DOCSLIB.ORG

The 6C RNA of Corynebacterium Glutamicum

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The 6C RNA of Corynebacterium Glutamicum C. glutamicum The 6C RNA of The 6C RNA Jennifer Pahlke Jennifer Gesundheit Health The 6C RNA of Corynebacterium glutamicum Jennifer Pahlke Gesundheit / Health Band/ Volume 76 ISBN 978-3-95806-003-6 76 Helmholtz Association Member of the Schriften des Forschungszentrums Jülich Reihe Gesundheit / Health Band / Volume 76 Forschungszentrum Jülich GmbH Institute of Bio- and Geosciences (IBG) Biotechnology (IBG-1) The 6C RNA of Corynebacterium glutamicum Jennifer Pahlke Schriften des Forschungszentrums Jülich Reihe Gesundheit / Health Band / Volume 76 ISSN 1866-1785 ISBN 978-3-95806-003-6 Bibliographic information published by the Deutsche Nationalbibliothek. The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.d-nb.de. Publisher and Forschungszentrum Jülich GmbH Distributor: Zentralbibliothek 52425 Jülich Tel: +49 2461 61-5368 Fax: +49 2461 61-6103 Email: [email protected] www.fz-juelich.de/zb Cover Design: Grafische Medien, Forschungszentrum Jülich GmbH Printer: Grafische Medien, Forschungszentrum Jülich GmbH Copyright: Forschungszentrum Jülich 2014 Schriften des Forschungszentrums Jülich Reihe Gesundheit / Health, Band / Volume 76 D 61 (Diss. Düsseldorf, Univ., 2014) ISSN 1866-1785 ISBN 978-3-95806-003-6 The complete volume is freely available on the Internet on the Jülicher Open Access Server (JuSER) at www.fz-juelich.de/zb/openaccess Neither this book nor any part of it may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Content Summary .............................................................................................................................. 1 Zusammenfassung .............................................................................................................. 3 1 Introduction .................................................................................................................. 5 1.1 Corynebacterium glutamicum, a model organism of biotechnological relevance ......... 5 1.2 Small RNAs in bacteria ......................................................................................................... 7 1.2.1 Cis-encoded antisense RNAs .......................................................................................... 8 1.2.2 Trans-encoded antisense RNAs .................................................................................... 10 1.2.3 sRNAs that modulate protein activity ............................................................................. 12 1.3 The 6C RNA family .............................................................................................................. 14 1.4 Aims of this work ................................................................................................................. 17 2 Materials and Methods ...............................................................................................19 2.1 Buffers and antibiotics ........................................................................................................ 19 2.2 Culture media ....................................................................................................................... 21 2.3 Oligonucleotides.................................................................................................................. 22 2.4 Bacterial strains and plasmids........................................................................................... 24 2.5 Plasmid constructions ........................................................................................................ 27 2.5.1 Construction of pk19mobsacB derivatives for the deletion of genes ............................ 27 2.5.2 Construction of pAN and pJC1 derivatives as expression plasmids for C. glutamicum 28 2.5.3 Construction of pUCBM21 derivatives as expression plasmids for E. coli .................... 28 2.6 Cultivation of bacteria ......................................................................................................... 28 2.6.1 Cultivation of E. coli ....................................................................................................... 28 2.6.2 Cultivation of C. glutamicum .......................................................................................... 29 2.6.3 Maintenance of bacteria ................................................................................................ 29 2.6.4 Determination of bacterial growth .................................................................................. 30 2.6.5 Determination of the cell dry weight (CDW) .................................................................. 30 2.6.6 Cultivation in microtiter scale in the BioLector system .................................................. 30 2.7 Molecular biology methods ................................................................................................ 31 2.7.1 Isolation of nucleic acid ................................................................................................. 31 2.7.2 Nucleic acid gel electrophoresis .................................................................................... 32 2.7.3 Determination of nucleic acid concentrations ................................................................ 33 2.7.4 Polymerase Chain Reaction (PCR) ............................................................................... 33 2.7.5 Purification of DNA fragments ....................................................................................... 