Metabolomic and Proteomic Analysis of ApoE4-Carrying H4 Neuroglioma Cells in Alzheimer's Disease Using OrbiSIMS and LC-MS/MS.

Growing clinical evidence reveals that systematic molecular alterations in the brain occur 20 years before the onset of AD pathological features. Apolipoprotein E4 (ApoE4) is one of the most significant genetic risk factors for Alzheimer's disease (AD), which is not only associated with the AD pathological features such as amyloid-ß deposition, phosphorylation of tau proteins, and neuroinflammation but is also involved in metabolism, neuron growth, and synaptic plasticity. Multiomics, such as metabolomics and proteomics, are applied widely in identifying key disease-related molecular alterations and disease-progression-related changes. Despite recent advances in the development of analytical technologies, screening the entire profile of metabolites remains challenging due to the numerous classes of compounds with diverse chemical properties that require different extraction processes for mass spectrometry. In this study, we utilized Orbitrap Secondary Ion Mass Spectrometry (OrbiSIMS) as a chemical filtering screening tool to examine molecular alterations in ApoE4-carried neuroglioma cells compared to wild-type H4 cells. The findings were compared using liquid chromatography (LC)-MS/MS targeted metabolomics analysis for the confirmation of specific metabolite classes. Detected alterations in peptide fragments by OrbiSIMS provided preliminary indications of protein changes. These were extensively analyzed through proteomics to explore ApoE4's impact on proteins. Our metabolomics approach, combining OrbiSIMS and LC-MS/MS, revealed disruptions in lipid metabolism, including glycerophospholipids and sphingolipids, as well as amino acid metabolism, encompassing alanine, aspartate, and glutamate metabolism; aminoacyl-tRNA biosynthesis; glutamine metabolism; and taurine and hypotaurine metabolism. Further LC-MS/MS proteomics studies confirmed the dysfunction in amino acid and tRNA aminoacylation metabolic processes, and highlighted RNA splicing alterations influenced by ApoE4.

Evaluation of candidate International Standards for meningococcal capsular polysaccharide groups W and Y.

Two candidate International Standards for meningococcal capsular group W and Y (MenW and MenY, respectively) polysaccharides were assessed for their suitability as quantitative standards in various physicochemical assays. The study was designed to evaluate the intended purpose of these standards, namely, to standardize the quantification of the respective polysaccharide content in meningococcal polysaccharide and conjugate vaccines and their intermediate components. Twelve laboratories from eleven different countries participated in the collaborative study of candidate preparations for International Standards for MenW and MenY polysaccharide (coded 16/152 and 16/206, respectively). Unitage was assigned using the Resorcinol assay. Our proposals, on the basis of data from the Resorcinol assay were: 1) candidate standard for MenW polysaccharide (16/152) to be assigned a content of 1.015 ± 0.071 mg MenW polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.13, corresponding to a 95 % level of confidence) and 2) candidate standard for MenY polysaccharide (16/206) be assigned a content of 0.958 ± 0.076 mg MenY polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.26, corresponding to a 95 % level of confidence). The amount of polysaccharide per ampoule remained consistent under all stability conditions over a 36-month period.

Evidence for Multiple Binding Modes in the Initial Contact Between SARS-CoV-2 Spike S1 Protein and Cell Surface Glycans.

Infection of host cells by SARS-CoV-2 begins with recognition by the virus S (spike) protein of cell surface heparan sulfate (HS), tethering the virus to the extracellular matrix environment, and causing the subunit S1-RBD to undergo a conformational change into the 'open' conformation. These two events promote the binding of S1-RBD to the angiotensin converting enzyme 2 (ACE2) receptor, a preliminary step toward viral-cell membrane fusion. Combining ligand-based NMR spectroscopy with molecular dynamics, oligosaccharide analogues were used to explore the interactions between S1-RBD of SARS CoV-2 and HS, revealing several low-specificity binding modes and previously unidentified potential sites for the binding of extended HS polysaccharide chains. The evidence for multiple binding modes also suggest that highly specific inhibitors will not be optimal against protein S but, rather, diverse HS-based structures, characterized by high affinity and including multi-valent compounds, may be required.

NMR Reveals Functionally Relevant Thermally Induced Structural Changes within the Native Ensemble of G-CSF.

