Molecular characterization of the ATP7A protein and selected
mutants
Reza Tondfekr
Submitted in total fulfilment of the requirements of
the degree of Doctor of Philosophy
March 2020
School of Chemistry
The University of Melbourne
Abstract
Copper is a vital but potentially toxic element for all living organisms. Therefore, it is necessary to maintain a balance between a deficiency and an excess level of copper. Cu-ATPase proteins are selective for Cu(I) and are crucial for removal of excess copper from cellular cytosols and for supply of essential copper to the required enzymes. Using the energy derived from ATP hydrolysis, the ATPase ATP7A (Menkes' protein, MNK) plays a key role in maturation of copper enzymes by inserting the essential metal cofactor into the nascent apo-forms. It also maintains copper homeostasis by controlling copper levels in cells. Mutations in the ATP7A gene appear to lead to neurodegenerative diseases such as Menkes' disease (MD), Occipital
Horn Syndrome (OHS), X-linked Distal Hereditary Motor Neuropathy (dHMNx), and Brachial
Amyotrophic Diplegia (BAD).
In the past decade, in vivo studies of the human copper pumps ATP7A/ATP7B have improved the understanding of their cellular functions. However, the investigation of the Cu-ATPase pump at the molecular level has fallen behind due to a number of challenges. In general, molecular characterization of transmembrane proteins requires large amounts of highly purified samples plus the challenge of producing a Cu pump in unmodified native form. It has also proven difficult to express and isolate the Cu pumps. This study details methods to address those challenges. This work also aims to undertake molecular characterisation of ATP7A and selected mutants using a 1H-NMR approach as a sensitive probe of ATPase activity.
Asp1044 is the primary phosphorylation site that is essential for ATP7A function and the variant D1044E served as a negative control for the functional assay. The two variants P1386S and M1311V are two mutations identified in patients with dHMNx and BAD diseases,
i respectively. A baculovirus expression system was used to express His-tagged wild-type protein ATP7Awt and the variants in fully functional form. When dissolved in the detergent n- dodecyl-ß-D-maltopyranoside (DDM) or embedded in Sf21 microsomes, the expressed proteins were each active in acyl-phosphorylation using the BODIPY® FL ATP-γ-S assay system. Rates varied with Cu(I) availability, ATP concentration, enzyme concentration and temperature, and other conditions were optimized. The effects of competition for Cu(I) by glutathione (GSH), bathocuproine disulfonate anion (BCS), the metal binding site 1 from the
E. coli analogue EcCopA (MBS1), metal binding site 6 of human Wilson disease protein
(WLN6) and antioxidant protein 1 (ATOX1) were investigated. The ATPase activity assays evaluated by 1H-NMR demonstrated that: ATPase activity is dependent upon Cu(I) availability to the enzyme; the ATP7Awt protein is most active in the presence of its native metallo- chaperone ATOX1.
The M1113V mutation does not affect ATPase activity significantly. Consequently, the problem of this mutation in BAD is likely related to the trafficking of Cu(I) rather than to its enzyme activity. The P1386S mutation associated with dHMNx, however, exhibited a reduced
ATPase activity.
ii Declaration
1. The thesis comprises only my original work towards the PhD except where indicated in the preface.
2. Due acknowledgements have been made in the text to all other materials used.
3. The thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices.
Reza Tondfekr
March 2020
iii Preface
The following work was performed collaboratively.
The plasmid pYHH019 used in chapter 3 was prepared by Dr. Ya Hui Hung (Florey Institute of Neuroscience and Mental Health) p-Venus-C1-ATP7A(M1311V) and p-Venus-C1-ATP7A(P1386S) plasmids were donated by, respectively, Professors Robert Bowser (Division of Neurobiology; Director, Gregory W.
Fulton ALS research center) and Kaler (Professor of Neurology and Neurobiology St. Joseph's
Hospital and Medical Center & Barrow Neurological Institute).
The Sf9/Sf21 insect host cells used in this work were donated by Dr. Heung-Chin Cheng
(University of Melbourne)
Red antibody used in this study was donated by Dr. Mark Greenough (an NHMRC-ARC
Dementia Research Fellow at the Florey).
The proteins WLN6 and ATOX1 used in chapter 5 were prepared by Drs Aswinie A.
Ukuwela and Chathuri J.K. Wijekoon.
All ATP7A transfer vectors (pYHH1705-9) and recombinant baculovirus expression vectors
(bYHH1705-9) were prepared by the author. Proteins (His-ATP7Awt, His-ATP7AD1044E, His-
ATP7AM1311V, His-ATP7AP1386S and MBS1) were prepared by the author.
iv Acknowledgments
First and for most, I would like to thank my supervisors Professor Anthony G. Wedd and Dr.
Zhiguang Xiao for giving me the opportunity to work in their laboratory, offering the resources, valuable advice and great support required for this challenging project. I feel honoured to be part of your team, Tony and Xiao.
I am grateful to Dr. Ya Hui Hung for day to day supervision in my first year of study and for teaching me many new skills, many of which are described in this thesis. Also, I would like to acknowledge a Melbourne Research Scholarship for financial support. Completion of my PhD would not have been possible without the generous help and scientific advice of the following people.
I wish to thank:-
Professor Paul Donnelly, for his valuable advice and support.
Professor Ashley I. Bush, for his support and for allowing me access to the Oxidation Biology
(OB) laboratory in Florey Institute.
Drs. Heung-Chin Cheng and Lung Yu, for donating the insect cells and for access to the Cheng laboratory.
Ashley Portbury, Celeste Mawal and everyone in the OB lab, for providing a homely atmosphere inside and outside the lab.
Dr. Sunnia Rajput, for teaching me how to use the 1H-NMR machines and their software.
Laura, Tessa, Chathuri, Nineveh and Sean, for make it fun and easy to work in the Wedd group.
Farrah, Linda, Mohammad, Brad (be tokhamam), Azadeh, Farnaz, my dear friends, for making
PhD life a little easier.
My family, for their love and support.
v Table of contents
ABSTRACT ...... I
DECLARATION ...... III
PREFACE ...... IV
ACKNOWLEDGMENTS ...... V
TABLE OF CONTENTS ...... VI
LIST OF TABLES ...... X
LIST OF FIGURES ...... XI
TERMS AND ABBREVIATIONS ...... XIII
CHAPTER 1: INTRODUCTION ...... 1
1.1 COPPER IN BIOLOGY ...... 1
1.2 COPPER UPTAKE, DISTRIBUTION AND EXPORT ...... 1
1.3 ATPASES ...... 2
1.4 P-TYPE ATPASES ...... 2
1.5 THE ATP7A PROTEIN ...... 3
1.6 ATP7A MUTATIONS ...... 6
1.7 THE P1386S MUTATION ...... 7
1.8 THE M1311V MUTATION ...... 8
1.9 PREVIOUS STUDIES ON CATALYTIC ACTIVITY OF THE ATP7A PROTEIN ...... 9
1.10 THE COPA PROTEIN FROM E. COLI ...... 10
1.11 CHALLENGES OF INVESTIGATING ATP7A ...... 11
1.12 AIMS AND STRUCTURE OF THIS STUDY ...... 12
CHAPTER 2: MATERIALS AND METHODS...... 16
2.1. CHEMICALS AND REAGENTS ...... 16
2.2. EXPRESSION OF ATP7A VARIANTS USING THE BACULOVIRUS EXPRESSION SYSTEM ...... 19
2.3 PLASMID DESIGN AND RECOMBINANT DNA TECHNIQUES ...... 19
vi 2.4 BACTERIAL STRAINS AND PLASMIDS ...... 19
2.5 DNA DIGESTIONS ...... 22
2.6 DNA LIGATION ...... 22
2.7 TRANSFORMATION OF DNA INTO E. COLI CELLS ...... 23
2.8 INTRODUCTION OF MUTATION AND HIS-TAG ...... 23
2.9 PLASMIDS EVALUATION AND SEQUENCING ...... 23
2.10 AGAROSE GEL ELECTROPHORESIS ...... 24
2.11 GENERATION OF ATP7A AND ITS VARIANTS' BACULOVIRUS ...... 24
2.12 CELL CULTURE AND MEDIUM ...... 24
2.13 ESTABLISHMENT OF SF9 AND SF21 CELL LINES...... 25
2.14 CELLS COUNTING ...... 25
2.15 CO-TRANSFECTION BETWEEN ACMNPV VIRUS AND ATP7A TRANSFER VECTORS ...... 25
2.16 WESTERN BLOTTING ...... 26
2.17 CONCENTRATIONS OF BACULOVIRUS CONSTRUCTS VIA QPCR ...... 27
2.19 WHOLE CELL PROTEIN EXTRACTION ...... 27
2.20 WHOLE PROTEIN QUANTITATION...... 28
2.21 MICROSOME PREPARATION OF ATP7A VARIANTS ...... 28
2.22 ACYL-PHOSPHORYLATION ASSAY ...... 29
2.22.1 Fluorescence labeling of microsomes containing ATP7A and variants ...... 29
2.22.2 Pulse chase ...... 30
2.23 AFFINITY PURIFICATION OF THE HIS-ATP7A VARIANTS ...... 30
2.24 SIZE EXCLUSION CHROMATOGRAPHY OF ATP7A VARIANTS ...... 31
2.25 ATPASE ACTIVITY ASSAY USING 1H-NMR ...... 31
CHAPTER 3: GENERATION OF THE ATP7A RECOMBINANT BACULOVIRUS EXPRESSION
SYSTEM ...... 32
3.1 INTRODUCTION ...... 32
3.2 MODIFICATION OF A BACULOVIRUS VECTOR FOR ATP7A EXPRESSION ...... 32
3.3 CONSTRUCTION OF THE BACULOVIRUS EXPRESSION VECTOR FOR ATP7AWT ...... 34
3.4 CONSTRUCTION OF ATP7A TRANSFER VECTORS ...... 35
3.4.1 Generation of ATP7A constructs in the pWSK29 vector ...... 36
vii 3.4.2 PARTIAL GENE SYNTHESIS TO INTRODUCE THE N-TERMINAL HIS8-TEV TAG ...... 38
3.4.3 Cloning of His-tagged ATP7A genes into the modified pBacPAK9 vector ...... 39
3.5 CO-TRANSFECTION OF SF9 CELLS WITH ATP7A TRANSFER VECTORS AND ATP7A VARIANTS INTO THE
BACULOVIRUS EXPRESSION SYSTEM...... 40
3.6 DETERMINATION OF THE CONCENTRATION OF BACULOVIRUS CONSTRUCTS ...... 40
