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...
The key step of N-glycosylation of proteins in the endoplasmic reticulum is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). It transfers the lipid-linked core-oligosaccharide to selected Asn-X-Ser/Thrsequences of nascent polypeptide chains. Biochemical and genetic approaches have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1p, Swp1p, Stt3p, Ost1p, Ost2p, Ost4p, Ost5p, Ostp3 and Ost6p. By blue native polyacrylamide electrophoresis we show that yeast OST consists of two isoforms with distinct functions differing only in the presence of the two related Ost3 and Ost6p proteins. The OST6-complex was found to be important for cell wall integrity and temperature stress. Ost3p and Ost6p are not essential for OST activity, and can in part displace each other in the complex when overexpressed, suggesting a dynamic regulation of the complex formation.
The molecular nature of a severe multisystemic disorder with a recurrent nonimmune hydrops fetalis was identified as deficiency of GDP-Man:GlcNAc(2)-PP-dolichol mannosyltransferase, the human orthologue of the yeast ALG1 gene (MIM 605907). The disease belongs to the group of congenital disorders of glycosylation (CDG) and is designated as subtype CDG-Ik. In patient-derived serum, the total amount of the glycoprotein transferrin was reduced. Moreover, a partial loss of N-glycan chains was observed, a characteristic feature of CDG type I forms. Metabolic labeling with [6-(3)H]glucosamine revealed an accumulation of GlcNAc(2)-PP-dolichol and GlcNAc(1)-PP-dolichol in skin fibroblasts of the patient. Incubation of fibroblast extracts with [(14)C]GlcNAc(2)-PP-dolichol and GDP-mannose indicated a severely reduced activity of the beta 1,4-mannosyltransferase, elongating GlcNAc(2)-PP-dolichol to Man(1)GlcNAc(2)-PP-dolichol at the cytosolic side of the endoplasmic reticulum. Genetic analysis of the patient's hALG1 gene identified a homozygous mutation leading to the exchange of a serine residue to leucine at position 258 in the hALG1 protein. The disease-causing nature of the hALG1 mutation for the glycosylation defect was verified by a retroviral complementation approach in patient-derived primary fibroblasts and was confirmed by the expression of wild-type and mutant hALG1 in the Saccharomyces cerevisiae alg1-1 strain.
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 ␣1,3-and the ␣1,6-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.Asparagine-linked glycosylation is an essential protein modification highly conserved in eukaryotes (1-4), and several features of this pathway even occur in prokaryotes (5-7). In eukaryotes, biosynthesis of N-glycans starts with the assembly of the common core oligosaccharide precursor Glc 3 Man 9 GlcNAc 2 -PP-Dol, the glycan moiety of which is subsequently transferred onto selected Asn-Xaa-(Ser/Thr) acceptor sites of the nascent polypeptide chain by the oligosaccharyl-transferase complex (8 -10). The initial steps of the dolichol pathway up to Man 5 GlcNAc 2 -PP-Dol take place on the cytosolic site of the endoplasmic reticulum (ER), 2 using sugar nucleotides as glycosyl donors. Upon translocation of the heptasaccharide to the luminal site, which is facilitated by Rft1 (11) and another not yet identified protein (12), it is extended by four mannose and three glucose residues deriving from Man-P-Dol and Glc-P-Dol. It has been demonstrated that the pathway operates sequentially in an ordered fashion based on differences in the substrate specificity of the various glycosyltransferases (13). In the yeast Saccharomyces cerevisiae, alg mutants (for asparagine-linked glycosylation) have been isolated, defective in lipid-linked oligosaccharide (LLO) assembly (14 -17), and shown to be invaluable to define the pathway as well as to isolate the genes encoding the respective glycosyltransferases by complementing a particular phenotype characteristic of the respective mutant. Likewise various mutant cell lines from mammalian origin have been described that produce truncated lipid-linked oligosaccharides (18 -20).One of the tempe...
We consider a circular deconvolution problem, in which the density f of a circular random variable X must be estimated nonparametrically based on an i.i.d. sample from a noisy observation Y of X. The additive measurement error is supposed to be independent of X. The objective of this work was to construct a fully data-driven estimation procedure when the error density ϕ is unknown. We assume that in addition to the i.i.d. sample from Y , we have at our disposal an additional i.i.d. sample drawn independently from the error distribution. We first develop a minimax theory in terms of both sample sizes. We propose an orthogonal series estimator attaining the minimax rates but requiring optimal choice of a dimension parameter depending on certain characteristics of f and ϕ, which are not known in practice. The main issue addressed in this work is the adaptive choice of this dimension parameter using a model selection approach. In a first step, we develop a penalized minimum contrast estimator assuming that the error density is known. We show that this partially adaptive estimator can attain the lower risk bound up to a constant in both sample sizes n and m. Finally, by randomizing the penalty and the collection of models, we modify the estimator such that it no longer requires any previous knowledge of the error distribution. Even when dispensing with any hypotheses on ϕ, this fully data-driven estimator still preserves minimax optimality in almost the same cases as the partially adaptive estimator. We illustrate our results by computing minimal rates under classical smoothness assumptions.
We estimate the distribution of a real-valued random variable from contaminated observations. The additive error is supposed to be normally distributed, but with unknown variance. The distribution is identifiable from the observations if we restrict the class of considered distributions by a simple condition in the time domain. A minimum distance estimator is shown to be consistent imposing only a slightly stronger assumption than the identification condition.
This study applied a procedure for the identification of potential emerging chemical risks in the food chain to substances registered under the REACH Regulation that was previously developed and tested in an EFSA‐sponsored pilot study. The selection was limited to substances that (a) were registered with a full registration, (b) met eligibility criteria (e.g. availability of a CAS number and a SMILES notation) and (c) were considered to be inside the applicability domain of the models used in this study (excluding e.g. ionisable compounds and metals). This selection reduced the number of substances from about 15 000 to 2 336 substances that were subsequently assessed in four blocks: environmental releases (based on tonnage and use pattern), biodegradation (using BIOWIN predictions assessed in a battery approach), bioaccumulation in food/feed (using ACC‐HUMANsteady modelling) and toxicity (based on classification for carcinogenicity, mutagenicity, reprotoxicity and repeated dose toxicity). A scoring system was applied with a maximum score of 10 in each of the four blocks. The procedure showed a good degree of differentiation in each block. Two weighting scenarios and pivot table selections were applied to the scores in the four blocks. An evaluation of both approaches led to the prioritisation of substances for their potential to represent ‘emerging chemical risks’ in the food chain. Following additional curation steps, 212 ‘potential emerging risks’ were identified that are considered to (a) be released to the environment and/or poorly biodegraded, (b) bioaccumulate in food/feed and (c) represent a chronic human health hazard. In this study, in‐depth evaluations were performed for ten ‘potential emerging risks’ that so far have not been assessed by an EU regulatory body for their presence in food via the investigated exposure pathway. The selection of these ten substances does not imply that the remaining 202 potential emerging risks are of lower priority
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.