Module 3-Cell surface Oligosaccharides (Lec 7-9) PDF

BIOC*4580 – Membrane Biochemistry Winter 2024 Cell surface oligosaccharides Cell identity and the extracellular matrix Lehninger Chapter 7 Essentials of Glycobiology, 4th ed. free online https://www.ncbi.nlm.nih.gov/books/NBK579918/ Sugars Lehninger 8th ed. Fig 7.1a Polyhydroxy aldehydes or polyhydr...

BIOC*4580 – Membrane Biochemistry Winter 2024 Cell surface oligosaccharides Cell identity and the extracellular matrix Lehninger Chapter 7 Essentials of Glycobiology, 4th ed. free online https://www.ncbi.nlm.nih.gov/books/NBK579918/ Sugars Lehninger 8th ed. Fig 7.1a Polyhydroxy aldehydes or polyhydroxy ketones Simple sugars have the chemical formula (CH2O)n D-aldohexoses Of these, glucose, galactose and mannose are important for mammalian cell surfaces Note: Mannose is the C-2 epimer of glucose; Galactose is the C-4 epimer of glucose Lehninger 8th ed. Fig 7.3 Fischer projections Fischer projections are used to show the chemical structure The carbon chain is written vertically, with the oxidized carbon (aldehyde or keton) nearer the top Substituents at each chiral carbon atom (H & OH) are written left or right depending on where they would appear with the carbon seen from this perspective Sugars are D- if bottom-most chiral OH is on the right, L- if it is on the left Lehninger 8th ed. Fig 7.2 Sugar cyclization- Converts sugars from linear structures (Fischer projections) to rings (Haworth perspectives) and forms either hemiacetals or hemiketals OH groups on the right in the Fischer- points down in Haworth (below the plane of the ring) OH groups on the left in the Fischer- points up in Haworth (above the plane of the ring) The terminal –CH2OH group projects upward for D-sugars; Downward for L- sugars Anomeric OH on the same side of the ring as the CH2OH – β; when it is on the opposite side from the CH2OH – α For D-sugars Lehninger 8th ed. Fig 7.6 LAB: Left Above Beta Pyranose rings are not planar but assume one of 2 “chair” conformations Conformations and configurations Conformations can be interconverted without breaking any bonds. The relative energies of each conformation is different and therefore, the 2 conformations do not readily interconvert. Another conformation, the “boat” is found only with very bulky substituents. Glucose derivatives Other hexoses have analogous variations, and are named by analogous schemas Amino sugars (e.g., D-glucosamine) are modified by replacing OH-2 with NH2-2 In N-acetyl-amine sugars (e.g., Nacetylglucosamine), this amine is acetylated, forming an amide bond Uronate sugars (e.g., glucuronate) have the terminal carbon (here C6) oxidized to carboxylate -onate sugars (e.g., gluconate) have the aldehyde oxidized to carboxylate These can cyclize to form lactones Lehninger 6th ed. Fig 7.9 Sialic acids Sialic acids are N- or O-substituted derivatives of neuraminic acid Neuraminic acid is a nine-carbon ketose (C2), with a carboxylate group at C1 Sialic acids form pyran rings, leaving a three-carbon hydroxylated “tail” e.g., Neu5Ac has an N-acetyl group at C5 Generally, only added as the last residue of saccharide chains Here their distinct properties are easily recognized Often function as a signal to not degrade a molecule or cell Hemagglutinin (HA) Neuraminidase (NA) Human Influenza virus (H#N#) Neuraminidase enzyme –cleaves neuraminic acid residues Neuraminic Acid Oligosaccharides and polysaccharides Lehninger 6th ed. Fig 7.12 Oligosaccharides typically have less than 20 sugar groups They are typically non-repetitive and branched Polysaccharides are larger These can be branched or unbranched Made of a single sugar or many Polysaccharides typically have monomers and linkages repeat in a simple pattern The complexity of oligosaccharides A typical oligosaccharide might contain 14 units Generally, one of four (Glc, Gal, Man, Neu5Ac) basic types, but other sugars are possible (e.g., fucose) Also, N, N-acetyl, uronic acid variations possible, also sulfation at various positions In total about 20 sugars utilized in mammal saccharides Various linkages possible - (1->2), (1->3), (1->4), (1->6)  or  anomeric configuration Much more diversity per residue is possible However, diversity in an organism is limited by the need for a separate enzyme to catalyze each linkage Glycosyltransferases transfer sugars UDP-glucose (an activated glucose) Glycosyltransferases (GTs) transfer carbohydrate monomers from an activated donor to an acceptor molecule The donor has a phosphate leaving group on the anomeric carbon Normally the PO4 is part of a nucleotide (or lipid phosphate) The acceptor can be a saccharide, a protein, or a small molecule (Inverting GTs invert the hand of the anomeric carbon) (Retaining GTs retain the hand at the anomeric carbon) Glycosaminoglycans are found in the extracellular matrix The extracellular matrix holds cells together and provide a porous pathway for diffusion of nutrients and oxygen It includes fibrous proteins (e.g., fibrillar collagens, elastins, fibronectins) The other major components are the complex polysaccharides known as glycosaminoglycans which are linear polymers made up of repeating disaccharide units One of the sugars is always either GlcNac or GalNAc and the other is typically a uronic acid (usually D-glucuronic or L-iduronic) SO422 Commonly esterified with sulfate groups (SO4 ) Highly negatively charged due to the sulfates and the –COO- of uronic acids Common glycosaminoglycans Hyaluronate forms very long chains of GlcA(1-3)-GlcNAc(1-4) (unsulfated) Important in synovial fluid in joints Chondroitin structurally similar, but GalNac4S instead of GlcNAc Keratan has Gal(1-4)GlcNac6S(1-3) No uronic acid, only 1 SO4, so minimal negative charge Chondroitin and keratan important in tendons and cartilage Lehninger 8th ed. Fig 7.19 Common glycosaminoglycans – Heparan sulfate Sulfation is variable IdoA is iduronate – unusually an Lsugar Four SO4 groups plus CO2- results in large negative charge -L-IdoA is the C-5 epimer of -Dglucuronate (C-5 determines L- vs D-, hence L-IdoA) Lehninger 8th ed. Fig 7.19 Heparin is an intracellular form of Heparan Sulfate that is synthesized mainly by the mast cells. Its physiological role is unclear. Purified heparin is used in medicine as an anticoagulant Glycosaminoglycans adopt extended conformations Lehninger 6th ed. Fig 7.22 Heparin has the highest charge density of any biological macromolecule The negatively charged groups repel one another leading to the chains assuming an extended rod like helical conformation The COO- occur on opposite sides of the rod and there is also maximum separation between the sulfates This also results in different strands preferring to not interact The Glycocalyx Most eukaryotic cells have an information rich carbohydrate layer extending from the cell surface The glycocalyx is several nm thick and extends well beyond the cell surface The carbohydrates of the glycocalyx are usually conjugated to proteins or lipids to form glycoconjugates. Glycoconjugates Glycoproteins are proteins modified by oligosaccharides Proteoglycans have glycosaminoglycans attached to a “core protein” Glycosphingolipids have oligosaccharides attached to sphingosine Lehninger 6th ed. Fig 7.24 (8th ed. 7-20) Proteoglycans Proteoglycans are found in the cell surface and in the ECM Act as tissue organizers They influence cellular processes such as attachment and growth factor activation Most are secreted, but some are transmembrane proteins Proteoglycans attach glycosaminoglycans covalently to a SGxG consensus motif within the protein A tetrasaccharide linker provides the attachment point for the glycosaminoglycan Lehninger 6th ed. Fig 7.25 (8th ed. 7-21) Membrane attached proteoglycans Two major families of membrane attached proteoglycans: Syndecans have single TM domains and 3-5 heparan sulfates & in some cases chondroitin sulfates Glypicans attach via a GPI anchor; 2-3 glycosaminoglycans attach near the membrane; Globular domain is stabilized by 7 conserved disulfide bonds Syndecans can be released from the membrane by proteolytic cleavage Glypicans can be released by a phospholipase Proteoglycan shedding allows cells to change their surface features quickly – Involved in cell-cell recognition & adhesion; cell proliferation & differentiation. Shedding is activated in cancer cells. Lehninger 6th ed. Fig 7.26 (8th ed. 7-22) Heparan sulfate is chemically variable NS-Highly sulfated domains; NA- Domains with unmodified GlcNAc and GlcA domains (NAcetylated or NA domains) NA domains have little sulfation, and have glucuronate (GlcA) NS domains (typically 3-8 disaccharides long) replace GlcA with iduronate, and are heavily sulfated 32 permutations of sulfation IdoA/GlcA Exact pattern of sulfation varies with protein and cell type Binding of extracellular proteins and signaling molecules to specific NS domains, modulate their activities Lehninger 6th ed. Fig 7.26 (8th ed. 7-22) Different types of protein interactions with NS domains of heparan sulfate 1. 