3.7 INFECTION OF SF21 CELLS FOR EXPRESSION OF HIS-ATP7A PROTEINS ...... 42
3.8 TIME-DEPENDENT EXPRESSION OF ATP7A PROTEINS ...... 44
3.9 SUMMARY AND CONCLUDING REMARKS ...... 45
CHAPTER 4: PURIFICATION AND PRELIMINARY CHARACTERIZATION OF ATP7A
PROTEINS ...... 46
4.1 INTRODUCTION ...... 46
4.2 SEMI-PURIFICATION OF ATP7A PROTEINS IN MICROSOMES ...... 46
4.3 AFFINITY CHROMATOGRAPHY ...... 49
4.4 SIZE EXCLUSION CHROMATOGRAPHY ...... 50
4.5 ACYL-PHOSPHORYLATION ACTIVITY OF ATP7A PROTEINS ...... 53
4.5.1 LABELLING ATP7A MICROSOMES WITH BODIPY® FL ATP-Γ-S...... 54
4.5.2 Pulse-chase of ATP7A proteins, labelled with BODIPY® FL ATP-γ-S ...... 55
4.6 ESTIMATION OF ATP7A PROTEIN CONCENTRATION IN PREPARED MICROSOMES USING WB ANALYSIS...... 57
4.7 SUMMARY AND CONCLUDING REMARKS ...... 60
CHAPTER 5: ATPASE ACTIVITY OF ATP7A PROTEINS USING...... 61
1H-NMR AS A DETECTION PROBE ...... 61
5.1 INTRODUCTION ...... 61
5.2 THE PRINCIPLE OF THE ATPASE ACTIVITY ASSAY BY 1H NMR ...... 62
5.3 OPTIMIZATION OF REACTION CONDITIONS FOR ATP7A ENZYMES ...... 64
5.3.1 Effect of enzyme concentration on the catalytic rate of ATP hydrolysis ...... 64
5.3.2 Effect of ATP concentration on the rate of ATP hydrolysis ...... 66
5.3.3 Effect of MgCl2 concentrations on the rate of ATP hydrolysis...... 67
5.3.4 Effect of temperature on ATPase activity ...... 68
5.3.5 Effect of Cu(I)-stabilising ligand GSH on ATPase activity ...... 69
viii 5.3.6 Effects of Cu(I) concentration on the rate of ATP hydrolysis ...... 70
5.4 ANALYSIS OF ATPASE ACTIVITY OF ATP7A PROTEINS UNDER DIFFERENT LEVELS OF CU AVAILABILITY...71
5.4.1 ATPase activity in the presence of GSH with/without copper ...... 72
5.4.2 ATPase activity in the presence of GSH with the addition of Bca ...... 75
5.4.3 ATPase activity in the presence of GSH with addition of Bcs ...... 76
5.4.4 ATPase activity in the presence of GSH with addition of WLN6 ...... 77
5.4.3 ATPase activity in the presence of GSH with addition of EcCopA MBD1 ...... 78
5.4.4 ATPase activity in the presence of GSH with addition of ATOX1 ...... 79
5.5 SUMMARY AND CONCLUDING REMARKS ...... 81
CHAPTER 6: CONCLUDING REMARKS ...... 84
6.1 MAIN PRINCIPLES ...... 84
6.2 FINAL CONCLUSION AND SUGGESTION FOR FUTURE RESEARCH ...... 87
REFERENCES ...... 88
APPENDIX ...... 97
ix List of Tables
Table 1.1 Classification of P-type ATPases, adapted from Axelsen and Palmgren19 ...... 3 Table 1.2 Selected examples of ATP7A and ATP7B mutations and their impact on function and regulation...... 7 Table 2.1 Chemicals and reagents ...... 16 Table 2.2 Plasmids ...... 20 Table 3.1 The ATP7A baculovirus’ concentration (copies/ml) ...... 42 Table 3.2 Infection of Sf21 cells under the conditions of Figure 3.7 with each culture volume of 50 ml...... 43 Table 4.1 Concentration of total protein embedded in Sf21/ATP7A microsomes ...... 48 Table 4.2 Quantification of ATP7A protein in Sf21/His-ATP7Awt and its variants...... 59 Table 5.1 ATPase activities of purified ATP7A and its variants dissolved in detergent DDM using 1H-NMR as the detection probe ...... 73 Table 5.2 ATPase activities of purified ATP7A and its variants isolated in microsomes using 1H-NMR as the detection probe ...... 74
x List of Figures
Figure 1.1 Topology of ATP7A, adapted from Skjørringe and et al.21...... 4 Figure 1.2 The Albers Albers-Post reaction cycle ATP7A, adapted from Skjørringe21 ……..5 Figure 1.3 Immunocytochemistry of ATP7A-P1386S, adapted from Kennerson and et al41 ... 8 Figure 1.4 The acyl-phosphate intermediate formation by the purified EE–MNK protein was time-dependent and transient...... 10 Figure 1.5 A) H-NMR detection of ATP hydrolysis catalysed by EcCopA in presence of bicinchoninic anion (BCA) ...... 11 Figure 1.6. Model structure of ATP7A obtained using the determined structure of LpCopA (PDB entry 3RFU) as a template...... 13 Figure 3.1 Modification of the baculovirus transfer vector, pBacPAK9 Using PCR………..33 Figure 3.2 Using the baculovirus system for co-transfection of ATP7A Transfer vectors and Digested BacPAK6 viral DNA in Sf9 cells as a host...... 34 Figure 3.3 construction of pWSK29-ATP7AD1044E...... 37 Figure 3.4 Designing partial gene synthesis to introduce N-terminal His8-TEV tag using ApE application...... 38 Figure 3.5 Verification of various baculovirus transfer vectors generated in this work via digestion with the selected restriction enzyme(s)...... 39 Figure 3.6 Measuring baculovirus titer using qPCR (ABI7500 Fast qPCR instrument) for the ATP7A and its variants expression...... 41 Figure 3.7 Evaluation of His-ATP7A variants expression in Sf9/Sf21 cells via WB analysis...... 43 Figure 3.8 WB analysis of time-dependent expression of ATP7A proteins in Sf21 cells...... 44 Figure 4.1 Detection of ATP7A proteins in microsomes generated from the infected Sf21 cells via Western blot analysis using CT77 antibody...... 47 Figure 4.2 SDS-PAGE analysis of protein fractions from the affinity column loaded with cell extract containing His-ATP7Awt...... 50 Figure 4.3 Size-exclusion chromatography (SEC) of affinity purified His-ATP7Awt...... 51 Figure 4.4 WB (Red, N-terminal antibody) and SDS (4 – 12% Bis-Tris gradient) gels of large- scale ATP7A variants purification...... 53 Figure 4.5 Labelling ATP7A proteins in microsomes with BODIPY® FL ATP-γ-S...... 54 Figure 4.6 Pulse chase of fluorescent labelled ATP7A proteins forming transient acyl- phosphate intermediate ...... 56
xi Figure 4.7 Estimation of ATP7A concentration in microsome constructs using WB analysis in the presence and absence of DDM...... 58 Figure 5.1 Schematic illustration of detection of ATP and ADP using 1H-NMR ...... 62 Figure 5.2 Linearity of the catalysis of purified His-ATP7A variants and Sf21/His-ATP7A microsomes with different concentrations of enzyme...... 65 Figure 5.3 Catalytic time course for purified His-ATP7Awt with different ATP concentrations ...... 66 Figure 5.4 Catalytic time course for Sf21/His-ATP7Awt microsomes with different ATP concentrations ...... 67
Figure 5.5. Optimization of MgCl2 concentration for ATP7A enzymes...... 68 Figure 5.6 Optimization of temperature for ATP7A enzyme purified His-ATP7A variant and Sf21/His-ATP7A microsomes...... 69 Figure 5.7 Catalytic time course for purified His-ATP7Awt and Sf21/His-ATP7Awt microsomes with and without GSH...... 70 Figure 5.8 Catalytic time course for purified His-ATP7Awt and Sf21/His-ATP7Awt microsomes with and without copper ...... 71 Figure 5.9 ATPase activity of ATP7A and its variants in presence of GSH with/without copper ………………………………………………………………………………………………..75 Figure 5.10 Catalytic time course for purified His-ATP7Awt with (100 M) and without BCA in presence of (GSH 100 M) ……………………………………………………………….76 Figure 5.11 Evaluation of His-ATP7Awt and its variants (dissolved in DDM and embedded in microsomes) in the presence and absence of BCS...... 77 Figure 5.12 Evaluation of His-ATP7Awt and its variants ATPase (dissolved in DDM and embedded in microsomes) in presence of WLN6...... 78 Figure 5.13 Evaluation of His-ATP7Awt and its variants ATPase (dissolved in DDM and embedded in microsomes) in presence of MSD1...... 79 Figure 5.14 Evaluation of His-ATP7Awt and its variants ATPase (dissolved in DDM and embedded in microsomes) in presence of ATOX1...... 80
xii Terms and Abbreviations
ADP Adenosine diphosphate
APP Amyloid precursor protein
ATP Adenosine triphosphate
ATP7A Menkes disease protein
ATP7B Wilson disease protein,
ATPase Adenosine triphosphatase
BCS Bathocuproine disulfonic acid anion
BCA Bicinchoninic anion
BSA Bovine serum albumin
CHO Chinese hamster ovary
CNS Central nervous system
D Diffusion coefficient
DDM n-dodecyl-ß-D-maltopyranoside
DMSO Dimethyl sulfoxide
DTT Dithiothreitol
EDTA Disodium ethylenediaminetetraacetate
EGFP Enhanced green fluorescent protein
Eq. Equation
FCS Fetal calf serum
FLIP Fluorescence loss in photobleaching
FPLC Fast performance liquid chromatography
GFP Green fluorescent protein
xiii GSH Glutathione
GST Glutathione S transferase
HPLC High performance liquid chromatography
HRP Horseradish peroxidase
IgG Immunoglobulin G
LUV Large unilamellar vesicle
MBS Metal binding site
MCS Multi-cloning site
MES 2-Morpholinoethane sulfonic acid
MNK Menkes disease protein min. Minute(s)
MOI Multiplicity of infection
MOPS 3-(N-Morpholino) propanesulfonic acid
MT Metallothionein
MW Molecular weight
NEM N-ethylmaleimide
NMR Nuclear magnetic resonance
NTP Nucleotide triphosphate
OD Optical density
OHS Occipital horn syndrome
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffer saline
PCR Polymerase chain reaction
PEG Polyethylene glycol pfu plaque forming unit(s)
xiv Pi Inorganic phosphate
PM Plasma membrane
PMCA Plasma membrane Ca2+-ATPase
PVDF Polyvinylidene fluoride
ROS Reactive oxygen species rpm Revolutions per minute
RFP Red fluorescent protein
SDS Sodium dodecyl sulfate sec. Second(s)
SERCA Sarco-endoplasmic reticulum Ca2+-ATPase
SOD Superoxide dismutase
Spodoptera frugiperda Sf (Sf21/Sf9)
SUV Small unilamellar vesicle
TCA Trichloroacetic acid
TGN Trans-Golgi network
TRIS Hydroxymethyl aminomethane
