Chemo-enzymatic synthesis of tetrasaccharide linker peptides to study the divergent step in glycosaminoglycan biosynthesis.

Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis.

Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.

Oligosaccharyltransferase (OST) is an essential membrane protein complex in the endoplasmic reticulum, where it transfers an oligosaccharide from a dolichol-pyrophosphate-activated donor to glycosylation sites of secretory proteins. Here we describe the atomic structure of yeast OST determined by cryo-electron microscopy, revealing a conserved subunit arrangement. The active site of the catalytic STT3 subunit points away from the center of the complex, allowing unhindered access to substrates. The dolichol-pyrophosphate moiety binds to a lipid-exposed groove of STT3, whereas two noncatalytic subunits and an ordered N-glycan form a membrane-proximal pocket for the oligosaccharide. The acceptor polypeptide site faces an oxidoreductase domain in stand-alone OST complexes or is immediately adjacent to the translocon, suggesting how eukaryotic OSTs efficiently glycosylate a large number of polypeptides before their folding.

Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains.

Phosphorus is a macronutrient taken up by cells as inorganic phosphate (P(i)). How cells sense cellular P(i) levels is poorly characterized. Here, we report that SPX domains--which are found in eukaryotic phosphate transporters, signaling proteins, and inorganic polyphosphate polymerases--provide a basic binding surface for inositol polyphosphate signaling molecules (InsPs), the concentrations of which change in response to P(i) availability. Substitutions of critical binding surface residues impair InsP binding in vitro, inorganic polyphosphate synthesis in yeast, and P(i) transport in Arabidopsis In plants, InsPs trigger the association of SPX proteins with transcription factors to regulate P(i) starvation responses. We propose that InsPs communicate cytosolic P(i) levels to SPX domains and enable them to interact with a multitude of proteins to regulate P(i) uptake, transport, and storage in fungi, plants, and animals.

Generation of hydrogen peroxide-resistant murine neuroblastoma cells: a target discovery platform for novel neuroprotective genes.

Oxidative stress has been suggested to play an important role in the pathogenesis of various neurodegenerative diseases including Alzheimer's disease (AD). Hydrogen peroxide (H2O2), one of the main reactive oxygen species, is converted into the highly toxic ·OH radical in the presence of redox-active transition metals, which then oxidises nucleic acids, lipids and proteins, leading to neurodegeneration and cell death. There is an urgent need to gain more knowledge about relevant therapeutic targets to combat oxidative stress and it neurotoxic effects, and how this knowledge can be utilized to develop novel neuroprotective therapies for AD. One way to identify new mechanisms combating oxidative stress was via the creation of H2O2-resistant cell lines and identification of the mechanisms responsible for their resistance. However, in most cases catalase overexpression or increased glutathione content was identified as the primary mode of H2O2 resistance in these cell lines. In this study, we have generated six different resistant neuronal cell lines or populations (from the same original murine Neuro2a neuroblastoma line) by exposing cells to increasing concentrations of H2O2 and performing continuous selection for survivors over a period of several months, which appear to have acquired H2O2 resistance based on other, novel mechanisms. These six populations showed a significant, but differential resistance against H2O2 when compared with the parental cell line. Using combinations of catalase-, glutathione synthesis- and glutathione peroxidase-inhibitors it was shown that the increased resistance of Neuro2a-HR cells is not solely based on an increased activity of catalase or the glutathione system, suggesting that their resistance might be based on yet unknown, novel defence mechanisms.

A quick, convenient and economical method for the reliable determination of methylglyoxal in millimolar concentrations: the N-acetyl-L-cysteine assay.

The determination of methylglyoxal (MG) concentrations in vivo is gaining increasing importance as high levels of MG are linked to various health impairments including complications of diabetes. In order to standardize the measurements of MG in body fluids, it is necessary to precisely determine the concentration of MG stock solutions used as analytical standards. The "gold standard" method for the determination of MG concentration in the millimolar range is an enzyme-catalyzed endpoint assay based on the glyoxalase I catalyzed formation of S-lactoylglutathione. However, as this assay used purified glyoxalase I enzyme, it is quite expensive. Another method uses a derivation reaction with 2,4-dinitrophenylhydrazine, but this substance is explosive and needs special handling and storage. In addition, precipitation of the product methylglyoxal-bis-2,4-dinitrophenylhydrozone during the reaction limits the reliability of this method. In this study, we have evaluated a new method of MG determination based on the previously published fast reaction between MG and N-acetyl-L-cysteine at room temperature which yields an easily detectable condensation product, N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)cysteine. When comparing these three different assays for the measurement of MG concentrations, we find that the N-acetyl-L-cysteine assay is the most favorable, providing an economical and robust assay without the need for the use of hazardous or expensive reagents.