Molecular Biology and Biochemistry, Department of

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Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INTRACELLULAR β-O-LINKED N-GLYCOLYLGLUCOSAMINE (GlcNGc), UDP-GlcNGc, AND THE BIOCHEMICAL AND STRUCTURAL RATIONALE FOR THE SUBSTRATE TOLERANCE OF β-O-LINKED β-N-ACETYLGLUCOSAMINIDASE

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2012-08-17
Abstract: 

TheO-GlcNAcmodificationinvolvestheattachmentofsingle-O-linkedN-acetylglucosamine residues to serine and threo-nine residues of nucleocytoplasmic proteins. Interestingly, pre-vious biochemical and structural studies have shown thatO-GlcNAcase (OGA), the enzyme that removesO-GlcNAc fromproteins, has an active site pocket that tolerates variousN-acylgroups in addition to theN-acetyl group of GlcNAc. Theremarkable sequence and structural conservation of residuescomprising this pocket suggest functional importance. Wehypothesized this pocket enables processing of metabolic vari-ants ofO-GlcNAc that could be formed due to inaccuracy withinthe metabolic machinery of the hexosamine biosynthetic path-way. In the accompanying paper (Bergfeld, A. K., Pearce, O. M.,Diaz, S. L.,Pham, T., and Varki, A. (2012)J. Biol. Chem.287,28865–28881),N-glycolylglucosamine (GlcNGc) wasshown to be acatabolite of NeuNGc. Here, we show that the hexosamine sal-vage pathway can convert GlcNGc to UDP-GlcNGc, which isthen used to modify proteins withO-GlcNGc. The kinetics of incorporation and removal ofO-GlcNGc in cells occur in adynamic manner on a time frame similar to that ofO-GlcNAc.Enzymatic activity ofO-GlcNAcase (OGA) toward a GlcNGcglycoside reveals OGA can process glycolyl-containing sub-strates fairly efficiently. A bacterial homolog (BtGH84) of OGA,from a human gut symbiont, also processesO-GlcNGc sub-strates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance andbinding of GlcNGc. Together, these results demonstrate thatanalogs of GlcNAc, such as GlcNGc, are metabolically viablespecies and that the conserved active site pocket of OGA likelyevolved to enable processing of mis-incorporated analogs ofO-GlcNAc and thereby prevent their accumulation. Such plas-ticity in carbohydrate processing enzymes may be a generalfeature arising from inaccuracy in hexosamine metabolicpathways.

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Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates*

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2012-05-01
Abstract: 

Cellular O-linked N-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine:polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for k(cat)/K(m) values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT (hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that K(m,app) values for UDP-GlcNAc varied considerably (from 1 μM to over 20 μM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of k(cat)/K(m) values obtained from hOGT and hOGA kinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGT and hOGA measured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity.

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Supervisor(s): 
Canadian Institutes of Health Research (CIHR)
Natural Sciences and Engineering Research Council of Canada (NSERC)

Multi-Omic Data Integration Allows Baseline Immune Signatures to Predict Hepatitis B Vaccine Response in a Small Cohort

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-11-30
Abstract: 

Background: Vaccination remains one of the most effective means of reducing the burden of infectious diseases globally. Improving our understanding of the molecular basis for effective vaccine response is of paramount importance if we are to ensure the success of future vaccine development efforts.

Methods: We applied cutting edge multi-omics approaches to extensively characterize temporal molecular responses following vaccination with hepatitis B virus (HBV) vaccine. Data were integrated across cellular, epigenomic, transcriptomic, proteomic, and fecal microbiome profiles, and correlated to final HBV antibody titres.

Results: Using both an unsupervised molecular-interaction network integration method (NetworkAnalyst) and a data-driven integration approach (DIABLO), we uncovered baseline molecular patterns and pathways associated with more effective vaccine responses to HBV. Biological associations were unravelled, with signalling pathways such as JAK-STAT and interleukin signalling, Toll-like receptor cascades, interferon signalling, and Th17 cell differentiation emerging as important pre-vaccination modulators of response.

Conclusion: This study provides further evidence that baseline cellular and molecular characteristics of an individual’s immune system influence vaccine responses, and highlights the utility of integrating information across many parallel molecular datasets.

