 11B).

Figure 11

A

B

The backbone is a hyaluronate molecule to which the bristles are covalently attached. The bristles

comprise of

a. A core protein to which GAGs are attached

b. Keratan sulfate or chondroitin sulfate are the most prevalent GAGs found attached to the core

protein

c. A link protein stabilizes the interaction between the GAGs and the backbone

d. Small polysaccharides are present near the site of attachment of the core protein to the backbone

e. These oligosaccharides are N-linked to the core protein through the N of the Asn residue

f. The GAGs are attached to the core protein via O-linked oligosaccharides

A single molecule of hyaluronate, which is the backbone can have about 100 core proteins attached

to it. There can be about 50 keratan sulfate and about 100 chondroitin sulfate chains attached to the

core protein. This results in generating proteoglycans that have high molecular weight.

3.2 Glycoproteins

Glycoproteins are carbohydrate-protein conjugates wherein the glycans are smaller, branched and

more diverse than the glycosaminoglycans of proteoglycans. Sugars are attached either to the

amide nitrogen atom in the side chain of asparagine (N-linked) or to the oxygen atom in the side

chain of serine or threonine (O-linked).

(GlcA GalNAc4S)n GlcA Gal Gal

Xyl

Ser

Gly

X

Gly

(β1 3) (β1 4) (β1 3) (β1 3) (β1 4)

Chondroitin sulfate

Carboxyl terminus

Amino terminus

Core protein

Proteoglycan structure depicting the

tetrasaccharide bridge

N-Linked oligosaccharides

Keratan sulfate

Chondroitin sulfate

Hyaluronate

Core protein

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N-linked glycoproteins

Glycoproteins, in which the GLcNAc is attached to the amide of an Asn residue, are referred to as

N-linked glycoproteins. The Asn is part of the sequence Asn-X-Ser/Thr, where X can be any

amino acid except Pro or Asp.

N-glycosylation occurs as the polypeptide chain is being synthesized (cotranslationally) while still

attached to the ribosome.

The synthesis is N-linked proteins is as depicted in Fig. 12.

Figure 12

The synthesis of N-linked glycoproteins is initiated when 9 mannose residues, 3 glucose and 2

GlcNac residues are attached to the Asn of a polypeptide chain that is being synthesized.

This is followed by removal of some of the sugar molecules and this process occurs in the

endoplasmic reticulum lumen. The removal of the sugar molecules continues in the Golgi

apparatus by glucosidases and mannosidases.

Glycosyltransferases present in the Golgi then enzymatically add GlcNac, galactose, fucose and

sialic acid residues to the already existing core oligosaccharide chain.

The processing is either limited, generating high mannose oligosaccharides or is extensive

generating large oligosaccharides containing different kinds of sugar molecules. This results in

the diversity observed in the oligosaccharides of the N-linked glycoproteins.

O-linked glycoproteins

These glycoproteins have oligosaccharides attached to the protein via Ser or Thr residues. All the

O-linked glycoproteins share a disaccharide core β-galactosyl-(1-3)-α-N-acteylgalactosamine to

the hydroxyl group of either Ser or Thr. Other sugar residues that can be rarely attached to Ser or

Thr are galactose, mannose and xylose.

14-residue

oligosaccharide is

attached to Asn of

a polypeptide.

Removal of monosaccharide

units produces a

(mannose)3(GlcNAc)2

oligosaccharide. This

(mannose)3(GlcNAc)2 core

is found in all N-linked

oligosaccharides.

Further trimming and addition of other

sugars yields a variety of N-linked

oligosaccharides.

Core

pentasaccharide

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The O-linked oligosaccharides are attached to the polypeptide chain after it has been synthesized,

that is, posttranslationally in the Golgi apparatus.

The transfer of GalNAc to a Ser or Thr residue on the polypeptide is the initiation step for the

synthesis of the O-linked oligosaccharides.

The Ser and Thr residues are not a part of a consensus sequence unlike the Asn of the N-linked

oligosaccharides. The point of attachment of the oligosaccharides is dictated by the secondary and

tertiary structures of the polypeptide. Glycosyltransferases add sugars in a stepwise manner to

increase the length of the chain.

Summary

Monosaccharides link through glycosidic linkages to generate polysaccharides.

Cellulose and chitin are structural polysaccharides with rigid and extended structures with β-

glycosidically linked glucose residues.

Starch and glycogen are storage polysaccharides with α-glycosidically linked glucose residues.

Glycosaminoglycans are unbranched polysaccharides that consist of uronic acid and amino sugars

that are often sulfated.

Proteoglycans are components of the extracellular matrix and consist of hyaluronate with attached

core proteins.

There are two types of glycoproteins; N-linked and O-linked glycoproteins.