Lecture 9 - Cell surface Oligosaccharides (March 22) PDF

Lecture 9 W24 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 L...

Lecture 9 W24 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) Multiadhesive Proteins (e.g. Fibronectin) Several ECM proteins have the conserved RGD motif – binds to integrins 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) Question Ricin, a potent toxin found in castor beans binds to GalNAc residues of cell surface oligosaccharides via a lectin to enter eukaryotic cells. A treatment for ricin poisoning is administration of GalNac itself or the oligosaccharide containing GalNac. Explain how this could be an effective treatment. 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 Human selectins are involved in a variety of diseases e.g. asthma, rheumatoid arthritis, psoriasis 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) What type of inhibition do you think is carried out by Oseltamivir? A. Un-competitive Inhibition B. Competitive Inhibition C. Non-Competitive Inhibition D. Mixed Inhibition 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