Advanced Biochemistry 高等生化學 Carbohydrates and Glycobiology 陳威戎

Carbohydrates and Glycobiology

Ah! Sweet mystery of life….. - Rida Johnson Young (lyrics) and Victor Herbert (music), 1910 I would feel more optimistic about a bright future for man if he spent less time proving that he can outwit Nature and more time tasting her sweetness and respecting her seniority. - E. B. White, “Coon Tree”, 1977 Preface

Most abundant biomolecules on Earth. - Photosynthesis: cellulose and other plant products - Dietary staple: sugar and starch - Oxidation: energy-yielding pathway - Structural and protective elements - Lubricate skeletal joints - Recognition and adhesion between cells - Signals determine the intracellular location or metabolic fate Structure and Functional Roles of Carbohydrates

Polyhydroxy aldehydes or ketones, or substances that yield such compounds on hydrolysis. Three major size classes: - Monosaccharides: glucose (dextrose) - Disaccharides: sucrose (cane sugar) - Polysaccharides: cellulose, glycogen Glycosidic linkage (glycosidic bond) Properties and Classification of Carbohydrates

1. Monosaccharides and Disaccharides 2. Polysaccharides 3. Glycoconjugates: Proteoglycans, Glycoproteins, and Glycolipids 4. Carbohydrates as Informational Molecules: The Sugar Code 5. Working with Carbohydrates Carbohydrates and Glycobiology

1. The two families of monosaccharides are aldoses and ketoses. 2. Monosaccharides have asymmetric centers. 3. The common monosaccharides have cyclic structures. 4. Organisms contain a variety of hexose derivatives. 5. Monosaccharides are reducing agents. 6. Disaccharides contain a glycosidic bond. Monosaccharides and Disaccharides

Colorless, crystalline solids, freely soluble in water. Backbone: unbranched carbon chains linked by single bonds Open chain form: one carbonyl group and many hydroxyl groups - aldose and ketose - simplest: two trioses - with four to seven carbon atoms: The two families of monosaccharides are aldoses and ketoses

Representative monosaccharides- two trioses

Representative monosaccharides- two hexoses

The pentose components of nucleic acids

Contain one or more asymmetric carbon: optically active isomeric forms With n chiral centers: have 2 n stereoisomers D, L designation: chiral center most distant from the carbonyl carbon - hydroxyl group on the right: D isomer - hexoses of living organisms: most are D isomers Monosaccharides have asymmetric centers

Three ways to represent the two stereoisomers of glyceraldehyde

Aldoses

Ketoses

Epimers

Sugars in aqueous solution occur predominantly as cyclic structures. Carbonyl + hydroxyl group: hemiacetals and hemiketals Additional chiral center at C-1 or C-2:  and  isomers Pyranose: six-membered ring compound Furanose: five-membered ring compound C-1 or C-2 isomers: anomers, mutarotation Common monosaccharides have cyclic structures

Formation of hemiacetals and hemiketals

Formation of the two cyclic forms of D-glucose

Pyranoses and Furanoses Haworth perspective formulas: Stereochemistry of ring forms of monosaccharides. The ring is not planar, but tends to assume either of two “chair” conformations.

Conformational formulas of pyranoses

Monosaccharides can be oxidized by mild oxidizing agents such as ferric (Fe 3+ ) or cupric (Cu 2+ ) ion. Carbonyl carbon is oxidized to a carboxylate group. Sugars capable of reducing ferric or cupric ion are called: reducing sugars. Fehling’s reaction: a qualitative test for the presence of reducing sugars. Sensitive methods for measuring blood and urine glucose. Monosaccharides are reducing agents

Sugars as reducing agents

Disaccharides consist of two monosaccharides joined by an O-glycosidic bond, formed between a hydroxyl of one sugar and the anomeric carbon of the other. N-glycosyl bonds: anomeric carbon + N in glycoproteins and nucleotides. Sugars with anomeric carbon involved in glycosidic bond: nonreducing sugar. Reducing end Disaccharides contain a glycosidic bond

Formation of maltose

1) Place nonreducing end to the left 2) Give configuration to the anomeric carbon (left) 3) Name the nonreducing residue, distinguish ring forms 4) Indicate the two carbons joined by glycosidic bond (in parenthesis with an arrow connecting the numbers) 5) Name the second residue 6) Use 3-letter abbreviations to shorten the description Maltose: Glc(  1→4)Glc ; Lactose: Gal(  1→4)Glc Naming rules for reducing disaccharides

