dc.contributor.author
Hu, Jing
dc.date.accessioned
2018-06-08T00:13:46Z
dc.date.available
2011-03-31T08:52:08.263Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/11656
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-15854
dc.description
Contents i 1 Introduction 1 1.1 GABA and GABA transmission 1 1.2 GABA and
Disease 3 1.3 The Family of Na+/Cl- GABA Tansporters (GATs) 3 1.3.1 History
and characterization of GATs 4 1.3.2 Localization of GATs 5 1.3.3 GATs
pharmacology 8 1.3.3.1 Pharmacological properties of GATs 8 1.3.3.2 Inhibitors
of GATs 9 1.4 GAT-1 12 1.4.1 Mechanism 12 1.4.2 Topology 15 1.4.3 Structure
and Function 17 1.4.3.1 Neurotransmitters binding site 18 1.4.3.2 Sodium
binding site 20 1.4.3.3 Chloride binding site 22 1.4.4 Conformational changes
23 1.4.5 N-Glycosylation of GAT1 27 1.5 Sialic acids 28 1.5.1 Structure and
occurrence of sialic acids 28 1.5.2 Biological Functions of sialic acids 29
1.5.2.1 Adhesion and cell-cell-interaction 29 1.5.2.2 Sialic acids as
recognition determinants for pathogens 31 1.5.2.3 Masking of antigenic
determinants by sialic acids 32 1.5.2.4 Influence of sialic acids on structure
and function of glycoconjugates and their carrier 33 1.5.2.5 Sialic acids and
carcinoma 33 2 Aim of this work 35 3 Results 37 3.1 The role of terminal
sialic acid of GAT1 in GABA uptake activity 37 3.1.1 The expression and
characterization of GAT1/GFP in CHO, CHO Lec3 and Hek293 cells 37 3.1.2 Effect
of deficiency of terminal sialic acid on GABA uptake activity of GAT1/GFP 40
3.1.2.1 Determination of sialic acid concentration in CHO and CHO Lec3 cells
40 3.1.2.2 Quantification of the influence of deficiency of terminal sialic
acid on the GABA uptake activity of GAT1/GFP 41 3.1.2.3 Influence of ManNAc
and ManNProp on GABA uptake activity of GAT1 in CHO Lec3 cells 43 3.1.3 Effect
of removal of terminal sialic acid on GABA uptake activity of GAT1/GFP 44
3.1.3.1 Reduction of GABA transport activity of GAT1/GFP by sialidase
treatment 44 3.1.3.2 Quantification of influence of removal of terminal sialic
acid on GABA uptake activity of GAT1/GFP 45 3.1.4 Effect of oxidation of
terminal sialic acid on GABA uptake activity of GAT1/GFP 47 3.1.5 Kinetic
analysis of GABA uptake activity of GAT1/GFP protein 48 3.1.5.1 Deficiency,
removal or oxidation of terminal sialic acid did not change the Km GABA values
of GAT1 49 3.1.5.2 Deficiency and removal of terminal sialic acid increased
KmNa+ values of GAT1 51 3.1.5.3 Oxidation of terminal sialic acid did not
change the KmNa+ value of GAT1 53 3.2 Influence of natural occurring and
chemical synthetic compounds on GABA uptake activity of GAT1 54 3.2.1 Effect
of hexosamines on GABA uptake activity 54 3.2.2 Effect of natural occurring on
GABA uptake activity 59 3.3. Expression, characterization and purification of
GAT1/GFP fusion protein 62 3.3.1 Purification of GAT1/GFP fusion protein in
Hek293 cells 62 3.3.1.1 Isolation of GAT1/GFP fusion protein in Hek293 cells
by ion-exchange chromatography 62 3.3.1.2 Isolation of GAT1/GFP fusion protein
in Hek293 cells by lectin-affinity chromatography 63 3.3.1.3 Purification of
GAT1/GFP fusion protein in Hek293 cells by immunoaffinity chromatography and