Structure-function relationships in proteins refer to a trade-off between stability and bioactivity, molded by evolution of the molecule. Identifying which protein amino acid residues jeopardize global or local stability for the benefit of bioactivity would reveal residues pivotal to this structure-function trade-off. Here, we use 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy to probe the microenvironment and dynamics of residues in granulocyte colony-stimulating factor (G-CSF) through thermal perturbation. From this analysis, we identified four residues (G4, A6, T133, and Q134) that we classed as significant to global stability, given that they all experienced large environmental and dynamic changes and were closely correlated to each other in their NMR characteristics. Additionally, we observe that roughly four structural clusters are subject to localized conformational changes or partial unfolding prior to global unfolding at higher temperature. Combining NMR observables with structure relaxation methods reveals that these structural clusters concentrate around loop AB (binding site III inclusive). This loop has been previously implicated in conformational changes that result in an aggregation prone state of G-CSF. Residues H43, V48, and S63 appear to be pivotal to an opening motion of loop AB, a change that is possibly also important for function. Hence, we present here an approach to profiling residues in order to highlight their potential roles in the two vital characteristics of proteins: stability and bioactivity.

MD simulation of the interaction between sialoglycans and the second sialic acid binding site of influenza A virus N1 neuraminidase.

The neuraminidases (NAs) of avian influenza viruses (IAVs) contain a second sialic acid-binding site (2SBS), historically known as the hemadsorption site, which is separated from the sialyl-hydrolase catalytic site and serves to facilitate NA catalytic activity towards multivalent sialyl-capped glycoconjugates. Transmission and adaptation of avian IAVs to humans decreases hemadsorption and catalytic activities of the NA. Here, we report the molecular recognition features of the NA 2SBS of two pandemic H1N1 IAVs, A/Brevig Mission /1/1918 (BM18) and A/California/04/2009 (CA09), differing by their 2SBS activity. Using explicit solvent MD simulation, molecular mechanics, and glycosidic conformation analysis we initially analyzed the interactions of BM18 2SBS with two sialyllacto-N-tetraose pentasaccharides, 3'SLN-LC and 6'SLN-LC, which are models for the glycan receptors of IAVs in birds and humans, respectively. These studies characterize the binding specificity of BM18 2SBS towards human-type and avian-type receptors and identifies the key amino acids that affects binding. We next compared the interactions of the 2SBSs of BM18 and CA09 with 6'SLN-LC, revealing the critical effect of amino acid 372 on binding. Our results expand the current knowledge of the molecular features of NA 2SBSs and its alteration during the adaptation of avian IAVs to humans.

Multivariate analysis applied to complex biological medicines.

A biological medicine (or biologicals) is a term for a medicinal compound that is derived from a living organism. By their very nature, they are complex and often heterogeneous in structure, composition and biological activity. Some of the oldest pharmaceutical products are biologicals, for example insulin and heparin. The former is now produced recombinantly, with technology being at a point where this can be considered a defined chemical entity. This is not the case for the latter, however. Heparin is a heterogeneous polysaccharide that is extracted from the intestinal mucosa of animals, primarily porcine, although there is also a significant market for non-porcine heparin due to social and economical reasons. In 2008 heparin was adulterated with another sulfated polysaccharide. Unfortunately this event was disastrous and resulted in a global public health emergency. This was the impetuous to apply modern analytical techniques, principally NMR spectroscopy, and multivariate analyses to monitor heparin. Initially, traditional unsupervised multivariate analysis (principal component analysis (PCA)) was applied to the problem. This was able to distinguish animal heparins from each other, and could also separate adulterated heparin from what was considered bona fide heparin. Taught multivariate analysis functions by training the analysis to look for specific patterns within the dataset of interest. If this approach was to be applied to heparin, or any other biological medicine, it would have to be taught to find every possible alien signal. The opposite approach would be more efficient; defining the complex heterogeneous material by a library of bona fide spectra and then filtering test samples with these spectra to reveal alien features that are not consistent with the reference library. This is the basis of an approach termed spectral filtering, which has been applied to 1D and 2D-NMR spectra, and has been very successful in extracting the spectral features of adulterants in heparin, as well as being able to differentiate supposedly biosimilar products. In essence, the filtered spectrum is determined by subtracting the covariance matrix of the library spectra from the covariance matrix of the library spectra plus the test spectrum. These approaches are universal and could be applied to biological medicines such as vaccine polysaccharides and monoclonal antibodies.

Subverting the mechanisms of cell death: flavivirus manipulation of host cell responses to infection.