34 2.7.6 Recombinant DNA techniques ...................................................................................... 34 2.7.7 DNA sequencing ............................................................................................................ 35 2.7.8 Generation and transformation of competent E. coli ..................................................... 35 2.7.9 Generation and transformation of competent C. glutamicum ........................................ 36 2.7.10 Chromosomal gene replacement using the pK19mobsacB system .............................. 36 2.7.11 RNA in vitro transcription ............................................................................................... 37 2.7.12 RNA purification by phenol chloroform extraction ......................................................... 37 2.7.13 Ethanol precipitation of RNA ......................................................................................... 38 2.7.14 Northern blot analysis .................................................................................................... 38 2.7.15 Quantification of 6C RNA molecules per cell ................................................................ 39 2.7.16 RNA-protein interaction studies ..................................................................................... 39 2.8 Global gene expression analysis using DNA microarrays ............................................. 41 2.8.1 Synthesis and labeling of cDNA .................................................................................... 41 2.8.2 C. glutamicum DNA microarray hybridization ................................................................ 42 2.8.3 DNA microarray fluorescence signal measurement and data analysis ......................... 43 2.9 Protein biochemical methods ............................................................................................ 44 2.9.1 Cell disruption methods ................................................................................................. 44 2.9.2 Determination of protein concentrations ........................................................................ 44 2.9.3 Protein precipitation ....................................................................................................... 45 2.9.4 SDS polyacrylamide gel electrophoresis ....................................................................... 45 2.9.5 Protein overexpression and purification ........................................................................ 45 2.9.6 Concentration and desalting of proteins ........................................................................ 47 2.9.7 DNA–protein interaction studies .................................................................................... 47 2.9.8 PGKCg enzyme activity assays ...................................................................................... 48 2.10 MALDI ToF mass spectrometry .......................................................................................... 49 2.11 Microscopy techniques ....................................................................................................... 50 2.12 Quantification of metabolites ............................................................................................. 50 2.12.1 Quantification of glucose and organic acids in culture supernatants ............................ 50 2.12.2 Quantification of amino acids ........................................................................................ 51 3 Results ........................................................................................................................53 3.1 The 6C RNA and its expression in C. glutamicum ........................................................... 53 3.1.1 The 6C RNA is very
Load more

Recommended publications
  • Down Regulation of Membrane-Bound Neu3 Constitutes a New
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Publications of the IAS Fellows IJC International Journal of Cancer Down regulation of membrane-bound Neu3 constitutes a new potential marker for childhood acute lymphoblastic leukemia and induces apoptosis suppression of neoplastic cells Chandan Mandal1, Cristina Tringali2, Susmita Mondal1, Luigi Anastasia2, Sarmila Chandra3, Bruno Venerando2 and Chitra Mandal1 1 Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, A Unit of Council of Scientific and Industrial Research, Govt of India, 4, Raja S. C. Mullick Road, Kolkata 700032, India 2 Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, and IRCCS Policlinico San Donato, San Donato, Milan, Italy 3 Department of Hematology, Kothari Medical Centre, Kolkata 700027, India Membrane-linked sialidase Neu3 is a key enzyme for the extralysosomal catabolism of gangliosides. In this respect, it regulates pivotal cell surface events, including trans-membrane signaling, and plays an essential role in carcinogenesis. In this report, we demonstrated that acute lymphoblastic leukemia (ALL), lymphoblasts (primary cells from patients and cell lines) are characterized by a marked down-regulation of Neu3 in terms of both gene expression (230 to 40%) and enzymatic activity toward ganglioside GD1a (225.6 to 30.6%), when compared with cells from healthy controls. Induced overexpression of Neu3 in the ALL-cell line, MOLT-4, led to a significant increase of ceramide (166%) and to a parallel decrease of lactosylceramide (255%). These events strongly guided lymphoblasts to apoptosis, as we assessed by the decrease in Bcl2/ Bax ratio, the accumulation of Neu3 transfected cells in the sub G0–G1 phase of the cell cycle, the enhanced annexin-V positivity, the higher cleavage of procaspase-3.