size exclusion-fast liquid chromatography (SE-FPLC) 64 3.3.1.3.1 Purification
of anti-GFP antibody 64 3.3.1.3.2 Purification of GAT1/GFP fusion protein in
Hek293 cells by immunoaffinity chromatography and SE-FPLC 65 3.3.2 Expression,
characterization and purification of GAT1/GFP fusion protein with BAC-TO-
BAC®-Baculovirus system 68 3.3.2.1 Cloning, Preparation and analysis of
GAT1-recombinant baculovirus 68 3.3.2.2 Expression and characterization of
GAT1/GFP fusion protein in insect cells 68 3.3.2.3 Isolation of GAT1/GFP
fusion protein in insect cells by ion-exchange chromatography 72 3.3.2.4
Purification of GAT1/GFP fusion protein in insect cells by immunoaffinity
chromatography and SE-FPLC 74 3.3.2.5 Transmission electron microscopy (TEM)
analysis 78 4 Discussion 87 4.1 Role of terminal sialic acid in the GABA
uptake activity of GAT1 87 4.1.1 Terminal sialic acid is essential for GABA
uptake activity of GAT1 87 4.1.2 Terminal sialic acid affects kinetics of
transport cycle 91 4.1.3 Terminal sialic acid in altering access transport
model 93 4.2 Influence of synthetic N-acyl hexosamines on GABA uptake activity
of GAT1 94 4.3 Potent inhibitors on GABA uptake activity of GAT1 from
naturally occurring compounds 95 4.4 Expression, characterization and
purification of GAT1/GFP recombinant protein 96 5 Future perspectives 100 6
Summary 101 7 Zusammenfassung 103 8 Materials and methods 105 8.1 Materials
105 8.1.1 Chemicals 105 8.1.2 Cells and bacteria 105 8.1.3 Cell culture
materials and mediums 106 8.1.4 Vectors 107 8.1.5 Primers 107 8.1.6 Markers
and Enzymes 107 8.1.7 Antibodies 107 8.1.8 Lectins 108 8.1.9 Kits 108 8.1.10
Laboratory equipments and instruments 108 8.2 Methods 110 8.2.1 Molecular
biological methods 110 8.2.1.1 Generation of comptent E.coli cells 110 8.2.1.2
Transformation of E.coli competent cells 110 8.2.1.3 Preparation of plasmid-
DNA from E.coli 111 8.2.1.4 Determination of DNA concentration 112 8.2.1.5
Digestion with restriction endonucleases 112 8.2.1.6 Agarose gel
electrophoresis 113 8.2.1.7 DNA sequencing 113 8.2.1.8 Polymerase Chain
Reaction (PCR) 114 8.2.1.9 Expression of recombinant proteins in Baculovirus
system 115 8.2.1.9.1 Cultivation of insect cells 116 8.2.1.9.2 Generation of
recombinant Bacmid DNA 117 8.2.1.9.3 Analysis of Bacmid DNA 117 8.2.1.9.4
Generation of recombinant virus 117 8.2.1.9.5 Amplification of virus 118
8.2.1.9.6 Plaque Assay 118 8.2.1.9.7 Expression of recombinant protein in
insect cells 119 8.2.2 Cell biological methods 119 8.2.2.1 General cultivation
conditions 119 8.2.2.2 Stable transfection of GAT1/GFP in CHO and CHO Lec3
cells 120 8.2.2.3 Stable transfection of GAT1/GFP in Hek293 cells 120 8.2.2.4
Selection of stable transfected cells by cloning 121 8.2.2.5 FACS and
Fluorescence microscopy analysis 121 8.2.2.6 Cell counting 121 8.2.3 Protein
biochemical and immunological methods 121 8.2.3.1 Immunoprecipitation 121
8.2.3.2 Biotinylation 122 8.2.3.3 Sialidase treatment 122 8.2.3.4 Sodium
periodate treatment 123 8.2.3.5 Mild sodium borohydride treatment 123 8.2.3.6
Alamar Blue Assay 123 8.2.3.7 Determination of protein concentration 123
