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, dolichyl-diphosphooligosaccharide biosynthesis and attachment
|Superclasses:||Biosynthesis → Carbohydrates Biosynthesis → Oligosaccharides Biosynthesis|
|Macromolecule Modification → Protein Modification → Protein Glycosylation|
Some taxa known to possess this pathway include : Saccharomyces cerevisiae
Expected Taxonomic Range: Eukaryota
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 branched 14-residue sugar (a tetradecasaccharide) precursor that contains 3 glucose, 9 mannose, and 2 N-acetylglucosamine residues, which is attached to a carrier molecule called dolichol phosphate. The structure of this precursor is common to most eukaryotes - animals, plants, most fungi, and the protist Euglena gracilis use this tetradecasaccharide precursor, although other protists are konwn to differ in the composition of the precursor [Ivatt84, De85, Samuelson05]. The pathway for the biosynthesis of the precursor is highly conserved.
While archaea use different oligosaccharides and use somewhat different dolichol carriers, the archaeal pathway is in general similar to the eukaryotic one, and there is even sequence similarity between some of the eukaryotic, bacterial and arachaeal glycosyltransferases involved in the process [Burda99] (see for example protein N-glycosylation (Methanococcus voltae)).
The eukaryotic biosynthetic pathway 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 a higher-mannose type, which would create a shorter glycoprotein half-life in humans [Wildt05].
In Saccharomyces cerevisiae, the polyisoprenol dolichol contains 14 to 18 isoprene units, with 15 or 16 as the predominant components ( [Jung73], and reviewed in [Burda99]). It is synthesized from farnesyl diphosphate produced by the mevalonate pathway (see trans, trans-farnesyl diphosphate biosynthesis and dolichol and dolichyl phosphate 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 [Burda99a], (in [Samuelson05, Gao04]), reviewed in [Burda99, Helenius04]. 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, Gao05d, 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". It has been initially thought that the flippase is encoded by the RFT1 gene, but that has proven incorrect, and the identity of the flippase is still unknown. 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]).
Note that the seven sugars added outside the ER are nucleoside-activated, while the seven sugars added inside the lumen are derived from individually charged and flipped phosphodolichol carriers.
This core lipid-linked oligosaccharide, (glucosyl)3(mannosyl)9-(N-acetylglucosaminyl)2-diphosphodolichol ( dolichyl diphosphooligosaccharide), is then transferred by the oligosaccharyl transferase complex (OST) to asparagine residues within the recognition sequence asparagine-x-threonine/serine (N-X-T/S) on nascent polypeptides [Karaoglu97, Karaoglu01]. During the transfer a dolichyl diphosphate 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 Saccharomyces 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]).
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
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
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
Gao05d: 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
Guan10: Guan Z, Naparstek S, Kaminski L, Konrad Z, Eichler J (2010). "Distinct glycan-charged phosphodolichol carriers are required for the assembly of the pentasaccharide N-linked to the Haloferax volcanii S-layer glycoprotein." Mol Microbiol 78(5);1294-303. PMID: 21091511
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
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
Kuntz97: Kuntz C, Sonnenbichler J, Sonnenbichler I, Sumper M, Zeitler R (1997). "Isolation and characterization of dolichol-linked oligosaccharides from Haloferax volcanii." Glycobiology 7(7);897-904. PMID: 9363431
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
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
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
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
Chi96: Chi JH, Roos J, Dean N (1996). "The OST4 gene of Saccharomyces cerevisiae encodes an unusually small protein required for normal levels of oligosaccharyltransferase activity." J Biol Chem 271(6);3132-40. PMID: 8621712
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
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
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
Karaoglu95: Karaoglu D, Kelleher DJ, Gilmore R (1995). "Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates." J Cell Biol 130(3);567-77. PMID: 7622558
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