Biochemistry II Lecture 1

Topic: Carbohydrates and Glycobiology

What are carbohydrates?

 Carbohydrates are the most abundant biomolecules on Earth.

 Each year, photosynthesis converts more than 100 billion metric tons of CO 2 and H2O
into cellulose and other plant products.
 Certain carbohydrates (sugar and starch) are a dietary staple in most parts of the world,
and the oxidation of carbohydrates is the central energy-yielding pathway in most non-
photosynthetic cells.
 Insoluble carbohydrate polymers serve as structural and protective elements in the cell
walls of bacteria and plants and in the connective tissues of animals.
 Other carbohydrate polymers lubricate skeletal joints and participate in recognition and
adhesion between cells.
 More complex carbohydrate polymers covalently attached to proteins or lipids act as
signals that determine the intracellular location or metabolic fate of these hybrid
molecules, called glycoconjugates.

What is Glycobiology?

 A term frequently attributed to Raymond Dwek, et al (circa 1990) to encompass the

body of research that contributes to understanding: The structure, biosynthesis, and
biology of saccharides
 Glycobiology: studies of the structure and functions of sugars attached to proteins and
lipids.
 Glycoconjugates: formed when monosaccharides, oligosaccharides or
polysaccharides are attached to proteins or lipids.
 Glycoproteins and glycolipids: proteins and lipids to which carbohydrates are
covalently attached, the mechanism of attachment is enzyme catalyzed in vivo.
 Glycan: the carbohydrate component of glycoproteins and glycolipids.

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Glycosylation and glycation

Glycosylation: enzyme catalyzed covalent modification of proteins and lipids; involved specific
sugar donors such as nucleotide and dolichol sugars and glycosyltransferases; glycosylated
products have specific structures and biological functions.

Glycation: process by which reducing sugars spontaneously (nonenzymatically) bind to proteins

and result in production of advanced glycation end-products (AGEs); chemical modification that
occurs in vivo; spontaneous, non-enzyme catalyzed; products are heterogeneous in structure and
are often deleterious to the organisms.

Thus, aspects of glycobiology impact a broad range of disciplines, to name a few:

1. Organic synthetic chemistry

2. Neurobiology
3. Protein biochemistry
4. Reproductive medicine
5. Enzymology
6. Endocrinology
7. Analytic chemistry
8. Cell signaling
9. Structural biochemistry
10. Stem cell biology
11. Cell biology
12. Membrane biophysics
13. Developmental biology
14. Microbiology
15. Genetics
16. Cancer biology
17. Genomics
18. Immunology
19. Proteomics
20. Parasitology
21. Biotechnology

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How do glycans impact

your work?

Depends on who you are:

– Protein biochemist
– Structural biochemist
– Synthetic chemist
– Geneticist
– Molecular Biologist

Monosaccharides

 Monosaccharides can be oxidized by relatively mild oxidizing agents such as ferric (Fe3)
or cupric (Cu2) ion.
 The carbonyl carbon is oxidized to a carboxyl group. Glucose and other sugars capable of
reducing ferric or cupric ion are called reducing sugars.

Disaccharides

 Disaccharides (such as maltose, lactose, and sucrose) consist of two monosaccharides

joined covalently by an O-glycosidic bond, which is formed when a hydroxyl group of
one sugar reacts with the anomeric carbon of the other.
 Glycosidic bonds are readily hydrolyzed by acid but resist cleavage by base.
 Thus, disaccharides can be hydrolyzed to yield their free monosaccharide components by
boiling with dilute acid.

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Polysaccharides-Glycans

 Most carbohydrates found in nature occur as polysaccharides, polymers of medium to

high molecular weight.

 Polysaccharides, also called glycans, differ from each other in the identity of their

recurring monosaccharide units, in the length of their chains, in the types of bonds linking

the units, and in the degree of branching.

 Glycans are saccharides that can be attached to a wide variety of biological molecules

through an enzymatic process called glycosylation to augment their function.

 Homopolysaccharides contain only a single type of monomer;

 Heteropolysaccharides contain two or more different kinds.

 Some homopolysaccharides serve as storage forms of monosaccharides that are used as

fuels; starch and glycogen are homopolysaccharides of this type.

