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2.4.99.18: dolichyl-diphosphooligosaccharide-protein glycotransferase

This is an abbreviated version!
For detailed information about dolichyl-diphosphooligosaccharide-protein glycotransferase, go to the full flat file.

Word Map on EC 2.4.99.18

Reaction

dolichyl diphosphooligosaccharide
+
[protein]-L-asparagine
=
dolichyl diphosphate
 +
a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine

Synonyms

AglB, AglB-L, AlgB, asparagine N-glycosyltransferase, DGL1, dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST2, dolichyl-diphosphooligosaccharide-protein glycosyltransferase, dolichyldiphosphooligosaccharide-protein oligosaccharyltransferase, EC 2.4.1.119, glycosyltransferase, dolichyldiphosphooligosaccharide-protein, glycosyltransferase, dolichylpyrophosphodiacetylchitobiose-protein, L.m.STT3, Lm STT3, oligomannosyltransferase, oligosaccharide transferase, oligosaccharyl transferase, oligosaccharyl transferase 16 kDa subunit, oligosaccharyl transferase subunit epsilon, oligosaccharyl transferase subunit OST2, oligosaccharyltransferase, oligosaccharyltransferase complex, oligosaccharyltransferase STT3, oligosaccharyltransferase, dolichyldiphosphoryloligosaccharide-protein, OST, OST-A, OST-B, OST-I, OST-II, OST-III, OST1, OST2, OST3, Ost3p, OST4, OST5, Ost6p, OSTC(I), OSTC(II), OSTC(III), OTase, PglB, PglB oligosaccharyltransferase, PglB1, PglL, PilO, STT3, STT3 protein, STT3-1, STT3-2, STT3-3, STT3-4, Swp1p, TbSTT3A, TbSTT3B, TbSTT3C, Wbp1p, yeast oligosaccharyl transferase

ECTree

     2 Transferases
         2.4 Glycosyltransferases
             2.4.99 Transferring other glycosyl groups
                2.4.99.18 dolichyl-diphosphooligosaccharide-protein glycotransferase

Cloned

Cloned on EC 2.4.99.18 - dolichyl-diphosphooligosaccharide-protein glycotransferase

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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
cloning of DNA encoding the full-length AfAglB-S1 gene, expression of N-terminally His6-tagged wild-type and selenomethionine-labeled AglB in Escherichia coli strains BL21(DE3) and C43(DE3), respectively
cloning of OST48, ribophorin I, ribophorin II and expression in COS-1 cells
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creation of a yeast strain in which the essential 64000 Da glycoprotein Nlt1p subunit of the oligosaccharyl transferase is modified by the addition of a 22-residue carboxy-terminal affinity tag, the tag includes both an 8-residue FLAG epitope and a 6-residue histidine motif
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despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui can readily replace their counterpart from Haloferax volcanii when introduced into Haloferax volcanii cells deleted of aglB
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despite processing distinct lipid-linked glycans in their native hosts, AglB from Halobacterium salinarum can readily replace their counterpart from Haloferax volcanii when introduced into Haloferax volcanii cells deleted of aglB
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despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloferax mediterranei can readily replace their counterpart from Haloferax volcanii when introduced into Haloferax volcanii cells deleted of aglB
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expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL cells
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expression in Escherichia coli
functionally expressed in Escherichia coli
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Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the Helicobacter pullorum PglB1 protein, but not PglB2, is able to transfer Campylobacter jejuni N-linked glycan onto an acceptor protein in Escherichia coli
Lm STT3 paralogues LmSTT3A, LmSTT3B, LmSTT3C, or LmSTT3D are expressed individually in Saccharomyces cerevisiae. Three complemented the yeast stt3 deletion. LmSTT3D expression suppresses deletions of other essential OTase subunits (Wbp1p, Ost1p, Ost2p, and Swp1p). Leishmania major STT3 paralogues are active enzymes that do not incorporate into the yeast OTase complex, but instead form dimers
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OST complex is composed of ribophorin I (Ost1p), ribophorin II (Swp1p), OST48 (Wbp1p), DAD1 (Ost2p), and an STT3 homologue
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PCR-amplification fused to GST, expressed in Escherichia coli BL21 (DE3)pLysS
PCR-amplification, expression of histidine-tagged enzyme in Escherichia coli and Salmonella enterica strains
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PCR-amplification, expression of the pure C-terminal domain of the subunit Stt3p in Escherichia coli BL21 (DE3)
PCR-amplification, FLAG-tag, expression in Saccharomyces cerevisiae
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PCR-amplification, TbSTT3A expression in Saccharomyces cerevisiae does not replace yeast SST3
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PCR-amplification, TbSTT3B expression in Saccharomyces cerevisiae can replace yeast SST3, mild temperature-sensitive growth at 37°C, wild-type growth at 23°C
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PCR-amplification, TbSTT3C expression in Saccharomyces cerevisiae can replace yeast SST3, severe growth defects at 23°C
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PglB is overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures
plasmids with subunit genes introduced into mutants lacking both subunits
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preparation of a membrane fraction from Escherichia coli in which PglB has been overexpressed
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replacement of Saccharomyces cerevisiae Stt3p by the Trypanosoma cruzi homologue
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STT3 from Leishmania is able to complement stt3 deficiency in yeast during vegetative growth, but only poorly during sporulation. The Leishmania STT3 homolog is functional mainly as a free enzyme with a broad specificity for the glycosyl donor. When incorporated into the OST complex, it accepts the common Glc3Man9GlcNAc2-PP-Dol donor. Three out of the four STT3 paralogs are able to functionally complement not only an stt3 mutant but also ost1, ost2, wbp1, or swp1 mutants. STT3 from Leishmania is not incorporated into the complex and most likely functions as a monomeric enzyme
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subcloning, overexpression, and a method of production of the pure C-terminal domain of Stt3p at 60-70 mg/l in Escherichia coli
the C-terminal domain of the STT3 protein of Pyrococcus furiosus is expressed in Escherichia coli cell BL21
the expression plasmid encoding the C-terminal soluble domain (residues 428–713) is constructed by inserting a PCR product from genomic DNA (ATCC700819D) into the pGEX-6P-1 vector (GE Healthcare). The GST-fused sPglB protein is expressed by the addition of isopropyl-beta-D-thiogalactopyranoside at 37°C in the Escherichia coli BL21(DE3)pLysS strain. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family reveals three types of oligosaccharyltransferases catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the oligosaccharyltransferases enzymes from eukarya, archaea, and bacteria
the large soluble domains of the subunits Nlt1p, Swp1p and Wbp1p are expressed in a baculovirus-infected insect cell system
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