    [Show full text]
  • Salmonella Degrades the Host Glycocalyx Leading to Altered Infection and Glycan Remodeling
    UC Davis UC Davis Previously Published Works Title Salmonella Degrades the Host Glycocalyx Leading to Altered Infection and Glycan Remodeling. Permalink https://escholarship.org/uc/item/0nk8n7xb Journal Scientific reports, 6(1) ISSN 2045-2322 Authors Arabyan, Narine Park, Dayoung Foutouhi, Soraya et al. Publication Date 2016-07-08 DOI 10.1038/srep29525 Peer reviewed eScholarship.org Powered by the California Digital Library University of California www.nature.com/scientificreports OPEN Salmonella Degrades the Host Glycocalyx Leading to Altered Infection and Glycan Remodeling Received: 09 February 2016 Narine Arabyan1, Dayoung Park2, Soraya Foutouhi1, Allison M. Weis1, Bihua C. Huang1, Accepted: 17 June 2016 Cynthia C. Williams2, Prerak Desai1,†, Jigna Shah1,‡, Richard Jeannotte1,3,§, Nguyet Kong1, Published: 08 July 2016 Carlito B. Lebrilla2,4 & Bart C. Weimer1 Complex glycans cover the gut epithelial surface to protect the cell from the environment. Invasive pathogens must breach the glycan layer before initiating infection. While glycan degradation is crucial for infection, this process is inadequately understood. Salmonella contains 47 glycosyl hydrolases (GHs) that may degrade the glycan. We hypothesized that keystone genes from the entire GH complement of Salmonella are required to degrade glycans to change infection. This study determined that GHs recognize the terminal monosaccharides (N-acetylneuraminic acid (Neu5Ac), galactose, mannose, and fucose) and significantly (p < 0.05) alter infection. During infection, Salmonella used its two GHs sialidase nanH and amylase malS for internalization by targeting different glycan structures. The host glycans were altered during Salmonella association via the induction of N-glycan biosynthesis pathways leading to modification of host glycans by increasing fucosylation and mannose content, while decreasing sialylation.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown Et Al
    US 20030082511A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown et al. (43) Pub. Date: May 1, 2003 (54) IDENTIFICATION OF MODULATORY Publication Classification MOLECULES USING INDUCIBLE PROMOTERS (51) Int. Cl." ............................... C12O 1/00; C12O 1/68 (52) U.S. Cl. ..................................................... 435/4; 435/6 (76) Inventors: Steven J. Brown, San Diego, CA (US); Damien J. Dunnington, San Diego, CA (US); Imran Clark, San Diego, CA (57) ABSTRACT (US) Correspondence Address: Methods for identifying an ion channel modulator, a target David B. Waller & Associates membrane receptor modulator molecule, and other modula 5677 Oberlin Drive tory molecules are disclosed, as well as cells and vectors for Suit 214 use in those methods. A polynucleotide encoding target is San Diego, CA 92121 (US) provided in a cell under control of an inducible promoter, and candidate modulatory molecules are contacted with the (21) Appl. No.: 09/965,201 cell after induction of the promoter to ascertain whether a change in a measurable physiological parameter occurs as a (22) Filed: Sep. 25, 2001 result of the candidate modulatory molecule. Patent Application Publication May 1, 2003 Sheet 1 of 8 US 2003/0082511 A1 KCNC1 cDNA F.G. 1 Patent Application Publication May 1, 2003 Sheet 2 of 8 US 2003/0082511 A1 49 - -9 G C EH H EH N t R M h so as se W M M MP N FIG.2 Patent Application Publication May 1, 2003 Sheet 3 of 8 US 2003/0082511 A1 FG. 3 Patent Application Publication May 1, 2003 Sheet 4 of 8 US 2003/0082511 A1 KCNC1 ITREXCHO KC 150 mM KC 2000000 so 100 mM induced Uninduced Steady state O 100 200 300 400 500 600 700 Time (seconds) FIG.