TM Transmembrane
WND Wilson disease protein
wt Wild type
xv Chapter 1: Introduction
1.1 Copper in Biology
Copper is an essential trace element required by all living organisms.1 It is the third most abundant transition metal element in cells after iron and zinc.2 Its Cu(II)/Cu(I) redox couple in various biological environments exhibits a broad range of reduction potentials between 200 and 800 mV.3 This redox-active nature has been exploited by organisms in many biological processes including antioxidant defence4 and acting as redox cofactor for enzymes such as lysyl oxidase (biogenesis of collagen)5, ceruloplasmin (iron homeostasis), and cytochrome c oxidase
(aerobic respiration).6 In these enzymes, copper mediates electron transfer or redox chemistry.7
The same chemistry that makes it a valuable nutrient also makes it cytotoxic when not controlled. Copper imbalance results in a number of fatal diseases in humans, such as Menkes’ disease (copper deficiency) and Wilson disease (copper overload).8, 9 Subsequently, cells have evolved elaborate regulatory machineries and mechanisms to strike a delicate balance to satisfy cellular requirements while minimizing toxicity.10
1.2 Copper uptake, distribution and export
Free copper ions are toxic and specific protein-protein interactions are responsible for minimizing intracellular free copper. Human copper transporter 1 (hCTR1) is considered to form a pore for Cu(I) permeation through the plasma membrane.11 Inside the cell, reduced
Cu(I) is transferred to Cu,Zn-SOD, cytochrome c oxidase, and ATP7A/7B by metallo- chaperones CCS, COX17 and ATOX1, respectively. ATP7A (Menkes’ disease protein, MNK) is the fundamental copper regulator and exporter in extrahepatic cells while the homologous
ATP7B (Wilson disease protein, WND) is the main copper transporter in hepatic cells.12
1 1.3 ATPases
ATPases are a large family of enzymes that catalyse the dephosphorylation of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) resulting in the release of chemical energy to drive other chemical processes.13 Transmembrane ATPases are imbedded in the biological membranes and transfer solutes against their concentration gradients. Based on function and structure, ATPases are divided into several groups including ABC transporters and P-type ATPases.14
1.4 P-type ATPases
P-type ATPases are a superfamily of transmembrane ion pumps that are of vital importance in all kingdoms of life. The name is based upon their ability to catalyse auto-phosphorylation within a conserved aspartate residue. They all appear to interconvert between at least two different conformations (E1 and E2) in their catalytic cycles. They play a significant role in many cellular processes including muscle contraction, absorption, and secretion; they generate and maintain critical electrochemical gradients across cellular membranes by translocating metal cations such as protons, Na+ and K+.15, 16 Others transport certain nutrients and toxins.
P-type ATPases are classified into five main groups based on phylogenetic analysis which are summarized in Table 1.1. The P1B subclass transfers heavy metal ions, playing the dual role of protecting cells from toxic levels of transition metals and of donating metal ions to the metal- required enzymes.17, 18 It is possible to divide this subclass into further subgroups based upon differences in conserved amino acid signatures in the transmembrane domain. However,
ATP7A and certain variants are the focus of this work.
2 Table 1.1 Classification of P-type ATPases, adapted from Axelsen and Palmgren.19
Type Sub-class Substrate specificity Example I A K(I) KdpB I B Cu(I); Ag(I); Cu(II); Cd(II); CopA; ATP7A; ATP7B; CadA; Zn(II); Pb(II); Co(II) ZntA II A Ca(II); Mn(II) SERCA; PacL II B Ca(II) PMCA II C Na(I), K(I); H(I), K(I) Na(I), K(I)-ATPase; H(I), K(I)- ATPase II D Ca(I); Na(I) CTA3 III A H(I) AHA1; PMA1 III B Mg(II) MgtA; MgtB
IV Phospholipids ATP10C; ATP11A V Unknown PfA TPase1;
1.5 The ATP7A protein
ATP7A (Menkes' protein, MNK) is a PIB-type copper-transporting transmembrane protein
(Table 1.1). It uses the energy derived from ATP hydrolysis to transport Cu(I) across cell membranes. It has 1500 amino acids (MW: 163.4 kDa) including twenty-six cysteine residues
(6.3 kDa) and is expressed in most extra-hepatic cells, such as heart, brain, lung, muscle and kidney, but at much lower levels in the liver.20 It is found in the Trans-Golgi Network (TGN) at low copper concentrations and in post-Golgi compartments and the plasma membrane at high copper concentrations.21
As is shown in Figure 1.1, ATP7A features three major functional domains:21, 22
1. An ATP-interaction domain consisting of: (i) the N domain for nucleotide binding; (ii) the P domain containing the D1044KTGT motif with the critical catalytic aspartate residue 1044;
(iii) the A domain containing the phosphatase motif T875GE.
3 2. Eight transmembrane (TM) segments that form a channel to transport Cu(I) through the membrane. It has been demonstrated that P-type ATPases bind their target substrate ion in specific transmembrane motifs to promote ion transfer across the cell membrane.23 A highly conserved CPC motif is characteristic of heavy metal transporting P-type ATPases.24, 25 It has been shown that Cu(I) can bind to the Cys residues of the CPC motif and this appears to be critical to its actual transfer across the cell membrane.26
3. A large N-terminal cytosolic domain contains six repeated Cu(I)-binding sites, each containing a GMTCXXC motif (H1-H6). These sites are required for various aspects of
ATP7A (or ATP7B) function, including copper translocation, protein localization and trafficking, protein-protein interactions and ATPase activity.27-33
Figure 1.1 Topology of ATP7A, adapted from Skjørringe et al.21 Several conserved motifs are present that are characteristic of members of the P -Type ATPase family such as ATP7B and CopA.23 They include the nucleotide binding domain (N-domain, shown in red), the phosphorylation domain (P-domain, purple), and the actuator domain (A-domain, yellow). Six metal binding sites (H1- 6, shown in blue) are present in the N-terminal tail.
4 It has been assumed that the Cu(I) transport process of ATP7A follows the Albers-Post reaction cycle developed initially for the Ca2+-ATPase (SERCA) from the sarco-endoplasmic reticulum found in muscle cells.21 In this model (Figure 1.2), the protein binds Cu(I) with high affinity within the TM domain when the protein is in a conformation termed the E1 state. Copper- binding and ATP-recognition allow auto-phosphorylation of the invariant D1044 in the P- domain leading to the E1P state and trapping of copper in the TM domain. Phosphorylation completion induces a large conformational change allowing copper release into the lumen side, giving the E2P state. De-phosphorylation leads to the E2 state followed by return to the E1 state to start the next reaction cycle.34
Figure 1.2 The Albers Albers-Post reaction cycle ATP7A, adapted from Skjørringe et al.21 Copper translocation by ATP7A is assumed to go through a general cycling model involving different steps: binding of the copper (E1), binding of ATP to N-domain followed by ATP hydrolysis and phosphorylation of the P-domain (E1P-ADP, E1P), translocation of the copper (E2P), dephosphorylation of the P-domain and that induces release of copper (E2P, E2).
5 Although it is possible to evaluate translocation of Cu(I) either using isolated membrane vesicles from cells expressing ATP7A or purified ATP7A reconstituted in liposomes, there is a need for studies to definitively characterize mechanism and the effect of mutations. For example, Figure 1.2 proposes transfer of two Cu(I) ions per ATP turnover, as demonstrated
Ca(II) transport by CERCA. However, the ATP7A analogue isolated from E. coli has been shown to transport one Cu(I) ion only per ATP turnover.35
1.6 ATP7A mutations
Menkes’ disease is an X-linked fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene. More than a hundred different mutations have been reported worldwide.36 Some of these and their impact on ATP7A are listed in Table 1.2. Many delete part of the gene, insert additional DNA base pairs or use the wrong base pairs and are predicted to produce proteins that do not function properly.37
The mutations can be divided into several sub-types. The majority of patients suffer from the classical form of Menkes disease but about 6% have milder forms such as Occipital Horn
Syndrome (OHS), X-linked Distal Hereditary Motor Neuropathy (dHMNx) and Brachial
Amyotrophic Diplegia (BAD) with longer survival periods.38
There is a need for studies to characterize the effect of mutations on the catalytic cycle and
ATPase activity of the protein.39, 40 The effect of disease-related mutations M131V and P1386S mutations on acyl- phosphorylation and ATPase activity of the ATP7A protein is investigated in this work. There
is a wider need to examine the effect of mutations on trafficking of the enzyme between the
TGN and the plasma membrane.
