Vertebrate polysialyltransferases [Elektronische Ressource] : a study on structure-function relationships / von Almut Christine Günzel

Almut Christine Guenzel

Sujets

• Biologie

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2008
• Nombre de lectures: 67
• Poids de l'ouvrage: 3 Mo

Extrait

Vertebrate Polysialyltransferases:
A Study on Structure-Function Relationships



Von der Naturwissenschaftlichen Fakultät der
Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades
Doktorin der Naturwissenschaften
Dr. rer. nat.



genehmigte Dissertation







von
Dipl.-Biochem. Almut Christine Günzel
geboren am 30. November 1979 in Gifhorn


2008

Referentin: Prof. Dr. Rita Gerardy-Schahn
Korreferent: Prof. Dr. Walter Müller
Tag der Promotion: Montag, 08.12.2008


















Schlagworte: Polysialyltransferasen, Polysialinsäure, Glykosyltransferase

Key words: polysialyltransferases, polysialic acid, glycosyltransferase






Erklärung zur Dissertation

Hierdurch erkläre ich, dass die Dissertation „Vertebrate Polysialyltransferases: A Study of
Structure-Function Relationships“ selbstständig verfasst und alle benutzten Hilfsmittel sowie
evtl. zur Hilfeleistung herangezogene Institutionen vollständig angegeben wurden.

Die Dissertation wurde nicht schon als Diplom- oder ähnliche Prüfungsarbeit verwendet.






















Table of Contents

Table of Contents
Zusammenfassung....................................................................................................................1
Abstract.................................................................................................................................... 2
General Introduction...............5
Polysialic acid ........................................................................................................................5
Function of polySia in vertebrates.........................6
Biosynthesis of polySia........................................................................................................10
Polysialyltransferases...........11
Objectives................................15
Chapter 1: Characterization of the eukaryotic polysialyltransferase ST8SiaII
from zebrafish .....................................................................................................16
1.1. Introduction.................16
1.2. Experimental Procedures...........................................................................................19
1.2.1. Materials.................................................19
1.2.2. Expression Plasmids..............................19
1.2.3. Site-directed mutagenesis of ST8SiaII cDNA .......................................................20
1.2.4. Cultivation of CHO cells ................................21
1.2.5. Transfection of CHO cells.....................21
1.2.6. Cultivation of Sf9 cells...........................................................22
1.2.7. In vitro assay for Auto- and NCAM polysialylation.............22
1.2.8. Immunofluorescence of zST8SiaII variants in CHO 2A10 cells ...........................23
1.2.9. zST8SiaII expression and purification from insect cells .......................................23
1.2.10. Size-exclusion chromatography...........................................25
1.2.11. SDS-PAGE analysis and immunoblotting ...........................................................26
1.2.12. Silver staining ......................................................................26
1.2.13. Soluble radioactive activity assay for polyST.....................27
1.2.14. Enzyme-linked immunosorbent assay for polysialyltransferase activity.............27
1.2.15. Nuclear magnetic resonance (NMR) spectroscopy..............................................28
1.2.16. Saturation-transfer-difference-(STD)-NMR spectroscopy..28
11.2.17. H-NMR spectroscopy based enzyme assay........................................................28
1.2.18. Crystallization trials carried out with ST8SiaII...................29



I Table of Contents


1.3. Results ..........................................................................................................................29
1.3.1. Comparison of zebrafish and murine ST8SiaII on primary sequence level ..........29
1.3.2. N-terminal truncation of zebrafish ST8SiaII.........................31
1.3.3. N-glycosylation site N95 is important for polysialylation activity........................32
1.3.4. Enzymatic activity of zST8SiaII variants in vivo..................................................34
1.3.5. Baculoviral expression of secreted zST8SiaII.......................36
1.3.6. Purification of Histidine-tagged zST8SiaII from insect cells ................................37
1.3.7. Insect cell expressed zST8SiaII?N86 is a monomeric protein and displays
enzymatic activity..................................................................39
1.3.8. Adaptation of assay condition for NMR-spectroscopy studies..............................40
1.3.9. Ligand binding analyses of zST8SiaII by STD-NMR spectroscopy.....................42
11.3.10. Activity studies for zST8SiaII?N86 by H-.........................46
1.3.11. Crystallization attempts of zST8SiaII?N86 ........................................................50
1.4. Discussion.....................................................................................51
Chapter 2: Engineering of murine ST8SiaII to a non N-glycosylated polysialyl-
transferase............................................................................................................56
2.1. Introduction.................56
2.2. Experimental Procedures...........................................................................................58
2.2.1. Materials.................................................58
2.2.2. Sequence analyses of polySTs ...............................................................................58
2.2.3. Site-directed mutagenesis of mST8SiaII ...............................................................58
2.2.4. Expression plasmids................................59
-2.2.5. Cultivation of LMTK and CHO cells...................................60
2.2.6. Cultivation of Sf9 cells...........................................................60
-2.2.7. Transfection of LMTK and CHO cells.61
-2.2.8. Immunofluorescence of ST8SiaII variants in LMTK cells...................................61
3 52.2.9. Recombinant expression of ST8SiaII Q AS EE in bacteria..61
3 52.2.10. NusA-Strep-purification of ST8SiaII Q AS EE ..................................................63
3 52.2.11. Maltose-Binding-Protein (MBP)-purification of ST8SiaII Q AS EE .................63
2.2.12. Size-exclusion chromatography...........................................63
3 52.2.13. mST8SiaII S AS EE expression and purification from insect cells ....................63
2.2.14. Enzyme linked immunosorbent assay for polyST activity..................................65
2.2.15. In vitro assay for NCAM polysialylation.............................................................66
II Table of Contents


2.2.16. SDS-PAGE analysis and immunoblotting ...........................................................67
2.2.17. Silver staining /coomassie staining......................................68
2.3. Results ..........................................................................................69
2.3.1. Phylogenetic display of the N-glycosylation patterns in the polysialyltransferase
ST8SiaII.................................................................................................................69
2.3.2. Engineering of minimally glycosylated mST8SiaII..............71
3 52.3.3. Recombinant expression of mST8SiaII Q AS EE in bacteria...............................74
3 52.3.4. Purification of ST8SiaII Q AS EE fusion proteins from bacteria .........................75
3 52.3.5. Size-exclusion chromatography of bacterial expressed MBP-ST8SiaII Q AS EE77
3 52.3.6. Recombinant Expression of N-glycan free mST8SiaII S AS EE in Sf9 cells .......78
3 52.3.7. Purification of histidine tagged mST8SiaII S AS EE expressed in Sf9 cells ........78
2.3.8. PolySia staining of glycosylation depleted mST8SiaII?31 variants in vitro.........79
2.4. Discussion.....................................................................................................................81
General Discussion.................86
References...............................................................................................................................90
Appendix 1 – Abbreviations..................................98 2 – curiculum vitae and publications100



III Zusammenfassung

Zusammenfassung

Polysialinsäure (PolySia) ist ein lineares Homopolysachcharid aus a2,8-verknüpften
Sialinsäureeinheiten. Als dynamisch regulierte posttranslationale Modifikation des Neuralen
Zelladhäsionsmoleküls NCAM übernimmt die PolySia eine wichtige Rolle in der Regul