DOCSLIB.ORG

Understanding and Manipulating Primary Cell Walls in Plant Cell Suspension Cultures

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Understanding and Manipulating Primary Cell Walls in Plant Cell Suspension Cultures Understanding and manipulating primary cell walls in plant cell suspension cultures Felicia Leijon Doctoral Thesis in Biotechnology School of Engineering Sciences in Chemistry, Biotechnology and Health Royal Institute of Technology Stockholm, Sweden 2019 © Felicia Leijon, Stockholm, 2019 TRITA-CBH-FOU-2019:2 ISBN 978-91-7873-074-2 KTH School of Engineering Sciences in Chemistry, Biotechnology and Health Royal Institute of Technology AlbaNova University Center 106 91 Stockholm Sweden Printed by Universitetsservice US-AB, Stockholm 2019 Abstract The cell wall is required for many aspects of plant function and development. It is also an accessible and renewable resource utilized both in unrefined forms and as raw material for further development. Increased knowledge regarding cell wall structure and components will contribute to better utilization of plants and the resources they provide. In this thesis aspects of the primary cell wall of Populus trichocarpa and Nicotiana tabacum are explored. In Publication I a method for isolation and biochemical characterization of plant glycosyltransferases using a spectrophotometric or a radiometric assay was optimized. The radiometric assay was applied in Publication II where the proteome of the plasmodesmata isolated from P. trichocarpa was analyzed. Proteins identified belonged to functional classes such as “transport”, “signalling” and “stress responses”. Plasmodesmata-enriched fractions had high levels of callose synthase activity under ion depleted conditions as well as with calcium present. The second part of the thesis comprises the alteration of the cell wall of N. tabacum cells and A. thaliana plants through in vivo expression of a carbohydrate binding module (CBM) (Publication III). In tobacco this resulted in cell walls with loose ultrastructure containing an increased proportion of 1,4-β-glucans. The cell walls were more susceptible to saccharification, possibly due to changes in the structure of cellulose or xyloglucan. Arabidopsis plants showed increased saccharification after mild pretreatment, suggesting that heterologous expression of CBMs is a promising method for cell wall engineering. In Publication IV cellulose microfibrils (CMFs) and nanocrystals (CNCs) were extracted from the transgenic cells. CNC preparation resulted in higher yields and longer CNCs. Nanopapers prepared from the CMFs of the CBM line demonstrated enhanced strength and toughness. Thus, changes to the ordered regions of cellulose were suggested to take place due to CBM expression. Keywords: Callose synthase, carbohydrate-binding module, cell wall engineering, cellulose microfibril, cellulose nanocrystal, glycosyltransferase, mass spectrometry, plasmodesmata, Populus, primary cell wall, radiometric assay, spectrophotometric assay. i Sammanfattning Cellväggen har en avgörande roll för många aspekter av funktion och utveckling hos växtcellen. Den utgör en tillgänglig och förnybar resurs både i sin obearbetade form och som råmaterial för vidare förädling. Ökad kunskap rörande cellväggens struktur och sammansättning skulle därför bidra till bättre nyttjande av växter och de råvaror de tillhandahåller. I den här avhandlingen undersöks olika aspekter av den primära cellväggen hos Populus trichocarpa och Nicotiana tabacum. I Publikation I optimeras en spektrofotometrisk och en radiometrisk metod för biokemisk karakterisering av gykosyltransferaser från växter. Den radiometriska metoden tillämpas sedan i Publikation II där proteomet hos plasmodesmata från P. trichocarpa celler kartlades. Identifierade proteiner tillhörde funktionella klasser så som ”transport”, ”signalering” och ”stress”. Plasmodesmata anrikade membranfraktioner hade förhöjda nivåer av kallos-syntas aktivitet både vid frånvaro av joner och med kalcium. Den andra delen av avhandlingen består av in vivo uttryck av en kolhydratbindande modul (CBM) i tobak och Arabidopsis plantor för att introducera förändringar i cellväggen (Publikation III). Detta resulterade i cellväggar med porös ultrastruktur innehållande mer 1,4-β- glukaner. Cellväggarna var lättare att bryta ner till kolhydratmonomerer vilket kan bero på att strukturen hos cellulosa eller xyloglukan förändrats. Arabidopsis plantor var också lättare att bryta ner efter en mild förbehandling vilket tyder på att heterologt uttryck