School of Chemistry - Research Publications
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ItemSelective Manipulation of Discrete Mannosidase Activities in the Endoplasmic Reticulum by Using Reciprocally Selective Inhibitors(WILEY-V C H VERLAG GMBH, 2017-06-01)Within the endoplasmic reticulum, immature glycoproteins are sorted into secretion and degradation pathways through the sequential trimming of mannose residues from Man9 GlcNAc2 to Man5 GlcNAc2 by the combined actions of assorted α-1,2-mannosidases. It has been speculated that specific glycoforms encode signals for secretion and degradation. However, it is unclear whether the specific signal glycoforms are produced by random mannosidase action or are produced regioselectively in a sequenced manner by specific α-1,2-mannosidases. Here, we report the identification of a set of selective mannosidase inhibitors and development of conditions for their use that enable production of distinct pools of Man8 GlcNAc2 isomers from a structurally defined synthetic Man9 GlcNAc2 substrate in an endoplasmic reticulum fraction. Glycan processing analysis with these inhibitors provides the first biochemical evidence for selective production of the signal glycoforms contributing to traffic control in glycoprotein quality control.
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ItemTotal Synthesis of Mycobacterium tuberculosis Dideoxymy-cobactin-838 and Stereoisomers: Diverse CD1a-Restricted T Cells Display a Common Hierarchy of Lipopeptide Recognition(WILEY-V C H VERLAG GMBH, 2017-01)Mycobacterium tuberculosis produces dideoxymycobactin-838 (DDM-838), a lipopeptide that potently activates T cells upon binding to the MHC-like antigen-presenting molecule CD1a. M. tuberculosis produces DDM-838 in only trace amounts and a previous solid-phase synthesis provided sub-milligram quantities. We describe a high-yielding solution-phase synthesis of DDM-838 that features a Mitsunobu substitution that avoids yield-limiting epimerization at lysine during esterification, and amidation conditions that prevent double-bond isomerization of the Z-C20:1 acyl chain, and provides material with equivalent antigenicity to natural DDM-838. Isomers of DDM-838 that varied in stereochemistry at the central lysine and the C20:1 acyl chain were compared for their ability to be recognised by CD1a-restricted T cell receptors (TCRs). These TCRs, derived from unrelated human donors, exhibited a similar spectrum of reactivity towards the panel of DDM-838 isomers, highlighting the exquisite sensitivity of lipopeptide-reactive T cells for the natural DDM stereochemistry.
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ItemNo Preview AvailableMannosidase mechanism: at the intersection of conformation and catalysis(CURRENT BIOLOGY LTD, 2020-06)Mannosidases are a diverse group of enzymes that are important in the biological processing of mannose-containing polysaccharides and complex glycoconjugates. They are found in 12 of the >160 sequence-based glycosidase families. We discuss evidence that nature has evolved a small set of common mechanisms that unite almost all of these mannosidase families. Broadly, mannosidases (and the closely related rhamnosidases) perform catalysis through just two conformations of the oxocarbenium ion-like transition state: a B2,5 (or enantiomeric 2,5B) boat and a 3H4 half-chair. This extends to a new family (GT108) of GDPMan-dependent β-1,2-mannosyltransferases/phosphorylases that perform mannosyl transfer through a boat conformation as well as some mannosidases that are metalloenzymes and require divalent cations for catalysis. Yet, among this commonality lies diversity. New evidence shows that one unique family (GH99) of mannosidases use an unusual mechanism involving anchimeric assistance via a 1,2-anhydro sugar (epoxide) intermediate.
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ItemA Family of Dual-Activity Glycosyltransferase-Phosphorylases Mediates Mannogen Turnover and Virulence in Leishmania Parasites(CELL PRESS, 2019-09-11)Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.
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ItemDistortion of mannoimidazole supports a B2,5 boat transition state for the family GH125 α-1,6-mannosidase from Clostridium perfringens(ROYAL SOC CHEMISTRY, 2019-09-14)Enzyme transition-state mimics can act as powerful inhibitors and allow structural studies that report on the conformation of the transition-state. Here, mannoimidazole, a mimic of the transition state of mannosidase catalyzed hydrolysis of mannosides, is shown to bind in a B2,5 conformation on the Clostridium perfringens GH125 α-1,6-mannosidase, providing additional evidence of a OS2-B2,5-1S5 conformational itinerary for enzymes of this family.