Some common disaccharides- Lactose

1) - 3) The same as above. 4) Indicate the two carbons joined by glycosidic bond (use double-headed arrow connecting to numbers) 5) Name the second sugar residue as glycoside 6) Use 3-letter abbreviations to shorten the description Sucrose: Glc(  1  2  )Fru Trehalose: Glc(  1  1  )Glc Naming rules for nonreducing disaccharides

Some common disaccharides- Sucrose

Some common disaccharides- Trehalose

1. Monosaccharides and Disaccharides 2. Polysaccharides 3. Glycoconjugates: Proteoglycans, Glycoproteins, and Glycolipids 4. Carbohydrates as Informational Molecules: The Sugar Code 5. Working with Carbohydrates Carbohydrates and Glycobiology

1. Some homopolysaccharides are stored forms of fuel. 2. Some homopolysaccharides serve structural roles. 3. Bacterial and algal cell walls contain structural heteropolysaccharides. 4. Glycosaminoglycans are heteropolysaccharides of the extracellular matrix. Polysaccharides (Glycans)

Homo- and heteropolysaccharides

The most important storage polysaccharides: 1) Starch in plant cells (tubers and seeds) 2) Glycogen in animal cells Both occur intracellularly as large clusters or granules. Both are heavily hydrated- why? Some homopolysaccharides are stored forms of fuel

Electron micrographs of starch granules

Electron micrographs of glycogen granules

Starch contains two types of glucose polymer: 1) amylose: unbranched ; thousands to millions (  1→4) linkage 2) amylopectin: highly-branched ; up to 100 million main chain: (  1→4) linkage branch points: (  1→6) linkage (every residues) Some homopolysaccharides are stored forms of fuel - Starch

The polysaccharides of starch- amylose

The polysaccharides of starch- amylopectin

A cluster of amylose and amylopectin

Like amylopectin, glycogen is a polymer of (  1→4)- linked glucose, with (  1→6)-linked branches. Glycogen is more extensively branched (every 8-12 residues) and more compact than starch. Abundant in the liver and skeletal muscle. One reducing end with as many nonreducing ends as it has branches. Why not store glucose in its monomeric form? Some homopolysaccharides are stored forms of fuel - Glycogen

Dextrans are bacterial and yeast polysaccharides: main chain: (  1→6)-linked poly-D-glucose branches: all (  1→3); some (  1→2) or (  1→4) Dental plaque: rich in dextrans Synthetic dextrans: ex: Sephadex - Chemically cross-linked insoluble materials of various porosities - Size-exclusion chromatography Some homopolysaccharides are stored forms of fuel - Dextrans

A fibrous, tough, water-insoluble substance found in the cell walls of plants. Linear and unbranched poly D-glucose (10,000-15,000 units) like amylose, but with (  1→4) linkage. Most animals:  -amylases only. Termites: readily digest cellulose with symbiotic Trichonympha that secretes cellulase. Ruminants: only vertebrate able to use cellulose with bacteria and protists in rumen that produce cellulase. Some homopolysaccharides serve structural roles - Cellulose

The structure of cellulose

Cellulose breakdown by wood fungi

A linear homopolysaccharide composed of N-acetylglucosamine residues in (  1→4) linkage. Only chemical difference from cellulose: replacement of the –OH group at C-2 with an acetylated amino group. Forms extended fibers and is the principle component of the hard exoskeleton or nearly a million species of arthropods. Some homopolysaccharides serve structural roles - Chitin

Chitin

The rigid component of bacterial cell wall, heteropolymer of alternating (  1→4)-linked N-acetyl-glucosamine and N-acetylmuramic acid residues. Cross-linked by short peptides, prevents swelling and lysis due to osmolarity changes. Lysozyme: hydrolyze the (  1→4)-linkage. Penicillin and related antibiotics: prevent synthesis of the cross-links. Bacterial and algal cell walls contain structural heteropolysaccharides - Peptidoglycan

Peptidoglycan

Certain marine red algae and seaweeds have cell walls that contain agar. Agar: Poly-[D-Gal(  1→4)3,6-anhydro-L-Gal2S] Two major component: agarose and agaropectin. The gel-forming property of agarose makes it useful in the biochemistry laboratory: - Agarose gel: for electrophoretic separation of N.A. - Agar: for the growth of bacterial colonies. - Capsules: for vitamin and drug package. Bacterial and algal cell walls contain structural heteropolysaccharides - Agar