av CBM är en lovande metod för att manipulera cellväggen. I Publikation IV extraherades mikrofibriller av cellulosa och nanokristallin cellulosa från de transgena tobakscellerna. Framställningen av nanokristallin cellulosa från celler som uttryckte CBM gav högre utbyte och kristallerna var längre. Nanopapper tillverkat från mikrofibrillerna visade på ökad styrka och seghet. Detta tolkades som att uttrycket av CBM hade lett till förändringar i de organiserade regionerna av cellulosa. Nyckelord: Glykosyltransferas, kallos-syntas, kolhydratbindande modul, masspektrometri, cellulosa mikrofibriller, nanokristallin cellulosa, plasmodesmata, Populus, primär cellvägg, radiometrisk analys, spektrofotometrisk analys. ii List of Publications Publication I: Brown, C., Leijon, F., and Bulone, V. (2012). Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis. Nat. Protoc. 7, 1634-1650. Publication II: Leijon, F., Melzer, M., Zhou, Q., Srivastava, V., and Bulone, V. (2018). Proteomic analysis of plasmodesmata from Populus cell suspension cultures in relation with callose biosynthesis. Front. Plant Sci. 9, 1681. Publication III: Leijon, F.*, Melida, H.*, Melzer, M., Larsson, T., Srivastava, V., Gomez, L., Guerriero, G., McQeen-Mason, S., and Bulone, V. The effect of carbohydrate-binding modules (CBMs) on plant cell wall properties: an in vivo approach. Manuscript. Publication IV: Butchosa, N., Leijon, F., Bulone, V., and Zhou, Q. (2018). Stronger cellulose microfibrils network structure through the expression of cellulose-binding modules in plant primary cell walls. Submitted to Cellulose. *Both authors contributed equally to the work. iii The Author´s Contribution Publication I: Felicia Leijon performed optimization experiments. Publication II: Felicia Leijon enriched membrane fractions, performed activity assays, product characterization and prepared samples for mass-spectrometric analysis. She also performed data analysis and contributed to the writing of the manuscript. Publication III: Felicia Leijon performed plant transformation, extraction of cell wall and cell wall carbohydrates, growth studies, xyloglucan assay, localisation studies, PCR, qPCR and contributed to the writing of the manuscript. Publication IV: Felicia Leijon performed plant transformation and preparation of plant cell cultures. She also contributed to the extraction of cellulose and preparation and testing of the nanopapers. iv List of Abbreviations BcsA Bacterial cellulose synthase CAZy Carbohydrate active enzymes CalS Callose Synthase CBM Carbohydrate-binding module CesA Cellulose synthase CipA Cellulosome-integrating protein A CMF Cellulose microfibril CNC Cellulose nanocrystal CNF Cellulose nanofibril DP Degree of polymerization DRM Detergent-resistant microdomain ER Endoplasmic reticulum GC-MS Gas chromatography–mass spectrometry GH Glycoside hydrolase GRP Glycine-rich protein Gsl Glucan synthase like GT Glycosyltransferase HPAEC High-performance anion-exchange chromatography HRGP Hydroxyproline-rich glycoprotein LPMO lytic polysaccharide monooxygenase MS Mass spectrometry NAD Nicotinamide adenine dinucleotide NDP Nucleoside diphosphate PRP Proline-rich protein PM Plasma membrane PD Plasmodesmata ROP Regulation of cell polarity protein SEL Size exclusion limit SuSy Sucrose synthase TC Terminal complex TMD Transmembrane domain UGT UDP-glucose transferring protein WT Wild type v Contents 1. Introduction ...................................................................................... 1 1.1. The plant cell wall ............................................................................ 2 1.1.1. Cell wall structure................................................................... 3 Middle lamella and pectins ................................................................ 3 Primary cell wall ................................................................................. 3 Secondary cell wall ............................................................................ 5 1.1.2. Cell wall growth ...................................................................... 6 1.1.3. The plasma membrane and the plasmodesmata and their relationship with the cell wall .................................................. 7 Plasma membrane ............................................................................. 7 Plasmodesmata ................................................................................. 7 1.1.4. Model organisms used for studying the plant cell wall .......... 9 Plant cell suspension cultures ......................................................... 11 1.2. Cell wall polysaccharides - Structure and function ................... 12 1.2.1. Cellulose .............................................................................. 13 Nanocellulose .................................................................................. 15 1.2.2. Callose ................................................................................. 17 1.2.3. Xyloglucan ........................................................................... 18 1.3. Biosynthesis of cell wall polysaccharides .................................. 21 1.3.1. Classification
