Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase

Silberstein, Susana; Gilmore, Reid

doi:10.1096/fasebj.10.8.8666161

Cited by 235 publications

(157 citation statements)

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“…Here it is elongated to Glc 3 Man 9 GlcNAc 2 -PP-dolichol by a set of glycosyltransferases that utilize Dol-P-Man and Dol-PGlc as donors. The precursor oligosaccharide Glc 3 Man 9 GlcNAc 2 is finally transferred by the oligosaccharyltransferase complex onto selected asparagine residues in nascent glycoproteins (35,36).…”

Section: Resultsmentioning

confidence: 99%

A New Type of Congenital Disorders of Glycosylation (CDG-Ii) Provides New Insights into the Early Steps of Dolichol-linked Oligosaccharide Biosynthesis

Thiel

Schwarz

Peng

et al. 2003

Journal of Biological Chemistry

113

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Deficiency of GDP-Man:Man 1 GlcNAc 2 -PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii. The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities. An accumulation of Man 1 GlcNAc 2 -PP-dolichol and Man 2 GlcNAc 2 -PP-dolichol was observed in skin fibroblasts of the patient. Incubation of patient fibroblast extracts with Man 1 GlcNAc 2 -PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man 1 GlcNAc 2 -PP dolichol. Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog. Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2. Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells. hALG2 was shown to act as an ␣1,3-mannosyltransferase. The resulting Man␣1,3-ManGlcNAc 2 -PP dolichol is further elongated by a yet unknown ␣1,6-mannosyltransferase.Congenital disorders of glycosylation (CDG) 1 compose a rapidly growing group of inherited multisystemic disorders in man, which are commonly associated with severe psychomotor and mental retardation (1). The characteristic biochemical feature of CDG is defective glycosylation of proteins due to mutations in genes required for the biosynthesis of N-linked oligosaccharides.The attachment of oligosaccharide chains onto newly synthesized proteins is one of the most widespread forms of co-and post-translational modifications and is found in animals, plants, and bacteria. Glycoproteins are located inside cells predominantly in subcellular organelles and in cellular membranes and most abundantly in extracellular fluids and matrices. The oligosaccharide moiety of the glycoproteins can affect their folding, their transport, as well as their biological activity and stability (2, 3). The complex process of protein glycosylation requires more than a hundred glycosyltransferases, glycosidases, and transport proteins. CDG are classified into two groups. Defects of the assembly of lipid-linked oligosaccharides or their transfer onto nascent glycoproteins compose CDG type I, whereas CDG type II includes all defects of trimming and elongation of N-linked oligosaccharides (4). In the past 7 years the molecular nature of eight CDG-I and four CDG-II types could be identified (5-24).Here we describe for the first time a molecular defect in glycoprotein biosynthesis in man which affects at the cytosolic side of the endoplasmic reticulum the transfer of mannosyl residues from GDP-Man to Man 1 Glc...

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Section: Resultsmentioning

confidence: 99%

A New Type of Congenital Disorders of Glycosylation (CDG-Ii) Provides New Insights into the Early Steps of Dolichol-linked Oligosaccharide Biosynthesis

Thiel

Schwarz

Peng

et al. 2003

Journal of Biological Chemistry

113

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“…However, glycosylation of B-Glyc-KDEL was found to be a relatively inefficient process that increased progressively with incubation time. N-Glycosylation normally occurs cotranslationally, and nascent proteins are presented to oligosaccharyltransferase activity while they are still in an unfolded state (32,47). Fully folded B-Glyc-KDEL may be a relatively poor substrate with respect to these criteria.…”

Section: Discussionmentioning

confidence: 99%

Retrograde Transport of KDEL-bearing B-fragment of Shiga Toxin

Johannes¹,

Tenza

Antony

et al. 1997

Journal of Biological Chemistry

187

254

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To investigate retrograde transport along the biosynthetic/secretory pathway, we have constructed a recombinant Shiga toxin B-fragment carrying an N-glycosylation site and a KDEL retrieval motif at its carboxyl terminus (B-Glyc-KDEL). After incubation with HeLa cells, B-Glyc-KDEL was progressively glycosylated in the endoplasmic reticulum (ER) and remained stably associated with this compartment. B-fragment with a nonfunctional KDEL sequence (B-Glyc-KDELGL) was glycosylated with about the same kinetics as B-Glyc-KDEL but localized at steady state to the Golgi apparatus. Morphological studies showed that B-Glyc-KDEL was delivered from the plasma membrane, via endosomes and the cisternae of the Golgi apparatus, to the ER. Moreover, the addition of a sulfation site allowed us to show that B-Glyc-KDEL on transit to the ER entered the Golgi apparatus through the trans-Golgi network. Transport of B-Glyc-KDEL to the ER was slowed down by nocodazole, indicating that microtubules are important for the retrograde pathway. Our results document the existence of a continuous pathway from the plasma membrane to the endoplasmic reticulum via the Golgi apparatus and show that a fully folded exogenous protein arriving in the endoplasmic reticulum via this pathway can undergo N-glycosylation.

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“…Dad1 comprises the oligosaccharyltransferase complex. This enzyme catalyses the transfer of an oligosaccharide from the dolichol carrier to a susceptible asparagine residue en bloc on a nascent protein, providing the common precursor of N-linked oligosaccharides (for review, see Silberstein & Gilmore 1996). Thus, we can infer that disruption of the Dad1 gene should lead to an absence of N-glycans.…”

Section: Discussionmentioning

confidence: 99%