Characterization of the D-amino acid oxidase interactome

228 Pages·2010·2.95 MB·English

by  M. Popiolek

Characterization of the D-Amino Acid Oxidase
Interactome

Michael Popiolek

A thesis submitted to the University College London for
the degree of Doctor of Philosophy in Pharmacology

April 2010
I, Michael Popiolek, confirm that the work presented in this thesis is my own.
Where the information has been derived from other sources, I confirm that this
has been indicated in the thesis.

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Abstract
Schizophrenia is a psychiatric disorder affecting about 1% of the world‟s
population and manifests itself as positive symptoms (eg. hallucinations),
negative symptoms (eg. social withdrawal) in conjunction with cognitive
impairments (eg. working memory). Evidence suggests that schizophrenia is, in
part, a heritable disease. Candidate susceptibility genes implicate the
glutamatergic neurotransmitter system, reinforcing clinical observations of N-
methyl-D-aspartate receptor (NMDAR) hypofunction in schizophrenia. One of
those genes, D-amino acid oxidase (DAO), is genetically associated with
schizophrenia and its function includes degradation of D-serine, the NMDAR co-
agonist. DAO is a candidate gene of high interest due to schizophrenic patients‟
manifestation of reduced D-serine levels, increased DAO expression and
increased DAO activity.
Despite mounting evidence for DAO involvement in schizophrenia, its
regulation is poorly understood. Characterization of DAO may lead to a more
thorough understanding of its function and biology and ultimately to the
identification of novel targets for this disorder. To this end, DAO-specific
antibodies were utilized to identify DAO-interacting proteins through co-
immunoprecipitation from rat cerebellum, where DAO expression is especially
high. Subsequent mass spectroscopy analysis of associating proteins yielded
twenty-four putative DAO interactors. The most abundant and interesting
interactors include known presynaptic active zone members such as bassoon
(BSN) and piccolo (PCLO). The DAO interaction with BSN was confirmed
through co-immunoprecipitation and both proteins were shown to localize in the
presynaptic junction. These data suggest that BSN is a novel DAO interactor and
defines a previously unappreciated localization of DAO perhaps as a result of a
physiologically important interaction with BSN. Furthermore, BSN was found to
partially inhibit DAO enzymatic activity in transiently transfected human
embryonic kidney (Hek293) cells. Collectively these novel findings suggest that
synaptic D-serine concentration may be under tight regulation by BSN via
proximally localized DAO. DAO may thus play a role in modulating the
functions of the presynaptic active zone via its interaction with BSN.
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Acknowledgements
I am very grateful to several people including Margaret Lie, Stan
Nawoschik and Dr. Kevin Pong who thought me key biochemistry and tissue
culture techniques even prior to enrollment in the doctorate program. Through
their assistance I was able to hit the ground running in respect to DAO research
detailed in this thesis. I am very thankful to Dr. John Ross for assistance with
identification of DAO interacting proteins through mass spectroscopy. His
expertise in designing suitable mass spectroscopy experimental controls was
critical in our identification of the DAO interactome. I am thankful to Brian
Strassle for teaching me everything in respect to imaging starting from animal
perfusion all the way to tissue preparation, staining and imaging. Brian‟s‟
expertise made my imaging experience very pleasurable indeed.
I am very deeply grateful to my Wyeth advisers: Dr. Mark Pausch and Dr.
Nick Brandon for selecting me as a doctorate degree candidate. Their support,
encouragement and advice throughout my studies allowed me to grow
scientifically and think independently. I am especially thankful to Dr. Brandon
for initiating the doctorate program and to Dr. Pausch for mentoring me during
the program and many years prior to it. Dr. Steve Moss has been instrumental to
my progress and I thank him for his advice and mentorship. I would also like to
thank the Brandon lab for their support.

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Contents

Title .………………………………………………………………………………1
Abstract .……………………………………………………………………….....3
Acknowledgements ……………………………………………………………...4
Contents ………………………………………………………………………….5
List of Tables …………………………………………………………………...10
List of Figures …………………………………………………………………..11
List of Abbreviations …………………………………………………………..15
References …………………………………………………………………..…190