8.2.3.7.1 Bradford assay 123 8.2.3.7.2 BCA methods 124 8.2.3.8 Endoglycosidase
H treatment 124 8.2.3.9 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 125
8.2.3.10 Silver staining 126 8.2.3.11 Coomassie blue staining 127 8.2.3.12
Analysis and identification of protein by MALDI-TOF-MS 127 8.2.3.13 Western
blot 129 8.2.3.14 Glycan staining 130 8.2.3.15 GABA uptake assay 131 8.2.3.16
Sialic acid concentration assay 131 8.2.3.17 Cell surface sialic acid analysis
by flow cytometry 132 8.2.3.18 Membrane preparation and solubilization 132
8.2.3.19 Ion exchange chromatography 133 8.2.3.20 Lectin affinity
chromatography 134 8.2.3.21 Immuno-affinity chromatography 134 8.2.3.21.1
Purification of the GFP-specific antiserum 134 8.2.3.21.2 Preparation of
immunoaffinity column 135 8.2.3.21.3 Isolation of GAT1/GFP by immunoaffinity
column 135 8.2.3.22 Size exclusion chromatography 136 8.2.3.23 Transmission
electron microscopy (TEM) analysis 136 8.2.3.23.1 Negative staining
preparation 136 8.2.3.23.2 Cryo-TEM preparation 136 8.2.3.23.3 Cryo-TEM 137 9
References 138 Appendix 155 Abbreviations 155 Restriction Map and Multiple
Cloning Site (MCS) of pEGFP-N1 Vector 157 Restriction Map and Multiple Cloning
Site (MCS) of pFASTBACTM 1-Vector 158 Curriculum Vitae 159 Publications 160
dc.description.abstract
GABA (gamma-aminobutyric acid) is the major inhibitory neurotransmitter in the
central nervous system (CNS). GABA re-uptake by GABA transporters from the
synaptic cleft is one important mechanism in the regulation of GABA
concentration in the synaptic cleft. GABAergic dysfunction is involved in a
lot of diseases such as Parkinson’s disease, epilepsy, chorea Huntingtone and
schizophrenia. The GABA transporter 1 (GAT1) belongs to the family of Na+ and
Cl--coupled transport proteins and possesses 12 putative transmembrane domains
and three N-glycosylation sites in the extracellular loop between the
transmembrane domain 3 and 4. Previous work showed that N-glycosylation, but
not terminal trimming of the N-glycan is involved in the attainment of a
correctly folded and stable conformation of GAT1, which influences on the
protein stability and trafficking to the plasma membrane. It also demonstrated
that N-linked oligosaccharides side chains of GAT1, in particular their
terminal structures, are involved in the GABA transport process of GAT1.
Sialic acids are negatively charged terminal sugar residues on the
oligosaccharide chains of cell surface or serum glycoconjugates, which are
involved in a broad range of biological and pathological processes. In this
work, we examined the effect of deficiency, removal or oxidation of surface
sialic acid residues on GABA uptake activity to investigate their role in the
GABA uptake of GAT1. We found that the reduced concentration of terminal
sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of
GAT1 in CHO Lec3 cells (mutant defective in sialic acid biosynthesis) in
comparison to CHO cells. Likewise, either enzymatic removal or chemical
oxidation of terminal sialic acids using sialidase or sodium periodate
(NaIO4), respectively, resulted in a strong reduction of GAT1 activity.