Load more

Recommended publications
  • Open Matthew R Moreau Ph.D. Dissertation Finalfinal.Pdf
    The Pennsylvania State University The Graduate School Department of Veterinary and Biomedical Sciences Pathobiology Program PATHOGENOMICS AND SOURCE DYNAMICS OF SALMONELLA ENTERICA SEROVAR ENTERITIDIS A Dissertation in Pathobiology by Matthew Raymond Moreau 2015 Matthew R. Moreau Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2015 The Dissertation of Matthew R. Moreau was reviewed and approved* by the following: Subhashinie Kariyawasam Associate Professor, Veterinary and Biomedical Sciences Dissertation Adviser Co-Chair of Committee Bhushan M. Jayarao Professor, Veterinary and Biomedical Sciences Dissertation Adviser Co-Chair of Committee Mary J. Kennett Professor, Veterinary and Biomedical Sciences Vijay Kumar Assistant Professor, Department of Nutritional Sciences Anthony Schmitt Associate Professor, Veterinary and Biomedical Sciences Head of the Pathobiology Graduate Program *Signatures are on file in the Graduate School iii ABSTRACT Salmonella enterica serovar Enteritidis (SE) is one of the most frequent common causes of morbidity and mortality in humans due to consumption of contaminated eggs and egg products. The association between egg contamination and foodborne outbreaks of SE suggests egg derived SE might be more adept to cause human illness than SE from other sources. Therefore, there is a need to understand the molecular mechanisms underlying the ability of egg- derived SE to colonize the chicken intestinal and reproductive tracts and cause disease in the human host. To this end, the present study was carried out in three objectives. The first objective was to sequence two egg-derived SE isolates belonging to the PFGE type JEGX01.0004 to identify the genes that might be involved in SE colonization and/or pathogenesis.
    [Show full text]
  • Fucosyltransferase 8 As a Functional Regulator of Nonsmall Cell Lung Cancer
    Fucosyltransferase 8 as a functional regulator of nonsmall cell lung cancer Chien-Yu Chena,b, Yi-Hua Janb, Yi-Hsiu Juanc, Chih-Jen Yangd, Ming-Shyan Huangd, Chong-Jen Yuc, Pan-Chyr Yangc, Michael Hsiaob, Tsui-Ling Hsub,1, and Chi-Huey Wongb,1 aInstitute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan; bGenomics Research Center, Academia Sinica, Taipei 115, Taiwan; cDepartment of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan; and dDepartment of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan Contributed by Chi-Huey Wong, November 26, 2012 (sent for review August 17, 2012) The up-regulation of fucosyltransferase 8 (FUT8), the only enzyme downstream signaling. Furthermore, the increase in core fuco- catalyzing α1,6-fucosylation in mammals, has been observed in sylation on E-cadherin has been shown to strengthen cell–cell several malignant cancers including liver, ovarian, thyroid, and co- adhesion (10). lorectal cancers. However, the pathological role and the regulatory Both transgenic and knockout mouse models have been gen- mechanism of FUT8 in cancers remain largely unknown. In the cur- erated to study the physiological role of FUT8 (7, 11, 12). Ec- rent study, we report that the expression of FUT8 is up-regulated in topic expression of FUT8 in mice results in an accumulation of nonsmall cell lung cancer (NSCLC) and correlates with tumor me- lipid droplets in hepatocytes and proximal renal tubular cells. tastasis, disease recurrence, and poor survival in patients with This steatosis-like phenotype observed in transgenic mice is NSCLC. Knocking down FUT8 in aggressive lung cancer cell lines linked to the activity of liver lysosomal acid lipase, which becomes significantly inhibits their malignant behaviors including in vitro inactive when over core-fucosylated (11), suggesting that excess invasion and cell proliferation, as well as in vivo metastasis and core fucosylation may lead to a breakdown of normal lipid me- tumor growth.