The structure of agarose

Extracellular matrix is composed of: - heteropolysaccharides: glycosaminoglycans - fibrous proteins: collagen, elastin, fibronectin, laminin. Glycosaminoglycans: a family of linear polymers of repeating disaccharide units. One is always either N- acetylglucosamine or N-acetylgalactosamine; the other is a uronic acid, usually D-glucuronic acid or L-iduronic acid. Some are esterified with sulfate. Glycosaminoglycans are heteropolysaccharides of the extracellular matrix

Repeating units of some common glycosaminoglycans - Hyaluronate

Repeating units of some common glycosaminoglycans - Chondroitin 4-sulfate

Repeating units of some common glycosaminoglycans – Keratan sulfate

Repeating units of some common glycosaminoglycans - Heparin

1)Hyaluronic acid - glassy and translucent - lubricants in joints, cartilage, and tendons - hyaluronidase in pathogenic bacteria and sperm 2) Chondroitin sulfate - cartilage, tendon, ligament, and walls of the aorta 3) Dermatan sulfate - skin, blood vessels, and heart valves 4) Keratan sulfate - cornea, bone, horn, hair, hoofs, nails and claws 5) Heparin - natural anticoagulant made in mast cells - bind antithrombin, then bind and inhibit thrombin Glycosaminoglycans are heteropolysaccharides of the extracellular matrix

Interaction between a glycosaminoglycan and its binding protein Heparin Fibroblast growth factor (FGF)

1. Monosaccharides and Disaccharides 2. Polysaccharides 3. Glycoconjugates: Proteoglycans, Glycoproteins, and Glycolipids 4. Carbohydrates as Informational Molecules: The Sugar Code 5. Working with Carbohydrates Carbohydrates and Glycobiology

1. Proteoglycans are glycosaminoglycan-containing macromolecules of the cell surface and extracellular matrix. 2. Glycoproteins have covalently attached oligosaccharides. 3. Glycolipids and Lipopolysaccharides are membrane components. Glycoconjugates: Proteoglycans, Glycoproteins, and Glycolipids

1) Destination labels 2) Mediators of specific cell-cell interactions 3) Cell-cell recognition and adhesion 4) Cell migration during development 5) Blood clotting 6) Immune response 7) Wound healing Biological roles of glycoconjugates

1) Proteoglycans - glycosaminoglycans + proteins - glycosaminoglycans: main site of biological activity - hydrogen bonding and electrostatic interactions - major components of connective tissues 2) Glycoproteins - oligosaccharides + proteins - outer face of membrane ; extracellular matrix, blood - Golgi complexes ; secretory granules ; lysosomes 3) Glycolipids – oligosaccharides + membrane lipids - oligosaccharides: hydrophilic head groups - specific sites for recognition Biologically active molecules of glycoconjugates

Proteoglycan superfamily: at least 30 types - tissue organizers - influence the development of specialized tissues - mediate activities of growth factors - regulate extracellular assembly of collagen fibrils Basic proteoglycan unit: core protein + trisaccharide linker + glycosaminoglycan Secreted into the extracellular matrix (basal lamina) Integral membrane protein (syndecans ; glypicans) Proteoglycans are glycosaminoglycan-containing macromolecules of the cell surface and extracellular matrix

Proteoglycan structure, showing the trisaccharide bridge

Proteoglycan structure of an integral membrane protein - Syndecan

Proteoglycan structure of an integral membrane protein

1) Conformational activation 2) Enhanced protein-protein interaction 3) Coreceptor for extracellular ligands 4) Cell surface localization/concentration Four types of protein interactions with S domains of heparan sulfate

Proteoglycan aggregate of the extracelluar matrix

Interactions between cells and the extracellular matrix - Anchor cells to the extracellular matrix - Direct cell migration in developing tissue - Convey information in both directions across the plasma membrane

Carbohydrate moiety in glycoproteins are smaller but more structurally diverse than the glycosaminoglycans of proteoglycans. O-linked: -OH of Ser or Thr(GalNAc) N-linked: amide nitrogen of Asn(GlcNAc) Membrane protein: glycophorin A Secreted proteins: immunoglobulins and certain hormones, lactalbumin, ribonuclease, etc. Proteins in lysosomes, Golgi complexes, and ER. Glycoproteins have covalently attached oligosaccharides