2. Lehninger 6th ed. Fig 7.27 (8th ed. 7-23) protein interactions with NS domains of heparan sulfate Cont., 3. 4. Lehninger 6th ed. Fig 7.27 (8th ed. 7-23) Molecular basis for the enhanced interaction between Thrombin and Antithrombin The binding pockets for heparan sulfate in both thrombin and antithrombin are rich in positively charged Arg and Lys residues (shown in blue) The negatively charged regions of heparan sulfate bind through electrostatic attractions to these regions This brings thrombin and antithrombin closer and enhances their interaction with one another The crystal structure of thrombin, antithrombin and a heparan sulfate-like polymer all crystalized together (Lehninger 8th ed. Figure 7.24, Page 250) Proteoglycan aggregates Aggrecan core protein (~250 kDa) is decorated with multiple chondroitin and keratan chains 100s of these 2 MDa structures are bound to hyaluronate via linker proteins Interacts with collagen, contributing to tensile strength and resilience of connective tissue Lehninger 6th ed. Fig 7.28 Interactions between cells and the ECM Fibronectin is an ECM protein It binds heparan sulfate, collagen It also binds integral membrane proteins known as integrins Integrins interact in turn with the cytoskeleton of the cell, linking it to the ECM Lehninger 6th ed. Fig 7.29 (8th ed. 7-26) Glycoproteins Proteins with covalently attached oligosaccharides The glycans of glycoproteins are: – branched – a lot smaller and – structurally diverse than the glycosaminoglycans of proteoglycans About half of all mammalian proteins are glycosylated The saccharide can account for anything from 1% to 70% of the mass of a glycoprotein O-linked oligosaccharides O-linked saccharides connect the anomeric carbon to the –OH of Ser or Thr No consensus target sequence, but insertion sites are generally Gly, Val & Pro rich Ser/Thr is connected to GalNAc via its anomeric carbon (in the figure shown.) Some proteins have one or a few O-linked sugars A variety of O-glycans are attached, ranging from very simple to complex Lehninger 6 ed. Fig 7.30 Many are antigenic th Mucins – O-linked glycans Mucins are common O-linked proteins with multiple saccharide chains added These are the most common O-linked proteins Mucins cover many epithelial surfaces of the body, e.g. the gastrointestinal, genitourinary, and respiratory tracts They shield the epithelial surfaces against physical and chemical damage and protect against infection by pathogens. Many mucins are secreted, and are important contributors to the viscosity and adhesiveness of mucus They can also be found as membrane glycoproteins N-linked oligosaccharides N-linked are attached to the amide N of asparagine in the consensus sequence Asn-X-Ser/Thr Sites have to be accessible within ER to be glycosylated First sugar is N-acetyl-glucosamine All N-linked oligosaccharides share a core branched structure (2 GlcNAc residues, followed by mannose with 2 branches, both mannose) N-linked saccharides are important for protein stability, immune cell targeting, signaling, neural development etc. Lehninger 6th ed. Fig 7.30 (8th ed. 7-27) Glycolipids and Lipopolysaccharides O-linked saccharides can also be attached to sphingolipids The oligosaccharide head groups of specific sphingolipids on the plasma membrane of red blood cells determine in part the human blood groups O, A and B. Fig 10-13 (Lehninger 8th ed) Lipopolysaccharides Outer membrane of gram-negative bacteria (e.g. E.coli, S. typhimurium) The O-specific chain is the main determinant of the bacterial serotype (immunological reactivity) Lipid A portion of some bacteria is known as endotoxin Lehninger 8th ed. Figure 7.28, Page 253 Roles of cell surface carbohydrates Communication between cells and their surroundings Label proteins for transport to specific cellular locations Label malformed proteins for destruction Act as recognition sites for extracellular signal molecules (e.g. growth factors) or for parasites such as bacteria or viruses. They also alter the polarity and water