6
Table 1.2 Selected examples of ATP7A and ATP7B mutations and their impact on function and regulation.
Mutation Catalytic ATPase activity Localization Protein-Protein interaction G591D Extensive ER mis Decreased interaction localization41 with ATOX142 H1069Q No ATP binding43, No Extensive ER mis ATPase activity43 localization44 R1115H Slightly decreased ATP binding 43 N1270S Decreased ATPase Normal,46 TGN activity45 L873R Increased formation of acyl Plasma membrane29 Decreased interaction phosphate intermediate29 with ATOX129 D1325V Protein absent in patient fibroblast cells47
ATOX1 is the native copper chaperone for the ATP7A protein.
1.7 The P1386S mutation
This missense mutation does not involve directly the copper transport functional domains and systemic copper metabolism does not appear to be compromised.48 The mutation targets highly conserved amino acids in the carboxyl half of the protein. It seems that conformational flexibility is affected and that normal trafficking into the secretory pathway is impaired.48
Figure 1.3 illustrates the reduced localization of ATP7AP1386S in the TGN compared to
ATP7Awt.48 Fibroblast protein with deletion of exons 20–23 from a patient with Menkes disease was used as a negative control.
7
Figure 1.3 Immuno-cytochemistry of ATP7A P1386S, adapted from Kennerson et al.48 p230 is the TGN. ATP7A is shown in green. In high concentration of copper, ATP7Awt relocated from the TGN to the plasma membrane. In contrast, ATP7AP1386S was mostly trapped in the TGN.
1.8 The M1311V mutation
Whole genome sequencing of a patient with BAD disease revealed a new inherited variant of unknown significance in the X-linked ATP7A gene.49 This amino acid alteration occurs next to the ATP binding site in the P-domain of the ATP7A protein. All MD residues identified around Met131121 are highly conserved (blue), but the residue Met1311(red) is not:
G1300DGINDSPALAM1311ANVG1315 (see Figure 1.6c). It may not disrupt protein expression but Met1311 could be in an important position for relaying energy transmission from ATP hydrolysis to the conformation changes in the TM domain required for driving Cu translocation. A functional comparison of this variant with D1044E (negative control) and wild type ATP7A protein (positive control) was anticipated to provide a better understanding of the effects of the M1311V mutation.
8 1.9 Previous studies on catalytic activity of the ATP7A protein
Cation translocation (e.g. H+, Na+, K+, Ca2+, Cu+ and Cd2+) and ATP hydrolysis, including transient acyl-phosphorylation, are two characteristics of the catalytic cycle of P-type ATPases.
Modelled on the SERCA mechanism, the catalytic mechanism of the ATP7A protein, including transient acyl-phosphorylation, remains imperfectly understood (Figure 1.2).
The phosphorylation reaction is proposed to result in a change in the enzyme conformation from E1 (high-affinity cation binding state) to E2 (low affinity state). It has been shown that the formation of acyl-phosphate in ATP7A was reversible in the presence of ADP (1 mM) or bicinchoninic anion (BCS; 1 mM), a ligand that binds Cu(I) with high affinity.30 Under the experimental conditions, at least 70 percent of the phosphorylated protein was present in the
ADP-sensitive E1-like state, while 30 percent of phosphorylated intermediate remained following pulse-chase with ADP.
In addition, a path-finding study of the catalytic activity of purified ATP7A protein has provided some understanding of the mechanism.40 This included observation of the acyl- phosphate intermediate during ATPase activity and vectorial Cu(I) translocation of the membrane-reconstituted enzyme (see Figure 1.4).
9
Figure 1.4 Functional assays on the ATP7A A.The acyl-phosphate intermediate formation by the purified ATP7A protein was time-dependent and transient. Purified protein was labelled with [γ -32 P] ATP for up to 15 min as indicated. B. Cu(I)-dependent acyl-phosphorylation activity. C. Copper-dependent ATPase activity. D. Active 64Cu transport. Adapted from Y.H. Hung et al.40
1.10 The CopA protein from E. coli
E. coli CopA (EcCopA) is also a Cu(I)-translocating P-type ATPase whose membrane topology is similar to human ATP7A and 7B.50 Recently, a 1H-NMR approach provided a quantitative ATPase activity assay for the CopA enzyme either dissolved in detergent or embedded in giant unilamellar vesicle (GUV) membranes (Figure 1.5).35 Figure 1.5A shows
1H-NMR detection of ATP hydrolysis catalyzed by EcCopA in the presence of the Cu(I)- binding ligand bicinchoninic acid (BCA). The hydrolysis rate increased proportionally with enzyme concentration (Figure 1.5B), allowing reliable estimation of the ATPase activity under a wide set of conditions. This approach has the potential to be applied to the ATP activity of wild type ATP7A and those with selected mutations.
10
Figure 1.5 A. 1H-NMR detection of ATP hydrolysis catalysed by EcCopA in the presence of bicinchoninic anion (BCA).
B. Linear correlation of reaction rate vs enzyme concentration in presence of 2- ligands: red: GSH plus vanadate ([HVO4] as an inhibitor); blue: MBD6; purple: BCA. C. Confocal image of CopA-GFP fusion protein embedded in a giant unilamellar vesicle.35
1.11 Challenges of investigating ATP7A
Although an understanding of the functions of ATP7A at the cellular level has advanced considerably during the past few years, a molecular understanding of this pump (which requires highly purified protein) has lagged. The main difficulties are associated with expression and handling of membrane proteins plus the need for sophisticated approaches to the copper chemistry. Success is dependent upon: -
1. Production of adequate quantities of purified and functionally active enzyme;
2. Quantification of the binding of Cu(I) and its link to ATPase activity.
As was seen in previous attempts51-53, lack of a highly effective approach for purification of the proteins of interest in their native forms emphasises the challenge of these requirements.
11 However, methods like polymer nanodiscs and cell free expression have both been effective in purification of membrane proteins.54 The presence of detergent is essential in purification of membrane proteins to prevent their denaturation and to reduce protein aggregation. On the other hand, the presence of detergents can lead to a significant loss in the final yield. Various affinity tags have been introduced to facilitate the isolation and purification of the recombinant proteins. The poly-histidine tag has been used in isolation and purification of Cu pumps including CopA from Legionalla pneumophila,55, 56 CopZ from Archaeoglobus fulgidus,57
ATP7B58, 59 and ATP7A.60-62
In addition, widely used protocols for estimation of ATPase activity rely on determination of inorganic phosphate (Pi) as a product of ATP hydrolysis (ATP→ ADP + Pi). However, there are issues with these protocols. Firstly, the procedures are tedious and require a large sample size.63 Secondly, there is high background activity because of the presence of Pi in media and the problem of non-enzymatic ATP hydrolysis under many experimental conditions. There are other approaches (such as γ−32P-ATP64 and BODIPY™ FL ATP-γ-S65) but they are all semi- quantitative but useful in isolation biochemistry. However, there is a need for better methods to replace these approaches in quantitative work.
1.12 Aims and structure of this study
Biochemical and molecular characterization of purified ATP7A enzyme and other Cu pumps, such as the bacterial CopA proteins, has provided a better understanding of their mechanisms of copper transport.35, 66 However, these path-finding studies were able to provide limited molecular characterisation only.
A number of molecular properties of the ATP7A protein and selected mutants were evaluated in the present study. The locations of the target mutations are modelled in Figure 1.6. The
M1311 residue is not conserved but is close to the ATP binding site. The M1311V mutation
12 (associated with BAD) is assumed to affect energy transmission from ATP hydrolysis via compromising conformation changes in the trans-membrane domain (TM) required for driving copper translocation. Comparative study of the ATP7AM1311V variant with D1044E (negative control) and wild type ATP7Awt (positive control) was aimed at characterising its specific properties. The P1386S is a missense mutation leading to a non-fatal form of motor neuron disorder, X-linked Distal Hereditary Motor neuropathy (dHMNX), without overt signs of systemic Cu deficiency.67
Figure 1.6. A) Model structure of ATP7A obtained using the determined structure of LpCopA (PDB entry 3RFU) as a template; B) Zoom-in view of molecular region for ATP7A phosphorylation and various mutation sites that affect ATP7A functions (left) and a 90o rotation view along the Z-axis; C) Sequence alignment of different Cu-ATPase transporters for residues harbouring the Met 1311 of ATP7A.
13 The overall aims of this study were:- 1. Generation of recombinant baculovirus expression vectors for ATP7A and selected
variants that include
• Cloning of ATP7A gene and selected variants into a modified pBacPAK9 transfer
vector. The targeted genes include His-ATP7AWT (positive control) plus native
ATP7AWT control without the His-tag, plus variants His-ATP7AD1044E (negative
control), His-ATP7AM1311V (BAD mutation) and His-ATP7AP1386S (dHMNX
mutation).
• Co-transfection of the generated transfer vectors with digested BacPAK6 viral DNA
into IPLB-Sf9-AE (Sf9) cells to produce recombinant baculovirus expression vector.
2. Expression of ATP7A protein and variants in Sf9/IPLB-Sf21-AE (Sf21) insect cells that
includes
• Infection of these cells with the generated recombinant baculovirus expression
vectors for expression of ATP7AWT protein and variants.
3. Isolation and purification of the ATP7A proteins via
• Semi-purification of ATP7A and variants as microsomes
• Affinity chromatography employing Co-affinity talon resin
• Size exclusion chromatography
4. Functional assays of ATP7A and variants in semi-purified (in microsomes) and purified (in
detergent DDM) forms for
• Semi-quantitative investigation of transient acyl-phosphorylation using fluorescence
BODIPY® FL ATP-γ-S (Life Technologies) technology.
• Quantitative ATPase activity assay using 1H-NMR (Bio600 - Bruker AVIII 600
MHz).
The thesis is organized into six self-contained chapters: -
14 Chapter 1 has reviewed the current understanding of Cu-ATPases and, more specifically, that of the ATP7A protein (structure and functions) and related studies and challenges corresponding to investigation of Cu pumps at the molecular level.
Chapter 2 lists chemicals and methods used in this work.
Chapter 3 details the generation of ATP7A and its variants by recombinant baculovirus techniques and their expression in Sf9 and Sf21insect host cells.
