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MetaCyc Pathway: dolichyl-diphosphooligosaccharide biosynthesis

This view shows enzymes only for those organisms listed below, in the list of taxa known to possess the pathway. If an enzyme name is shown in bold, there is experimental evidence for this enzymatic activity.

Synonyms: mannosyl-chito-dolichol biosynthesis, eukaryotic N-linked glycosylation

Superclasses: Biosynthesis Carbohydrates Biosynthesis Oligosaccharides Biosynthesis

Some taxa known to possess this pathway include ? : Saccharomyces cerevisiae

Expected Taxonomic Range: Eukaryota

Summary:
N-linked glycosylation is an important process found in eukaryotes and archaea, and very rarely in bacteria. During this process certain oligosaccharides are attached to an asparagine residue in the polypeptide chain of the target protein. It has been shown that the glycosylation is essential for the proper folding of some eukaryotic proteins.

In Eukaryotes, most N-linked oligosaccharides derive from a 14-residue sugar (tetradecasaccharide) branched precursor that contains 3 glucose, 9 mannose, and 2 N-acetylglucosamine residues, which is attached to a carrier molecule called dolichol. The structure of this precursor is common to most eukaryotes - animals, plants, most fungi,and Euglena gracilis use this tetradecasaccharide precursor, although other protists glycosylate the precursor differently [Samuelson05, Ivatt84, De85]. The pathway for the biosynthesis of the precursor is highly conserved. The archaeal pathway is similar to the eukaryotic one, and there is even sequence similarity between some of the eukaryotic glycosyltransferases and bacterial transferases involved in murein and lipopolysaccharide biosynthesis (reviewed in [Burda99]).

The biosynthetic pathway for this lipid-linked precursor begins with the phosphorylation of dolichol, followed by stepwise addition of sugar residues to form the product dolichyl-diphosphooligosaccharide. This pathway is of particular interest in humans, because defects in the glycosyltransferases involved lead to congenital disorders of glycosylation. The genetic analysis of this pathway in a model organism, the budding yeast Saccharomyces cerevisiae, has helped to elucidate the molecular basis of these disorders (reviewed in [Aebi01]). Humanization of the N-glycosylation pathway in yeast may allow production of therapeutically useful glycoproteins. Humanization is necessary because N-glycosylation in yeast is of the high-mannose type, which would create a shorter glycoprotein half-life in humans [Wildt05].

In Saccharomyces cerevisiae, the polyisoprenol dolichol (see a dolichol) contains 14 to 18 isoprene units, with 15 or 16 as the main components ([Jung73], and reviewed in [Burda99]). It is synthesized from farnesyl diphosphate produced by the mevalonate pathway (see also trans, trans-farnesyl diphosphate biosynthesis). Dolichol can be phosphorylated de novo to dolichyl phosphate by the CTP-mediated dolichol kinase, the product of gene SEC59 [Bernstein89, Heller92]. Dolichyl phosphate can also be regenerated by other reactions (reviewed in [Burda99]).

Dolichyl phosphate is the essential carrier lipid for glycosyl residues. Each of the glycosyl residues is added by a specific glycosyltransferase, although not all have been defined [Burda99a], (in [Samuelson05, Gao04]), reviewed in [Burda99, Helenius04]. Reactions in this pathway without defined or putative yeast enzymes are indicated as hypothetical. The substrate specificity of these enzymes results in a highly ordered assembly of glycosyl residues [Burda99a]. In Saccharomyces cerevisiae, Alg (Asparagine-linked glycosylation) mutants of the glycosyltransferases have been isolated and the accumulated dolichyl-diphospho-glycan intermediates analyzed. On the cytosolic side of the endoplasmic reticulum, glycosyltransferase Alg7 adds phospho-N-acetylglucosamine to dolichol-P; Alg13 and Alg14 form a complex that adds N-acetylglucosamine [Bickel05, Gao05c, Chantret05]; Alg1 adds the first mannose residue; Alg2 adds the second and third mannose residues [OReilly06]; and Alg11 adds the fourth and fifth mannose residues [OReilly06], creating the (mannosyl)5-(N-acetylglucosaminyl)2-diphosphodolichol intermediate [Cipollo01], (reviewed in [Burda99]). There is evidence that Alg1, Alg2 and Alg11 are physically associated in oligomeric complexes, suggesting a possible regulatory mechanism [Gao04].