Viruses exploit host metabolic and defence machinery for their own replication. The flaviviruses, which include Dengue (DENV), Yellow Fever (YFV), Japanese Encephalitis (JEV), West Nile (WNV) and Zika (ZIKV) viruses, infect a broad range of hosts, cells and tissues. Flaviviruses are largely transmitted by mosquito bites and humans are usually incidental, dead-end hosts, with the notable exceptions of YFV, DENV and ZIKV. Infection by flaviviruses elicits cellular responses including cell death via necrosis, pyroptosis (involving inflammation) or apoptosis (which avoids inflammation). Flaviviruses exploit these mechanisms and subvert them to prolong viral replication. The different effects induced by DENV, WNV, JEV and ZIKV are reviewed. Host cell surface proteoglycans (PGs) bearing glycosaminoglycan (GAG) polysaccharides - heparan/chondroitin sulfate (HS/CS) - are involved in initial flavivirus attachment and during the expression of non-structural viral proteins play a role in disease aetiology. Recent work has shown that ZIKV-infected cells are protected from cell death by exogenous heparin (a GAG structurally similar to host cell surface HS), raising the possibility of further subtle involvement of HS PGs in flavivirus disease processes. The aim of this review is to synthesize information regarding DENV, WNV, JEV and ZIKV from two areas that are usually treated separately: the response of host cells to infection by flaviviruses and the involvement of cell surface GAGs in response to those infections.

19F labelled glycosaminoglycan probes for solution NMR and non-linear (CARS) microscopy.

Studying polysaccharide-protein interactions under physiological conditions by conventional techniques is challenging. Ideally, macromolecules could be followed by both in vitro spectroscopy experiments as well as in tissues using microscopy, to enable a proper comparison of results over these different scales but, often, this is not feasible. The cell surface and extracellular matrix polysaccharides, glycosaminoglycans (GAGs) lack groups that can be detected selectively in the biological milieu. The introduction of 19F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing 19F NMR spectroscopy is followed. Furthermore, the ability of 19F labelled GAGs to be imaged using CARS microscopy is demonstrated. 19F labelled GAGs enable both 19F NMR protein-GAG binding studies in solution at the molecular level and non-linear microscopy at a microscopic scale to be conducted on the same material, essentially free of background signals.

Nuclear Magnetic Resonance and Molecular Dynamics Simulation of the Interaction between Recognition Protein H7 of the Novel Influenza Virus H7N9 and Glycan Cell Surface Receptors.

Avian influenza A viruses, which can also propagate between humans, present serious pandemic threats, particularly in Asia. The specificity (selectivity) of interactions between the recognition protein hemagglutinin (HA) of the virus capsid and the glycoconjugates of host cells also contributes to the efficient spread of the virus by aerosol between humans. Some avian origin viruses, such as H1N1 (South Carolina 1918), have improved their selectivity for human receptors by mutation in the HA receptor binding site, to generate pandemic viruses. Molecular details and dynamics of glycan-HA interactions are of interest, both in predicting the pandemic potential of a new emerging strain and in searching for new antiviral drugs. Two complementary techniques, 1H saturation transfer difference (1H STD) nuclear magnetic resonance and molecular dynamics (MD) simulation, were applied to analyze the interaction of the new H7 (A/Anhui/1/13 H7N9) with LSTa [Neu5Ac α(2→3) Gal ß(1→3) GlcNAc ß(1→3) Gal ß(1→4) Glc] and LSTc [Neu5Ac α(2→6) Gal ß(1→4) GlcNAc ß(1→3) Gal ß(1→4) Glc] pentasaccharides, models of avian and human receptor glycans. Their interactions with H7 were analyzed for the first time using 1H STD and MD, revealing structural and dynamic behavior that could not be obtained from crystal structures, and contributing to glycan-HA specificity. This highlighted aspects that could affect glycan-HA recognition, including the mutation H7 G228S, which increases H2 and H3 specificity for the human receptor. Finally, interactions between LSTc and H7 were compared with those between LSTc and H1 of H1N1 (South Carolina 1918), contributing to our understanding of the recognition ability of HAs.

Atomic Details of the Interactions of Glycosaminoglycans with Amyloid-ß Fibrils.

