Essentials of GlycobiologySecond EditionN-Glycans Dr. Lianchun WangEssentials of GlycobiologySecond EditionCommon Classes of Animal GlycansEssentials of GlycobiologySecond EditionN-glycans• 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 theirconformation, solubility, antigenicity, and recognition by glycan-bindingproteins.• Defects in N-glycan synthesis lead to a variety of human diseases• N-glycans are covalently attached toprotein at asparagine (Asn) residues by anN-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 arecandidates for receiving an N-glycanEssentials of GlycobiologySecond EditionTypes of N-glycansTypes of N-glycans. N-glycans added to protein at Asn-X-Ser/Thr sequons are of three generaltypes in a mature glycoprotein: oligomannose, complex, and hybrid. Each N-glycan contains thecommon core Man" 1–6(Man" 1–3)Man! 1–4GlcNAc! 1–4GlcNAc! -Asn (Man3GlcNAc2Asn).Essentials of GlycobiologySecond EditionN-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 sequenceencoded by a cDNA, they are not identified categorically as N-glycan sites, but arereferred to as potential N-glycan sites. Proof that an N-glycan is actually present ata 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 sideof the endoplasmic reticulum (ER) membrane while the protein moiety is beingsynthesized on ER-bound ribosomes and is translocating through the translocon inthe ER membraneEssentials of GlycobiologySecond EditionN-glycan Isolation & Analysis• Release- Peptide-N-glycosidase F (PNGase F): remove oligomannose, hybrid andcomplex 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 notcomplex N-glycans.- Endoglycosidase F: release simple biantennary N-glycans, but notoligomannose or hybrid N-glycans- Hydrazinolysis- Protease• Purification and analysis- ion-exchange and size-exclusion chromatography, high-pressure liquidchromotography (HPLC) methods, and affinity chromatograph- composition, linkage and sequenceEssentials of GlycobiologySecond EditionSynthesis of N-glycanI. Synthesis of the Dolichol-P-P-Glycan PrecursorII. Transfer of the Dolichol-linked Precursor to NascentProteinsIII. Early Processing Steps: Glc3Man9GlcNAc2Asn toMan5GlcNAc2AsnIV. Late Processing Steps: From Man5GlcNAc2Asn toHybrid and Complex N-GlycansV. Maturation of N-GlycansEssentials of GlycobiologySecond EditionDolichol Phosphate Dolichol is a polyisoprenol lipid comprised of five-carbon isopreneunits linked linearly in a head-to-tail fashion. The number of isoprene units in dolichol varies within cells andbetween cell types and organisms. Dol-P is used in N-glycan synthesis N-Glycan synthesis begins by the transfer of GlcNAc-1-P from UDP-GlcNAc to Dol-P to generate dolichol pyrophosphate N-acetylglucosamine (Dol-P-P-GlcNAc). This reaction is inhibited bytunicamycin.Essentials of GlycobiologySecond EditionSynthesis of dolichol-P-P-GlcNAc2Man9Glc3Chapter 8, Figure 3Essentials of GlycobiologySecond EditionDolichol (red squiggle) phosphate (Dol-P) located on the cytoplasmic face of the ER membrane receives GlcNAc-1-P from UDP-GlcNAc in the cytoplasm to generate Dol-P-P-GlcNAc. Dol-P-P-GlcNAc is extended to Dol-P-P-GlcNAc2Man5 using GDP-Man asprecusor before being “flipped” across the ER membrane to the lumenal side. On the lumenal face of the ER membrane, fourmannose residues are added from Dol-P-Man and three glucose residues from Dol-P-Glc. Dol-P-Man and Dol-P-Glc are alsomade on the cytoplasmic face of the ER and “flipped” onto the lumenal face. Yeast mutants defective in an ALG gene have beenused to identify the gene that encodes the enzyme responsible for each transfer. Some reactions affected in congenital disorders ofglycosylation (CDG) are noted.ALG= Altered in glycosylationEssentials of GlycobiologySecond EditionProcessing and maturation of an N-glycanThe mature Dol-P-P-glycan is transferred to Asn-X-Ser/Thr sequonsduring protein synthesis as proteins are being translocated into theER. Following transfer of the 14-sugar Glc3Man9GlcNAc2 glycan toprotein, glucosidases in the ER remove the three glucose residues,and ER mannosidase removes a mannose residue. These reactionsare intimately associated with the folding of the glycoproteinassisted by the lectins calnexin and calreticulin, and theydetermine whether the glycoprotein continues to the Golgi or isdegraded. Another lectin, termed EDEM, binds to mannose residueson misfolded glycoproteins and escorts them via retrotranslocationinto the cytoplasm for degradation. The removal of the first glucose(and therefore all glucose) can be blocked by castanospermin. Formost glycoproteins, additional mannose residues are removed in thecis compartment of the Golgi until Man5GlcNAc2Asn is generated.The mannosidase inhibitor deoxymannojirimycin blocks the removalof these mannose residues. The action of GlcNAcT-1 onMan5GlcNAc2Asn in the medial-Golgi initiates the first branch of anN-glycan. This reaction is blocked in the Lec1 CHO mutant in whichGlcNAcT-I is inactive, leaving Man5GlcNAc2Asn, which is not furtherprocessed. " -Mannosidase II removes two outer mannose residuesin a reaction that is blocked by the inhibitor swainsonine. The actionof !-mannosidase II generates the substrate for GlcNAcT-II. Theresulting biantennary N-glycan is extended by the addition of fucose,galactose, and sialic acid to generate a complex N-glycan with twobranches. The addition of galactose does not occur in the Lec8 CHOmutant, which has an inactive UDP-Gal transporter. In Lec8 mutants,complex N-glycans terminate in N-acetylglucosamine. The addition ofsialic acid does not