Chapter 4 details the isolation and purification protocol and functional assay (acyl- phosphorylation) of semi-purified ATP7Awt and its variants in microsomes generated from the insect host cells.
Chapter 5 reports ATPase activities of ATP7A protein and its variants (either dissolved in
DDM detergent or embedded in microsomes), using 1H-NMR under different conditions.
Chapter 6 summarises the conclusions derived from this study and makes suggestions for future progression of this topic.
15 Chapter 2: Materials and Methods
2.1. Chemicals and reagents
General chemicals and reagents (analytical grade) were purchased from various commercial sources (Table 2.1) and all were used as received.
Table 2.1 Chemicals and reagents
Name Vendor Catalog #
SalI New England Biolabs (NEB) R0138S
NdeI " R0111S
BglII " R0144S
XhoI " R0146S
BamHI-HF " R3136S
XmaI " R0180S
SphI-HF " R3182S
EcoRV-HF " R3195S
BclI " R0160S
XbaI " R0145S
dNTP Mix Promega U1511
Nuclease-Free Water " P1193
T4 DNA Ligase NEB M0202S
TriDye 2- Log DNA Ladder " N3270S
SYBR Safe DNA Gel Stain Life Technologies S33102
TriDye 2- Log DNA Ladder - 125 gel NEB N3270S lanes
16 SYBR Safe DNA Gel Stain Life Technologies S33102
Wizard Plus SV Minipreps DNA Promega A1330 Purification System
Wizard SV Gel and PCR Clean-Up " A9281 System
Low melting agarose Scientifix 9040A
BacPAK™6 DNA (Bsu36 I digest) Clontech 631401
Baculovirus Titration, BacPAK™ " 631414 qPCR, 200 Rxns
Sf-900 TM II SFM Life Technologies 10902096
Grace's medium " 11605094
PhosphoSafe™ Extraction Reagent Merck 71296-3
Pierce™ BCA Protein Assay Kit Thermo Fisher 23225
Bol 4-12% Bis-Tris Plus Gels, 17-well Life Technologies NW04127BOX
NuPAGE 4-12% Bis-Tris Midi " WG1403BOX Protein Gels, 26-well
Bolt 4-12% 10 well gels " NW04120BOX iBlot® 2 Transfer Stacks, PVDF, mini " IB24002
Gel Loading Dye Blue (6X) NEB B7021S
His Tag Antibody Sapphire Bioscience Pty. Ltd OAEA00010
PANSORBIN® Cells Merck 507858-1GM
BODIPY™ FL ATP-γ-S, Thioester Life Technologies A22184 (Adenosine5'-O-(3-Thiotriphosphate), BODIPY™ FL Thioester, Sodium Salt)
TALON® Metal Affinity Resin Scientifix 6355003
17 Corning® Erlenmeyer baffled cell Sigma-Aldrich CLS431407- culture flasks 250 mL Erlenmeyer 50EA Flask w/ Vent Cap, polycarbonate, sterile, 50/cs
Deuterium oxide,99.9 atom % D " 151882-25G
Adenosine5′-triphosphate (ATP) " 34369-07-8 disodium salt hydrate
Polyclonal CT77 antibody Hycult Biotech HP8040
18 2.2. Expression of ATP7A variants using the baculovirus expression system
The ATP7A baculovirus systems were constructed via co-transfection of the various ATP7A transfer vectors and genetically-engineered nuclear polyhedrosis viral DNA virus from the moth Autographa californica (AcMNPV, Clontech). ATP7A protein was expressed by infection of insect cells with the constructed virus. The viral DNA was purchased from
Clontech and used as received. Genes of ATP7A and its variants were cloned into pBacPAK9 transfer vectors (Clontech).
2.3 Plasmid design and recombinant DNA techniques
The plasmids for this project were designed initially by the ApE software (a plasmid editor v2.0.51). They were constructed using standard recombinant DNA procedures, as described previously.68 Plasmid DNA from transformed DH5a bacterial cells was extracted using the
Wizard Plus SV Miniprep DNA purification kit (Promega), according to the manufacturer’s instructions. A PCR Clean-up Kit (Promega) was used for ligated DNA fragments in low melt agarose gels (Scientifix).
2.4 Bacterial strains and plasmids
DH5a bacterial cells strains were used as host cells to construct ATP7A baculovirus transfer vectors. DH5a cells were grown in Luria Broth (LB) medium at 37 C. An appropriate antibiotic (Ampicillin (Ap), 50 g /mL) was added to the culture. Plasmids were stored either in their host strain (Glycerol in LB 50% v/v) or in nuclease-free H2O at -80 C. The plasmids used in this project are listed in Table 2.2.
19 Table 2.2 Plasmids
Plasmid ID Relevant characteristics Source/Ref
pBacPAK9 Baculovirus transfer vector: pUC and M13 Clonthech origin of replication pWSK29 low-copy bacterial cloning vector Wang/Kushner69 pJFM77 low-copy mammalian IRES vector La Fontaine70
pJFM19 4.7kb BamHI-BamHI fragment containing La Fontaine70 (pWSK29-ATP7A) ATP7A cDNA in pWSK29 pYHH019 modified pBacPAK9 with pUC origin Hung et al40
(pBacPAK9low) replaced with low copy pSC101 origin of pWSK29 pJFM134 4.7kb Spel-SalI fragment containing Pertis et al71 ATP7A(D1044E) cDNA cloned into Nhel- SalI in pJFM77 p-Venus-C1- 4.7kb SalI-ApaI fragment containing Bowser ATP7A(M1311V) ATP7A(M1311V) cDNA cloned into pVenus-C1 p-Venus-C1- 4.7kb SalI-ApaI fragment containing Kaler72 ATP7A(P1386S) ATP7A(P1386S) cDNA cloned into pVenus- C1 pBHA-His-TEV- XbaI-SphI fragment containing 937bp Bioneer ATP7A(XbaI-SphI) ATP7A cDNA with N-terminal His8-TEV tag cloned into pHBA pYHH1601 4.7kb BamHI-BamHI fragment containing This work (pWSK29- ATP7A cDNA (D1044E) from pJFM134 ATP7A(D1044E)) cloned into pWSK29 pYHH1602 3.7kb SphI-XmaI fragment containing This work (pWSK29- ATP7A cDNA (M1311V) from pVenus- ATP7A(M1311V)) C1(M1311V) replacing the SphI-XmaI fragment in pJFM19
20 pYHH1603 3.7kb SphI-XmaI fragment containing This work (pWSK29- ATP7A cDNA (P1386S) from pVenus- ATP7A(P1386S)) C1(P1386S) replacing the SphI-XmaI fragment in pJFM19 pYHH1604 XbaI-SphI fragment in ATP7A cDNA from This work (pWSK29-His-TEV- pJFM19 replaced with XbaI-SphI fragment ATP7A) containing 937 bp ATP7A with N-terminal His8-TEV tag from pBHA-His8-TEV- ATP7A(XbaI-SphI) pYHH1605 XbaI-SphI fragment in ATP7A(D1044E) This work (pWSK29-His-TEV- cDNA from pYHH1601 replaced with XbaI- ATP7A(D1044E)) SphI fragment 937 bp ATP7A cDNA with N- terminal His8-TEV tag from pBHA-His8- TEV-ATP7A(XbaI-SphI) pYHH1701 1,983 bp EcoRV-XmaI fragment in This work (pYHH019- ATP7A(M1311V) cDNA from pYHH1602 ATP7A(M1311V)) cloned via EcoRV-XmaI pYHH019 partial pYHH1702 1,983 bp EcoRV-XmaI fragment in This work (pYHH019- ATP7A(P1386S) cDNA from pYHH1602 ATP7A(P1386S)) cloned via EcoRV-XmaI pYHH019 partial pYHH1703 EcoRV-XmaI fragment in ATP7A cDNA This work (pWSK29-His-TEV- from pYHH1604 replaced with EcoRV-XmaI ATP7A(M1311V)) fragment containing 2,030 bp ATP7A (M1311V) cDNA pYHH1704 EcoRV-XmaI fragment in ATP7A cDNA This work (pWSK29-His-TEV- from pYHH1604 replaced with EcoRV-XmaI ATP7A(P1386S)) fragment containing 2,030 bp ATP7A (P1386S) cDNA
21 pYHH1705 4,746 bp XbaI-XhoI fragment of His-TEV- This work (pYHH019-His-TEV- ATP7A cDNA from pYHH1604 cloned via ATP7A) XbaI-XhoI in pYHH019 pYHH1706 4,746 bp XbaI-XhoI fragment of His-TEV- This work (pYHH019-His-TEV- ATP7A(D1044E) cDNA from pYHH1605 ATP7A(D1044E)) cloned via XbaI-XhoI in pYHH019 pYHH1707 3,706 bp SphI-XhoI fragment of His-TEV- This work (pYHH019-His-TEV- ATP7A(M1311V) cDNA from pYHH1703 ATP7A(M1311V)) cloned via SphI-XhoI in pYHH1705 pYHH1708 3,706 bp SphI-XhoI fragment of His-TEV- This work (pYHH019-His-TEV- ATP7A(P1386V) cDNA from pYHH1704 ATP7A(P1386S)) cloned via SphI-XhoI in pYHH1705 pYHH1709 4,698 bp XbaI-XhoI fragment cDNA from This work (pYHH019 -ATP7A) pJFM19 cloned via XbaI-XhoI in pYHH019
*Ampicillin resistance
2.5 DNA digestions
Appropriate restriction enzymes (see Table 2.1) were used. Restriction endonuclease digestions of DNA were performed according to the manufacturer’s instructions (New England
Biolabs). The reactions were incubated for 2-3 hours at the appropriate temperature for optimum activity.
2.6 DNA ligation
To increase the chance of successful ligation, DNA fragments were mixed in the ratios 3:1 to
5:1 insert:vector in reaction mixture (10 l). T4 DNA ligase (0.5-1 unit; Promega) and ligation buffer (1x) were added and the mixture incubated at 16 C overnight.