 Other homopolysaccharides (cellulose and chitin, for example) serve as structural

elements in plant cell walls and animal exoskeletons.

 Heteropolysaccharides provide extracellular support for organisms of all kingdoms. For

example, the rigid layer of the bacterial cell envelope (the peptidoglycan) is composed in

part of a heteropolysaccharide built from two alternating monosaccharide units.

 In animal tissues, the extracellular space is occupied by several types of

heteropolysaccharides, which form a matrix that holds individual cells together and

provides protection, shape, and support to cells, tissues, and organs

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Glycans

 The most important storage polysaccharides are starch in plant cells and glycogen in

animal cells. Both polysaccharides occur intracellularly as large clusters or granules

 Of the four fundamental building blocks of life, proteins, carbohydrates (glycans), lipids

and nucleic acids, glycans have received the least attention from researchers.

 Glycans are found in Archaea, bacteria and eukaryotes, and their diverse functions

contribute to physical and structural integrity, extracellular matrix formation, signal

transduction, protein folding and information exchange between cells (and pathogens).

 Authors Stevan Springer and Pascal Gagneux discussed how evolution shaped glycan

diversity. The regulatory capacity and structural diversity of glycans surpasses those of

other biological molecules.

 The authors argue that glycan diversity stems from their role as mediators of cellular

interaction.

 Glycans are the predominant molecule on the cell surface and serve as the first point of

contact between a cell and other cells, the extracellular matrix and pathogens.

 Cells recognize one another because of the saccharides attached to cell surfaces.

 They are present usually as oligosaccharides associated through covalent links to lipids

and/or proteins forming

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Glycoconjugates

 The lipid or protein part is integrated into the cell membrane structure, with the
saccharide part towards the external membrane surface.
 Membrane carbohydrates (2-10% of the membranes) are on the extracellular surface
bounded to lipids or proteins of the membrane, forming glycoconjugates that serve as
docking sites in cell recognition, adhesion and receptor action.
 These sugars include mainly glucose, galactose, mannose, fucose, Nacetyl galactosamine
and N-acetyl glucosamine.

The different kinds of Glycoconjugates include:

Proteoglycans: In the Proteoglycans, the Glycosaminoglycan moiety forms the greater fraction
of the molecule (typically a proteoglycan consists of 95 % of carbohydrates) and is the main site
of biological activity, providing multiple binding sites. They are found mainly in the
extracellular matrix. They are major components of connective tissue.

Glycoproteins: Membrane bound glycoproteins participate in a wide range of cellular

phenomena, including cell recognition, cell surface antigenicity, etc. In the glycoproteins, the
majority of the molecule consist of proteins; they have one or more oligosaccharides attached to
a protein, and they usually are branched and do not have serial repeats, so they are rich in
information, forming highly specific sites for recognition and high affinity binding by other
proteins

Glycolipids: are membrane lipids in which the hydrophilic head groups are oligosaccharides.

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 As in glycoproteins, glycolipids act as specific sites for recognition by carbohydrate

binding proteins.
 The four types of human RBC have different oligosaccharides (antigens) in their cell
membranes. Blood groups depends on the gangliosides (a kind of sphingolipid) in the
surface of the RBC.

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 Glycoproteins and glycolipids on plasma membranes mediate cell identity,

communication, adhesion and/or growth
 Most polysaccharides attached to proteins extend away from the protein’s surface and
probably do not affect the protein structure significantly

Extracellular matrix

 The extracellular space in the tissues of multicellular animals is filled with a gel-like
material, the extracellular matrix, also called ground substance, which holds the cells
together and provides a porous pathway for the diffusion of nutrients and oxygen to
individual cells.
 The extracellular matrix is composed of an interlocking meshwork of
heteropolysaccharides and fibrous proteins such as collagen, elastin, fibronectin, and
laminin.
 These heteropolysaccharides, the glycosaminoglycans, are a family of linear polymers
composed of repeating disaccharide units

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Cellular interactions

Interactions between cells and the extracellular

matrix.

 The association between cells and the

proteoglycan of the extracellular matrix is
mediated by a membrane protein (integrin) and by
an extracellular protein (fibronectin in this
example) with binding sites for both integrin and
the proteoglycan.
 Note the close association of collagen fibers with
the fibronectin and proteoglycan.