After the fifth mannose residue is added, the dolichol-diphospho-glycan is translocated (flipped) into the lumen of the endoplasmic reticulum by a "flippase", the Rft1 protein [Helenius02]. Mannosyltransferases in the lumen of the endoplasmic reticulum, Alg3, Alg9, and Alg12 use dolichyl β-D-mannosyl phosphate, synthesized by the product of gene DPM1, as a sugar donor to add other mannose residues. Also in the lumen of the endoplasmic reticulum, dolichyl β-D-glycosyl phosphate, synthesized by Alg5, is the sugar donor to add the three glucose residues via Alg6, Alg8, and Alg10 ([Frank05] and reviewed in [Burda99]).

This core lipid-linked oligosaccharide, (glucosyl)3(mannosyl)9-(N-acetylglucosaminyl)2-diphosphodolichol (dolichyl-diphosphooligosaccharide), is then transferred by an oligosaccharyltransferase, OST, to asparagine residues within the recognition sequence asparagine-x-threonine/serine (N-X-T/S) on nascent polypeptides [Karaoglu97, Karaoglu01]. After transfer, dolichyl diphosphate (dolichyl pyrophosphate) is released in the lumen of the endoplasmic reticulum where it is cleaved by a dolichyl diphosphate phosphatase to a dolichyl phosphate. Mechanisms for recycling of this dolichyl phosphate have been proposed [Rush08].

In higher eukaryotes, further processing of the glycosylated, nascent polypeptide by enzymatic glucose addition and removal serves as a quality control mechanism for improperly folded proteins in the endoplasmic reticulum, although this mechanism appears to be modified in S. cerevisiae [Jakob98]. Final glycosylation of properly folded proteins occurrs in the golgi (reviewed in [Herscovics93]). In Saccharomyces cerevisiae, expression of the alg genes may also have a role in regulation of the cell cycle (reviewed in [Kukuruzinska99]).

Citations: [Overbeek98]

Credits:
Revised 25-Sep-2006 by Fulcher CA , SRI International
Revised 23-Jan-2009 by Fulcher CA , SRI International


References

Aebi01: Aebi M, Hennet T (2001). "Congenital disorders of glycosylation: genetic model systems lead the way." Trends Cell Biol 11(3);136-41. PMID: 11306275

Bernstein89: Bernstein M, Kepes F, Schekman R (1989). "Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae." Mol Cell Biol 9(3);1191-9. PMID: 2657387

Bickel05: Bickel T, Lehle L, Schwarz M, Aebi M, Jakob CA (2005). "Biosynthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Alg13p and Alg14p form a complex required for the formation of GlcNAc(2)-PP-dolichol." J Biol Chem 280(41);34500-6. PMID: 16100113

Burda99: Burda P, Aebi M (1999). "The dolichol pathway of N-linked glycosylation." Biochim Biophys Acta 1426(2);239-57. PMID: 9878760

Burda99a: Burda P, Jakob CA, Beinhauer J, Hegemann JH, Aebi M (1999). "Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases." Glycobiology 9(6);617-25. PMID: 10336995

Chantret05: Chantret I, Dancourt J, Barbat A, Moore SE (2005). "Two proteins homologous to the N- and C-terminal domains of the bacterial glycosyltransferase Murg are required for the second step of dolichyl-linked oligosaccharide synthesis in Saccharomyces cerevisiae." J Biol Chem 280(10);9236-42. PMID: 15615718

Cipollo01: Cipollo JF, Trimble RB, Chi JH, Yan Q, Dean N (2001). "The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum." J Biol Chem 276(24);21828-40. PMID: 11278778

De85: De La Canal, L., Parodi, A.J. "Glycosylation of proteins in the protozoan Euglena gracilis." Comp. Biochem. Physiol. 81B:803-805 1985.

Fedorow05: Fedorow H, Pickford R, Hook JM, Double KL, Halliday GM, Gerlach M, Riederer P, Garner B (2005). "Dolichol is the major lipid component of human substantia nigra neuromelanin." J Neurochem 92(4);990-5. PMID: 15686500

Frank05: Frank CG, Aebi M (2005). "ALG9 mannosyltransferase is involved in two different steps of lipid-linked oligosaccharide biosynthesis." Glycobiology 15(11);1156-63. PMID: 15987956

Gao04: Gao XD, Nishikawa A, Dean N (2004). "Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum." Glycobiology 14(6);559-70. PMID: 15044395

Gao05c: Gao XD, Tachikawa H, Sato T, Jigami Y, Dean N (2005). "Alg14 recruits Alg13 to the cytoplasmic face of the endoplasmic reticulum to form a novel bipartite UDP-N-acetylglucosamine transferase required for the second step of N-linked glycosylation." J Biol Chem 280(43);36254-62. PMID: 16100110