22 2.7 Transformation of DNA into E. coli cells
The DNA ligation product was transformed into E. coli cells (DH5) using the TSS
73 transformation method. The cells were harvested by centrifugation at OD600 0.4 - 0.5. Cells
(10 mL) were made to be competent by resuspension in ice-cold TSS solution (1 ml, 1% tryptone, 0.5% yeast extract, 1% NaCl, 10% PEG-3350, 5% (v/v) DMSO). Competent cells
(200 l) were added to DNA in TSS enhancement solution (100 l, 100 mM KCl, 30 mM
CaCl2, 50 mM MgCl2). The transformation mixture was then incubated for 20 minutes on ice, followed by 20 minutes at room temperature. LB medium (700 l, Ap 50 g /mL) was added and the mixture incubated for 1 h at 37 C. Cells were plated onto LB-agar plates containing
Ap and the plates incubated overnight at 37 C.
2.8 Introduction of mutation and His-Tag p-Venus-C1-ATP7A(M1311V) and p-Venus-C1-ATP7A(P1386S) plasmids were donated, respectively, by Professors Robert Bowser (division of neurobiology; director, Gregory W.
Fulton ALS research center) and Kaler (Neurology and Neurobiology St. Joseph's Hospital and
Medical Center & Barrow Neurological Institute). ATP7A mutant genes that were confirmed by DNA sequencing (AGRF Sanger Service, Melbourne, Australia) were amplified by PCR.
2.9 Plasmids evaluation and sequencing
To identify positive colonies, random colonies from LB-agar plates were selected, added to LB media (15 ml) containing Ap (50 g /mL) and grown at 37 C overnight. Plasmid DNA from the culture was extracted using a Wizard Plus SV Miniprep DNA purification Kit (Promega) and digested with appropriate restriction enzymes (see Table 2.1). Alternatively, PCR was used to verify positive colonies and appropriate primers were used in each set of experiments. Either
23 the digested DNAs or PCR products were loaded onto agarose gel and compared with expected results predicted by the ApE app.
2.10 Agarose gel electrophoresis
Electrophoresis was performed in gels (0.5-1.2%) to separate DNA fragments within the size range of 300 to 19 kbp. TAE buffer (40 mM TRIS-acetate, 1 mM EDTA, pH 8.3) was used at
80-100 V in ice (if required). DNA fragments were stained with ethidium bromide (1g/ml) and visualised using an ultra-violet trans illuminator and Bio-Rad Chemi-Doc™ MP Imager.
2.11 Generation of ATP7A and its variants' baculovirus
Baculoviruses were constructed via co-transfection using the BacPAK6 kit (Clonthech, containing Bsu36 I digested BacPAK6 (viral DNA)) and ATP7A transfer vectors according to the manufacturer’s instructions.
2.12 Cell culture and medium
IPLB-Sf-9AE (abbreviated as Sf9) or IPLB-Sf21-AE (Sf21) insect cells were used as host cells for both co-transfection and protein expression. These cells were obtained from Associate
Professor Heung-Chin Cheng’s laboratory (Biochemistry and Molecular Biology Centre,
University of Melbourne). Cell lines were cultured either as monolayers or in suspension at 27
C. They were propagated in Grace’s insect medium (Thermo Fisher Scientific) supplemented with fetal bovine serum (10%, FBS, Scientific) and Sf-900™ II SFM medium (Thermo Fisher
Scientific).
24 2.13 Establishment of Sf9 and Sf21 cell lines
A vial of cells was collected from a liquid nitrogen storage tank and thawed at 37 C in a pre- warmed water bath for approximately 2 min. The thawed cells were transferred to a sterile
Falcon tube (50 ml) containing pre-warmed (27 C) Grace’s insect medium (10 ml) or Sf-
900™ II SFM medium. The cell suspension was centrifuged at 750 rpm for 5 minutes. The cell pellets were resuspended gently into the same medium (20 ml) and transferred into sterile Sharp bottles (100 ml, for suspension cell line) or T25 flasks (for monolayer cell line). Incubation at
27C followed, either in a shaking incubator at 120 rpm (for suspension cells) or static incubator (for monolayer cells).
2.14 Cell counting
Viability was evaluated by a hemocytometer or automatic cell counter (Life Technologies,
Countess II FL) with the cell suspension diluted (1:1 ratio) with Trypan Blue dye in both methods.
2.15 Co-transfection between AcMNPV virus and ATP7A transfer vectors
Recombinant baculovirus expression vector was generated by co-transfection between modified bacterial ATP7A transfer vectors and Bsu36 I digested bacPAK6 (AcMNPV viral
DNA) using Sf9 as host cells. The cells (1x106 cells/ml) were seeded in a six-well tissue culture tray and allowed to attach for 1-2 hours at 27 C. They were then washed gently with serum- free Grace’s medium and incubated at room temperature. Meanwhile, the transfection mixture containing bacfectin (liposomal preparation), an ATP7A-containing transfer vector and viral
DNA BacPAK6 was prepared and incubated at room temperature for 15 min (to allow the transfection reagent to form a complex with the DNA). The mixture was then added dropwise
25 to the Sf9 cells and incubated at 27 C. After 5 hours, Grace’s medium supplemented with FBS
(10%) was added and incubation continued at 27 C for 3 to 5 days.
The supernatant containing the primary ATP7A variants recombinant baculovirus expression vectors (defined as P0) was filtered through a syringe filter (2 m) and transferred to Eppendorf tubes (2 ml). P0 baculovirus of ATP7A variants (300 l) was added to Sf21 cell culture (50 ml).
The supernatant (P1) was collected and filtered through a syringe filter (2 m). The P0 and P1 samples were aliquoted and stored at both 4 C and -80 C. Cell pellets (P0 and P1) were analyzed via western blot for evaluation of ATP7A protein expression.
2.16 Western blotting
Proteins were separated by SDS-polyacrylamide gel electrophoresis (Bol 4-12% Bis-Tris Plus gel, Life Technologies) in 1x MES SDS running buffer (50 mM MES, 50 mM Tris Base, 0.1%
SDS, 1 mM EDTA, pH 7.3). Proteins were transferred from the gel to a PVDF membrane using iBlot® 2 Transfer Stacks (Life Technologies) via the iBlot® 2 Dry blotting system, according to the manufacturer’s instructions.
Membranes were blocked with 5% skim milk/T-BST (0.15 M sodium chloride, 0.050 M TRIS-
HCl buffer; 0.05% Tween 20; pH 7.6) at 4 C overnight or 1 h at room temperature. For detection of protein by chemiluminescence, blocked membranes were incubated with T-BST/ primary antibody (1:5000 ratio) at room temperature for 1 h followed by an anti-rabbit secondary antibody. Protein bands were visualized by a luminescent image analyser
(FUJIFILM LAS-4000).
26 2.17 Concentrations of baculovirus constructs via qPCR
Generally, viral supernatant is collected and then, using a viral DNA purification kit, genomic
DNA is extracted from a small aliquot of the supernatant. Serial dilutions of the viral DNA sample are subjected to qPCR to determine threshold cycle (Ct) values for each dilution.
ROXTM Reference Dye may be used to normalize fluorescent signal intensity between reactions when using qPCR instruments that are equipped with the option to do so. The DNA genome copy number in a sample dilution is determined by finding the copy number that corresponds to its Ct value on a standard curve generated from serial dilutions of the calibrated
BacPAK DNA Control Template.
These were estimated by the BacPAK qPCR Titration Kit (Clonthech) according to the manufacturer’s instructions. The supernatant from each construct (150 μl) was purified using the NucleoSpin Virus Kit to a final volume of 50 μl. DNA template from the kit and all samples were diluted sequentially (1-4 times) with Easy Dilution Buffer. Each sample (1μl) was added to a master mix (H2O 3.4 l, forward Primer 0.2 l, reverse primer 0.2 l, ROX
(carboxyrhodamine) dye 50 x 0.2 l, SYBER green 5l, total 9 μl). The mixture was pipetted into a 96-well plate. Amplification plots and standard curves were created by using an ABI7500
Fast qPCR instrument (Applied Biosystems).
2.19 Whole cell protein extraction
PhosphoSafe Extraction Reagent (Merck) was used to extract proteins from Sf9/Sf21 insect cells according to the manufacturer’s instructions. Alternatively, cells were collected by low speed centrifugation (5 min, 2500 g) and protein extraction was carried out by resuspending the cells in ice-cold RIPA buffer (25 mM TRIS, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100 or NP-40, EDTA-free protease inhibitor tablet (1 tablet
27 per 50 mL of buffer)). After incubation at 4 C for 30 min, the cell lysate was collected by centrifugation (5 min, 16,000 g, 4°C).
2.20 Whole protein quantitation
Total protein concentrations in extracts from whole cells were estimated using a Pierce™ BCA
Protein Assay Kit (Thermo Fisher) according to the manufacturer’s instructions. An eight-point standard curve was prepared using BSA standard (1 mg/mL) covering the range 0 – 14 g.
Protein samples were diluted so that the total protein concentration was within the range of the standard curve. The reaction was initiated by the addition of BCA Protein Assay Kit to diluted samples and incubated at 60 C for 15-20 minutes. The dye absorbance was measured at 595 nm.
2.21 Microsome preparation of ATP7A variants
ATP7A/Sf21 cell pellet was thawed on ice and resuspended in buffer A (10 mM TRIS-HCl, pH 7.4; 250 mM sucrose; 0.5 mM EDTA; 1 mM DTT; anti-protease cocktail (50 L/50 mL culture)). Cells homogenized using a Teflon pestle (1200 rpm) were centrifuged at 1000 g at
4 C for 10 min. Supernatants were transferred to polyallomer tubes and centrifuged by ultracentrifuge at 10,000 g at 4 C for 10 min. The pellet was rinsed carefully with a solution of sucrose (250 mM) in TRIS-HCl (10 mM, pH 7.4) and resuspended in buffer A. The suspension was transferred to fresh polyallomer tubes and centrifuged at 13,000 g at 4 C for
70 minutes. The new pellet was resuspended in buffer A and aliquoted for storage at – 80 C.
