dc.contributor.author
Martínez Font, Jacobo
dc.date.accessioned
2018-06-08T01:28:41Z
dc.date.available
2011-12-01T12:21:21.607Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/13408
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-17606
dc.description
INDEX Abbreviations III Acknowledgments V 1\. INTRODUCTION 1 1.1.
Contextualization of glycan and sialic acids 1 1.2. Sialic acids history and
definition 2 1.3. Differential features of the sialic acid family 3 1.4.
Sialic acids diversity 4 1.5. Sias general biological functions 5 1.6. Sias
recognition/antirecognition duality function 6 1.7. The biosynthetic pathway
of Sias in vertebrates 10 1.8. The UDP-GlcNAc-2-Epimerase/ManNAc Kinase
(GNE/MNK or GNE) 12 1.9. Alternative roles of GNE 14 1.10. GNE related
diseases 15 1.11. Previous available structural information on hGNE 16 1.12.
The MNK domain of GNE belongs to the ROK family 17 1.13. Starting point and
aims of this thesis 18 2\. MATERIALS 19 2.1. Instrumentation 19 2.2.
Crystallization 20 2.3. Chemicals buffers and media 21 2.4. Plasmids and
bacterial strains 22 2.5. Primers 22 2.6. Enzymes, kits and ladders 23 3\.
METHODS 24 3.1. Cloning 24 3.1.1. Polymerase chain reaction (PCR) 24 3.1.2.
Agarose gel DNA extraction 25 3.1.3. DNA digestion 25 3.1.4. DNA ligation 25
3.1.5. Preparation of chemically competent cells 25 3.1.6. Transformation
(Heat shock) 26 3.1.7. Colony PCR 26 3.1.8. Plasmid preparation 26 3.1.9. DNA
Sequencing 27 3.1.10. Clone and plasmid storage 27 3.1.11. In vitro DNA-
Mutagenesis 27 3.2. Protein expression and purification 28 3.2.1. Expression
test (small scale expression) 28 3.2.2. Pre-culture 28 3.2.3. Protein
overexpression 28 3.2.4. Cell disruption 29 3.2.5. Ultracentrifugation 29
3.2.6. Chromatographic purification 29 3.2.6.1. Ni-NTA affinity chromatography
29 3.2.6.2. Size exclusion chromatography 30 3.2.7. hMNK digestion with TEV or
Thrombin 30 3.3. General biochemical methods 30 3.3.1. Agarose gel
electrophoresis of DNA 30 3.3.2. Discontinuous denaturing sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of Proteins 31 3.3.3.
DNA concentration determination 32 3.3.4. Protein concentration determination
32 3.3.4.1. UV-absorption 32 3.3.4.2. Bradford test 32 3.4. Protein
characterization 33 3.4.1. Kinase activity test 33 3.4.2. Circular Dichroism
Spectroscopy (CD-Spectroscopy) 34 3.4.3. Isothermal Titration calorimetry
(ITC) 34 3.5. Crystallographic methods 35 3.5.1. Crystallization 35 3.5.2.
Soaking experiments 35 3.5.3. Data collection and processing 36 3.5.4. Solving
the phase problem 36 3.5.5. Model refinement and validation 36 3.5.6.
Structural comparison and picture preparation 37 3.6. Preparation of
6-O-Acetyl ManNAc 37 4\. RESULTS 38 4.1. Cloning and protein purification 38
4.2. Protein Characterization 40 4.2.1. Circular dichroism spectroscopy 40
4.2.2. Isothermal titration calorimetry (ITC) 42 4.2.3. Enzymatic activity and
specificity. 43 4.3. Crystal structure of the complex hMNK-ManNAc 44 4.3.1.
Crystal reproduction and soaking 44 4.3.2. Co-crystallization of the complex
hMNK-ManNAc 46 4.3.3. X-ray data collection and processing 46 4.3.4.
Determination of the hMNK-ManNAc structure 47 4.4. The overall structure of
the hMNK-ManNAc monomer 49 4.5. Multimeric state 49 4.6. Sequential and
structural homology search and family classification 51 4.7. hMNK requires
Zinc binding for structural stability and activity 54 4.8. The ManNAc binding
site 55 4.9. Domain movements upon ManNAc binding 57 4.10. Crystal structure
of the ternary complexes hMNK/ManNAc/ADP, hMNK/ManNAc-6P/ADP and
hMNK/ManNAc/AMPPCP 58 4.10.1. Soaking experiments 58 4.10.2. Structure
determination 58 4.11. The nucleotide binding site 60 4.12. The
MNK/ManNAc-6-P/ADP structure 62 4.13. The MNK/ManNAc/AMPPCP complex 65 4.14.
Surface representation and electrostatic potential 67 4.15. hMNK inhibition 69
5\. DISCUSSION 70 5.1. The catalytic mechanism 71 5.1.1. General
considerations (based on [117]) 71 5.1.2. Proposed catalytic mechamism for the
ManNAc kinase 74 5.1.2.1. D517 has a catalytic role in hMNK 74 5.1.2.2. The
phosphorylation of ManNAc proceeds by an associative SN2-like mechanism 75
5.1.2.3. hMNK is a magnesium dependent kinase 78 5.1.2.4. hMNK needs a
conformational rearrangement upon ManNAc binding for catalysis 79 5.2. hMNK
specificity 80 5.3. Structural Mapping of HIBM related mutations 82 5.4. The
search for an hMNK inhibitor 83 6\. SUMMARY 85 6\. ZUSAMMENFASSUNG 86 7\.