solubility of proteins Erythrocyte surface – David Goodsell Lectins are proteins that specifically recognize carbohydrates Lectins show moderate (millimolar) to high (micromolar) affinity They make multiple interactions with the target sugar, so specificity is very high Lectins are often polyvalent (a single lectin has multiple carbohydrate binding domains (CBDs)), allowing avidity to drive tight binding Lectins play diverse roles in cell-cell recognition, adhesion, and intracellular targeting of newly sorted proteins Lectin Affinity Chromatography Applications of lectins include ELISA measurements, glycoconjugate purification, cell selection or sorting, cell agglutination, enzyme assays. Stryer 5th ed. Roles of oligosaccharide recognition by lectins Act as receptors for bacteria, viruses and toxins Surface carbohydrates play an important role in cell-cell recognition, especially with the immune system Within the cell, Mannose-6-PO4 binding targets proteins to the lysosome Lehninger 6th ed. Fig 7.37 (8th ed. 7-33) Saccharide recognition Galactose binding Lehninger 6th ed. Fig 7.36 (8th ed. 7-32) Many sugars have a more polar face (the ring O and multiple OHs) and a less polar face The polar side can hydrogen bond with lectins The less polar side interacts with non-polar amino acid residues through the hydrophobic effect Trp rings are ideal, but Phe, Tyr, Val & Leu also work well Mannose-6-PO4 binding by receptor Man-6-PO4 serves as an intracellular sorting signal, directing proteins for degradation in the lysosome Each hydroxyl group forms a hydrogen bond with the receptor (a lectin) Note that the pocket is very basic (blue) to bind the negative charge of the PO4 group Lehninger 6 ed. Fig 7.35 (8 ed. 7-31) th th Lectin-ligand interactions allow leucocytes to target inflammation Lehninger 6th ed. Fig 7.32 Leucocyte travelling in a capillary is slowed by transient weak interactions mediated by selectin (a lectin) Once slowed, strong interactions by integrin (recognizing epitopes on epithelial cells) bring it to a stop After adhesion, leucocyte can invade to reach site of inflammation Neuraminadase Hemagglutinin (a lectin) Neuraminadase Influenza virus recognizes target cells by binding Neu5Ac containing oligosaccharides on the cell surface Freshly budded virus particles will stick to these groups on the host cell These particles have an enzyme called neuraminadase that cleaves off the sialic acid The importance of this enzyme makes it a useful drug target Neuraminadase inhibitors Oseltamivir and Zanamivir are small molecules that resemble Neu5Ac. They bind tightly to the Neu5Ac binding site of influenza neuraminidase. Lehninger 6th ed. Fig 7.33 (8TH ed. 7-30) Lehninger 6th ed. Fig 7.33 Inhibition and resistance This structural mimicry allows these drugs to block influenza from infecting the next generation of cells. The His274Tyr mutation weakens Oseltamivir binding by subtly modifying the shape of the binding site. This mutation yields a drug resistant virus. Characterizing oligosaccharides Lehninger 6th ed. Fig 7.38 (8th ed. 7-34) Characterizing oligosaccharides Saccharides can be hydrolysed and the individual monosaccharides identified, along with abundances Exhaustive methylation: Methylation (by CH3I + base) converts all free OHs to acid-stable methyl ethers Acid hydrolysis to release free monosaccharides Any free OHs in the released sugars are those that were involved in glycosidic linkages. Exoglycosidases (enzymes that cleave off a single sugar from the non-reducing end) of known specificity (saccharide and linkage) can be used to remove one sugar at a time to determine the sequence of monosaccharide residues. Based on the specificity can tell the terminal sugar, and linkage of that sugar Lehninger 6th ed. Fig 7.39 Mass spectrometry of oligosaccharides Oligosaccharides can be identified by differences in their mass You can reconstruct branching patterns from the masses of breakdown products Linkage sites and sugar chirality are not resolved. Question Lehninger pg.277 Pg.261 (8th ed) The amount of branching (number of α(1→6) glycosidic bonds) in amylopectin was determined by exhaustively methylating a sample of amylopectin. All the glycosidic bonds in the treated sample are then hydrolyzed in aqueous acid, and the amount of 2,3-di-O-methylglucose so formed is determined. (a) Explain