Chapter 1
1.1 Schizophrenia symptoms, diagnosis, and costs …………………………..18
1.2 Major pathways associated with schizophrenia ………………………….20
1.2.1 Dopamine hypothesis of schizophrenia …………………………………20
1.2.2 Glutamate and schizophrenia …………………………………………....22
1.2.2.1 N-methyl-D-aspartate receptor and schizophrenia ……………………...23
1.3 Brain regions implicated in schizophrenia ……………………………….28
1.3.1 DLPFC …………………………………………………………………..28
1.3.2 Hippocampus ……………………………………………………………30
1.3.3 Cerebellum ………………………………………………………………31
1.4 Treatment of Schizophrenia ……………………………………………….33
1.4.1 Advantages of allosteric modulation of ionotropic glutamate receptor …35
1.5 Genetics and Aetiology of Schizophrenia ………………………………...36
1.6 Astrocytic involvement in NMDAR activity ……………………………...38
1.7 D-Serine: the NMDAR obligatory co-agonist ……………………………40
1.7.1 Schizophrenia and D-serine …………………………………….……….42
1.7.2 D-serine synthesis: Serine racemase ……………………………….……43
1.7.3 D-serine uptake: alanine-serine-cysteine transporter-1 ……………........45
1.7.4 D-serine degradation: D-amino acid oxidase (DAO)………………....…46
1.7.4.1 G72 and DAO protein-protein interaction suggests DAO involvement
in schizophrenia ……………………………………………………....…51
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1.8 Objectives and aims of this thesis ……………………………………....…52

Chapter 2: Generation of a DAO stable cell line and purification of DAO
protein from bacterial cells

2.1 AmplexRed functional assay for analysis of DAO activity …………..…55
2.2 Selection of host cell type line for the DAO stable lines ….…………..…57
2.3 Identification of DAO stable cell lines ………………………..……..……59
2.4 Purification of DAO from BL21 bacteria strain …………….……….….60
2.5 Conclusions ……………………………………..………………………….64

Chapter 3: Production and characterization of DAO polyclonal antibodies to
identify DAO interactors and investigate DAO localization

3.1 Identification of DAO specific sites for immunization ….………………69
3.2 Generation of DAO specific antibodies ……………………….…………72
3.3 Characterization of crude DAO polyclonal antibody..……………….….72
3.4 Purification of DAO antibody against immunizing peptide ……………75
3.5 Purification of DAO antibody against purified human DAO enzyme….77
3.6 DAO antibody validation against DAO KO mice lysate ………..………80
3.7 Optimization of DAO immunoprecipitation procedures ……….………82
3.8 Conclusions ………………………..……………………………………….88

Chapter 4: Identification of putative DAO interactors through DAO
antibody pull-down from rat cerebellum
4.1 Introduction ………………………………………………………..………89
4.2 Schematic flow of the proposed immunoprecipitation study ……….......92
4.3 Rabbit IgG and peptide blocked DAO antibody as negative controls …93
4.4 Coomassie stained gels of the eluates ………………………………….…95
4.5 Identification of novel putative DAO interactors through mass
spectroscopy using PBS as a washing buffer ……………………………96
4.6 Optimization of harsh washing conditions …………………….…….…105
4.6.1 Presynaptic active zone interacting proteins ………………………...…110
4.6.2 Vesicle-associated membrane proteins (VAMPs) …………….….……112
4.6.3 Syntaxin interacting proteins ………………………………………..…113
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4.6.4 Rab interacting proteins ……………………………………………..…114
4.6.5 Nicotinamide adenine dinucleotide dehydrogenase (NADH)
interacting proteins …………………………………………………..…116
4.6.6 ATPase/H+ transporting subunits interacting proteins ………...………118
4.7 Conclusions ………………………………………………………………118

Chapter 5: The Synapse
5.1 Introduction ………………………………………………………………120
5.2 Bassoon ………………………………………………………………...…121
5.3 Co-immunoprecipitation of BSN with DAO from rat cerebellum ..…..124
5.4 Does bassoon serve as a scaffold for DAO, SRR and ASC-1? ……...…126
5.5 Immunohistochemistry localization of bassoon and DAO in
rat cerebellum slices ………………...………………………………..…128
5.6 Cerebellar fractionation and localization of DAO in synaptic
junction membrane fraction ………………………………...……….…130
5.7 Immunohistochemistry localization studies in cerebellar
granule neurons ………………..……………………………………...…133
5.8 Immunoprecipitation from Hek293 cells …………………………….…138
5.9 Functional effect of rat bassoon on rDAO enzymatic activity ……...…139
5.10 Conclusions …...……………………………………………………….…139

Chapter 6: Yeast-two-hybrid screen for identification of DAO interacting
proteins from human fetal brain
6.1 Introduction ………………………………………………………………148
6.2 The Matchmaker Pretransformed Library …………………………….150
6.3 Yeast-two-hybrid Control Experiments ………………………..………152
6.4 DAO Truncation Mutants ………………………………………………152
6.5 Results from the Mating Screen ………………………...………………154
6.6 Characterization of DAO and WBP-2 interaction …………..…………155
6.7 Y2H screening of human adult brain library ………………….………..159
6.8 Conclusions …………………………………………………………….…160
6.9 Conclusions and Future Studies ………………………………………...160
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Chapter 7: Discussion
7.1: Dynein: a potential DAO interacting protein ………………………….163
7.2: Discrepancies with published literature ………………………………..163
7.3: Additional imaging studies ……………………………………………..166
7.4: Thesis summary ………………………………………………………….166
7.5: Relevance of our findings ………………………………………………..170