    [Show full text]
  • Estudi Cinètic De L'activitat Enzimàtica De GT-MG517
    PART EXPERIMENTAL - Materials i mètodes – - 229 - - 230 - - 231 - - 232 - Capítol 8: Protocols de biologia molecular - 233 - - 234 - Capítol 8: Protocols de biologia molecular 8 Protocols de biologia molecular 8.1 Capítol 2: Estudi de l’essencialitat de les possibles glicosiltransferases de Mycoplasma genitalium 8.1.1 Preparació de medi SP4 La preparació de medi SP4, medi complex que s’usa per al creixement de Mycoplasma genitalium, té lloc com s’explica a continuació. A la Taula 8.1 se’n presenten els components i els volums corresponents per a la preparació de 500 mL de medi líquid. Taula 8.1. Composició i preparació del medi SP4. Quantitat per Compost a 500 mL Preparació de la base PPLO (Difco) 1.75 g Triptona (Difco) 5 g Bactopeptona (Difco) 2.65 g Glucosa (Sigma-Aldrich) 2.5 g Aigua destil·lada Fins a 312 mL Preparació dels complements Yestolate 2 % autoclavat (Difco) 50 mL Roig de fenol 0.1 % pH 7 autoclavat 7 mL Extracte de llevat fresc 25 % 17.5 mL CMRL 10x (Invitrogen) 25 mL Sèrum boví fetal (Invitrogen) 85 mL Glutamina 29.2 mg/mL 1.71 mL Inicialment es mesclen tots els components corresponents a la base del medi i s’ajusta el pH a 7.8 amb NaOH 1 M. El conjunt s’autoclava 15 min a 120 ºC i es deixa refredar abans d’afegir-hi els complements. Un cop aquests s’han addicionat, cal comprovar que el pH del medi es manté entre 7.6 i 7.8. La preparació de l’extracte de llevat té lloc a partir de la dilució de 250 g de llevat fresc en 1 L d’aigua destil·lada.
    [Show full text]
  • Sugar Nucleotides Sugar Nucleotides Are Known As Activated Sugars and Are Composed of a Nucleoside and a Monosaccharide Residue Linked by a Pyrophosphate Moiety
    Sugar Nucleotides Sugar nucleotides are known as activated sugars and are composed of a nucleoside and a monosaccharide residue linked by a pyrophosphate moiety. They are key substrates for the synthesis of complex polysaccharides and the glycosylation of peptides and lipids. The enzymatic formation of glycosidic linkages using Leloir glycosyltransferases allows quick access to complex carbohydrates and forms an attractive alternative to synthetic methods. These enzymes are regio- and stereoselective and do not require cumbersome protecting group strategies. GDP-β-L-fucose α-(1,3)-fucosyltransferase V LacNAc Sialyl Lewis X trisaccharide GDP We offer a wide range of sugar nucleotides as useful building blocks for the preparation of biologically relevant oligosaccharides and glycoconjugates. Also, a range of modified sugar nucleotides is available for the study of biosynthetic pathways, such as Lipid A or (Sia)LeX biosynthesis. Code Product Activity Quantity Price ($) MG31129 GDP-D-Glc Substrate for glucosyltransferase 1 mg 50.00 MU08960 UDP-D-Glc disodium salt Substrate for β-glucosyltransferase 500 mg 60.00 MG01912 GDP-β-L-Fuc Substrate for fucosyltransferase 1 mg 85.00 MG05610 GDP-D-Man disodium salt Substrate for mannosyltransferase 5 mg 75.00 MU07658 UDP-α-D-Xyl Substrate for xylosyltransferase 1 mg 150.00 MU07955 UDP-GlcNAc disodium salt (other salts available) Substrate for acetylglucosaminyltransferase 25 mg 75.00 MU06699 UDP-α-D-Gal disodium salt (other salts available) Substrate for galactosyltransferase 10 mg 68.30 MU04515 UDP-GalNAc
    [Show full text]
  • Anchored Cellulase from Poplar
    Functional studies of a membrane- anchored cellulase from poplar Ulla Jonsson Rudsander Royal Institute of Technology School of Biotechnology Department of Wood Biotechnology Stockholm, 2007 Copyright © Ulla J. Rudsander Stockholm, 2007 ISBN 978-91-7178-790-3 Royal Institute of Technology AlbaNova University Center School of Biotechnology SE – 106 91 Stockholm Sweden Printed at Universitetsservice US-AB Box 700 14 100 44 Stockholm Sweden Cover illustration: 2 weeks old Arabidopsis thaliana seedlings. From the left: wildtype, korrigan1-1 mutant, korrigan1-1 complemented with construct pUR19, korrigan1-1 complemented with construct pUR20, korrigan1-1 complemented with construct pUR21, see section 8 (courtesy of Junko Takahashi). Ulla J. Rudsander (2007). Functional studies of a membrane-anchored cellulase from poplar. Doctoral thesis in Wood Biotechnology. School of Biotechnology, Department of Wood Biotechnology, Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden. ISBN 978-91-7178-790-3 ABSTRACT Cellulose in particular and wood in general are valuable biomaterials for humanity, and