Document type: 
Article
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Alpine Snow Algae Microbiome Diversity in the Coast Range of British Columbia

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-07-28
Abstract: 

Snow algae blooms contain bacteria, fungi, and other microscopic organisms. We surveyed 55 alpine snow algae blooms, collecting a total of 68 samples, from 12 mountains in the Coast Range of British Columbia, Canada. We used microscopy and rDNA metabarcoding to document biodiversity and query species and taxonomic associations. Across all samples, we found 173 algal, 2,739 bacterial, 380 fungal, and 540 protist/animalia operational taxonomic units (OTUs). In a previous study, we reported that most algal species were distributed along an elevational gradient. In the current study, we were surprised to find no corresponding distribution in any other taxa. We also tested the hypothesis that certain bacterial and fungal taxa co-occur with specific algal taxa. However, despite previous evidence that particular genera co-occur, we found no significant correlations between taxa across our 68 samples. Notably, seven bacterial, one fungal, and two cercozoan OTUs were widely distributed across our study regions. Taken together, these data suggest that any mutualisms with algae may not be taxon specific. We also report evidence of snow algae predation by rotifers, tardigrades, springtails, chytrid fungi, and ciliates, establishing the framework for a complex food web.

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Article
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Sticking With It: ER-PM Membrane Contact Sites as a Coordinating Nexus for Regulating Lipids and Proteins at the Cell Cortex

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-07-22
Abstract: 

Membrane contact sites between the cortical endoplasmic reticulum (ER) and the plasma membrane (PM) provide a direct conduit for small molecule transfer and signaling between the two largest membranes of the cell. Contact is established through ER integral membrane proteins that physically tether the two membranes together, though the general mechanism is remarkably non-specific given the diversity of different tethering proteins. Primary tethers including VAMP-associated proteins (VAPs), Anoctamin/TMEM16/Ist2p homologs, and extended synaptotagmins (E-Syts), are largely conserved in most eukaryotes and are both necessary and sufficient for establishing ER-PM association. In addition, other species-specific ER-PM tether proteins impart unique functional attributes to both membranes at the cell cortex. This review distils recent functional and structural findings about conserved and species-specific tethers that form ER-PM contact sites, with an emphasis on their roles in the coordinate regulation of lipid metabolism, cellular structure, and responses to membrane stress.

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Article
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Substrate‐Guided Front‐Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N‐Acetylgalactosaminyltransferase 2

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014-06-20
Abstract: 

The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNActransferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomicelectronic level of detail. Our study provides a detailed structural rationale for an ordered bi–bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues andenforce a front-face SNi-type reaction in which the substrate Nacetyl sugar substituent coordinates efficient glycosyl transfer.

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Article
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Multivalency To Inhibit and Discriminate Hexosaminidases

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2017-05-26
Abstract: 

A set of multivalent polyhydroxylated acetamidoazepanes based on ethylene glycol, glucoside, or cyclodextrin scaffolds was prepared. The compounds were assessed against plant, mammalian, and therapeutically relevant hexosaminidases. Multimerization was shown to improve the inhibitory potency with synergy, and to fine tune the selectivity profile between related hexosaminidases.

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Article
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A Divergent Synthesis to Generate Targeted Libraries of Inhibitors for Endo-N-Acetylglucosaminidases

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2017-12-18
Abstract: 

Cell active inhibitors of glycoside processing enzymes are valuable research tools that help us understand the physiological roles of this diverse class of enzymes. endo-N-Acetylglucosaminidases have gained increased attention for their important roles in both mammals and human pathogens; however, metabolically stable cell active inhibitors of these enzymes are lacking. Here, we describe a divergent synthetic strategy involving elaboration of a thiazoline core scaffold. We illustrate the potential of this approach by using the copper catalysed azide-alkyne click (CuAAC) reaction, in combination with a suitable catalyst to avoid poisoning by the thiazoline moiety, to generate a targeted panel of candidate inhibitors of endo-N-acetylglucosaminidases and chitinases

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Article
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A Convenient Approach to Stereoisomeric Iminocyclitols: Generation of Potent Brain‐Permeable OGA Inhibitors

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015-11-06
Abstract: 

Pyrrolidine‐based iminocyclitols are a promising class of glycosidase inhibitors. Reported herein is a convenient epimerization strategy that provides direct access to a range of stereoisomeric iminocyclitol inhibitors of O‐GlcNAcase (OGA), the enzyme responsible for catalyzing removal of O‐GlcNAc from nucleocytoplasmic proteins. Structural details regarding the binding of these inhibitors to a bacterial homologue of OGA reveal the basis for potency. These compounds are orally available and permeate into rodent brain to increase O‐GlcNAc, and should prove useful tools for studying the role of OGA in health and disease.

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Article
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A Selective Inhibitor Gal‐PUGNAc of Human Lysosomal β‐Hexosaminidases Modulates Levels of the Ganglioside GM2 in Neuroblastoma Cells

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2009-01-28
Abstract: 

Gal‐PUGNAc (see picture), a highly selective inhibitor for β‐hexosaminidases HEXA and HEXB is cell‐permeable and modulates the activity of HEXA and HEXB in tissue culture, increasing ganglioside GM2 levels. Gal‐PUGNAc should allow the role of these enzymes to be studied at the cellular level without generating a complex chemical phenotype from concomitant inhibition of O‐GlcNAcase.

Document type: 
Article
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