Analysis of free oligosaccharides (fOS) from wild-type Saccharomyces cerevisiae (Baker’s yeast) using two different extraction methods

The glycomic profiles of free oligosaccharides (fOS) derived from misfolded N- and O-linked glycoproteins and lipid-linked oligosaccharides are important molecular signatures in various biological processes and serve as a readout of functional properties such as glycosidase inhibition. Several glycan extraction methods are available based on different sorbent chemistries that may influence the analytical profiles obtained. However, there is limited availability of studies comparing the effects of sorbent chemistries on glycan profiles. Therefore, in our study, the fOS profiles from wild-type Saccharomyces cerevisiae (Baker’s yeast) extracted using two common methods namely mixed-bed ion-exchange (MBIE) [AG50W-X12 (H+) and AG2-X8 (Cl-)] and reversed-phase (C18) sorbents were compared using total carbohydrate (phenol sulfuric acid) and total protein (bicinchoninic acid, BCA) assays, thin-layer chromatography (TLC) and high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) analyses. MBIE extraction contained higher oligosaccharide and protein (0.26 mg/mL and 1.8 mg/mL) content than C18 extraction (0.11 mg/mL and 0.2 mg/mL). TLC analysis (butanol: ethanol: water = 6:3:1 and 5:4:1) showed the presence of fOS in both the MBIE and C18 extracts based on the detection of orcinol active (UV-inactive) spots. Similar peaks were present in the HPLC-ELSD chromatograms for both extractions methods with MBIE showing higher abundance. Glycan unit (GU) analysis of the dextran standard using HPLC-ELSD showed that the largest possible oligosaccharide structures detected were only di/trisaccharides. Based on all these results, MBIE extraction is a more suitable carbohydrate extraction technique compared to C18 extraction for subsequent profiling and functional studies of fOS

Structure of human endo-a-1,2-mannosidase (MANEA), an antiviral host-glycosylation target

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-a-1,2-mannosidase (MANEA) is the sole endoacting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation

Minimal in vivo efficacy of iminosugars in a lethal Ebola virus guinea pig model

The antiviral properties of iminosugars have been reported previously in vitro and in small animal models against Ebola virus (EBOV); however, their effects have not been tested in larger animal models such as guinea pigs. We tested the iminosugars N-butyl-deoxynojirimycin (NB-DNJ) and N-(9-methoxynonyl)-1deoxynojirimycin (MON-DNJ) for safety in uninfected animals, and for antiviral efficacy in animals infected with a lethal dose of guinea pig adapted EBOV. 1850 mg/kg/day NB-DNJ and 120 mg/kg/day MON-DNJ administered intravenously, three times daily, caused no adverse effects and were well tolerated. A pilot study treating infected animals three times within an 8 hour period was promising with 1 of 4 infected NB-DNJ treated animals surviving and the remaining three showing improved clinical signs. MON-DNJ showed no protective effects when EBOV-infected guinea pigs were treated. On histopathological examination, animals treated with NB-DNJ had reduced lesion severity in liver and spleen. However, a second study, in which NB-DNJ was administered at equally-spaced 8 hour intervals, could not confirm drug-associated benefits. Neither was any antiviral effect of iminosugars detected in an EBOV glycoprotein pseudotyped virus assay. Overall, this study provides evidence that NB-DNJ and MON-DNJ do not protect guinea pigs from a lethal EBOV-infection at the dose levels and regimens tested. However, the one surviving animal and signs of improvements in three animals of the NB-DNJ treated cohort could indicate that NB-DNJ at these levels may have a marginal beneficial effect. Future work could be focused on the development of more potent iminosugars

T cell phenotypes in COVID-19 - a living review

COVID-19 is characterized by profound lymphopenia in the peripheral blood, and the remaining T cells display altered phenotypes, characterized by a spectrum of activation and exhaustion. However, antigen-specific T cell responses are emerging as a crucial mechanism for both clearance of the virus and as the most likely route to long-lasting immune memory that would protect against re-infection. Therefore, T cell responses are also of considerable interest in vaccine development. Furthermore, persistent alterations in T cell subset composition and function post-infection have important implications for patients’ long-term immune function. In this review, we examine T cell phenotypes, including those of innate T cells, in both peripheral blood and lungs, and consider how key markers of activation and exhaustion correlate with, and may be able to predict, disease severity. We focus on SARS-CoV-2-specific T cells to elucidate markers that may indicate formation of antigen-specific T cell memory. We also examine peripheral T cell phenotypes in recovery and the likelihood of long-lasting immune disruption. Finally, we discuss T cell phenotypes in the lung as important drivers of both virus clearance and tissue damage. As our knowledge of the adaptive immune response to COVID-19 rapidly evolves, it has become clear that while some areas of the T cell response have been investigated in some detail, others, such as the T cell response in children remain largely unexplored. Therefore, this review will also highlight areas where T cell phenotypes require urgent characterisation