cellulose is now also in the spotlight as a starting material for the production of biofuel. Understanding the processes of wood formation and cellulose biosynthesis could therefore be rewarding, and genomics and proteomics approaches have been initiated to learn more about wood biology. For example, the genome of the tree Populus trichocarpa has been completed during 2006. A single-gene approach then has to follow, to elucidate specific patterns and enzymatic details. This thesis depicts how a gene encoding a membrane-anchored cellulase was isolated from Populus tremula x tremuloides Mich, how the corresponding protein was expressed in heterologous hosts, purified and characterized by substrate analysis using different techniques.
    [Show full text]
  • Advances in Understanding Glycosyltransferases from A
    Available online at www.sciencedirect.com ScienceDirect Advances in understanding glycosyltransferases from a structural perspective Tracey M Gloster Glycosyltransferases (GTs), the enzymes that catalyse commonly activated nucleotide sugars, but can also be glycosidic bond formation, create a diverse range of lipid phosphates and unsubstituted phosphate. saccharides and glycoconjugates in nature. Understanding GTs at the molecular level, through structural and kinetic GTs have been classified by sequence homology into studies, is important for gaining insights into their function. In 96 families in the Carbohydrate Active enZyme data- addition, this understanding can help identify those enzymes base (CAZy) [1 ]. The CAZy database provides a highly which are involved in diseases, or that could be engineered to powerful predictive tool, as the structural fold and synthesize biologically or medically relevant molecules. This mechanism of action are invariant in most of the review describes how structural data, obtained in the last 3–4 families. Therefore, where the structure and mechanism years, have contributed to our understanding of the of a GT member for a given family has been reported, mechanisms of action and specificity of GTs. Particular some assumptions about other members of the family highlights include the structure of a bacterial can be made. Substrate specificity, however, is more oligosaccharyltransferase, which provides insights into difficult to predict, and requires experimental charac- N-linked glycosylation, the structure of the human O-GlcNAc terization of individual GTs. Determining both the transferase, and the structure of a bacterial integral membrane sugar donor and acceptor for a GT of unknown function protein complex that catalyses the synthesis of cellulose, the can be challenging, and is one of the reasons there are most abundant organic molecule in the biosphere.
    [Show full text]
  • GDP-Glo™ Glycosyltransferase Assay Instructions for Use of Products VA1090, VA1091 and VA1092
    TECHNICAL MANUAL GDP-Glo™ Glycosyltransferase Assay Instructions for Use of Products VA1090, VA1091 and VA1092 10/17 TM505 GDP-Glo™ Glycosyltransferase Assay All technical literature is available at: www.promega.com/protocols/ Visit the web site to verify that you are using the most current version of this Technical Manual. E-mail Promega Technical Services if you have questions on use of this system: [email protected] 1. Description .........................................................................................................................................1 2. Product Components and Storage Conditions ........................................................................................6 3. Preparing for the GDP-Glo™ Glycosyltransferase Assay .........................................................................9 3.A. Preparing the GDP Detection Reagent ..........................................................................................9 3.B. Generating a Standard Curve for GDP ........................................................................................ 10 4. GDP-Glo™ Glycosyltransferase Assay Protocols ................................................................................... 11 4.A. GDP-Glo™ Glycosyltransferase Assay Protocol ............................................................................ 11 4.B. Optimizing Glycosyltransferase Reaction Conditions ................................................................... 12 4.C. Determining Km Value for FUT7 Substrate GDP-Fucose ..............................................................