BIBLIOGRAPHY 87 Curriculum vitae 95
dc.description.abstract
Sialic acids are essential components of membrane glycoconjugates. They are
responsible for the interaction, structure and functionality of all
deuterostome cells and have major functions in cellular processes and
diseases. The key enzyme of the biosynthesis of sialic acid is the
bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase
(GNE) that transforms UDP-N-acetylglucosamine to
N-acetylmannosamine-6-phosphate and has a direct impact on the sialylation of
the cell surface. In this thesis three high resolution crystal structures of
the recombinant human acetylmannosamine kinase domain (hMNK) from GNE are
presented. hMNK converts N-acetylmannosamine to
N-acetylmannosamine-6-phosphate, a precursor of sialic acid, and has a direct
impact on the sialylation of the cell surface, which is of medical interest
because sialylation of tumor cell surface glycans is at a higher level than
that of healthy cells. The complexes formed by hMNK with its substrate
N-acetylmannosamine (ManNAc), with ManNAc and ADP, and with ManNAc-6-phosphate
and ADP, show structural details of reaction educt and product. hMNK belongs
to the ROK family of proteins, and this study presents the first human member
of this family in complex with its cognate substrate. The interactions between
hMNK and ManNAc explain the high substrate specificity of hMNK and suggest a
key active site residue, Asp517, that was mutated and shown to be crucial for
enzymatic activity. Since the structure of the complex formed between hMNK and
ManNAc also indicated the structural properties of a possible inhibitor, we
synthesized ManNAc-6-O-acetate that proved to be a modest inhibitor and may be
taken as lead for more potent inhibitors that could open novel ways for sialic
acid research, glycan bioengineering and cancer therapy.
de
dc.description.abstract
Sialinsäuren (Sias) sind essentielle Komponenten der Membran-Glycokonjugate.
Sias sind zuständig für die Interaktion, Struktur und Funktionalität aller
Deuterostomia-Zellen und spielen eine wichtige Rolle bei zahlreichen
zellulären Prozessen und Krankheiten. Das Schlüsselenzym der
Sialinsäurebiosynthese ist die bifunktionelle
UDP-N-Acetylglukosamin-2-Epimerase/N-Acetylmannosamin Kinase (GNE), die
UDP-N-Acetylglucosamin in N-Acetylmannosamin-6-phosphate umwandelt und einen
direkten Impakt auf der Sialylierung der Zelloberfläche hat. Dies ist von
medizinischem Interesse, denn Tumorzellen weisen eine höhere Sialylierung der
Zelloberflächeglykane im Vergleich zu gesunden Zellen auf. In der vorliegenden
Dissertation, werden drei hochaufgelöste Kristallstrukturen der rekombinanten
menschlichen N-acetyl Mannosamine Kinase Domäne (hMNK) von GNE präsentiert.
hMNK wandelt N-Acetylmannosamin in N-Acetylmannosamin-6-Phosphat (eine
Vorläufersubstanz von Sias) um. Die Komplexe zwischen hMNK und ihrem
natürlichen Substrate N-Acetylmannosamin (ManNAc), zwischen hMNK, ManNAc und
ADP und zwischen hMNK, ManNAc-6-Phosphat and ADP zeigen strukturelle Details
von Reaktions-edukt und –produkt. hMNK gehört zur ROK Proteinfamilie und diese
Arbeit präsentiert die erste Struktur eines menschlichen Proteins dieser
Familie im Komplex mit seinem Substrat. Die Interaktionen zwischen hMNK und
ManNAc erklären die hohe Substratspezifität von hMNK und schlagen einen
aktiven Aminosäuren Rest vor, Asp517, der mutiert wurde und eine entscheidende
Rolle für die Katalyse zeigte. Da die Struktur des Komplexes hMNK/ManNAc die
strukturellen Eigenschaften von einem möglichen Inhibitor vorgeschlagen hatte,
wurde ManNAc-6-O-Acetat synthetisiert und zeigte eine schwache Inhibition.
Diese Verbindung kann daher genommen werden als Startpunkt für die Herstellung
von neuen und besseren hMNK- Inhibitoren, die guten Alternativen, sowohl für
Sialinsäure Forschung, als auch für Glykan Bioengineering und die Krebs
Therapie bieten sollten.
de
dc.format.extent
IX, 95 S.
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
N-acetyl mannosamine kinase
dc.subject
Crystal structure
dc.subject.ddc
500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::572 Biochemie
dc.title
Structural and functional studies concerning human N-acetyl mannosamine kinase
dc.contributor.contact
jacobomf@zedat.fu-berlin.de
dc.contributor.inspector
Prof. Christoph Schalley
dc.contributor.inspector
Prof. Ulrich Abram
dc.contributor.firstReferee
Prof. Wolfram Saenger
dc.contributor.furtherReferee
Prof. Udo Heinemann
dc.date.accepted
2011-11-10
dc.identifier.urn
urn:nbn:de:kobv:188-fudissthesis000000034692-8
dc.title.translated
Struktur- und Funktionsuntersuchungen zur humanen N-Acetylmannosaminkinase
de
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000034692
refubium.mycore.derivateId
FUDISS_derivate_000000010311
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access