    [Show full text]
  • Characterization of the Human Sialidase Neu4 Gene Promoter
    Turkish Journal of Biology Turk J Biol (2014) 38: 574-580 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1401-63 Characterization of the human sialidase Neu4 gene promoter 1, 2 Volkan SEYRANTEPE *, Murat DELMAN 1 Department of Molecular Biology and Genetics, İzmir Institute of Technology, Urla İzmir, Turkey 2 Biotechnology and Bioengineering Graduate Program, İzmir Institute of Technology, Urla, İzmir, Turkey Received: 21.01.2014 Accepted: 08.05.2014 Published Online: 05.09.2014 Printed: 30.09.2014 Abstract: There are 4 different sialidases that have been described in humans: lysosomal (Neu1), cytoplasmic (Neu2), plasma membrane (Neu3), and lysosomal/mitochondrial (Neu4). Previously, we have shown that Neu4 has a broad substrate specificity and is active against glyco-conjugates, including GM2 ganglioside, at the acidic pH of 3.2. An overexpression of Neu4 in transfected neuroglia cells from a Tay–Sachs patient shows a clearance of accumulated GM2, indicating the biological importance of Neu4. In this paper, we aimed to characterize a minimal promoter region of the human Neu4 gene in order to understand the molecular mechanism regulating its expression. We cloned 7 different DNA fragments from the human Neu4 promoter region into luciferase expression vectors for a reporter assay and also performed an electrophoretic mobility shift assay to demonstrate the binding of transcription factors. We demonstrated that –187 bp upstream of the Neu4 gene is a minimal promoter region for controlling transcription from the human Neu4 gene. The electrophoretic mobility shift assay showed that the minimal promoter region recruits a c-myc transcription factor, which might be responsible for regulation of Neu4 gene transcription.
    [Show full text]
  • Brain Lipid Profiling of Triply Mouse Model with the Deficiencies of Sialidase Neu1, Neu4 and Β-Hexosaminidase a Enzymes
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by DSpace@IZTECH Institutional Repository BRAIN LIPID PROFILING OF TRIPLY MOUSE MODEL WITH THE DEFICIENCIES OF SIALIDASE NEU1, NEU4 AND β-HEXOSAMINIDASE A ENZYMES A Thesis Submitted to the Graduate School of Engineering and Sciences of İzmir Institute of Technology in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in Molecular Biology and Genetics by Zehra Kevser PEKMEZCİ December 2011 İZMİR We approve the thesis of Zehra Kevser PEKMEZCİ ____________________________ Assoc. Prof. Dr. Volkan SEYRANTEPE Supervisor _________________________ Assoc. Prof. Dr. Yusuf BARAN Committee Member ______________________________ Assoc. Prof. Dr. Şermin GENÇ Committee Member 20 December 2011 _________________________________ ______________________________ Assoc. Prof. Dr. Ahmet KOÇ Prof. Dr. R. Tuğrul SENGER Head of the Department of Molecular Dean of the Graduate School of Biology and Genetics Engineering and Sciences ACKNOWLEDGMENTS First of all, I would like to indicate my deepest regards and thanks to my supervisor Assoc. Prof. Dr. Volkan SEYRANTEPE for his encouragement, understanding, guidance, and excellent support during my graduate studies. I would like to thank to Assist. Prof. Dr. Alper Arslanoğlu to let me use his laboratory during my thesis studies. And I would also like to thank to Assist. Prof. Dr. Ayten Nalbant, Assist. Prof. Dr. Bünyamin Akgül, Assoc. Prof. Dr. Sami Doğanlar, Assoc. Prof. Dr. Ahmet Koç, Prof. Dr. Serdar Özçelik and Assoc. Prof. Dr. Mehtap Demirağ to let me use their laboratory during my studies. Also I would like to express my grateful thanks to my committee members Assoc.
