Carbohydrates - Oligosaccharides

Carbohydrates A. Definition B. Roles C. Monosaccharides-Chemistry 1. Chirality a. One or more asymmetric carbons b. Linear and ring forms 2. Derivatives: the chemistry of carbohydrates a. Oxidation b. Reduction c. d. Ester formation Amino sugars i. ii. i. ii. C1 C6 C1/C2 Other carbons 3. Polymerization a. The Glycosidic Bond b. Non-covalent bonds in macro-molecular structure D. Oligosaccharides 1. 2. 3. 4. Glycoproteins & glycolipids O-linked N-linked Sequence determination-ABO E. Polysaccharides 1. Polymers of glucose 2. Polymers of disaccharides Carbohydrates Oligosaccharides: O-linked • Specific glycosyltransferases use “activated” sugars • Activated by attaching to nucleotides: UDP, CDP, etc. Sugar 1-P + UTP ⇌ UDP-sugar + PPi EXAMPLE: Gal b(1à3) glycosyl transferase Sia a(2à6) glycosyl transferase Fuc a(1à2) glycosyl transferase Carbohydrates Oligosaccharides: O-linked • Specific glycosyltransferases use “activated” sugars • Activated by attaching to nucleotides: UDP, CDP, etc. EXAMPLE: Sugar 1-P + UTP ⇌ UDP-sugar + PPi Gal b(1à3) glycosyl transferase Sia a(2à6) glycosyl transferase Fuc a(1à2) glycosyl transferase Carbohydrates Oligosaccharides: N-linked • Larger, more complex than Olinked • All have a “Core” containing GlcNAc & Man • Added as a unit, then modified before adding to Asn groups on proteins; all in the ER/Golgi • Use an isoprene, called Dolichol, to build core • Examples: Always the same linkages Let’s look at this Dolichol & its use in biosynthesis more closely…. Carbohydrates Oligosaccharides: N-linked • Activated by attaching two phosphates, one using CTP (step 13) • Again, uses NDP-sugar precursors H Dolichol pyrophosphate sugar Carbohydrates Oligosaccharides: N-linked Specific glycosidases Specific glycosidases Specific glycosyl transferases Carbohydrates Oligosaccharides: Determination of Sequence • Whole different problem compared to proteins and nucleic acids…. Its branched!! • Moreover, a given residue can have several (and stereo-specific) ways of attaching to a neighboring residue. • Need to use a combination of methods: – Chemical – Hydrolysis & chromatography to identify sugars – Exhaustive methylation & hydrolysis, then chromatography to identify what positions were not methylated – Biochemical – Use of enzymes that stereo-specifically hydrolyze glyosidic bonds (from the non-reducing end) EXAMPLE: First, just like protein sequencing, you need to purify glyco-protein or lipid. Lets say we isolate the glycolipid from a person’s RBC’s who is O-positive. Treat it with a ceramidase to hydrolyze the lipid from the sugar. Second, take an aliquot and just hydrolyze (like what was done for amino acid analysis). This gets the composition and stoichiometry. Carbohydrates Oligosaccharides: Determination of Sequence • Whole different problem compared to proteins and nucleic acids…. Its branched!! • Moreover, a given residue can have several (and stereo-specific) ways of attaching to a neighboring residue. • Need to use a combination of methods: – Chemical – Hydrolysis & chromatography to identify sugars – Exhaustive methylation & hydrolysis, then chromatography to identify what positions were not methylated – Biochemical – Use of enzymes that stereo-specifically hydrolyze glyosidic bonds (from the non-reducing end) EXAMPLE: First, just like protein sequencing, you need to purify glyco-protein or lipid. Lets say we isolate the glycolipid from a person’s RBC’s who is O-positive. Treat it with a ceramidase to hydrolyze the lipid from the sugar. Second, take an aliquot and just hydrolyze (like what was done for amino acid analysis). This gets the composition and stoichiometry. Carbohydrates Oligosaccharides: Determination of Sequence a-glycosidase ③ Glycosylation yielding only Fuc + Disacc. means there is an a-linkage followed by a b-linkage ① Fuc b-glycosidase ② ③ Disacch. Trisacch. No reaction with the original (no reaction) trisacch means that there b-glycosidase are not sugars in b-linkage at the non-reducing end. ③ 1 Gal Gal •Methylation of C1-OH means it’s a reducing