    [Show full text]
  • Ep 1 117 822 B1
    Europäisches Patentamt (19) European Patent Office & Office européen des brevets (11) EP 1 117 822 B1 (12) EUROPÄISCHE PATENTSCHRIFT (45) Veröffentlichungstag und Bekanntmachung des (51) Int Cl.: Hinweises auf die Patenterteilung: C12P 19/18 (2006.01) C12N 9/10 (2006.01) 03.05.2006 Patentblatt 2006/18 C12N 15/54 (2006.01) C08B 30/00 (2006.01) A61K 47/36 (2006.01) (21) Anmeldenummer: 99950660.3 (86) Internationale Anmeldenummer: (22) Anmeldetag: 07.10.1999 PCT/EP1999/007518 (87) Internationale Veröffentlichungsnummer: WO 2000/022155 (20.04.2000 Gazette 2000/16) (54) HERSTELLUNG VON POLYGLUCANEN DURCH AMYLOSUCRASE IN GEGENWART EINER TRANSFERASE PREPARATION OF POLYGLUCANS BY AMYLOSUCRASE IN THE PRESENCE OF A TRANSFERASE PREPARATION DES POLYGLUCANES PAR AMYLOSUCRASE EN PRESENCE D’UNE TRANSFERASE (84) Benannte Vertragsstaaten: (56) Entgegenhaltungen: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU WO-A-00/14249 WO-A-00/22140 MC NL PT SE WO-A-95/31553 (30) Priorität: 09.10.1998 DE 19846492 • OKADA, GENTARO ET AL: "New studies on amylosucrase, a bacterial.alpha.-D-glucosylase (43) Veröffentlichungstag der Anmeldung: that directly converts sucrose to a glycogen- 25.07.2001 Patentblatt 2001/30 like.alpha.-glucan" J. BIOL. CHEM. (1974), 249(1), 126-35, XP000867741 (73) Patentinhaber: Südzucker AG Mannheim/ • BUTTCHER, VOLKER ET AL: "Cloning and Ochsenfurt characterization of the gene for amylosucrase 68165 Mannheim (DE) from Neisseria polysaccharea: production of a linear.alpha.-1,4-glucan" J. BACTERIOL. (1997), (72) Erfinder: 179(10), 3324-3330, XP002129879 • GALLERT, Karl-Christian • DE MONTALK, G. POTOCKI ET AL: "Sequence D-61184 Karben (DE) analysis of the gene encoding amylosucrase • BENGS, Holger from Neisseria polysaccharea and D-60598 Frankfurt am Main (DE) characterization of the recombinant enzyme" J.
    [Show full text]
  • Analyzing the Complex Machinery of Cell Wall Biosynthesis
    Analyzing the complex machinery of cell wall biosynthesis Jaap Timmers Thesis committee Thesis supervisor Prof. dr. R.G.F. Visser Professor of Plant Breeding Wageningen University Thesis co-supervisor Dr.ir. L.M. Trindade Assistant professor at the Laboratory of Plant Breeding Wageningen University Dr.ir. J.-P. Vincken Assistant professor at the Laboratory of Food Chemistry Wageningen University Other members Prof.dr.ir. A.G.J. Voragen Wageningen University Dr. H. Höfte INRA Centre de Versailles, France Dr.ir. G.H. Immink Plant Research International Prof.dr. M.E. Janson Wageningen University This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences. Analyzing the complex machinery of cell wall biosynthesis Jaap Timmers Thesis submitted in partial fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof.dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Doctorate Board to be defended in public on Friday 4 December 2009 at 11 AM in the Aula. Timmers, J.F.P Analyzing the complex machinery of cell wall biosynthesis Ph.D. thesis Wageningen University, the Netherlands, 2009 with summary in Dutch ISBN 978-90-8585-516-3 Contents Chapter 1 7 General introduction Chapter 2 21 Implementation and optimization of a system to characterize protein complexes involved in plant cell wall biosynthesis Chapter 3 35 Interactions between membrane bound cellulose synthases involved in the synthesis of the secondary cell
    [Show full text]
  • Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation
    molecules Review Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation Birgitte Zeuner , David Teze , Jan Muschiol and Anne S. Meyer * Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Kgs Lyngby, Denmark; [email protected] (B.Z.); [email protected] (D.T.); [email protected] (J.M.) * Correspondence: [email protected]; Tel.: +45-45252600 Academic Editor: Ramón J. Estévez Cabanas Received: 30 April 2019; Accepted: 26 May 2019; Published: 28 May 2019 Abstract: Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite 1 mutations, to unique and novel transplantations of designed − peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite 1 modification, acceptor binding site matching, and loop engineering, − including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.