Helenius02: Helenius J, Ng DT, Marolda CL, Walter P, Valvano MA, Aebi M (2002). "Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein." Nature 415(6870);447-50. PMID: 11807558

Helenius04: Helenius A, Aebi M (2004). "Roles of N-linked glycans in the endoplasmic reticulum." Annu Rev Biochem 73;1019-49. PMID: 15189166

Heller92: Heller L, Orlean P, Adair WL (1992). "Saccharomyces cerevisiae sec59 cells are deficient in dolichol kinase activity." Proc Natl Acad Sci U S A 89(15);7013-6. PMID: 1323123

Herscovics93: Herscovics A, Orlean P (1993). "Glycoprotein biosynthesis in yeast." FASEB J 7(6);540-50. PMID: 8472892

Ivatt84: Ivatt RL, Das OP, Henderson EJ, Robbins PW (1984). "Glycoprotein biosynthesis in dictyostelium discoideum: developmental regulation of the protein-linked glycans." Cell 38(2);561-7. PMID: 6088086

Jakob98: Jakob CA, Burda P, te Heesen S, Aebi M, Roth J (1998). "Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo." Glycobiology 8(2);155-64. PMID: 9451025

Jung73: Jung P, Tanner W (1973). "Identification of the lipid intermediate in yeast mannan biosynthesis." Eur J Biochem 37(1);1-6. PMID: 4580884

Karaoglu01: Karaoglu D, Kelleher DJ, Gilmore R (2001). "Allosteric regulation provides a molecular mechanism for preferential utilization of the fully assembled dolichol-linked oligosaccharide by the yeast oligosaccharyltransferase." Biochemistry 40(40);12193-206. PMID: 11580295

Karaoglu97: Karaoglu D, Kelleher DJ, Gilmore R (1997). "The highly conserved Stt3 protein is a subunit of the yeast oligosaccharyltransferase and forms a subcomplex with Ost3p and Ost4p." J Biol Chem 272(51);32513-20. PMID: 9405463

Kukuruzinska99: Kukuruzinska MA, Lennon-Hopkins K (1999). "ALG gene expression and cell cycle progression." Biochim Biophys Acta 1426(2);359-72. PMID: 9878828

OReilly06: O'Reilly MK, Zhang G, Imperiali B (2006). "In vitro evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-linked glycoprotein biosynthesis." Biochemistry 45(31);9593-603. PMID: 16878994

Overbeek98: Overbeek, R, Larsen, N, Selkov, EE, Maltsev, M "The WIT Database." 1998 WWW URL http://www.cme.msu.edu/WIT/.

Rush08: Rush JS, Gao N, Lehrman MA, Waechter CJ (2008). "Recycling of dolichyl monophosphate to the cytoplasmic leaflet of the endoplasmic reticulum after the cleavage of dolichyl pyrophosphate on the lumenal monolayer." J Biol Chem 283(7);4087-93. PMID: 18077451

Samuelson05: Samuelson J, Banerjee S, Magnelli P, Cui J, Kelleher DJ, Gilmore R, Robbins PW (2005). "The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases." Proc Natl Acad Sci U S A 102(5);1548-53. PMID: 15665075

Sato01: Sato M, Fujisaki S, Sato K, Nishimura Y, Nakano A (2001). "Yeast Saccharomyces cerevisiae has two cis-prenyltransferases with different properties and localizations. Implication for their distinct physiological roles in dolichol synthesis." Genes Cells 6(6);495-506. PMID: 11442630

Wildt05: Wildt S, Gerngross TU (2005). "The humanization of N-glycosylation pathways in yeast." Nat Rev Microbiol 3(2);119-28. PMID: 15685223

Other References Related to Enzymes, Genes, Subpathways, and Substrates of this Pathway

Aebi96: Aebi M, Gassenhuber J, Domdey H, te Heesen S (1996). "Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae." Glycobiology 6(4);439-44. PMID: 8842708

Albright89: Albright CF, Orlean P, Robbins PW (1989). "A 13-amino acid peptide in three yeast glycosyltransferases may be involved in dolichol recognition." Proc Natl Acad Sci U S A 86(19);7366-9. PMID: 2678101

Albright90: Albright CF, Robbins RW (1990). "The sequence and transcript heterogeneity of the yeast gene ALG1, an essential mannosyltransferase involved in N-glycosylation." J Biol Chem 265(12);7042-9. PMID: 2182636

