N Glycans Dr Lianchun Wang Essentials of Glycobiology Second Edition Common Classes of Animal Glycans Essentials of Glycobiology Second Edition N glycans N glycans are covalently attached to protein at asparagine Asn residues by an N glycosidic bond Five different N glycan linkages are known of which N acetylglucosamine to asparagine GlcNAc 1 Asn is the most common Asn X Ser Thr sequons in a protein are candidates for receiving an N glycan Complicated biosynthesis Dolichol phosphate Dol P as carrier for N glycan biosynthesis N glycan synthetic pathway is conserved in all of the metazoa in plants and in yeast Other linkages to Asn glucose N acetylgalactosamine GalNAc rhamnose And linkage to argnine glucose N glycans affect many properties of glycoproteins including their conformation solubility antigenicity and recognition by glycan binding proteins Essentials of Glycobiology Second Edition Defects in N glycan synthesis lead to a variety of human diseases Types of N glycans Types of N glycans N glycans added to protein at Asn X Ser Thr sequons are of three general types in a mature glycoprotein oligomannose complex and hybrid Each N glycan contains the Essentials of Glycobiology common core Man 1 6 Man 1 3 Man 1 4GlcNAc 1 4GlcNAc Asn Man3GlcNAc2Asn Second Edition N glycan Sites N glycans occurs only on the Asn X Ser Thr sequon About two thirds of protein contain the Asn X Ser Thr consensus sequence Among which more than two thirds of those sequons are likely to be N glycosylated when Asn X Ser Thr sequons are present in a deduced amino acid sequence encoded by a cDNA they are not identified categorically as N glycan sites but are referred to as potential N glycan sites Proof that an N glycan is actually present at a potential site requires experimental evidence Occasionally N glycans occurs at Asn X Cys The transfer of N glycans to Asn X Ser Thr sequons occurs on the lumenal side of the endoplasmic reticulum ER membrane while the protein moiety is being synthesized on ER bound ribosomes and is translocating through the translocon in the ER membrane Essentials of Glycobiology Second Edition N glycan Isolation Analysis Release Peptide N glycosidase F PNGase F remove oligomannose hybrid and complex N glycan from ASN but N glycan core needs not to be modified PNGase A remove all N glycan from Asn Endoglycosidase H release oligomannose and hybrid N glycans but not complex N glycans Endoglycosidase F release simple biantennary N glycans but not oligomannose or hybrid N glycans Hydrazinolysis Protease Purification and analysis ion exchange and size exclusion chromatography high pressure liquid chromotography HPLC methods and affinity chromatograph composition linkage and sequence Essentials of Glycobiology Second Edition Synthesis of N glycan I Synthesis of the Dolichol P P Glycan Precursor II Transfer of the Dolichol linked Precursor to Nascent Proteins III Early Processing Steps Glc3Man9GlcNAc2Asn to Man5GlcNAc2Asn IV Late Processing Steps From Man5GlcNAc2Asn to Hybrid and Complex N Glycans V Maturation of N Glycans Essentials of Glycobiology Second Edition Dolichol Phosphate Dolichol is a polyisoprenol lipid comprised of five carbon isoprene units linked linearly in a head to tail fashion The number of isoprene units in dolichol varies within cells and between cell types and organisms Dol P is used in N glycan synthesis N Glycan synthesis begins by the transfer of GlcNAc 1 P from UDPGlcNAc to Dol P to generate dolichol pyrophosphate Nacetylglucosamine Dol P P GlcNAc This reaction is inhibited by tunicamycin Essentials of Glycobiology Second Edition Synthesis of dolichol P P GlcNAc2Man9Glc3 ALG Altered in glycosylation Dolichol red squiggle phosphate Dol P located on the cytoplasmic face of the ER membrane receives GlcNAc 1 P from UDPGlcNAc in the cytoplasm to generate Dol P P GlcNAc Dol P P GlcNAc is extended to Dol P P GlcNAc2Man5 using GDP Man as precusor before being flipped across the ER membrane to the lumenal side On the lumenal face of the ER membrane four mannose residues are added from Dol P Man and three glucose residues from Dol P Glc Dol P Man and Dol P Glc are also made on the cytoplasmic face of the ER and flipped onto the lumenal face Yeast mutants defective in an ALG gene have been Essentials of Glycobiology used to identify the gene that encodes the enzyme responsible for each transfer Some reactions affected in congenital disorders of Chapter 8 Figure 3 Second Edition glycosylation CDG are noted Processing and maturation of an N glycan Essentials of Glycobiology Second Edition The mature Dol P P glycan is transferred to Asn X Ser Thr sequons during protein synthesis as proteins are being translocated into the ER Following transfer of the 14 sugar Glc3Man9GlcNAc2 glycan to protein glucosidases in the ER remove the three glucose residues and ER mannosidase removes a mannose residue These reactions are intimately associated with the folding of the glycoprotein assisted by the lectins calnexin and calreticulin and they determine whether the glycoprotein continues to the Golgi or is degraded Another lectin termed EDEM binds to mannose residues on misfolded glycoproteins and escorts them via retrotranslocation into the cytoplasm for degradation The removal of the first glucose and therefore all glucose can be blocked by castanospermin For most glycoproteins additional mannose residues are removed in the cis compartment of the Golgi until Man5GlcNAc2Asn is generated The mannosidase inhibitor deoxymannojirimycin blocks the removal of these mannose residues The action of GlcNAcT 1 on Man5GlcNAc2Asn in the medial Golgi initiates the first branch of an N glycan This reaction is blocked in the Lec1 CHO mutant in which GlcNAcT I is inactive leaving Man5GlcNAc2Asn which is not further processed Mannosidase II removes two outer mannose residues in a reaction that is blocked by the inhibitor swainsonine The action of mannosidase II generates the substrate for GlcNAcT II The resulting biantennary N glycan is extended by the addition of fucose galactose and sialic acid to generate a complex N glycan with two branches The addition of galactose does not occur in the Lec8 CHO mutant which has an inactive UDP Gal transporter In Lec8 mutants complex N glycans terminate in N acetylglucosamine The addition of sialic acid does not occur in the Lec2 CHO mutant which has an inactive CMP sialic acid transporter In Lec2 mutants complex Nglycans terminate with galactose Complex N glycans can