the basis of this procedure for determining the number of α(1→6) branch points in amylopectin. What happens to the unbranched glucose residues in amylopectin during the methylation and hydrolysis procedure? (b) A 258 mg sample of amylopectin treated as described above yielded 12.4 mg of 2,3-di-O-methylglucose. Determine what percentage of the glucose residues in amylopectin contained an (α1→6) branch. (Assume the average molecular weight of a glucose residue in amylopectin is 162 g/mol). Note: determine the H2C OH MW of 2,3-di-O-methylglucose based on its structure. C O H H C O CH H C 3 HO C C OH H 2,3-di-O-methylglucose H O CH3 1. Shown on the right is the Fischer structure of D-glucose. Identify the correct statement for the Haworth projection of α-D-glucopyranose. A. The terminal —CH2OH group projects downward. B. The anomeric hydroxyl is on the opposite side from C-6. C. The hydroxyl group on C-2 is placed pointing up. D. The cyclic structure is a five-membered ring. 2. Identify the incorrect statement regarding homopolysaccharides and heteropolysaccharides. A. Homopolysaccharides contain a single monomeric sugar species. B. Some homopolysaccharides serve as structural elements in animal exoskeletons. C. Heteropolysaccharides serve as storage forms of monosaccharides that are used as fuel. D. In animal tissues, the extracellular space is occupied by several types of heteropolysaccharides. 3. A. B. C. D. Identify the incorrect statement about glycosaminoglycans. found in extracellular matrix always contain sulfates are heteropolysaccharides are disaccharide repeat units 4. A. B. C. D. The glycosaminoglycan hyaluronate is sulfated. consists of alternating residues of D-glucuronic acid and N-acetylglucosamine. is covalently linked to specific proteins. is a much shorter polymer than heparin. 5. Which statement about glycoconjugates is false? A. The glycosaminoglycan chain of the proteoglycan can bind to extracellular proteins through electrostatic interactions. B. Glycolipids are found in specific organelles, such as Golgi complexes. C. Glycosphingolipids play a role in signal transduction. D. The oligosaccharide portions of glycoproteins are very heterogeneous. 6. Which statement about proteoglycans is false? A. The glycosaminoglycans are linked to the protein through a Ser residue in an SGxG consensus motif B. They can help localize lipoprotein lipase to the cell surface C. They are only found as secreted proteins in the extracellular matrix D. Some proteoglycans are integral membrane proteins 7. In glycoproteins, the oligosaccharide chains are attached to the protein via which of the following amino acids? A. Ser, Thr, and Tyr B. Ser, Thr, and Asn C. Trp, Tyr, and Phe D. Lys, His, and Arg 8. Glycoproteins: A. contain unbranched oligosaccharides. B. The oligosaccharides are much more structurally diverse than the glycosaminoglycans of proteoglycans C. have oligosaccharides covalently attached to aspartate residues. D. are a small fraction of the total number of proteins in a human cell. 9. Which of these is NOT a glycoconjugate? A. lactose B. syndecan C. glypican D. glycosphingolipid 10. Oligosaccharides are: A. never found in mucins. B. classified as a N- or O-linked when found in gangliosides. C. found in bacterial lipopolysaccharides. D. never attached to hormones. 11. Which statement about selectins is false? A. They mediate cell-cell recognition. B. They are involved in the movement of immune system cells. C. They interact with specific oligosaccharides on cell surface glycoproteins of leukocytes. D. They are intracellular. 12. Lectins: A. often bind their ligands via multiple weak interactions. B. bind their ligands with relatively low specificity. C. prevent viruses from binding to their target cells. D. are carbohydrates that bind to receptor proteins. 13. Identify which of the following is determined when characterizing oligosaccharides and polysaccharides. A. sequence of monosaccharides B. Positions of glycosidic bonds C. configurations of glycosidic bonds D. All of the above 14. N linked oligosaccharides of glycoproteins attach to the protein via the side chain N atom of Asn. If a given protein has 10 Asn residues in its sequence, 3 of which act as glycosylation points, can you identify these 3 Asn residues by looking at the amino acid sequence of the protein? Explain.