Carbohydrates and Glycobiology

Carbohydrates

Carbohydrates are aldehydes or ketones with at least two hydroxyl groups, or substances that yield such compounds upon hydrolysis.
Many carbohydrates have the empirical formula $(CH<em>2O)</em>n$ .

Classes of Carbohydrates

Monosaccharides: Simple sugars consisting of a single polyhydroxy aldehyde or ketone unit. Example: D-glucose.
Oligosaccharides: Short chains of monosaccharide units (residues) joined by glycosidic bonds.
Disaccharides: Oligosaccharides with two monosaccharide units. Example: sucrose (D-glucose and D-fructose).
Polysaccharides: Sugar polymers with 10 or more monosaccharide units. Examples: cellulose (linear), glycogen (branched).

Principles of Carbohydrate Chemistry

Principle 1: Carbohydrates can have multiple chiral carbons. The configuration of groups around each carbon atom determines how the compound interacts with other biomolecules. Biological evolution selected the D-series for sugars.
Principle 2: Monosaccharides serve as building blocks of large carbohydrate polymers. The specific sugar, the way the units are linked, and whether the polymer is branched determine its properties and function.
Principle 3: Storage of low molecular weight metabolites in polymeric form avoids high osmolarity that would result from storing them as individual monomers. High concentrations of monomeric glucose would cause cells to swell and lyse due to water entry by osmosis.
Principle 4: Sequences of complex polysaccharides are determined by the intrinsic properties of biosynthetic enzymes that add each monomeric unit to the growing polymer. This is different from DNA, RNA, and proteins, which are synthesized on templates.
Principle 5: Polysaccharides assume three-dimensional structures with the lowest-energy conformations, determined by covalent bonds, hydrogen bonds, charge interactions, and steric factors. Starch folds into a helical structure stabilized by internal hydrogen bonds, while cellulose assumes an extended structure with more important intermolecular hydrogen bonds.
Principle 6: Molecular complementarity is central to function. The recognition of oligosaccharides by sugar-binding proteins (lectins) results from a perfect fit between lectin and ligand.
Principle 7: An almost infinite variety of discrete structures can be built from a small number of monomeric subunits. Even short polymers, when arranged in different sequences, joined through different linkages, and branched to specific degrees, present unique faces recognized by their molecular partners.

Monosaccharides and Disaccharides

Stereoisomerism in Sugars

Sugar stereoisomers arise because many of the carbon atoms with hydroxyl groups are chiral centers.
Enzymes that act on sugars are stereospecific.

Aldoses and Ketoses

Monosaccharide backbones are unbranched carbon chains with single bonds linking all carbon atoms.
One carbon atom is double-bonded to an oxygen atom to form a carbonyl group.
Other carbon atoms are bonded to a hydroxyl group.
Aldose: Carbonyl group is at an end of the carbon chain (aldehyde group).
Ketose: Carbonyl group is at any other position (ketone group).

Types based on number of carbons:

Trioses: Simplest monosaccharides with a three-carbon backbone.
Tetroses: Four-carbon backbone.
Pentoses: Five-carbon backbone, component of RNA and DNA.
Hexoses: Six-carbon backbone.
Heptoses: Seven-carbon backbone.

Sweetness

TAS1R2 and TAS1R3 encode sweet-taste receptors.
Binding of a compatible molecule generates a "sweet" electrical signal in the brain, requiring a steric match.

Asymmetric Centers in Monosaccharides

All monosaccharides (except dihydroxyacetone) contain one or more chiral carbon atoms.
Occur in optically active isomeric forms.
Enantiomers: Two different optical isomers that are mirror images.
A molecule with $n$ chiral centers can have $2^n$ stereoisomers.

Fischer Projection Formulas

Used to represent three-dimensional sugar structures on paper.
Horizontal bonds project out of the plane of the paper.
Vertical bonds project behind the plane of the paper.

D and L Isomers

Reference carbon: Chiral center most distant from the carbonyl carbon.
D isomers: Configuration at the reference carbon is the same as D-glyceraldehyde (right in a projection formula); most hexoses in living organisms are D isomers.
L isomers: Configuration at the reference carbon is the same as L-glyceraldehyde (left in a projection formula).

Numbering Carbons of a Sugar

Carbons are numbered beginning at the end of the chain near the carbonyl group.

Cyclic Forms of D-Glucose

Reaction between the aldehyde group at C-1 and the hydroxyl group at C-5 forms a hemiacetal linkage.
Mutarotation: The interconversion of α and β anomers.

Hexose Derivatives

Glucose family: Glucose, Galactosamine, Mannosamine, Glucosamine, N-Acetyl-Glucosamine
Deoxy sugars: Fucose, Rhamnose
Acidic sugars: Glucuronate, Gluconate, Glucono--lactone, N-Acetylneuraminic acid (a sialic acid)

Phosphorylated Derivatives

Sugar intermediates can be phosphate esters (e.g., glucose 6-phosphate).
Stable at neutral pH and bear a negative charge.
Trap sugar inside the cell because most cells do not have membrane transporters for phosphorylated sugars.