carbon at the GlcNAc reducing end •Lack of methylation of C4-OH means it was in glycosidic linkage with another sugar 1,3,6 trimethyl-GlcNAc •Lack of methylation of C1-OH means its anomeric carbon was in ① Chemical: Hydrolysis a glycosidic bond 3,4,6 trimethy-Gal •Lack② of methylation Chemical: of C2-OH Exhaustive means that it was in glycosidic methylation & hydrolysis linkage with another sugar 2,3,4 trimethyl-Fuc Biochemical: •This ③ sugar is in the middle glyosidase ① 1 GlcNAc •Again, lack of methylation of C1-OH. •That all possible hydroxyls are methylated (recall Fuc is a 6-deoxy sugar); this sugar is at a nonreducing end (no other sugars attached to it) 1 Fuc Carbohydrates Oligosaccharides: Determination of Sequence ① 1 Gal 1 GlcNAc 1 Fuc ① Chemical: Hydrolysis ② Chemical: Exhaustive methylation & hydrolysis ③ Biochemical: glyosidase Carbohydrates Oligosaccharides: Determination of Sequence ① Chemical: Hydrolysis ② Chemical: Exhaustive methylation & hydrolysis ③ Biochemical: glyosidase ② •Methylation of C1-OH means it’s a reducing carbon at the reducing end •Lack of methylation of C4-OH means it was in glycosidic linkage with another sugar 1,3,6 trimethyl-GlcNAc •Lack of methylation of C1-OH means its anomeric carbon was in a glycosidic bond 3,4,6 trimethy-Gal •Lack of methylation of C2-OH means that it was in glycosidic linkage with another sugar 2,3,4 trimethyl-Fuc •This sugar is in the middle ① •Again, lack of methylation of C1-OH. •That all possible hydroxyls are methylated (recall Fuc is a 6-deoxy sugar); this sugar is at a nonreducing end (no other sugars attached to it) Carbohydrates Oligosaccharides: Determination of Sequence a-glycosidase ③ Hydrolysis yielding only Fuc + Disacc. means there is an a-linkage followed by a blinkage Fuc b-glycosidase ③ Disacch. Trisacch. No reaction with the original (no reaction) trisacch means that there b-glycosidase are not sugars in b-linkage at the non-reducing end. ③ Gal GlcNAc ① Chemical: Hydrolysis ② Chemical: Exhaustive methylation & hydrolysis ③ Biochemical: glyosidase Carbohydrates Oligosaccharides: Determination of Sequence – In vertebrates, ganglioside carbohydrate composition determines blood groups. CH2OH OH 6CH OH 2 O–Cer OH OH OH OH OH OH OH OH L-Fuc a(1à2)-D-Gal b(1à4) D-GlcNAc b-Ceramide O blood group D-Gal a(1à3) D-GalNAc a(1à3) B blood group A blood group Carbohydrates Oligosaccharides: Determination of Sequence – In vertebrates, ganglioside carbohydrate composition determines blood groups. CH2OH OH 6CH OH 2 O–Cer OH OH OH OH OH OH OH OH L-Fuc a(1à2)-D-Gal b(1à4) D-GlcNAc b-Ceramide O blood group D-Gal a(1à3) D-GalNAc a(1à3) B blood group A blood group Glycoconjugates: Analysis Carbohydrates Polysaccharides Carbohydrates Polysaccharides • The majority of natural carbohydrates are usually found as large polymers. • These polysaccharides can be: – homopolysaccharides (one monomer unit) – heteropolysaccharides (multiple monomer units) – linear (one type of glycosidic bond) – branched (multiple types of glycosidic bonds) • Polysaccharides do not have a defined molecular weight. – This is in contrast to proteins because, unlike proteins, no template is used to make polysaccharides. – Polysaccharides are often in a state of flux; monomer units are added and removed as needed by the organism. Carbohydrates Polysaccharides: Polymers of Glucose Homopolymers of Glucose: - Starch - Glycogen - Cellulose - Chitin • Glycogen and Starch are the main storage polysaccharides for energy. – Glycogen and starch are insoluble due to their high molecular weight and often form granules in cells. – Molecular weight reaches several millions (~200 x 106)(can see in microscope). • Glycogen is a branched homopolysaccharide of glucose. – Glucose monomers form a(1 ® 4) linked chains. – There are branch points with a(1 ® 6) linkers every 8–12 residues. • Starch is a mixture of two homopolysaccharides of glucose. – Amylopectin is like glycogen, but the branch points (a(1 ® 6) linkages) occur every 24–30 residues. – Amylose is an unbranched polymer of a(1 ® 4) linked residues. Carbohydrates