    [Show full text]
  • Function Study of Arabidopsis Thaliana Core Alpha1,3-Fucosyltransferase (Fucta) Peter Both
    Structure – function study of Arabidopsis thaliana core alpha1,3-fucosyltransferase (FucTA) Peter Both To cite this version: Peter Both. Structure – function study of Arabidopsis thaliana core alpha1,3-fucosyltransferase (FucTA). Biomolecules [q-bio.BM]. Université Joseph-Fourier - Grenoble I, 2009. English. tel- 00449431 HAL Id: tel-00449431 https://tel.archives-ouvertes.fr/tel-00449431 Submitted on 21 Jan 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITE JOSEPH FOURIER (GRENOBLE I ) ECOLE DOCTORALE CHIMIE ET SCIENCES DU VIVANT UNIVERSITE SLOVAQUE DE TECHNOLOGIE DE BRATISLAVA (STUBA) THESE Pour l’obtention du Diplôme de DOCTEUR DE L’UNIVERSITE JOSEPH FOURIER Discipline: Biologie Présentée et soutenue publiquement le 29 octobre 2009 par PETER BOTH ETUDE STRUCTURE-FUNCTION D’UNE FUCOSYLTRANSFERASE (FUCT-A) DE ARABIDOPSIS THALIANA JURY Rapporteurs : Prof. Abderrahman MAFTAH, Université de Limoges Dr Eva HOSTINOVA, Slovak Academy of Sciences, Bratislava Examinateurs : Dr Eva KUTEJOVA, Slovak Academy of Sciences, Bratislava Dr Serge PEREZ, ESRF, Grenoble Dr Jan MUCHA, Slovak Academy of Sciences, Bratislava Prof. Christelle BRETON, Université Grenoble I Thèse préparée au CERMAV (Grenoble) et ICHSAS (Bratislava) 1 I would like to express my gratitude to all those who gave me the possibility to complete this thesis.
    [Show full text]
  • A Cloned Human Cdna Determines Expression of a Mouse Stage-Specific Embryonic Antigen and the Lewis Blood Group (1,3/1,4)-Fucosyltransferase
    Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group (1,3/1,4)-fucosyltransferase Jolanta F. Kukowska-Latallo, ~ Robert D. Larsen, ~ Rajan P. Nair, ~ and John B. Lowe ~,2,s tHoward Hughes Medical Institute and the Department of Pathology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0650 USA The stage-specific embryonic antigen SSEA-I is a cell-surface oligosaccharide molecule expressed with temporal precision during the murine preimplantation period and implicated in adhesive events involving the process of compaction. We used a mammalian transient expression system to isolate a cloned human cDNA that determines expression of the SSEA-I molecule. The cDNA sequence predicts a type II transmembrane protein with a domain structure similar to mammalian glycosyltransferases, but without primary sequence similarity to these enzymes. The carboxy-terminal domain of this protein was shown to be catalytically active as a fucosyltransferase when expressed in COS-I cells as a portion of a secreted protein A fusion peptide. The enzyme is an exceptional glycosyltransferase in that it can use both type I and type II oligosaccharides as acceptor substrates to generate subterminal Fucc~(l,4)- and Fuc~(l,3)-linkages, respectively, in a manner analogous to the human Lewis blood group fucosyltransferase. Southern blot analysis shows that the cDNA corresponds to sequences syntenic to the Lewis locus on chromosome 19. These results indicate that this cDNA is the product of the human Lewis blood group locus, provide genetic confirmation of the hypothesis that this enzyme can catalyze two distinct transglycosylation reactions, and outline an approach to the isolation of other sequences that determine expression of developmentally regulated oligosaccharide antigens.
    [Show full text]