The amyloid plaques associated with Alzheimer's disease (AD) comprise fibrillar amyloid-ß (Aß) peptides as well as non-protein factors including glycosaminoglycan (GAG) polysaccharides. GAGs affect the kinetics and pathway of Aß self-assembly and can impede fibril clearance; thus, they may be accessory molecules in AD. Here we report the first high-resolution details of GAG-Aß fibril interactions from the perspective of the saccharide. Binding analysis indicated that the GAG proxy heparin has a remarkably high affinity for Aß fibrils with 3-fold cross-sectional symmetry (3Q). Chemical synthesis of a uniformly (13)C-labeled octasaccharide heparin analogue enabled magic-angle spinning solid-state NMR of the GAG bound to 3Q fibrils, and measurements of dynamics revealed a tight complex in which all saccharide residues are restrained without undergoing substantial conformational changes. Intramolecular (13)C-(15)N dipolar dephasing is consistent with close (<5 Å) contact between GAG anomeric position(s) and one or more histidine residues in the fibrils. These data provide a detailed model for the interaction between 3Q-seeded Aß40 fibrils and a major non-protein component of AD plaques, and they reveal that GAG-amyloid interactions display a range of affinities that critically depend on the precise details of the fibril architecture.

Differentiation of generic enoxaparins marketed in the United States by employing NMR and multivariate analysis.

The U.S. Food and Drug Administration defines criteria for the equivalence of Enoxaparin with Lovenox, comprising the equivalence of physiochemical properties, heparin source material and mode of depolymerization, disaccharide building blocks, fragment mapping and sequence of oligosaccharide species, biological and biochemical assays, and in vivo pharmacodynamic profile. Chemometric analysis of the NMR spectra, utilizing both (1)H and (1)H-(13)C HSQC NMR experiments, of Lovenox and Enoxaparin, the latter being the generic version of the former, revealed that Lovenox and the four Enoxaparin compounds produced by Sandoz (Enoxaparin and Fibrinox), Winthrop, and Amphastar exhibit dissimilarities in terms of their composition. All of the collected samples had expiry dates between 2012 and 2015. These studies, in addition to chromatographic analysis, highlighted signatures that differentiated the branded material from the generic products.

CDApps: integrated software for experimental planning and data processing at beamline B23, Diamond Light Source.

The B23 Circular Dichroism beamline at Diamond Light Source has been operational since 2009 and has seen visits from more than 200 user groups, who have generated large amounts of data. Based on the experience of overseeing the users' progress at B23, four key areas requiring the most assistance are identified: planning of experiments and note-keeping; designing titration experiments; processing and analysis of the collected data; and production of experimental reports. To streamline these processes an integrated software package has been developed and made available for the users. The subsequent article summarizes the main features of the software.

CDApps: integrated software for experimental planning and data processing at beamline B23, Diamond Light Source. Corrigendum.

In the paper by Hussain et al. [(2015), J. Synchrotron Rad. 22, 465-468], Daniel Myatt is missing from the list of authors. The complete list of authors should be Rohanah Hussain, Kristian Benning, Daniel Myatt, Tamas Javorfi, Edoardo Longo, Timothy R. Rudd, Bill Pulford and Giuliano Siligardi.

Heparan sulfate phage display antibodies recognise epitopes defined by a combination of sugar sequence and cation binding.

Phage display antibodies are widely used to follow heparan sulfate (HS) expression in tissues and cells. We demonstrate by ELISA, that cations alter phage display antibody binding profiles to HS and this is mediated by changes in polysaccharide conformation, demonstrated by circular dichroism spectroscopy. Native HS structures, expressed on the cell surfaces of neuroblastoma and fibroblast cells, also exhibited altered antibody binding profiles following exposure to low mM concentrations of these cations. Phage display antibodies recognise conformationally-defined HS epitopes, rather than sequence alone, as has been assumed, and resemble proteins in being sensitive to changes in both charge distribution and conformation following binding of cations to HS polysaccharides.

Insights into the human glycan receptor conformation of 1918 pandemic hemagglutinin-glycan complexes derived from nuclear magnetic resonance and molecular dynamics studies.

The glycan receptor binding and specificity of influenza A viral hemagglutinin (HA) are critical for virus infection and transmission in humans. However, ambiguities in the interpretation of the receptor binding specificity of hemagglutinin from human- and avian-adapted viruses have prevented an understanding of its relationship with aerosol transmissibility, an exclusive property of human-adapted viruses. A previous conformational study, which we performed, indicated that human and avian receptors sample distinct conformations in solution. On the basis of detailed nuclear magnetic resonance (NMR) studies provided herein, we offer evidence of the distinct structural constraints imposed by hemagglutinin receptor binding sites on the glycan conformational space upon binding. The hemagglutinin from the SC18 virus, which has efficient aerosol transmissibility in humans (human-adapted), imposed the most stringent constraints on the conformational space of the human glycan receptor (LSTc), compared to single (NY18) or double (AV18) amino acid HA mutants, a property correlating to the ligand-HA binding strength. This relationship was also observed for the avian-adapted HA, where the high affinity binding partner, AV18, imposed the most stringent conformational constraints on the avian receptor, compared to those imposed by NY18. In particular, it is interesting to observe how different HAs when binding to human or avian glycosidic receptors impose significantly different conformational states, in terms of the states sampled by the glycosidic backbone and/or the entire molecule shape (linear or bent), when compared to the corresponding unbound glycans. Significantly, we delineate a "characteristic NMR signature" for the human adapted hemagglutinin (SC18) binding to human glycan receptors. Therefore, the conformational space constraints imposed by the hemagglutinin receptor binding site provide a characteristic signature that could be a useful tool for the surveillance of human adaptation of other (such as H7N9 and H5N1) deadly influenza viruses.

