Mass spectrometric characterisation of immunologically important glycosylation

Glycosylation is a vital modification for many macro-biomolecules to obtain structural heterogeneity and functional diversity. In the immune system, nearly all engaged molecules are glycosylated, and their glycosylation status directly correlates with their functions. Therefore, structural studies of glycosylation are important and fundamental to allow understanding of the basic mechanisms underlying the immune system. In this thesis, comprehensive mass spectrometric based glycomic and glycoproteomic strategies were applied to investigate the glycosylation of the following immunologically important glycoproteins and animal models: pregnancy specific glycoprotein 1 (PSG1), canine melanoma, fragment crystallisable (Fc) fusion protein and cryoglobulins. Glycomic and glycoproteomic studies of native PSG1 showed that four of the seven N-glycosylation sites on this protein were occupied by glycans, which were dominated by mono- and bi-antennary complex structures with sialylation and partial core-fucosylation. N-glycosylation of recombinant N-terminal domain of PSG1 from Chinese Hamster Ovary (CHO) systems differs from the native materials for universal core-fucosylation and extensive poly-LacNAc elongation. N-glycosylation of the new Expi-CHO system was characterised as abundant truncated multi-antennary complex structures. Analysis of the glycoprotein and glycolipid glycans expressed by stage III canine melanoma cells indicates that the glycosylation of this animal model is highly consistent with that of human melanoma with an exception of species-specific expression of Gal-α-Gal epitopes. These results support the idea that canine melanoma should be an excellent animal model for the pre-clinical studies of melanoma. Glycomic studies of the Fc fusion protein with engineered additional N-glycosylation sites indicated an improvement of sialylation and branching on N-glycans in a CHO cell system. Identical fusion proteins expressed in a HEK293 cell system exhibited more diverse epitopes and considerable antennary truncation. Finally, analysis of the N-glycosylation of cryoglobulin indicated that glycosylation might not play a vital role in the formation of the cryoprecipitate of mixed-type cryoglobulins.Open Acces

Choice of Host Cell Line Is Essential for the Functional Glycosylation of the Fc Region of Human IgG1 Inhibitors of Influenza B Viruses

Abs are glycoproteins that carry a conserved N-linked carbohydrate attached to the Fc whose presence and fine structure profoundly impacts on their in vivo immunogenicity, pharmacokinetics, and functional attributes. The host cell line used to produce IgG has a major impact on this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. In this study, we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the human endothelial kidney 293-F or Chinese hamster ovary–K1 systems. We show that the types of N-linked glycans between matched pairs of Fc mutants vary greatly and in particular, with respect, to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B–mediated agglutination of human erythrocytes when expressed in Chinese hamster ovary–K1, but not in human endothelial kidney 293-F cells

Structural Evolution of Layered Manganese Oxysulfides during Reversible Electrochemical Lithium Insertion and Copper Extrusion.

The electrochemical lithiation and delithiation of the layered oxysulfide Sr2MnO2Cu4-δS3 has been investigated by using a combination of in situ powder X-ray diffraction and ex situ neutron powder diffraction, X-ray absorption and 7Li NMR spectroscopy, together with a range of electrochemical experiments. Sr2MnO2Cu4-δS3 consists of [Sr2MnO2] perovskite-type cationic layers alternating with highly defective antifluorite-type [Cu4-δS3] (δ ≈ 0.5) anionic layers. It undergoes a combined displacement/intercalation (CDI) mechanism on reaction with Li, where the inserted Li replaces Cu, forming Li4S3 slabs and Cu+ is reduced and extruded as metallic particles. For the initial 2-3% of the first discharge process, the vacant sites in the sulfide layer are filled by Li; Cu extrusion then accompanies further insertion of Li. Mn2.5+ is reduced to Mn2+ during the first half of the discharge. The overall charging process involves the removal of Li and re-insertion of Cu into the sulfide layers with re-oxidation of Mn2+ to Mn2.5+. However, due to the different diffusivities of Li and Cu, the processes operating on charge are quite different from those operating during the first discharge: charging to 2.75 V results in the removal of most of the Li, little reinsertion of Cu, and good capacity retention. A charge to 3.75 V is required to fully reinsert Cu, which results in significant changes to the sulfide sublattice during the following discharge and poor capacity retention. This detailed structure-property investigation will promote the design of new functional electrodes with improved device performance

Chk1 Inhibition Ameliorates Alzheimer's Disease Pathogenesis and Cognitive Dysfunction Through CIP2A/PP2A Signaling

Alzheimer's disease (AD) is the most common neurodegenerative disease with limited therapeutic strategies. Cell cycle checkpoint protein kinase 1 (Chk1) is a Ser/Thr protein kinase which is activated in response to DNA damage, the latter which is an early event in AD. However, whether DNA damage-induced Chk1 activation participates in the development of AD and Chk1 inhibition ameliorates AD-like pathogenesis remain unclarified. Here, we demonstrate that Chk1 activity and the levels of protein phosphatase 2A (PP2A) inhibitory protein CIP2A are elevated in AD human brains, APP/PS1 transgenic mice, and primary neurons with A beta treatment. Chk1 overexpression induces CIP2A upregulation, PP2A inhibition, tau and APP hyperphosphorylation, synaptic impairments, and cognitive memory deficit in mice. Moreover, Chk1 inhibitor (GDC0575) effectively increases PP2A activity, decreases tau phosphorylation, and inhibits A beta overproduction in AD cell models. GDC0575 also reverses AD-like cognitive deficits and prevents neuron loss and synaptic impairments in APP/PS1 mice. In conclusion, our study uncovers a mechanism by which DNA damage-induced Chk1 activation promotes CIP2A-mediated tau and APP hyperphosphorylation and cognitive dysfunction in Alzheimer's disease and highlights the therapeutic potential of Chk1 inhibitors in AD

