7 1 MCB 450 Lecture 7 O Some Explanations Glycoconjugates Proteoglycans Glycoproteins Glycan Synthesis Examples of Importance of Glycans 7 2 Why does the linkage in sucrose look like an 7 3 How to spot the anomeric carbon in a pyranose or furanose CH2OH C C HO OH OH C CH2OH C C OH O H C C HO O OH C C OH OH l anomeric C has 2 oxygens bonded to it C 7 4 Glycoconjugates NOTE THE MODIFICATIONS WITH SUGARS ARE ALL ON EXTRACYTOPLASMIC PORTIONS OF PROTEINS AND LIPIDS 7 5 Definitions proteoglycan vs glycoprotein Proteoglycan Protein Uronic acid and or SO42 containing polysaccharide glycosaminoglycan Protein Glycosaminoglycan GAG Glycoprotein Protein Oligosaccharide not GAG covalently attached to peptide backbone Two major types of linkage to extracytoplasmic domains of proteins Occur with variations in all eukaryotes Examples in bacteria and archaea 7 6 Glycosaminoglycan repeats 1 One sugar always GalNAc GlcNAc or GlcN often esterified with sulfate The other usually a uronic acid e g glucuronate GlcA GalNAc 7 7 Glycosaminoglycan repeats 2 Ionized carboxyl sulfate groups give molecule a ve charge Assume e x t e n d e d c o n f o r m a t i o n in solution GlcA GalNAc Gal GlcA GlcNAc GlcNAc Highly hydrated forms viscous solution is lubricant in joints 7 8 Cartilage proteoglycan Central filament of hyaluronate SUPRAMOLECULAR COMPLEX OF GLYCOSAMINOGLYCAN CHAINS PROTEINS Aggrecan Electron micrograph Mass 2 x 106 daltons 7 9 Cartilage proteoglycan Can spring back after being deformed so cartilage can cushion compressive forces HIGHLY HYDRATED 7 10 Definitions proteoglycan vs glycoprotein Proteoglycan Protein Uronic acid and or SO42 containing oligosaccharide glycosaminoglycan Protein Glycosaminoglycan Glycoprotein Protein Oligosaccharide not GAG covalently attached to peptide backbone Two major types of linkage to extracytoplasmic domains of proteins Occur with variations in all eukaryotes Examples in bacteria and archaea 7 11 Two major types of protein saccharide linkage in glycoproteins 7 12 Asparagine linked N glycoproteins Added to proteins in lumen of rough ER Branched oligosaccharide transferred from lipid carrier glycan assembly platform 2 6 2 3 3 2 3 2 6 2 4 4 3 GlcNAc is the first sugar of a branched oligosaccharide GlcNAc Man Glc N glycosidic linkage involving Asn amide N 7 13 Oligosaccharide is built up on and transferred from a carrier lipid 2 6 2 3 3 2 3 2 6 2 4 4 3 Dolichol a polyisoprenoid C80 100 Transfer to selected asparagines in lumen of endoplasmic reticulum H 7 14 N glycosylation is a highly conserved pathway for modification of secretory proteins in the rough ER MEMBRANE 14 sugar oligosaccharide transferred to protein post translationally Helenius A Aebi M 2001 Science 291 2364 2369 7 15 N linked saccharides are trimmed further and new sugars are added back in the Golgi Helenius A Aebi M 2001 Science 291 2364 2369 7 16 A great variety of N linked glycan structures can be generated in the Golgi Oligosacch trimmed back to varying degrees 2 6 2 Different sugars added back 3 3 6 3 2 2 2 4 4 3 in many linkages in Golgi Great structural diversity possible w different sugars different linkages between them and branching VaRiability between protein cell tissue type developmental stage organism important in intermolecular recognition information rich also for stability of some proteins NAc 3 3 3 3 3 3 3 3 3 3 3 3 6 2 6 2 3 4 4 NAc Fucose 7 17 Complex glycans with sialic acid a k a N acetylneuraminic acid HO l ll H C C N l H OH OH OH O COO OH OH C 2 anomeric C 7 18 N linked glycans are important Help promote proper folding of freshly translated polypeptides in the lumen of the ER Can help stabilize protein conformation and protect against proteolysis Can help direct proteins to various membrane compartments e g lysosome in mammalian cells Even subtle underglycosylation of asparagines i e an Asn glycosylation site is missed is associated with human developmental defects Congenital Disorders of Glycosylation 7 19 O linked glycoproteins Mostly added to proteins in lumen of Golgi Single sugar transferred initially Subsequent ones added individually 7 20 Mucins are heavily O glycosylated RICH IN Ser Thr Variable number tandem repeats 7 21 Mucins polymerize S S bonding The highly glycosylated domains of mucins dimerization are l o n g e x t e n d e d structures much less flexible than unglycosylated random coils Oligosaccharides contribute to stiffness by limiting rotation around peptide bonds charge repulsion among neighboring vely charged oligosacch groups O linked saccharides Long extended mucin molecules have a much greater solution volume than native proteins w little or no carbohydrate Mucins therefore form viscous solutions or gels that protect against infection by microbes that bind cell surface carbohydrates Perez Vilar J Hill R L 1999 J Biol Chem 274 31751 7 22 Contribution of carbohydrate to protein structure Protein covalently attached sugar chain N glycosidically linked GPI anchors glycoprotein in membrane covalently attached sugars O glycosidically linked force protein into extended conformation 7 23 Energetics of glycosidic bond formation Formation of a glycosidic bond between two sugars Sugar Sugarn Sugarn 1 energetically unfavorable General strategy for making glycans use an activated sugar donor compounds in which anomeric carbon is activated by attachment to a nucleotide through a phosphodiester linkage e g UDP glucose ke n i l d 7 24 Different sugar nucleotides serve as sugar donors for different glycosyltransferases UDP Glc UDP GlcNAc UDP Gal GDP Man CMP sialic acid Luis Leloir Nobel Prize in Chemistry 1970 for his discovery of sugar nucleotides and their role in the biosynthesis of carbohydrates 7 25 Elongation of an 1 4 linked glycogen chain Glc added at non reducing end of chain UDP Nucleophilic displacement of UDP from C 1 of UDPGlc by C 4 OH of glucose at non reducing end of chain UDP excellent leaving group facilitates nucleophilic attack by activating the sugar carbon to which it is attached 7 26 Nucleophiles and leaving groups Nucleophile from nucleus loving or ve charge loving reactant that provides a pair of electrons to form a new covalent bond ENZYME TAKES THE H CH2OH H O O R Leaving group accepts the electron pair from the nucleophile http www masterorganicchemistry com 2012 06 05 nucleophiles and electrophiles P P R O MAKES NUCLEOPHILIC ATTACK l N ACTIVATION BY UDP MAKES C 1 WANT TO ACCEPT ELECTRONS 7 27 Cellulose synthase catalyzes Glc transfer and
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