    [Show full text]
  • Cell Surface Sialylated N-Glycan Alterations During Development
    REVIEW ARTICLE Eur J Biol 2017; 76(2): 79-88 Cell Surface Sialylated N-Glycan Alterations during Development Sabire Karacali Ege Universiy, Faculty of Science, Department of Biology, Izmir, Turkey Please cite this article as: Karacali S. Cell Surface Sialylated N-Glycan Alterations during Development. Eur J Biol 2017; 76(2): 79-88 ABSTRACT This brief survey focuses on the comparison of sialylated N-glycans of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and of differentiated cells. In addition, the impact of sialic acid (Sia) deficiency on cell surfaces during development is summarized. The most common Sia is N-acetylneuraminic acid (Neu5Ac). The branched structures of complex- and hybrid- type N-glycans are the carrier for Sia. Transmembrane adhesive proteins, voltage-gated ion channels and many ligand-activated receptors are some examples of heavily sialylated N-glycan bearing membrane proteins. Their oligosaccharide extensions provide an important contribution to glycocalyx glycans. ESCs and iPSCs are characterized with high mannose-type and biantennary complex-type core structures. Two branches terminate with α2,6- linked Sia. MSCs contain high mannose, hybrid- and complex- type N-glycans. Linear poly-N-acetyllactosamine (poly-Galβ1-4GlcNAc, poly-LacNAc) chains are the characteristic structures. Both α2,3- and α2,6- linked Sias are seen in a species-specific manner in MSCs. α2,6- linked Sia is probably a marker associated with the multipotency of human MSCs. Differentiated healthy cells contain the most abundant 2-branched complex structures. The bisecting branch on the core structure appears as a differentiation marker. poly-LacNAc chains are terminated with α2,3- and α2,6- linked Sia, with the former being higher.
    [Show full text]
  • Characterization of Transgenic Mouse Model of Humanized Sialidase
    TRANSGENIC MOUSE MODEL OF HUMANIZED SIALIDASE CHRACTERIZATION OF TRANSGENIC MOUSE MODEL OF HUMANIZED SIALIDSAE By Ghada A. Tarbah, B.Sc. A Thesis submitted to the School of Graduate Studies in Partial fulfillment of the Requirements for the Degree Master of Science McMaster University © Copyright by Ghada Tarbah, June 2009 MASTER OF SCIENCE (2008) McMaster University (Biology) Hamilton, Ontario M.Sc. thesis - G. Tarbah McMaster - Biology TITLE: Characterization of Transgenic Mouse Model of Humanized Sialidase AUTHOR: Ghada A. Tarbah, B.Sc. SUPERVISOR: Suleiman Igdoura, PhD, Associate Professor, McMaster University Number of Pages: XI, 59 ii M.Sc. thesis - G. Tarbah McMaster - Biology ABSTRACT Ganglosidoses are a group of fatal neurological disorders which primarily affect children. Pathologically, these disorders need more characterization. The recent development of mouse models of these diseases makes it possible to identify progressive pathological changes within the CNS at the molecular level. Tay Sachs disease is a lysosomal storage disorder characterized by accumulation of GM2 Ganglioside. Targeted Knockout of ~-hexosaminidase A (hex a) was generated as a mouse model of Tay Sachs disease; however, the mouse showed little clinical phenotype. The molecular mechanism which allows the hexa-/- mouse to escape the disease involves a metabolic bypass facilitated by lysosomal Sialidase (neu l). In this study we hypothesize that the reason humans have the infantile Tay-Sachs whereas mice do not, is a difference in specificity between human and mouse sialidases. This specificity difference makes the human sialidase unable to hydrolyze GM2 to GA2, which leads to the accumulation of GM2 in neurons and consequently a resulting Tay-Sachs phenotype.