Structural Basis of Chemokine Sequestration by CrmD, a Poxvirus-Encoded Tumor Necrosis Factor Receptor

Pathogens have evolved sophisticated mechanisms to evade detection and destruction by the host immune system. Large DNA viruses encode homologues of chemokines and their receptors, as well as chemokine-binding proteins (CKBPs) to modulate the chemokine network in host response. The SECRET domain (smallpox virus-encoded chemokine receptor) represents a new family of viral CKBPs that binds a subset of chemokines from different classes to inhibit their activities, either independently or fused with viral tumor necrosis factor receptors (vTNFRs). Here we present the crystal structures of the SECRET domain of vTNFR CrmD encoded by ectromelia virus and its complex with chemokine CX3CL1. The SECRET domain adopts a β-sandwich fold and utilizes its β-sheet I surface to interact with CX3CL1, representing a new chemokine-binding manner of viral CKBPs. Structure-based mutagenesis and biochemical analysis identified important basic residues in the 40s loop of CX3CL1 for the interaction. Mutation of corresponding acidic residues in the SECRET domain also affected the binding for other chemokines, indicating that the SECRET domain binds different chemokines in a similar manner. We further showed that heparin inhibited the binding of CX3CL1 by the SECRET domain and the SECRET domain inhibited RAW264.7 cell migration induced by CX3CL1. These results together shed light on the structural basis for the SECRET domain to inhibit chemokine activities by interfering with both chemokine-GAG and chemokine-receptor interactions

Analysis on the Influence Mechanism of Cooling Water on Turbocharger and Optimum Coolant Mass Flow Rate Intelligent Prediction

Due to the high speed and high temperature of engine exhaust, the turbocharger bears very high heat load. The heat dissipation of turbocharger is an important factor to determine the service life and performance of turbocharger. In this paper, a mathematical model of the fluid-structure interaction heat transfer of the water-cooled bearing body of turbocharger was established and the cooling performance of a 1.8 L gasoline engine turbocharger was analyzed. The effects of cooling water inlet flow, engine exhaust temperature, cooling water inlet temperature, and wall roughness of cooling water chamber on the cooling performance of important parts of the bearing body were analyzed by the numerical simulation method. In addition, the cooling water flow required by bearing body with a different structure under different working conditions was studied based on the orthogonal test method. The predicted result shows a good agreement with the experiment result, which could provide a reference for relevant production design and cooling strategy. In the range larger than the thickness of laminar flow bottom layer of the cooling water chamber wall, the increase of wall roughness height can enhance the heat transfer between the fluid and the solid

Investigation on Gas-Soot Flow Distribution Characteristic of Soot Capture Process in the Wall-Flow Diesel Particulate Filter

In order to investigate the distribution characteristics of gas-particle two-phase flow in the diesel particulate filter in the capture process, a mathematical model of gas-particle two-phase flow for inside-and-outside filter had been established in the capture process according to the mass conservation equation, momentum conservation equation, and k-ε turbulence equation. The model verification was carried out with the experimental and simulated of flow distribution characteristics of gas-particle two-phase. The obtained results showed that the static pressure gradient along the radial distribution was greater at the inlet of the filter in capture process in the diesel particulate filter, which could easily lead to causing eventual fatigue damage due to stress concentration in the front-end of filter; moreover, the weaker the vortex strength of gas-particle formed in expansion pipe was, the better uniformity of flow velocity and soot concentration distribution were. Therefore, the established mathematical model can be used for predicting gas-particle flow velocity distribution in the diesel particulate filter

Analysis of Network Robustness in Weighted and Unweighted Approaches: A Case Study of the Air Transport Network in the Belt and Road Region

Aviation transport is one of the most important and critical infrastructures in today’s global economy. Failure in its proper operations can seriously impact regional economic development, which is why it is important to evaluate network robustness. Previous analyses of robustness have mainly been conducted with an unweighted approach. In the development of air transport, however, the demand for route configuration has gradually decreased, while the demand for flight adjustments has increased. Consequently, the aviation network has developed unevenly, so adhering to a uniform approach for evaluating network robustness may lead to inaccurate results. Therefore, we examined which centrality sequence is the most sensitive to network robustness in both unweighted and weighted approaches. The air transport network selected for the case study comprised the six subregions of the Eurasian landmass of the Belt and Road region. The study results showed the following: (a) in the network constructed as an unweighted one, betweenness, and degree centrality had higher priorities in preserving network functionalities than eigenvector and closeness centrality; (b) in the network constructed as a weighted one, recursive power had a higher priority in preserving network functionalities than recursive centrality; and (c) no particular centrality measurement had a significant advantage in representing the totality of robustness. The betweenness centrality sequence was sensitive to the average shortest path length and global efficiency; the recursive power sequence was sensitive to the clustering coefficient, while degree centrality was sensitive to graph diversity. The findings of this study support the decisions about managing air transportation in the Belt and Road region