Kinetic analysis revealed that deficiency, removal or oxidation of terminal
sialic acids did not affect the KmGABA values. However, deficiency and removal
of terminal sialic acids of GAT1 reduced the VmaxGABA values with a reduced
apparent affinity for extracellular Na+, suggesting a reduced affinity of GAT1
for Na+ and slowed kinetics of the transport cycle. Oxidation of cell surface
sialic acids also strongly reduced VmaxGABA without affecting both affinities
of GAT1 to GABA and Na+, respectively, indicating further that not only is the
negative charge involved, but also the unique structure of sialic acid itself
is crucial for the GABA uptake process. These results demonstrated for the
first time that the terminal sialic acid of N-linked oligosaccharides of GAT1
is directly involved in regulation of GABA uptake process of GAT1. Based on
the correlation between the reduction of the GABA uptake activity and the
reduction of the terminal sialic acid concentration of GAT1/GFP, a primary
screening model using GAT1 transfected cell culture was established for the
selection of potent inhibitors of GAT1 activity by regulating N-glycan
trimming or sialic acid biosynthesis. The influence of the candidate compounds
on GAT1 activity can quickly determined by performing GABA uptake assay in
GAT1/GFP stable trasfected Hek293 cells. Thus the compounds which have
inhibitory effect on GAT1 activity could be selected. In this work, several
synthetic N-acyl hexosamines, such as GlcNProp, GlcNCyclo, GlcNHex, GlcNAc-
Acetamido and 3-O-Met-GlcNAc was found to have inhibitory effect on GABA
uptake activity of GAT1 as inhibitors of N-glycan trimming or sialic acid
biosynthesis. Besides, several naturally occurring compounds were used for
selection of potent inhibitors on GABA uptake activity of GAT1. Resveratrol
was found to exhibit a typical non-competitive inhibition on GABA uptake of
GAT1. In order to perform structural analysis of GAT1 protein, GAT1/GFP fusion
protein was functionally expressed in mammalian (Hek293) cells, as well as in
insect Sf9 cells by BAC-TO-BACTM-Baculovirus expression system. Different
chromatography methods including affinity chromatography, ion-exchange
chromatography and size exclusion chromatography were tested for the
purification of this protein. A two-step purification procedure for GAT1/GFP
fusion protein from insect Sf9 cells was established containing immuno-
affinity chromatography using self-prepared anti-GFP and size exclusion-FPLC.
Certain amount (200-300 g per 400-600 mL culture) GAT1/GFP protein can be
purified, which was analysed by transmission electron microscopy (TEM)
analysis. Different buffer conditions were tested to obtain homogenous
GAT1/GFP fusion protein. TEM results showed different formations of purified
GAT1/GFP fusion protein with different detergents and certain amount of the
monomers of GAT1/GFP fusion protein has been isolated.
de
dc.description.abstract
GABA (gamma-Aminobuttersäure) ist der wichtigste inhibitorische
Neurotransmitter des Zentralnervensystems (ZNS). Die Wiederaufnahme von GABA
durch GABA-Transporter ist ein bedeutender Mechanismus zur Regulation der
Konzentration dieser Substanz im synaptischen Spalt. Störungen in der GABA-
Regulation werden mit vielen Krankheiten wie beispielsweise Parkinson,
Epilepsie, Chorea Huntington oder Schizophrenie in Zusammenhang gebracht. Der
GABA-Transporter 1 (GAT1) gehört zu der Familie der Na+/Cl- gekoppelten
Transporter. Es besitzt 12 vermeintliche Transmembrandomänen (TMD) und drei
N-Glykosylierungstellen in der extrazellulären Schleife zwischen der dritten
und vierten TMD. Frühere Arbeiten haben gezeigt, dass N-Glykosylierungen und
nicht das terminale Trimmen von N-Glykanen für die korrekte Faltung und
stabile Konformation von GAT1 eine Rolle spielen; was wiederum die
Proteinstabilität und den Transport an die Plasmamembran beeinflussen. Es
wurde zudem gezeigt, dass N-Glykan Seitenketten von GAT1, insbesondere die
terminalen Oligosaccharide, am GABA-Transportprozess von GAT1 beteiligt sind.