Oligosaccharide linkages in glycoproteins

Gangliosides: membrane lipids whose polar head group is a complex oligosaccharide containing sialic acid and other monosaccharides. Blood group type determination: identical oligosaccharide moieties as those found in certain glycoprotein, also are blood group type determinants. Lipopolysaccharides: dominant surface feature of the outer membrane of G(-) bacteria. - prime targets of the antibodies - determinants of the serotypes of bacterial strains Glycolipids and lipopolysaccharides are membrane components

Bacterial lipopolysaccharides

1. Monosaccharides and Disaccharides 2. Polysaccharides 3. Glycoconjugates: Proteoglycans, Glycoproteins, and Glycolipids 4. Carbohydrates as Informational Molecules: The Sugar Code 5. Working with Carbohydrates Carbohydrates and Glycobiology

1. Lectins are proteins that read the sugar code and mediate many biological processes. 2. Lectin-carbohydrate interactions are very strong and highly specific. Carbohydrates as Informational Molecules: The Sugar Code

Glycobiology: the study of the structure and function of glycoconjugates. 20 different monosaccharides: x different hexameric oligosaccharides 20 common amino acids: x 10 7 (20 6 ) different hexapeptides 4 nucleotides: - 4,096 (4 6 ) different hexanucleotides Oligosaccharides are enormously rich in structural information- the sugar code. Glycobiology and the sugar code

Lectins: proteins that bind carbohydrate with high affinity and specificity. Cell-cell recognition, signaling, adhesion processes, and intracellular targeting of newly synthesized proteins. Useful reagents for detecting and separating glycoproteins with different oligosaccharide moieties. Lectins are proteins that read the sugar code and mediate many biological processes

1) Lectin receptor of hepatocytes- peptide hormones 2) Asialoglycoprotein receptors- ceruloplasmin 3) Mechanisms for removing old erythrocytes 4) HA of influenza virus- bind sialic acid- viral infection 5) HS-1 and HS-2 of HSV- bind heparan sulfate 6) Selectins- cell-cell recognition and adhesion ex: Movement of T lymphocytes through the capillary wall, from blood to tissues, at sites of infection or inflammation. Lectins are proteins that read the sugar code and mediate many biological processes

Role of lectin-ligand interactions in T lymphocyte movement to the site of an infection or injury

1) Mediate bacterial adhesion to host cells - Helicobacter pylori vs. Le b oligosaccharide on the membrane glycoproteins of gastric epithelial cells - Treatment- chemically synthesized Le b 2) Mediate toxin entry into host cells - Cholera toxin (Vibrio cholerae) vs. GM1 ganglioside on the surface of intestinal epithelial cells - Pertussis toxin (Bordetella pertussis) - Treatment- toxin analogs as vaccines Lectins of microbial pathogens

Role of lectin-ligand interactions in lymphocyte movement to the site of an infection or injury

Oligosaccharides containing mannose 6-phosphate marks newly synthesized proteins in the Golgi complex for transfer to the lysosome. Recognized by the cation-dependent mannose 6- phosphate receptor, a membrane-associated lectin on the lumenal side of the Golgi complex. Many degradative enzymes (hydrolases) of the lysosome are targeted and delivered by this mechanism. Lectins acting intracellularly

1) Sialoadhesin (siglec-1) of mouse macrophages vs. certain sialic acid-containing oligosaccharides. -  sandwich domain - each ring substituent is involved in the interaction 2) Bovine mannose 6-phosphate receptor vs. mannose 6-phosphate - specificity of the binding - necessity for a divalent cation in the interaction Lectin-carbohydrate interactions are very strong and highly specific

Sialic acid-specific lectin vs. sialic acid

Each ring substituent is involved in the interaction between sugar and lectin

Details of lectin-carbohydrate interaction

Many carbohydrates have a more polar and a less polar side. General interactions- - more polar side: hydrogen bonds with lectin - less polar side: hydrophobic interactions with nonpolar amino acid residues Lectin-carbohydrate interactions are very strong and highly

Hydrophobic interactions of sugar residues

Roles of oligosaccharides in recognition and adhesion at the cell surface

Methods of carbohydrate analysis