Babczinski80: Babczinski P, Haselbeck A, Tanner W (1980). "Yeast mannosyl transferases requiring dolichyl phosphate and dolichyl phosphate mannose as substrate. Partial purification and characterization of the solubilized enzyme." Eur J Biochem 105(3);509-15. PMID: 6989607

Barnes84: Barnes G, Hansen WJ, Holcomb CL, Rine J (1984). "Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step." Mol Cell Biol 4(11);2381-8. PMID: 6096695

Burda96: Burda P, te Heesen S, Brachat A, Wach A, Dusterhoft A, Aebi M (1996). "Stepwise assembly of the lipid-linked oligosaccharide in the endoplasmic reticulum of Saccharomyces cerevisiae: identification of the ALG9 gene encoding a putative mannosyl transferase." Proc Natl Acad Sci U S A 93(14);7160-5. PMID: 8692962

Burda98: Burda P, Aebi M (1998). "The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation." Glycobiology 8(5);455-62. PMID: 9597543

Chantret03: Chantret I, Dancourt J, Dupre T, Delenda C, Bucher S, Vuillaumier-Barrot S, Ogier de Baulny H, Peletan C, Danos O, Seta N, Durand G, Oriol R, Codogno P, Moore SE (2003). "A deficiency in dolichyl-P-glucose:Glc1Man9GlcNAc2-PP-dolichyl alpha3-glucosyltransferase defines a new subtype of congenital disorders of glycosylation." J Biol Chem 278(11);9962-71. PMID: 12480927

Cipollo00: Cipollo JF, Trimble RB (2000). "The accumulation of Man(6)GlcNAc(2)-PP-dolichol in the Saccharomyces cerevisiae Deltaalg9 mutant reveals a regulatory role for the Alg3p alpha1,3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing." J Biol Chem 275(6);4267-77. PMID: 10660594

Couto84: Couto JR, Huffaker TC, Robbins PW (1984). "Cloning and expression in Escherichia coli of a yeast mannosyltransferase from the asparagine-linked glycosylation pathway." J Biol Chem 259(1);378-82. PMID: 6368538

Haselbeck82: Haselbeck A, Tanner W (1982). "Dolichyl phosphate-mediated mannosyl transfer through liposomal membranes." Proc Natl Acad Sci U S A 79(5);1520-4. PMID: 6951194

Haselbeck89: Haselbeck A (1989). "Purification of GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase from Saccharomyces cerevisiae." Eur J Biochem 181(3);663-8. PMID: 2659345

Heesen94: Heesen S, Lehle L, Weissmann A, Aebi M (1994). "Isolation of the ALG5 locus encoding the UDP-glucose:dolichyl-phosphate glucosyltransferase from Saccharomyces cerevisiae." Eur J Biochem 224(1);71-9. PMID: 8076653

Huffaker83: Huffaker TC, Robbins PW (1983). "Yeast mutants deficient in protein glycosylation." Proc Natl Acad Sci U S A 80(24);7466-70. PMID: 6369318

Jackson93: Jackson BJ, Kukuruzinska MA, Robbins P (1993). "Biosynthesis of asparagine-linked oligosaccharides in Saccharomyces cerevisiae: the alg2 mutation." Glycobiology 3(4);357-64. PMID: 8400550

Jadid11: Jadid N, Mialoundama AS, Heintz D, Ayoub D, Erhardt M, Mutterer J, Meyer D, Alioua A, Van Dorsselaer A, Rahier A, Camara B, Bouvier F (2011). "DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 mediates the biogenesis of isoprenyl-linked glycans and influences development, stress response, and ammonium hypersensitivity in Arabidopsis." Plant Cell 23(5);1985-2005. PMID: 21558543

Kampf09: Kampf M, Absmanner B, Schwarz M, Lehle L (2009). "Biochemical characterization and membrane topology of Alg2 from Saccharomyces cerevisiae as a bifunctional alpha1,3- and 1,6-mannosyltransferase involved in lipid-linked oligosaccharide biosynthesis." J Biol Chem 284(18);11900-12. PMID: 19282279

Kukuruzinska87: Kukuruzinska MA, Robbins PW (1987). "Protein glycosylation in yeast: transcript heterogeneity of the ALG7 gene." Proc Natl Acad Sci U S A 84(8);2145-9. PMID: 3031666

Latendresse13: Latendresse M. (2013). "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."

Lazarowski03: Lazarowski ER, Shea DA, Boucher RC, Harden TK (2003). "Release of cellular UDP-glucose as a potential extracellular signaling molecule." Mol Pharmacol 63(5);1190-7. PMID: 12695547

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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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