28 2.22 Acyl-phosphorylation assay
The activity of Sf21 microsomes containing ATP7A and its variants were assayed using the fluorophore BODIPY® FL ATP-γ-S (Life Technologies). The fluorescence pixel intensity was measured using ImageJ software (version 2.0.0, http://imagej.net). The copper-dependent kinetics were analyzed using GraphPad PrismTM Software (version 8.0.1, http://www.graphpad.com).
2.22.1 Fluorescence labeling of microsomes containing ATP7A and variants
Microsomes of ATP7A and selected variants were labelled with fluorescent ATP for various time periods. Reactions were initiated by addition of fluorophore ATP mixture (1 M, 1 L
BODIPY® FL ATP-γ-S, 5 L 10 M ATP) to microsome mixture (45 l) containing ATP7A protein (10 g total) and MOPS buffer (20 mM, pH 6.8, 150 mM NaCl, 5 mM MgCl2, fresh
50 M DTT supplemented 5 M CuCl2). After the mixtures were incubated in ice for different time periods, the reactions were stopped by adding SDS (5 L, 20% stock). The target ATP7A protein was immuno-precipitated using anti-ATP7A (Red) antibody and Pansorbin (Merck) as a source of protein A. The immunoprecipitated pellet was rinsed once with MOPS buffer (20 mM, pH 6.5 containing 0.1% NP-40), followed by two rinses in MOPS buffer (20 mM, pH
6.5). The proteins were solubilized by resuspension of the pellet in the sample buffer (65 mM
TRIS-phosphate, pH 6.5; 8 M urea, 5% SDS, 5 mM EDTA, 0.5 M DTT, 0.005% bromophenol blue) and resolved by SDS-PAGE on an acid polyacrylamide gel (5.5%). The gels were incubated in gel fixing buffer (70% H2O, 20% EtOH, 10% acetic acid) for 24 hours at 4 C and were exposed to Typhoon FLA 9500 imager (Life Science) at emission maximum 509 nm. An increase in acyl-phosphorylation corresponded to longer labelling times.
29 2.22.2 Pulse chase
Microsomes of ATP7A and selected variants were labelled with fluorescent ATP for 25 sec.
Reactions were initiated by addition of ATP mixture (1 M, 1 L BODIPY® FL ATP-γ-S, 10
M) to ATP7A microsome protein (10 g total) in MOPS buffer (20 mM, pH 6.8, 150 mM
NaCl, 5 mM MgCl2, fresh 50 M DTT supplemented 5 M CuCl2). To pulse chase, ATP (5 l of 10 M) per reaction was added to the mixture and incubated for various time periods.
Reactions were stopped by adding SDS (5 L of 20% stock). The samples were provided as described in the previous section and resolved by SDS-PAGE on an acid polyacrylamide gel
(5.5%). The gels were incubated in gel-fixing buffer and were exposed to Typhoon FLA 9500 imager (Life Science).
2.23 Affinity purification of the His-ATP7A variants
An affinity column preloaded with His GraviTrap TALON Superflow Co2+ resin was used to purify histidine-tagged ATP7A proteins (6xHis at the N-terminal). The column was equilibrated with buffer (50 mM MOPS-TRIS, pH 7.5 containing 150 mM NaCl, 10% glycerol,
2 mM beta-mercaptoethanol (βME)). Membrane proteins were solubilised (50 mM MOPS-
TRIS buffer, pH 7.5, containing 10 mM DDM, 150 mM NaCl, 10% glycerol, 2 mM βME, and
EDTA-free protease inhibitor) and loaded onto the column which was incubated at 4 oC for 2-
4 hours. The column was washed several times with buffer (50 mM MOPS-TRIS, pH 7.5 containing 150 mM NaCl, 10-15 mM imidazole). The ATP7A protein was eluted (50 mM
MOPS-TRIS buffer, pH 7.5 containing 150 mM NaCl, 10 mM imidazole).
30 2.24 Size exclusion chromatography of ATP7A variants
The protein fractions from affinity chromatography that contained the target proteins were concentrated and applied to a Superdex 10/300 HR column and eluted (50 mM MOPS-TRIS buffer, pH 7.5 containing 2 mM DDM, 200 mM NaCl, 10% glycerol, and 2 mM βME).
2.25 ATPase activity assay using 1H-NMR
To increase detection sensitivity, all assays were carried out using a Bruker 600 MHz spectrometer equipped with cryogenic probes and water suppression. The following general protocol was adapted and used for each experiment.35, 74 The reaction mixture (0.8 mL) contained the following components, depending on the variables targeted in the assay: ATP
(50-400 M,), MgCl2 (50-400 M), NaCl (50 mM), D2O (10 % v/v), CuSO4 (10 μM), BCA
(25-100 μM), GSH (25-100 M), ascorbate (500 μM, as a reductant), Na-MOPS buffer (50 mM, pH 7.5). The reaction mixture and a stock of the His-ATP7A enzymes (either dissolved in DDM detergent or embedded in Sf21 microsomes) were incubated separately at 37 oC. The
ATP7A enzymes (0.03-0.05 M) were added to the mixture which was immediately transferred into an NMR tube and the reaction followed by NMR spectroscopy. To increase the signal to noise (S/N) ratio, 8 and 16 consecutive scans were taken for each reading at a total scan time of approximately 1.03-1.57 min for each complete spectrum. Using the TopSpin
4.0.6 App, peak areas corresponding to ATP and ADP were integrated to calculate the percentage of ATP hydrolysis.74
31 Chapter 3: Generation of the ATP7A recombinant
baculovirus expression system
3.1 Introduction
Study of membrane proteins is challenging due to their low abundance relative to the total cell protein content. Commonly, heterologous expression systems such as E. coli, yeast, insect and mammalian cells are used for large-scale protein production. Mammalian expression systems are ideal for expressing eukaryotic proteins. The baculovirus gene expression system is a popular and effective method. Post-translational processing of eukaryotic proteins expressed in insect host cells is usually similar to protein processing in mammalian cells.75 Consequently, the biological activities of proteins expressed in this way are comparable to proteins expressed in mammalian cells. Autographa californica nuclear polyhedrosis virus (AcMNPV) is the baculovirus commonly uses to over-express proteins.76 It is propagated in insect cells derived from the ovary of the fall armyworm, Spodoptera frugiperda (Sf9 or Sf21 cells). Powerful viral polyhedrin or p10 promotors leads to a high level of protein expression.77 The present work utilised a baculovirus expression system to obtain high functional expression of the ATP7A protein and its variants. This chapter describes generation of the ATP7A and its variants and their expression in a baculovirus system using Sf9/Sf21 as insect host cells.
3.2 Modification of a baculovirus vector for ATP7A expression
The intrinsic instability of ATP7A cDNA requires the use of low-copy number plasmids when it is propagated in E. coli cells.70 The baculovirus transfer vector from BacPAK9 Baculovirus
Expression System (Clontech, Mountain View, CA, USA) contains a pUC high-copy number origin of replication. Therefore, it was necessary to modify pBacPAK9 to enable manipulation and cloning of ATP7A cDNA in E. coli cells. The pBacPAK9 high-copy number origin of
32 replication was replaced with the low-copy number pSC101 origin of replication from pWSK29 (Figure 3.1).69
Figure 3.1 Modification of the baculovirus transfer vector pBacPAK9 using PCR. pBacPAK9 was amplified to exclude the high-copy number pUC origin of replication, and to introduce BclI restriction sites at the ends. The BglII fragment of the low-copy number pSC101 origin of replication from pWSK29 was ligated to pBacPAK9 via BclI restriction sites. The resulting eight kb plasmid was designated pBacPAKlow (pYHH019).
33 As shown in Figure 3.1, the pBacPAK9 plasmid was amplified by PCR using the oligonucleotides 5’-GCT GGC CTT TTG ATC ACA TGT TC-3’ and 5’-GGG TCT GAT CAT
CAG TGG AAC G-3’ to exclude the pUC origin of replication. The oligonucleotides contain engineered BclI sites to enable subcloning of the BglII fragment containing the pSC101 origin of replication from pWSK29. The resulting 8 kb low-copy number baculovirus transfer vector was designated pBacPAK9low (pYHH019).
3.3 Construction of the baculovirus expression vector for ATP7AWT
Ayres et al. demonstrated that it is challenging to directly clone the heterologous gene of interest into the large (134 kb) baculovirus AcMNPV genome.78 Consequently, recombinant baculovirus expression vectors are constructed in two steps (Figure 3.2).
Figure 3.2 Using the baculovirus system for co-transfection of ATP7A transfer vectors and digested BacPAK6 viral DNA in host Sf9 cells. Genetically-engineered AcMNPV viral DNA is linearized by digestion with Bsu36 I, which partially deletes the ORF1629 gene essential for viral propagation. The transfer of the target gene into the viral DNA via homologous recombination restores the complete ORF1629 for viral propagation. Adapted from the BacPAKTM Baculovirus Expression System User Manual (Clonetech).
34 Firstly, the ATP7A gene was cloned into a modified polyhedrin locus contained in the pBacPAK9low transfer vector. A multiple cloning site (MCS) was replaced with a partly deleted polyhedrin coding sequence and the ATP7A gene was inserted into this MCS, between the polyadenylation signals and the polyhedrin promotor. In addition, the transfer vector
(pBacPAK9low) contained an antibiotic resistance gene and a plasmid origin of replication for propagation in E. coli; however, they cannot replicate in Sf9/Sf21 insect host cells. In the second step, a viral expression vector (AcMNPV) and the pBacPAK9low transfer vector were co- transfected into Sf9 insect host cells.
Double recombination between the viral sequence in the pBacPAK9low transfer vector and the corresponding sequences in the AcMNPV viral DNA transferred the ATP7A gene into the viral genome. BacPAK6 (Clontech) is a specifically engineered virus that facilitates construction and selection of recombinant expression vectors and that is present in the BacPAK baculovirus expression system. It has a necessary gene adjacent to the polyhedrin locus that provides selection for recombinant viruses.79 Two fragments are released upon digestion of BacPAK6 with Bsu36 I. One carries part of ORF1629 which is vital for viral replication,80 whereas the transfer vector carries the remaining ORF1629 sequence.