An unusual antithrombin-binding heparin octasaccharide with an additional 3-O-sulfated glucosamine in the active pentasaccharide sequence.

The 3-O-sulfation of N-sulfated glucosamine is the last event in the biosynthesis of heparin/heparan sulfate, giving rise to the antithrombin-binding pentasaccharide sequence AGA*IA, which is largely associated with the antithrombotic activity of these molecules. The aim of the present study was the structural and biochemical characterization of a previously unreported AGA*IA*-containing octasaccharide isolated from the very-low-molecular-mass heparin semuloparin, in which both glucosamine residues of the pentasaccharide moiety located at the non-reducing end bear 3-O-sulfate groups. Two-dimensional and STD (saturation transfer difference) NMR experiments clearly confirmed its structure and identified its ligand epitope binding to antithrombin. The molecular conformation of the octasaccharide-antithrombin complex has been determined by NMR experiments and docking/energy minimization. The presence of the second 3-O-sulfated glucosamine in the octasaccharide induced more than one order of magnitude increase in affinity to antithrombin compared to the pentasaccharide AGA*IA.

Human (α2→6) and avian (α2→3) sialylated receptors of influenza A virus show distinct conformations and dynamics in solution.

Differential interactions between influenza A virus protein hemagglutinin (HA) and α2→3 (avian) or α2→6 (human) sialylated glycan receptors play an important role in governing host specificity and adaptation of the virus. Previous analysis of HA-glycan interactions with trisaccharides showed that, in addition to the terminal sialic acid linkage, the conformation and topology of the glycans, while they are bound to HA, are key factors in regulating these interactions. Here, the solution conformation and dynamics of two representative avian and human glycan pentasaccharide receptors [LSTa, Neu5Ac-α(2→3)-Gal-ß(1→3)-GlcNAc-ß(1→3)-Gal-ß(1→4)-Glc; LSTc, (Neu5Ac-α(2→6)-Gal-ß(1→4)-GlcNAc-ß(1→3)-Gal-ß(1→4)-Glc] have been explored using nuclear magnetic resonance and molecular dynamics simulation. Analyses demonstrate that, in solution, human and avian receptors sample distinct conformations, topologies, and dynamics. These unique features of avian and human receptors in solution could represent distinct molecular characteristics for recognition by HA, thereby providing the HA-glycan interaction specificity in influenza.

Diversification of the structural determinants of fibroblast growth factor-heparin interactions: implications for binding specificity.

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K(D) values varying between 38 nM (FGF-18) and 620 nM (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 M(-1) s(-1) and FGF-9, 130,000 M(-1) s(-1)). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.

Unravelling structural information from complex mixtures utilizing correlation spectroscopy applied to HSQC spectra.

The first use of statistical correlation spectroscopy to extract chemical information from 2D-HSQC spectra, termed HSQC correlation spectroscopy (HSQCcos), is reported. HSQCcos is illustrated using heparin, a heterogeneous polysaccharide, whose diverse composition causes signals in HSQC spectra to disperse. HSQCcos has been used to probe the chain modifications that cause this effect and reveals hitherto unreported structural details. An interesting finding was that the signal for position 2 of trisulfated glucosamine [N-, 3-O-, and 6-O-sulfated] (A*) is bifurcated, owing to the presence of A* residues in both the "normal" antithrombin binding site and also at the nonreducing end of the molecule, which is reported in intact heparin for the first time. The method was also applied to investigating the environment around other rare sequences/disaccharides, suggesting that the disaccharide; 2-O-sulfated iduronic acid linked to 6-O-sulfated N-glucosamine, which contains a free amine at position 2, is adjacent to the heparin linkage region. HSQCcos can extract chemically related signals from information-rich spectra obtained from complex mixtures such as heparin.