    [Show full text]
  • RT² Profiler PCR Array (Rotor-Gene® Format) Human Glycosylation
    RT² Profiler PCR Array (Rotor-Gene® Format) Human Glycosylation Cat. no. 330231 PAHS-046ZR For pathway expression analysis Format For use with the following real-time cyclers RT² Profiler PCR Array, Rotor-Gene Q, other Rotor-Gene cyclers Format R Description The Human Glycosylation RT² Profiler PCR Array profiles the expression of 84 key genes encoding enzymes that post-translationally add and remove sugar residues to and from proteoglycans and glycoproteins. The process of generating and altering mature N-linked and O-linked glycans essential for proteoglycan and glycoprotein function requires not only glycosyltransferase activity for de novo oligosaccharide synthesis, but also both glycosidase and glycosyltransferase activity for remodeling. Increased expression of cell surface and secreted proteins, whether by stimulation of cells to differentiate or proliferate or by exogenous over-expression, requires more glycosyltransferase and glycosidase activity, contributed at least in part by their own increased gene expression. This array includes glycosyltransferase and glycosidase genes for several important sugars: galactose, glucose, mannose, N-acetylgalactosamine, N-acetylglucosamine, fucose, and sialic acid. Some of the represented enzymes also act on glycosphingolipids. Using real-time PCR, you can easily and reliably analyze the expression of a focused panel of genes involved in protein glycosylation with this array. For further details, consult the RT² Profiler PCR Array Handbook. Shipping and storage RT² Profiler PCR Arrays in the Rotor-Gene format are shipped at ambient temperature, on dry ice, or blue ice packs depending on destination and accompanying products. For long term storage, keep plates at –20°C. Note: Ensure that you have the correct RT² Profiler PCR Array format for your real-time cycler (see table above).
    [Show full text]
  • An Intrinsic Mechanism of Secreted Protein Aging and Turnover
    An intrinsic mechanism of secreted protein aging and turnover Won Ho Yanga,b, Peter V. Aziza,b, Douglas M. Heithoffc, Michael J. Mahanc, Jeffrey W. Smithb, and Jamey D. Martha,b,c,1 aCenter for Nanomedicine, University of California, Santa Barbara, CA 93106; bSanford–Burnham–Prebys Medical Discovery Institute, La Jolla, CA 92037; and cDepartment of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106 Edited by Kevin P. Campbell, University of Iowa Carver College of Medicine, Iowa City, IA, and approved September 23, 2015 (received for review August 4, 2015) The composition and functions of the secreted proteome are con- activity levels that change in disease (9). We investigated whether trolled by the life spans of different proteins. However, unlike the normal activities of circulating glycosidases may hydrolyze intracellular protein fate, intrinsic factors determining secreted pro- N-glycan linkages attached to secreted proteins, thereby generat- tein aging and turnover have not been identified and characterized. ing multivalent ligands of endocytic lectin receptors in contribut- Almost all secreted proteins are posttranslationally modified with the ing to a mechanism of secreted protein aging and turnover. covalent attachment of N-glycans. We have discovered an intrinsic Results mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-Glycan Remodeling in the Aging of Secreted Proteins. Most se- N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), creted proteins are modified with N-glycans before entry into cir- neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosamini- culatory systems. We isolated secreted proteins among platelet-poor dase B (HexB), possess hydrolytic activities that temporally remodel plasma following i.v.
    [Show full text]
  • Keeping It Trim: Roles of Neuraminidases in CNS Function
    Glycoconjugate Journal (2018) 35:375–386 https://doi.org/10.1007/s10719-018-9837-4 REVIEW Keeping it trim: roles of neuraminidases in CNS function Alexey V. Pshezhetsky1,2 & Mila Ashmarina1 Received: 1 June 2018 /Revised: 25 July 2018 /Accepted: 27 July 2018 /Published online: 7 August 2018 # The Author(s) 2018 Abstract The sialylated glyconjugates (SGC) are found in abundance on the surface of brain cells, where they form a dense array of glycans mediating cell/cell and cell/protein recognition in numerous physiological and pathological processes. Metabolic genetic blocks in processing and catabolism of SGC result in development of severe storage disorders, dominated by CNS involvement including marked neuroinflammation and neurodegeneration, the pathophysiological mechanisms of which are still discussed. SGC patterns in the brain are cell and organelle-specific, dynamic and maintained by highly coordinated processes of their biosynthesis, trafficking, processing and catabolism. The changes in the composition of SGC during development and aging of the brain cannot be explained based solely on the regulation of the SGC-synthesizing enzymes, sialyltransferases, suggesting that neuraminidases (sialidases) hydrolysing the removal of terminal sialic acid residues also play an essential role. In the current review we summarize the roles of three mammalian neuraminidases: neuraminidase 1, neuraminidase 3 and neuraminidase 4 in processing brain SGC. Emerging data demonstrate that these enzymes with different, yet overlapping expression patterns, intracellular localization and substrate specificity play essential roles in the physiology of the CNS. Keywords Sialic acid . Polysialic acid . Lysosome . CNS . Ganglioside . Neuraminidase Sialoglycoconjugates in mammalian brain SGC strongly depends on the cell and tissue type, and changes during development [5].
