Lecture 8 - Cell surface Oligosaccharides

Lecture 8 - W24 Monday, March 18 See Fig 7-9 for structures of fucose, Rhamnose 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...

Lecture 8 - W24 Monday, March 18 See Fig 7-9 for structures of fucose, Rhamnose 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) a or b 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(b1-3)-GlcNAc(b1-4) (unsulfated) Important in synovial fluid in joints Chondroitin structurally similar, but GalNac4S instead of GlcNAc Keratan has Gal(b1-4)GlcNac6S(b1-3) 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 (a-L-IdoA is the C-5 epimer of b-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)