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Dolichol is not a necessary moiety for lipid-linked oligosaccharide substrates of the mannosyltransferases involved in in vitro N-linked-oligosaccharide assembly.

ABSTRACT: Dolichol is utilized in vivo as an unusually large anchor on which the precursor for N-linked oligosaccharides is assembled by a series of glycosyltransferases. The role of dolichol in enzyme substrate recognition is investigated. Thus the biosynthetic intermediate NN'-diacetylchitobiose was chemically linked to either dolichol or the much shorter fully saturated tetraisoprenoid phytanol. Both lipids were used as substrates by a recombinant, soluble beta-1,4-mannosyltransferase. beta-[3H]Mannosylated lipids from this reaction were then used as substrates for the subsequent mannosyltransferases from yeast or rat liver microsomes. It was found that both the dolichyl- and phytanyl-linked substrates were easily mannosylated to form Man5GlcNAc2, with some further mannosylation to Man7GlcNAc2 and Man9GlcNAc2 at low concentrations of lipid-linked substrate. It is concluded that dolichol is not necessary in vitro as part of the substrate for the mannosyltransferases in the biosynthetic pathway for N-glycosylation.

SUBMITTER: Wilson IB

PROVIDER: S-EPMC1135982 | biostudies-other | 1995 Sep

REPOSITORIES: biostudies-other

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Dolichol-linked oligosaccharide selection by the oligosaccharyltransferase in protist and fungal organisms.

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The lipid-linked oligosaccharide donor specificities of Trypanosoma brucei oligosaccharyltransferases.

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Inhibition of lipid-linked oligosaccharide synthesis by aryl phosphates.

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Translation attenuation by PERK balances ER glycoprotein synthesis with lipid-linked oligosaccharide flux.

Project description:Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc(3)Man(9)GlcNAc(2) (G(3)M(9)Gn(2)) from the lipid-linked oligosaccharide (LLO) glucose(3)mannose(9)N-acetylglucosamine(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) on Ser(51) by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G(3)M(9)Gn(2)-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2alpha Ser(51) phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser(51) kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway.

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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.

Project description:N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc(3)Man(9) GlcNAc(2)-PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the alpha1,3- and the alpha1,6-mannose-linked residue from GDP-mannose to Man(1)GlcNAc(2)-PP-Dol forming the Man(3)GlcNAc(2)-PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N- or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX(7)E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys(230), as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.

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Metabolically programmed quality control system for dolichol-linked oligosaccharides.

Project description:The glycolipid Glc3Man9GlcNAc2-pyrophosphate-dolichol serves as the precursor for asparagine (N)-linked protein glycosylation in mammals. The biosynthesis of dolichol-linked oligosaccharides (DLOs) is arrested in low-glucose environments via unknown mechanisms, resulting in abnormal N-glycosylation. Here, we show that under glucose deprivation, DLOs are prematurely degraded during the early stages of DLO biosynthesis by pyrophosphatase, leading to the release of singly phosphorylated oligosaccharides into the cytosol. We identified that the level of GDP-mannose (Man), which serves as a donor substrate for DLO biosynthesis, is substantially reduced under glucose deprivation. We provide evidence that the selective shutdown of the GDP-Man biosynthetic pathway is sufficient to induce the release of phosphorylated oligosaccharides. These results indicate that glucose-regulated metabolic changes in the GDP-Man biosynthetic pathway cause the biosynthetic arrest of DLOs and facilitate their premature degradation by pyrophosphatase. We propose that this degradation system may avoid abnormal N-glycosylation with premature oligosaccharides under conditions that impair efficient DLO biosynthesis.

| S-EPMC3845098 | biostudies-literature

Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody.

Project description:PglK is an ABC transporter that flips a lipid-linked oligosaccharide (LLO) that serves as a donor in protein N-glycosylation. Previous structures revealed two inward-facing conformations, both with very large separations of the nucleotide binding domains (NBDs), and a closed, ADP-bound state that featured an occluded cavity. To investigate additional states, we developed conformation-sensitive, single-domain camelid nanobodies (Nb) and studied their effect on PglK activity. Biochemical, structural, and mass spectrometric analyses revealed that one inhibitory Nb binds as a single copy to homodimeric PglK. The co-crystal structure of this Nb and ADP-bound PglK revealed a new, narrowly inward-open conformation. Rather than inducing asymmetry in the PglK homodimer, the binding of one Nb results in steric constraints that prevent a second Nb to access the symmetry-related site in PglK. The Nb performed its inhibitory role by a "sticky-doorstop" mechanism, where inhibition of ATP hydrolysis and LLO flipping activity occurs due to impaired closing of the NBD interface, which prevents PglK from converting to an outward-open conformation. This inhibitory mode suggests tight conformational coupling between the ATPase sites, which may apply to other ABC transporters.

| S-EPMC5395944 | biostudies-literature

Nogo-B receptor is necessary for cellular dolichol biosynthesis and protein N-glycosylation.

Project description:Dolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite the recognition of cis-IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood. Here, we identify Nogo-B receptor (NgBR) as an essential component of the Dol-P biosynthetic machinery. Loss of NgBR results in a robust deficit in cis-IPTase activity and Dol-P production, leading to diminished levels of dolichol-linked oligosaccharides and a broad reduction in protein N-glycosylation. NgBR interacts with the previously identified cis-IPTase hCIT, enhances hCIT protein stability, and promotes Dol-P production. Identification of NgBR as a component of the cis-IPTase machinery yields insights into the regulation of dolichol biosynthesis.

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