Polysaccharides: Glycogen & Starch Glycosidic Linkages in Glycogen and Starch (Glc-a(1à4)Glc)n with a(1à6) branching, where n≈106 Non-reducing à reducing Haworth projections are not good to show the actual shape, which is helical! Carbohydrates Polysaccharides: Glycogen & Starch n≈150 I2 2 3 5 1 4 6 Axial = a-OH 4 6 3 5 2 1 • Granules contain enzymes that synthesize and degrade these polymers. • Glycogen and amylopectin have one reducing end but many nonreducing ends. • Enzymatic processing occurs simultaneously in many nonreducing ends. Carbohydrates Polysaccharides: Glycogen & Starch n≈150 I2 2 3 5 1 4 6 Axial = a-OH 4 6 3 5 2 1 Carbohydrates Polysaccharides: Glycogen & Starch Mixture of Amylose and Amylopectin in Starch Carbohydrates Polysaccharides: Homopolymers of Glucose: - Starch - Glycogen • Cellulose is a linear homopolysaccharide of glucose. – Glucose monomers form b(1 ® 4) linked chains. - Cellulose – Hydrogen bonds form between adjacent monomers. – There are additional H-bonds between chains. - Chitin – Structure is now tough and water insoluble. – It makes up almost 50% of plant mass; in cell walls. – Cotton is nearly pure fibrous cellulose. • Chitin is a linear homopolysaccharide of Nacetylglucosamine (GlcNAc). – N-acetylglucosamine monomers form b(1 ® 4)linked chains. – forms extended fibers that are similar to those of cellulose – hard, insoluble, cannot be digested by vertebrates – structure is tough but flexible, and water insoluble – found in cell walls in mushrooms and in exoskeletons of insects, spiders, crabs, and other arthropods Carbohydrates Polysaccharides: Cellulose & Chitin Glc b 1 OH 4 Glc OH Glycosidic Linkages in Cellulose & Chitin (Glc-b(1à4)Glc)n with NO branching, n ≈ 104 Notice that each chair is alternatively flipped 3 Equitorial = b-OH 4 THE most abundant biological molecule in the world: ~300 trillion kg 2 5 6 1 Carbohydrates Polysaccharides: Cellulose & Chitin • The fibrous structure and water insolubility make cellulose a difficult substrate to act upon. • Most animals cannot use cellulose as a fuel source because they lack the enzyme to hydrolyze b(1®4) linkages (b-Amylase or cellulase). • Fungi, bacteria, and protozoa secrete cellulase, which allows them to use wood as source of glucose. • Ruminants and termites live symbiotically with microorganisms that produce cellulase and are able to absorb the freed glucose into their bloodstreams. • Cellulases hold promise in the fermentation of biomass into biofuels. Carbohydrates Polysaccharides: Cellulose & Chitin b b 4 1 4 (GlcNAc-b(1à4)-GlcNAc)n Carbohydrates Polysaccharides: Polymers of Disaccharides First, need to describe the Extracellular Matrix (ECM) • • • Material outside the cell Strength, elasticity, and physical barrier in tissues (varies tremendously) Main components – proteoglycans – collagen & elastin fibers • Proteoglycans – Different glycosaminoglycans are O-linked to the “core protein.” – Linkage from anomeric carbon of xylose to serine hydroxyl – Our tissues have many different core proteins; aggrecan is the best studied. ECM is a barrier for tumor cells seeking to invade new tissues. Carbohydrates Polysaccharides: Polymers of Disaccharides Extracellular Matrix (ECM) Carbohydrates Polysaccharides: Polymers of Disaccharides Glycosaminoglycans (the carbohydrate part of proteoglycans) • • Linear polymers of repeating disaccharide units (sugarX-sugarY)n One monomer (sugarX) is either sugar acid or Gal – uronic acids (C6 oxidation) – Most have sulfate esters • One monomer (sugarY) is either: – N-acetyl-glucosamine (GlcNAc) or N-acetyl-galactosamine (GalNAc) – Also sulfate esters • Extended hydrated molecule – Negatively charged – minimizes charge repulsion • Forms meshwork with fibrous proteins to form extracellular matrix – connective tissue – lubrication of joints • Form huge (Mr > 2 • 108) noncovalent aggregates (Hyaluronan and Aggrecan). – They hold a lot of water (1000´ its weight) and provide lubrication. – Very low friction material – Covers joint surfaces: articular cartilage o reduced friction & load balancing