C-mannosylation supports folding and enhances stability of thrombospondin repeats

Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging

NMR spectroscopic characterization of the C‐mannose conformation in a thrombospondin repeat using a selective labeling approach

Despite the great interest in glycoproteins, structural information reporting on conformation and dynamics of the sugar moieties are limited. We present a new biochemical method to express proteins with glycans that are selectively labeled with NMR‐active nuclei. We report on the incorporation of 13C‐labeled mannose in the C‐mannosylated UNC‐5 thrombospondin repeat. The conformational landscape of the C‐mannose sugar puckers attached to tryptophan residues of UNC‐5 is characterized by interconversion between the canonical 1C4 state and the B03 / 1S3 state. This flexibility may be essential for protein folding and stabilization. We foresee that this versatile tool to produce proteins with selectively labeled C‐mannose can be applied and adjusted to other systems and modifications and potentially paves a way to advance glycoprotein research by unravelling the dynamical and conformational properties of glycan structures and their interactions

Effects of a Partially Perforated Flooring System on Ammonia Emissions in Broiler Housing—Conflict of Objectives between Animal Welfare and Environment?

A partially (50%) perforated flooring system showed positive effects on health- and behavior-based welfare indicators without affecting production performance. Ammonia (NH3) is the most common air pollutant in poultry production, with effects on animal welfare and the environment. The objectives of animal welfare and environmental protection are often incompatible. Therefore, this study addresses the question of how a partially perforated flooring system affects NH3 emissions. According to German regulations, three fattening periods were carried out with 500 Ross 308 broilers per barn (final stocking density: 39 kg m−2). The experimental barn was equipped with an elevated perforated area in the supply section, accessible by perforated ramps. The remaining area in the experimental barn and the control barn were equipped with wood shavings (600 g m−2). Besides the different floor types, management was identical. Air temperature (Temp), relative air humidity (RH), NH3 concentration, and ventilation rate (VR) were measured continuously. Furthermore, dry matter (DM) content, pH, and litter quality were assessed. Towards the end of the fattening periods, the NH3 emission rate (ER) of the partially perforated flooring system was higher compared with that of the littered control barn (all p 3 concentrations, which are promoted by the lack of compaction underneath the elevated perforated area and the increase in pH value under aerobic conditions. Nevertheless, the partially perforated flooring system offers different approaches for NH3 reduction that were previously not feasible, potentially contributing equally to animal welfare and environmental protection

C-mannosylation supports folding and enhances stability of thrombospondin repeats

Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging

Der zweite Arbeitsmarkt in Ost-Deutschland Forschungsergebnisse eines sozialoekonomischen Projektes

UuStB Koeln(38)-960106651 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

C. elegans DPY-19 is a C-mannosyltransferase glycosylating thrombospondin repeats

Item does not contain fulltextAmong the different types of protein glycosylation, C-mannosylation of tryptophan residues stands out because of the unique linkage formed between sugar and protein. Instead of the typical O- or N-glycosidic linkage, a C-C bond is used for attachment of a single mannose. C-mannose is characteristically found in thrombospondin type 1 repeats and in the WSXWS motif of type I cytokine receptors. The genetic base of the enzymatic activity catalyzing C-mannosylation was not known. Here we demonstrate that Caenorhabditis elegans DPY-19 is a C-mannosyltransferase. DPY-19 exhibits topological and sequential homology to the N-glycan oligosaccharyltransferase, highlighting an evolutionary link between N- and C-glycosylation. We show that the C. elegans surface receptors MIG-21 and UNC-5 are acceptor substrates of DPY-19 and that C-mannosylation is essential for the secretion of soluble MIG-21. Thereby, our data provide an explanation for the previously described identical Q neuroblast migration phenotypes of dpy-19 and mig-21 mutants