O-Glycosidic Bonds

Covalent linkage joining two monosaccharides.
Formed when a hydroxyl group of one sugar molecule reacts with the anomeric carbon of the other.
Readily hydrolyzed by acid.

Common Disaccharides

lactose: (ß-D-galactopyranosyl-(1-4)-ß-D-glucopyranose) - Gal(ß1-4)Glc
Sucrose: (B-D-fructofuranosyl a-D-glucopyranoside) - Fru(2ßa1)Glc = Glc(a12ß) Fru
trehalose: (a-D-glucopyranosyl a-D-glucopyranoside) - Glc(a11a)Glc

Polysaccharides

Most carbohydrates in nature occur as polysaccharides (Mr > 20,000), also called glycans.

Homopolysaccharides and Heteropolysaccharides

Homopolysaccharides: Contain only a single monomeric sugar species; serve as storage forms and structural elements.
Heteropolysaccharides: Contain two or more kinds of monomers; provide extracellular support.

Lengths and Molecular Weights

Polysaccharides generally do not have defined lengths or molecular weights.
This distinction between proteins and polysaccharides is a consequence of the mechanisms of assembly.
There is no template for polysaccharide synthesis.
The program for polysaccharide synthesis is intrinsic to the enzymes that catalyze the polymerization of monomer units.

Storage Forms of Fuel

Storage polysaccharides include starch in plant cells and glycogen in animal cells.
Starch and glycogen molecules are heavily hydrated because they have many exposed hydroxyl groups available to hydrogen bond.

Starch and Glycogen

Starch: Contains two types of glucose polymer, amylose and amylopectin.
- Amylose: Long, unbranched chains of D-glucose residues connected by ( $\alpha$ 1→4) linkages.
- Amylopectin: Larger than amylose with ( $\alpha$ 1→4) linkages between glucose residues and highly branched due to ( $\alpha$ 1→6) linkages.
Glycogen: Polymer of ( $\alpha$ 1→4)-linked glucose subunits, with ( $\alpha$ 1→6)-linked branches; more extensively branched and more compact than starch.

Storage of Glucose and Osmolarity

Hepatocytes in the fed state store glycogen equivalent to a glucose concentration of 0.4 M.
0.4 M glucose in the cytosol would elevate the osmolarity, and the resulting osmotic entry of water might rupture the cell.

Structural Roles

Cellulose: Tough, fibrous, water-insoluble substance; linear, unbranched homopolysaccharide consisting of 10,000 to 15,000 D-glucose units; glucose residues have the β configuration linked by (β1→4) glycosidic bonds; animals do not have the enzyme to hydrolyze (β1→4) glycosidic bonds.
Chitin: Linear homopolysaccharide composed of N-acetylglucosamine residues in (β1→4) linkage; the acetylated amino group makes chitin more hydrophobic and water-resistant than cellulose.

Steric Factors and Hydrogen Bonding

Three-dimensional structures stabilized by weak interactions within or between molecules. Hydrogen bonding is especially important due to the high number of hydroxyl groups in polysaccharides.
Free rotation about both C—O bonds linking the residues is limited by steric hindrance by substituents.

Helical Structure

Most stable three-dimensional structure for the ( $\alpha$ 1→4)-linked chains of starch and glycogen with six residues per turn.

Linear Structure

Most stable conformation for cellulose is a straight, extended chain. Each chair is turned 180° relative to its neighbors.

Polysaccharides in bacteria and extracellular matrix

Peptidoglycan: Rigid component of bacterial cell walls; heteropolymer of alternating (β1→4)- linked N- acetylglucosamine and N-acetylmuramic acid residues; cross-linked by short peptides.
Glycosaminoglycans: Are Heteropolysaccharides of the Extracellular Matrix in ECM – linear polymers composed of repeating disaccharide units; one monosaccharide is always either N-acetylglucosamine or N-acetylgalactosamine and the other is usually a uronic acid – unique to animals and bacteria – some contain esterified sulfate groups
Hyaluronan (hyaluronic acid): alternating residues of D-glucuronic acid and N-acetylglucosamine
chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate: heparan sulfate differ from hyaluronan in three respects: – generally much shorter polymers – covalently linked to specific proteins (proteoglycans) – one or both monomer units differ from hyaluronan. provide viscosity, adhesiveness, and tensile strength to the extracellular matrix
Heparan: highly sulfated, intracellular form of heparan sulfate produced primarily by mast cells, used as a therapeutic agent to inhibit coagulation of blood through its capacity to bind the protease inhibitor antithrombin
Glycosaminoglycans alternating residues of D-glucuronic acid and N-acetylglucosamine

Glycoconjugates: Proteoglycans, Glycoproteins, and Glycolipids

Glycoconjugate = biologically active molecule consisting of an informational carbohydrate joined to a protein or lipid.
Proteoglycans
Glycoproteins
Glycosphingolipids

Proteoglycans

Macromolecules of the cell surface or ECM consisting of one or more sulfated glycosaminoglycan chains joined covalently to a membrane protein or secreted protein.
Major component of all extracellular matrices.