Bacterial and Algal Cell Walls Contain Structural Heteropolysaccharides.

 The rigid component of bacterial cell walls is a heteropolymer of alternating (1n4)-linked

Nacetylglucosamine and N-acetylmuramic acid residues
 The linear polymers lie side by side in the cell wall, crosslinked by short peptides, the
exact structure of which depends on the bacterial species.
 The peptide cross-links weld the polysaccharide chains into a strong sheath that envelops
the entire cell and prevents cellular swelling and lysis due to the osmotic entry of water.
 The enzyme lysozyme kills bacteria by hydrolyzing the (1n4) glycosidic bond between
Nacetylglucosamine and Nacetylmuramic acid.
 Lysozyme is notably present in tears, presumably as a defense against bacterial infections
of the eye.

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Peptidoglycan

Polymer of disaccharide:

 network of disaccaharide chains cross linked by short

peptides
 Each disaccahride in this very large molecule composed
of 2 monosaccharide :
 N-acetylglucosamine (NAG) and
 N-acetylmuramic acid (NAM)

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(A.) Oligosaccharides with unique structures (represented as strings of hexagons), components of a

variety of glycoproteins or glycolipids on the outer surface of plasma membranes, interact with
high specificity and affinity with lectins in the extracellular milieu.

 Some
bacteria, such
as H. pylori,
adhere to and
then colonize
or infect
animal cells.

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 Selectins (lectins) in the plasma membrane of certain cells mediate cell-cell interactions,
such as those of T lymphocytes with the endothelial cells of the capillary wall at an
infection site.
 The mannose 6-phosphate receptor/lectin of the trans Golgi complex binds to the
oligosaccharide of lysosomal enzymes, targeting them for transfer into the lysosome.

Working with Carbohydrate

 Establishing the complete structure of oligosaccharides and polysaccharides requires

determination of branching positions, the sequence in each branch, the configuration of
each monosaccharide unit, and the positions of the glycosidic links—a more complex
problem than protein and nucleic acid analysis.
 The structures of oligosaccharides and polysaccharides are usually determined by a
combination of methods: specific enzymatic hydrolysis to determine stereochemistry and
produce smaller fragments for further analysis; methylation analysis to locate glycosidic
bonds; and stepwise degradation to determine sequence and configuration of anomeric
carbons.
 Mass spectrometry and high-resolution NMR spectroscopy, applicable to small samples
of carbohydrate, yield essential information about sequence, configuration at anomeric
and other carbons, and positions of glycosidic bonds.
 Penicillin and related antibiotics kill bacteria by preventing synthesis of the cross-links,
leaving the cell wall too weak to resist osmotic lysis.
 Certain marine red algae, including some of the seaweeds, have cell walls that contain
agar, a mixture of sulfated heteropolysaccharides made up of D-galactose and an L-
galactose derivative etherlinked between C-3 and C-6.
 The two major components of agar are the unbranched polymer agarose (Mr ~120,000)
and a branched component, agaropectin.
 The remarkable gel-forming property of agarose makes it useful in the biochemistry
laboratory. When a suspension of agarose in water is heated and cooled, the agarose
forms a double helix: two molecules in parallel orientation twist together with a helix
repeat of three residues; water molecules are trapped in the central cavity.

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 These structures in turn associate with each other to form a gel— a three-dimensional
matrix that traps large amounts of water.
 Agarose gels are used as inert supports for the electrophoretic separation of nucleic acids,
an essential part of the DNA sequencing process.

Lipopolysaccharides

 The surface feature of gram-negative bacteria outer membrane

 Targets for antibodies

Immune Attack

 Selectins a family of lectins that mediate cell-cell recognition and adhesion (e.g. P-
selectin).
 Neutrophil (white blood cell that initiates immune response) possesses ligands with weak
affinity for integrin and P-selectin.
 Neutrophil flows through blood, but is slowed down when it weakly binds to integrin or
Pselectin expressed on the surface of capillary endothelial cells.
 At site of inflammation/infection there is a high concentration of integrin and P-selectin.
 High concentration overcomes low affinity (kD = µM-mM) for Neutrophil and stops its
movement through the blood.
 The Neutrophil begins extravasation (leaves blood vessel) and enters
inflammation/infected site to initiate immune attack.

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