The role and uses of antibodies in COVID-19 infections: a living review

Coronavirus disease 2019 has generated a rapidly evolving field of research, with the global scientific community striving for solutions to the current pandemic. Characterizing humoral responses towards SARS-CoV-2, as well as closely related strains, will help determine whether antibodies are central to infection control, and aid the design of therapeutics and vaccine candidates. This review outlines the major aspects of SARS-CoV-2-specific antibody research to date, with a focus on the various prophylactic and therapeutic uses of antibodies to alleviate disease in addition to the potential of cross-reactive therapies and the implications of long-term immunity

Cellular Mechanism of Glycosylation Inhibition by Imino Sugars

Glycosylation is the most common posttranslational modification of proteins. It is a complex process involving many functional proteins and resulting'in a great diversity of structures. The retention of glucose residues on N-linked oligosaccharides following ER a-glucosidase inhibition by imino sugars such as N-butyl deoxynojirimycin, increases the protein misfolding and the amount of glycoprotein destined for degradation via the endoplasmic reticulum associated degradation (ERAD) pathway. Intracellular peptide N-glycosidases (PNGases) act generating free oligosaccharides (FOS) and the protein is targeted for digestion. Free oligosaccharides were extracted from cells treated with NB-DNJ and subjected to ion-exchange chromatography before fluorescence labelling with 2-AA (anthranilic acid) and lectin-affinity chromatography. Separation of labelled FOS by NP-HPLC provided a rapid and sensitive method for the detection of all FOS species resulting from the degradation of glycoprotein exported from the ER. A. robust, cellular-based assay for ER a-glucosidase activity in the presence of inhibitor was developed that provided meaningful kinetics for a-glucosidase-mediated hydrolysis ofN-linked oligosaccharides as proteins are folded in the ER. Furthermore, the origin of FOS generation was studied, to determine the relative amount of FOS produced as a result of ERAD (protein derived) relative to the amount produced by a possible hydrolytic activity of oligosaccharyltransferase (lipid linked derived). A dual localisation of PNGase activity and non-proteasomal and proteasomal ERAD were demonstrated. Analysis of FOS in endomannosidase negative/non-utilised cells revealed the extent of ER/Golgi recycling of glycoproteins and the role of this enzyme in qua~ity control pathways in cells. Oral administration of NB-DNJ results in the production of glucosylated free oligosaccharide in vivo. Further to cellular based assays these glucosylated free oligosaccharides were detected in murine and human samples. The observed differences in the free oligosaccharides produced in different tissues can be explained according to hypothesises generated from culture cell studies. The effects observed with NB-DNJ, a therapeutic with considerable potential for treating lysosomal storage disorders and reducing viral infectious processes, was dose and time dependent so enabling the pharmokinetics of NB-DNJ to be observed. These studies also elucidated a transrenal method of clearance of glucosylated FOS and demonstrate that glucosylated FOS are excellent non-invasive in vivo biomarkers for a-glucosidase inhibition and protein misfolding in the ER.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins

Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc3Man7GlcNAc2 FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions

Amine-linked diglycosides: Synthesis facilitated by the enhanced reactivity of allylic electrophiles, and glycosidase inhibition assays

Diglycose derivatives, consisting of two monosaccharides linked at non-anomeric positions by a bridging nitrogen atom, have been synthesised. Conversion of one of the precursor monosaccharide coupling components into an unsaturated derivative enhances its electrophilicity at the allylic position, facilitating coupling reactions. Mitsunobu coupling between nosylamides and 2,3-unsaturated-4-alcohols gave the 4-amino-pseudodisaccharides with inversion of configuration as single regio- and diastereoisomers. A palladium-catalysed coupling between an amine and a 2,3-unsaturated 4-trichloroacetimidate gave a 2-amino-pseudodisaccharide as the major product, along with other minor products. Derivatisation of the C=C double bond in pseudodisaccharides allowed the formation of Man(N4–6)Glc and Man(N4–6)Man diglycosides. The amine-linked diglycosides were found to show weak glycosidase inhibitory activity

A Novel Iminosugar UV-12 with Activity against the Diverse Viruses Influenza and Dengue (Novel Iminosugar Antiviral for Influenza and Dengue)

Iminosugars are capable of targeting the life cycles of multiple viruses by blocking host endoplasmic reticulum α-glucosidase enzymes that are required for competent replication of a variety of enveloped, glycosylated viruses. Iminosugars as a class are approved for use in humans with diseases such as diabetes and Gaucher’s disease, providing evidence for safety of this class of compounds. The in vitro antiviral activity of iminosugars has been described in several publications with a subset of these demonstrating in vivo activity against flaviviruses, herpesviruses, retroviruses and filoviruses. Although there is compelling non-clinical in vivo evidence of antiviral efficacy, the efficacy of iminosugars as antivirals has yet to be demonstrated in humans. In the current study, we report a novel iminosugar, UV-12, which has efficacy against dengue and influenza in mouse models. UV-12 exhibits drug-like properties including oral bioavailability and good safety profile in mice and guinea pigs. UV-12 is an example of an iminosugar with activity against multiple virus families that should be investigated in further safety and efficacy studies and demonstrates potential value of this drug class as antiviral therapeutics