Chapter 8: Materials and Methods
8.1 Molecular Biology ……………………………………………………..…171
8.1.1 Bacterial and yeast strains …………………………………………...…171
8.1.2 Growth media and agar plates ……………………………………….…171
8.1.3 Chemical transformation protocol …………………………………..…172
8.1.4 DNA electrophoresis ………………………………………………...…172
8.1.5 Ethanol precipitation of DNA ……………………………………….…172
8.1.6 Gel extraction ……………………………………………………..……173
8.1.7 PCR purification ……………………………………………………….173
8.1.8 Subcloning with TOPO TA and Zero Blunt TOPO ……………………173
8.1.9 DNA Maxi- and mini-preparations …………………………………….174
8.1.10 DNA digestion with restriction endonucleases ……………………...…175
8.1.11 DNA ligation …………………………………………………………...175
8.1.12 Polymerase chain reaction (PCR) ………………………………..…….175
8.1.12.1 Preparation of vector fragments ………………………….……176
8.1.12.2 Preparation of inserts synthesized by PCR ………………..…...176
8.1.13 Sequencing ………………………………………………………..……178
8.1.14 Preparation of competent yeast cells ……………………………..…….178
8.1.15 Yeast transformation ………………………………………..………….178
8.1.16 Yeast mating ………………………………………………………..….179
8.1.17 Yeast DNA extraction ……………………………………………….....179
8.2 Biochemistry ………………………………………………………..……..179
8.2.1 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
(SDS-PAGE) ………………………………………………………...…179
8.2.1.1 Coomassie Blue staining …………………………………………….....180
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8.2.1.2 Silver stain ………………………………………………………..……180
8.2.2 Western blotting ……………………………………………….…….…180
8.2.2.1 Primary antibodies used in this study for western blotting ………….....181
8.2.3 Synaptic junction membrane fractionation experiments ……………….181
8.2.4 Amplex Red DAO assay ……………………………………………….182
8.2.5 Bacterial culture preparation and chitin column hDAO purification ......182
8.2.6 Protein assay ……………………………………………...……………183
8.3 Cell Culture …………………………………………..…………………...183
8.3.1 Maintenance of HEK293 cells ………………………………..………..183
8.3.2 Transient transfection of Hek293 ……………………………..………..184
8.3.3 Stable line generation ………………………………………………..…184
8.3.4 Culturing of cerebellar granule neurons …………………………….....185
8.4 Immunofluorescence ……………………………………………….……..185
8.4.1 Cerebellar granule neurons preparation for immunofluorescence …..…185
8.4.2 Rat cerebellar immunofluorescence preparation …………………........186
8.5 Immunoprecipitation …………………………………………………..…187
8.5.1 Application of Dynal Protein A beads ……………………………...….187
8.5.2 Application of Dynal Protein G beads ………………………………....188
8.6 Mass spectroscopy …………………………………………………….….188

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List of Tables
Table 1.1: DSM-IV Diagnostic criteria for schizophrenia ………………..…….19
Table 1.2: Antipsychotic effects of glycine site potentiators …………………...29
Table 1.3: MATRICS Consensus Cognitive Battery ………………………..….35
Table 1.4: Candidate schizophrenia susceptibility genes ……………………….39
Table 2.1: hDAO purified enzyme yield from the intein column purification …63
Table 2.2: Specific activity of the purified hDAO fractions ……………………68
Table 3.1: Rabbit immunization schedule for DAO antibody generation ……..73
Table 4.1: Methods used in identification of protein-protein interactions ……...90
Table 4.2: Putative DAO interacting proteins identified from
co-immunoprecipitation experiments …………………………………………...98
Table 4.3: List of the top 24 DAO-interacting-proteins ……………………….105
Table 4.4: Summary of the presynaptic active zone proteins identified
by the mass spectroscopy from the DAO immunoprecipitation experiments …112
Table 4.5: Percent protein coverage for the presynaptic active zone
constituents ………………………………………………………………….…111
Table 4.6: Summary of the vesicle-associated membrane proteins
identified by the mass spectroscopy from the DAO immunoprecipitation
experiments …………………………………………………………………….113
Table 4.7: Summary of the syntaxins identified by the mass spectroscopy
from the DAO immunoprecipitation experiments ……………………….….…114
Table 4.8: Summary of the Rabs identified by the mass spectroscopy
from the DAO immunoprecipitation experiments ………………………..……115
Table 4.9: Summary of the NADH dehydrogenase proteins identified
by the mass spectroscopy from the DAO immunoprecipitation experiments …117
Table 4.10: Summary of ATPase/H+ transporting proteins identified
by the mass spectroscopy from the DAO immunoprecipitation experiments …119

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