    [Show full text]
  • Activity of Plasma Membrane Β-Galactosidase and Β-Glucosidase
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 583 (2009) 2469–2473 journal homepage: www.FEBSLetters.org Activity of plasma membrane b-galactosidase and b-glucosidase Massimo Aureli, Anie Priscilla Masilamani, Giuditta Illuzzi, Nicoletta Loberto, Federica Scandroglio, Alessandro Prinetti, Vanna Chigorno, Sandro Sonnino * Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milano, 20090 Segrate, Italy article info abstract Article history: Human fibroblasts produce ceramide from sialyllactosylceramide on the plasma membranes. Siali- Received 4 May 2009 dase Neu3 is known to be plasma membrane associated, while only indirect data suggest the plasma Revised 28 May 2009 membrane association of b-galactosidase and b-glucosidase. To determine the presence of b-galacto- Accepted 26 June 2009 sidase and b-glucosidase on plasma membrane, cells were submitted to cell surface biotinylation. Available online 4 July 2009 Biotinylated proteins were purified by affinity column and analyzed for enzymatic activities on Edited by Felix Wieland artificial substrates. Both enzyme activities were found associated with the cell surface and were up-regulated in Neu3 overexpressing cells. These enzymes were capable to act on both artificial and natural substrates without any addition of activator proteins or detergents and displayed a Keywords: Glycohydrolases trans activity in living cells. Plasma membrane Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. b-Galactosidase b-Glucosidase trans activity 1. Introduction demonstrated to be different enzymes with respect the corre- sponding lysosomal glycohydrolases [6,7].
    [Show full text]
  • Insights Into Isoenzyme Selectivity
    Molecular dynamics simulations of viral neuraminidase inhibitors with the human neuraminidase enzymes: Insights into isoenzyme selectivity Michele R. Richards, Tianlin Guo, Carmanah D. Hunter, Christopher W. Cairo* Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada Dedicated to Professor Laura L. Kiessling on the receipt of the 2017 Tetrahedron Prize *Correspondence, Tel.: 780 492 0377; fax: 780 492 8231; e-mail: [email protected] Abstract: Inhibitors of viral neuraminidase enzymes have been previously developed as therapeutics. Humans can express multiple forms of neuraminidase enzymes (NEU1, NEU2, NEU3, NEU4) that share a similar active site and enzymatic mechanism with their viral counterparts. Using a panel of purified human neuraminidase enzymes, we tested the inhibitory activity of 2-deoxy-2,3- dehydro-N-acetylneuraminic acid (DANA), zanamivir, oseltamivir, and peramivir against each of the human isoenzymes. We find that, with the exceptions of DANA and zanamivir, these compounds show generally poor activity against the human neuraminidase enzymes. To provide insight into the interactions of viral inhibitors with human neuraminidases, we conducted molecular dynamics simulations using homology models based on coordinates reported for NEU2. Simulations revealed that an organized water is displaced by zanamivir in binding to NEU2 and NEU3 and confirmed the critical importance of engaging the binding pocket of the C7–C9 glycerol sidechain. Our results suggest that compounds designed
    [Show full text]