Sialinsäuren sind terminale negativ geladene Zuckerstrukturen an
Oligosaccharideketten auf Zelloberflächen oder Serumglykokonjugate, die an
vielfältigen biologischen und pathologischen Prozessen beteiligt sind. In der
vorliegenden Arbeit untersuchten wir die Rolle von Sialinsäuren auf die GABA-
Wiederaufnahmeaktivität von GAT1. Wir stellten fest, dass die Erniedrigung der
Sialinsäurekonzentration auf Zelloberflächen mit der Abnahme der GABA-
Wiederaufnahmeaktivität korreliert: In CHO Lec3-Zellen mit defekter
Sialinsäurebiosynthese war diese Aktivität im Vergleich zu normalen CHO Zellen
erniedrigt. Auch den enzymatischen Verdau durch Sialidase sowie chemische
Oxidation von terminalen Sialinsäuren durch Natriumperiodat (NaIO4) führte zur
Reduktion der GAT1-Aktivität. Kinetische Analysen ergaben, dass das Fehlen von
terminalen Sialinsäuren - sei es durch Verdau oder Oxidation - keinen Einfluss
auf den Km-Wert für GABA hat. Vielmehr wurde dadurch der Vmax für GABA
reduziert; einhergehend mit einer offensichtlichen Reduktion der Affinität für
extrazelluläres Natrium. Das Fehlen von terminalen Sialinsäuren führt demnach
zu einer reduzierten Affinität von GAT1 zu Na+ und einer verlangsamten Kinetik
des Transportzyklus. Die Oxidation von Oberflächensialinsäuren reduzierte den
Vmax von GAT1 stark, ohne dabei die Affinität für GABA und Na+ zu
beeinflussen. Diese Tatsache weist darauf hin, dass nicht nur die negative
Ladung von Sialinsäuren, sondern auch ihre Struktur eine bedeutende Rolle beim
GABA-Wiederaufnahmeprozess spielt. Mit dieser Arbeit konnte zum ersten Mal
gezeigt werden, dass terminale Sialinsäuren auf N-Glykanen am GABA-
Wiederaufnahmeprozeß von GAT1 direkt beteiligt sind. Aufgrund der Korrelation
zwischen der Reduktion der terminalen Sialinsäuren auf GAT1 und dessen GABA-
Wiederaufnahmeaktivität wurden erste Screenings hinsichtlich der Wirkung von
mutmaßlichen Sialinsäurenbiosyntheseinhibitoren auf die GAT1-Aktivität in
GAT1- transfizierten HEK293-Zellen durchgeführt. In dieser Arbeit wurden
einige synthetische N-Acylhexoamine auf ihre inhibitorische Wirkung
hinsichtlich der GABA-Wiederaufnahme hin überprüft. Verbindungen wie GlcNProp,
GlcNCyclo, GlcHex, GlcNAc-Acetamido und 3-O-Met-GlcNAc wurden als Inhibitor
der Sialinsäurebiosynthese und GABA-Aktivität von GAT1 identifiziert. Neben
vielen natürlichen Substanzen, die hier ebenfalls untersucht wurden, zeigte
Resveratrol eine typische nicht-kompetitive Inhibition der GAT1-Aktivität. Um
strukturelle Analysen des GAT1-Proteins durchzuführen, wurde GAT1/GFP als
funktionelles Fusionsprotein sowohl in die Säugerzelllinie HEK293 als auch die
Insektenzelllinie Sf9 über das BAC-TO-BAC™-Baculovirus-System exprimiert.
Unterschiedliche Chromatographiemethoden einschließlich
Affinitätschromatographie, Ionaustauschchromatographie und Gelfitration wurden
für die Reinigung dieses Proteins getestet. Eine zweistufige
Reinigungsprozedur mit GFP-Immunoaffinitätschromatographie und Gelfiltration
konnte für das GAT1/GFP Fusionsprotein von Sf9 Zellen erfolgreich etabliert,
und das so aufgereinigte GAT1/GFP unter dem Transmissionselektronenmikroskop
(TEM) analysiert werden. Unterschiedliche Pufferbedingungen wurden getestet,
um homogenes GAT1-GFP Fusionsprotein zu erhalten. TEM Ergebnisse zeigten
unterschiedliche Formationen von gereinigtem GAT1-GFP in unterschiedlichen
Detergenzien und eine bestimmte Menge von monomerem GAT1-GFP Fusionsprotein
konnten isoliert werden.
de
dc.format.extent
V, 161 S.
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
GABA transporter 1
dc.subject
characterization
dc.subject
structural analysis
dc.subject.ddc
500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::572 Biochemie
dc.title
Role of terminal sialic acid of GABA transporter 1 in GABA uptake &
purification and characterization of this transporter for structural analysis
dc.contributor.firstReferee
P.D. Dr. Hua Fan
dc.contributor.furtherReferee
Prof. Dr. Gerd Multhaup
dc.date.accepted
2011-03-17
dc.identifier.urn
urn:nbn:de:kobv:188-fudissthesis000000022083-6
dc.title.translated
Die Rolle der terminalen Sialinsäure von GABA-Transporter 1 in GABA-Aufnahme &
Reinigung und Charakterisierung dieses Transporters für die Strukturanalyse
de
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000022083
refubium.mycore.derivateId
FUDISS_derivate_000000009288
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access