Glycoproteins

Have one or several oligosaccharides joined covalently to a protein.
Found on the outer face of the plasma membrane, in ECM, in blood, and in organelles (Golgi complexes, secretory granules, and lysosomes).
Oligosaccharide portions are heterogeneous and rich in information.

Glycolipids and Glycosphingolipids

Plasma membrane components in which the hydrophilic head groups are oligosaccharides.
Glycosphingolipids are a class of glycolipids with specific backbone structure.
Neurons are rich in glycosphingolipids.
Play a role in signal transduction.

Proteoglycan Unit

Core protein with covalently attached glycosaminoglycan(s).
Tetrasaccharide linker connects to glycosaminoglycan to a Ser residue of the protein.

Membrane Heparan Sulfate Proteoglycans

Syndecans: Single transmembrane domain and an extracellular domain bearing 3–5 chains of heparan sulfate and chondroitin sulfate.
Glypicans: Attached to the membrane by a GPI anchor (a glycosylated derivative of the membrane lipid phosphatidylinositol).

NS Domains

Highly sulfated domains that alternate with domains having unmodified GlcNAc and GlcA residues.

Protein Interactions

Conformational activation.
Enhanced protein-protein interaction.
Coreceptor for extracellular ligands.
Cell surface localization/concentration.

Heparan Sulfate Enhancement

Antithrombin binds to and inhibits the protease thrombin only in the presence of heparan sulfate.
Both proteins are rich in Arg and Lys residues and interact electrostatically with the sulfates of the glycosaminoglycans.

Proteoglycan Aggregates

Supramolecular assemblies of many core proteins all bound to a single molecule of hyaluronan.
Aggrecan interacts strongly with collagen in the ECM of cartilage.

ECM Interactions

Fibronectin has separate domains to bind fibrin, heparan sulfate, and collagen; contains the conserved RGD sequence (Arg–Gly–Asp) to bind integrins.
Integrins mediate signaling between cell interior and ECM molecules.

Attachments of Glycoproteins

O-linked: A glycoside bond joins the anomeric carbon of a carbohydrate to the —OH of a Ser or Thr residue.
N-linked: An N-glycosyl bond joins the anomeric carbon of a sugar to the amide nitrogen of an Asn residue.

Examples of Glycoproteins

Mucins: Secreted or membrane glycoproteins; can contain large numbers of O-linked oligosaccharide chains; present in most secretions.
Proteins of the blood: Immunoglobulins (antibodies), follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone.
Milk proteins: Major whey protein α-lactalbumin.

Glycomics

The systematic characterization of all carbohydrate components of a given cell or tissue, including those attached to proteins and to lipids.

Advantages of Oligosaccharides

Covalently attached oligosaccharides:
- Influence the folding and stability of the proteins.
- Provide critical information about the targeting of newly synthesized proteins.
- Allow specific recognition by other proteins.

Membrane Components

Gangliosides are membrane lipids of eukaryotic cells in which the polar head group is a complex oligosaccharide containing a sialic acid and other monosaccharide residues.
Lipopolysaccharides are the dominant surface feature of the outer membrane of gram-negative bacteria.

Carbohydrates as Informational Molecules: The Sugar Code

Glycobiology

The study of the structure and function of glycoconjugates.
The challenge is to understand how cells use specific oligosaccharides to encode information about:
- intracellular targeting of proteins
- cell-cell interactions
- cell differentiation and tissue development
- extracellular signals

Oligosaccharide Structures

Branched structures, not found in nucleic acids or proteins, are common in oligosaccharides.
Almost limitless variety of oligosaccharides due to differences in:
- stereochemistry and position of glycosidic bonds
- type and orientation of substituent groups
- the number and type of branches

Lectins

Bind carbohydrates with high specificity and with moderate to high affinity.
Functions:
- cell-cell recognition
- signaling
- adhesion
- intracellular targeting of newly synthesized proteins

Selectins

Family of plasma membrane lectins that mediate cell-cell recognition and adhesion in a wide range of cellular processes.
Move immune cells through the capillary wall.
Mediate inflammatory responses.
Mediate the rejection of transplanted organs.

Lectin-Carbohydrate Interactions

Highly specific and often multivalent.
Subtle molecular complementarity allows interaction only with the lectin’s correct carbohydrate binding partners.

Lectin Multivalency

Single lectin molecule has multiple carbohydrate binding domains, which increases effective affinity.

Hydrophobic Effect

Many sugars have a more polar side and a less polar side.