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ItemDiscovery of N-Aryloxypropylbenzylamines as Voltage-Gated Sodium Channel NaV1.2-Subtype-Selective Inhibitors(WILEY-V C H VERLAG GMBH, 2019-03-05)We previously reported that a lipophilic N-(4'-hydroxy-3',5'-di-tert-butylbenzyl) derivative (1) of the voltage-gated sodium channel blocker mexiletine, was a more potent sodium channel blocker in vitro and in vivo. We demonstrate that replacing the chiral methylethylene linker between the amine and di-tert-butylphenol with an achiral 1,3-propylene linker (to give (2)) maintains potency in vitro. We synthesized 25 analogues bearing the 1,3-propylene linker and found that minor structural changes resulted in pronounced changes in state dependence of blocking human NaV 1.2 and 1.6 channels by high-throughput patch-clamp analysis. Compared to mexiletine, compounds 1 and 2 are highly selective NaV 1.2 inhibitors and >500 times less potent in inhibiting NaV 1.6 channels. On the other hand, a derivative (compound 4) bearing 2,6-dimethoxy groups in place of the 2,6-dimethyl groups found in mexiletine was found to be the most potent inhibitor, but is nonselective against both channels in the tonic, frequency-dependent and inactivated states. In a kindled mouse model of refractory epilepsy, compound 2 inhibited seizures induced by 6 Hz 44 mA electrical stimulation with an IC50 value of 49.9±1.6 mg kg-1 . As established sodium channel blockers do not suppress seizures in this mouse model, this indicates that 2 could be a promising candidate for treating pharmaco-resistant epilepsy.
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ItemGas-Phase Intercluster Thiyl-Radical Induced C-H Bond Homolysis Selectively Forms Sugar C2-Radical Cations of Methyl D-Glucopyranoside: Isotopic Labeling Studies and Cleavage Reactions(SPRINGER, 2017-07)A suite of isotopologues of methyl D-glucopyranosides is used in conjunction with multistage mass spectrometry experiments to determine the radical site and cleavage reactions of sugar radical cations formed via a recently developed 'bio-inspired' method. In the first stage of CID (MS2), collision-induced dissociation (CID) of a protonated noncovalent complex between the sugar and S-nitrosocysteamine, [H3NCH2CH2SNO + M]+, unleashes a thiyl radical via bond homolysis to give the noncovalent radical cation, [H3NCH2CH2S• + M]+. CID (MS3) of this radical cation complex results in dissociation of the noncovalent complex to generate the sugar radical cation. Replacement of all exchangeable OH and NH protons with deuterons reveals that the sugar radical cation is formed in a process involving abstraction of a hydrogen atom from a C-H bond of the sugar coupled with proton transfer to the sugar, to form [M - H• + D+]. Investigation of this process using individual C-D labeled sugars reveals that the main site of H/D abstraction is the C2 position, since only the C2-deuterium labeled sugar yields a dominant [M - D• + H+] product ion. The fragmentation reactions of the distonic sugar radical cation, [M - H•+ H+], were studied by another stage of CID (MS4). 13C-labeling studies revealed that a series of three related fragment ions each contain the C1-C3 atoms; these arise from cross-ring cleavage reactions of the sugar. Graphical Abstract ᅟ.
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ItemNo Preview AvailableStructure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target( 2020-07-01)ABSTRACT 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-α-1,2-mannosidase (MANEA) is the soleendo-acting glycoside hydrolase involved in N-glycan trimming and unusually 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 upon substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. The structures inspired the development of new inhibitors that disrupted host protein N-glycan processing of viral glycans and reduced infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts of developing broad-spectrum antiviral agents and bring about a more detailed view of the biology of mammalian glycosylation. SIGNIFICANCE STATEMENT The glycosylation of proteins is a major protein modification that occurs extensively in eukaryotes. Glycosidases in the secretory pathway that trim N-linked glycans play a key role in protein quality control and in the specific modifications leading to mature glycoproteins. Inhibition of glucosidases in the secretory pathway is a proven therapeutic strategy, and one with great promise in the treatment of viral disease. The enzyme endo-α-1,2-mannosidase, MANEA, provides an alternative processing pathway to evade glucosidase inhibitors. We report the 3D structure of human MANEA and complexes with enzyme inhibitors that we show act as antivirals for bovine viral diarrhea and human dengue viruses. The structure of MANEA will support inhibitor optimization and the development of more potent antivirals.
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