3.4 Construction of ATP7A transfer vectors
Expression of protein via the baculovirus expression system requires construction of pBacPAK9low transfer vectors carrying the genes of interest. As discussed in previous sections
(3.2, 3.3), it was essential to change the high-copy number origin of replication of pBacPAK9 with the low-copy number origin of pWSK29.
35 Cloning of ATP7A and its variants was accomplished in a few steps. Firstly, the ATP7A gene was introduced into the backbone of pWSk29,69 using pJFM19 and pJFM134 as templates to introduce pWSK29-ATP7Awt and pWSK29-ATP7AD1044E respectively. Secondly, pWSK29-
ATP7AM1311V and pWSK29-ATP7AP1386S were generated using templates of the SphI-XmaI fragment (4,836 bp) from pVenus-C1-ATP7AM1311V and pVenus-C1-ATP7AP1386S, respectively.72 The 6x His-tag (Sapphire Bioscience Pty. Ltd) was introduced to the N-termini of the genes for purification purposes. Finally, the His-tagged ATP7A genes were cloned into modified pBacPAk9. Plasmid details are listed in Table 2.2.
3.4.1 Generation of ATP7A constructs in the pWSK29 vector
As discussed, the pWSK29 vector was chosen due to its low copy origin of replication. The
ATP7A genes from the different templates (Table 2.2) were subcloned into the vector. As an example, construction of the pWSK29-ATP7A-D1044E (pYHH1601) variant is described in this section. All other constructs were generated following the same method (data not shown).
As it is shown in Figure 3.3, pWSK29 and pJFM134(ATP7AD1044E) were digested with
BamHI. Expected fragments (5434 bp from pWSK29 and 4635 bp from pJFM134) were cut from electrophoresis gels (low melt agarose l/1x TAE buffer (1.2%)). Ligation was initiated with T4 ligase enzyme and T4 ligase buffer with a ratio of 1:5 of vector (pWSK29) to insert
(pJFM134(ATP7AD1044E)) at room temperature overnight. E. coli strain of DH5 was used as the competent cells to transfer the new construct to an agar plate containing antibiotic Amp.
Positive colonies were picked and grown overnight. New plasmids were extracted from the culture and digested by XbaI, SphI and BglII to confirm the generation of pWSK29-
ATP7AD1044E.
36
Figure 3.3 Construction of pWSK29-ATP7AD1044E. A. Cloning of pJFM134 into low copy pWSk29 was designed using the ApE program. B. Theoretical gel patterns of DNA fragments predicted by ApE program for the digestion of pWSk29-ATP7AD1044E with XbaI, SphI and BglII to confirm the generation pYHH1601. C. Experimental gel (1% agarose in TAE buffer) patterns for;
1. DNA ladder 2. pYHH1601 (His-pwSK29-ATP7AD1044E), XbaI, SphI 3. pYHH1601 (His-pwSK29-ATP7AD1044E), BglII
37 3.4.2 Partial gene synthesis to introduce the N-terminal His8-TEV tag
To facilitate downstream purification of the ATP7A proteins, a His8-tag was introduced to the
N-terminus.81 The N-terminal His8-TEV tag in pBHA vector (2002 bp) was obtained from
Bioneer and its sequence checked after digestion by SphI and XbaI restriction enzymes (Figure
3.4). Vector pJFM19 and pBHA ATP7A-His8-TEV tag (as insert) were digested by SphI and
XbaI. Fragments (9186 bp from pJFM19 and 937 bp from pBHA) were cut from electrophoresis gels (low melt agarose /1x TAE buffer (1%)) and added in a ratio of ~five parts of insert to one part of vector into 1 l ligation buffer and 1 unit of T4 DNA ligase enzyme.
The mixture was incubated at room temperature overnight. DNA ligation products were transferred into DH5 cells and grown overnight in LB medium containing Amp. New plasmids were extracted using the Wizard Plus SV Miniprep DNA purification system and digested by BamHI to confirm the generation of pWSK29-ATP7A-His8-TEV.
Figure 3.4 Designing the partial gene synthesis to introduce N-terminal His8-TEV tag using ApE application. Theoretical cloning of Bioneer partial synthetic ATP7A-His8-TEV (SphI, XbaI) into pJFM19 (ATP7A) to generate His-Tev-ATP7Awt (pHYY1604) is shown.
38 3.4.3 Cloning of His-tagged ATP7A genes into the modified pBacPAK9 vector
Various His-ATP7A genes from different templates were subcloned into the pBacPAK9low
(pYHH019) vector to generate the respective transfer vectors. For example, pYHH019
(modified low-copy pBacPAK9) and pYHH1604 (pWSK29-ATP7A-His-TEV) were digested by XbaI/XhoI and XbaI/XhoI/NdeI, respectively, to generate pYHH019-His-ATP7Awt
(pYHH1705). Fragments (7984 bp from pYHH1604, 4746 bp from pYHH1604) were cut from electrophoresis gels (low melt agarose gel/1x TAE buffer (1.2%)) and mixed in a ratio of ~five parts of insert to one part of vector in ligation buffer (1 l); T4 DNA ligase enzyme (1 unit)).
Other constructs were generated similarly. The products were digested with the appropriate restriction enzyme(s) to verify generation of the right constructs (Figure 3.5). The constructs were confirmed by DNA sequencing.
Figure 3.5 Verification of various baculovirus transfer vectors generated in this work via digestion with selected restriction enzyme(s).
A. Experimental gel (1% agarose in TAE buffer) patterns for; 1. pYHH1705 (His-ATP7Awt baculovirus transfer vector), Bam-HI, NdeI 2. pYHH1706 (His-ATP7AD1044E baculovirus transfer vector), Bam-HI, NdeI 3. pYHH1707 (His-ATP7AM1311V baculovirus transfer vector), Bam-HI, NdeI 4. pYHH1708 (His-ATP7AP1386S baculovirus transfer vector), Bam-HI, NdeI 5. pYHH1709 (ATP7Awt baculovirus transfer vector), Bam-HI 6. DNA Ladder B. Theoretical gel patterns of DNA fragments predicted by ApE program for the experiments in (A).
39 3.5 Co-transfection of Sf9 cells with ATP7A transfer vectors and ATP7A variants into the baculovirus expression system. The five His-ATP7A transfer vectors (pYHH1705-9) were co-transfected with the linearized
BacPAK6 (Clontech) viral DNA (AcMNPV) into Sf9/Sf21 host cells using the Bacfectin liposomal preparation. The resulting recombinant His-ATP7A systems were produced by an in vivo double recombination event (P0). The presence of the desired baculoviruses in the cell medium (P1, bYHH1705-9) was tested by WB analysis of the corresponding Sf9/Sf21 cells using different antibodies against the C-terminal (Ct77) and the N-terminal (Red, 6x His) domains (data not shown). The highest expressing recombinant baculovirus was selected and purified using a BacPAK qPCR Titration Kit (Clonthech).
3.6 Determination of the concentration of baculovirus constructs
The BacPAK qPCR Titration Kit (Clonthech, Cat. No. 631414) provides a fast and simple method for titrating baculoviral stocks generated from AcMNPV-based baculoviral vectors, including all of Clontech’s BacPAK vectors. The kit employs a quick DNA purification step and allows determination of viral DNA genome content in just 4 hours, using qPCR and
SYBR® Green (Clonthech, Cat. No. 639676) detection technologies. Because qPCR titration is so fast, target cells can be infected with accurately titrated virus on the same day when the virus is harvested. This method makes it possible to infect cells at a known multiplicity of infection (MOI).
For consistent expression levels, it is essential to know the concentration of constructs. Sf9/Sf21 cells were infected with specific concentrations of the particular ATP7A baculovirus.
Concentrations (copies/ml) were measured by a BacPAK qPCR Titration Kit (Clonthech, Cat.
No. 631414) according to the manufacturer’s instructions. An amplification plot and standard
40 curve was created by using ABI7500 Fast qPCR instrument (Figure 3.6). The results are summarized in Table 3.1. Baculovirus concentrations (copies/ml) from various target ATP7A constructs (P1) and from the host cell control were calculated separately for each batch using the standard curve and threshold cycle (Ct) point for each batch using the following formula: -
푙 (푟푎푤 푐표푝푦 푛푢푚푏푒푟 (푄푢푎푛푡푖푡푖푦 푖푛 푠푡푎푛푑푎푟푑 푐푢푟푣푒)(푐표푝푖푒푠))(1000 )(푒푙푢푡푖표푛 푣표푙푢푚푒 (푙)) 퐶표푝푖푒푠/푚푙 = 푚푙 (푠푎푚푝푙푒 푠푖푧푒 (푙))(푣표푙푢푚푒 푝푒푟 푤푒푙푙 (푙)) where elution volume = 50 μl; sample size: 150 μl; volume per well: 1 μl.
Figure 3.6 Estimation of the concentration of baculovirus constructs using qPCR (ABI7500 Fast qPCR instrument) for expression of ATP7A genes.
A. Amplification plots of qPCR reactions using different dilutions of BacPAK DNA control templates (2.8 108- 2.8 103).
B. Amplification plots of qPCR reactions for bYHH1705 (His-ATP7Awt).
C. Standard curve made from the plots presented in panel A (different dilutions of BacPAK DNA control templates and the BacPAK qPCR titration kit) which shows a linear correlation between the Ct values and the DNA copy number (log scale).
D. Quantity of bYHH1705 (His-ATP7Awt, obtained from B), the positive control (obtained from A) and negative control (Sf9) on the standard curve.
41
Table 3.1 Baculovirus construct concentrations (copies/ml)
Construct Approximate conc. (x1010 copies/ml)
Sf9 cells (non-infected) 2.0 x10-6
Sf9-His-Tag-ATP7Awt 1.3
Sf9-His-Tag-ATP7AD1044E 1.4
Sf9-His-Tag-ATP7AM1311V 1.3
Sf9-His-Tag-ATP7AP1386E 1.0
Sf9-ATP7Awt 1.3
3.7 Infection of Sf21 cells for expression of His-ATP7A proteins
Although both Sf9 and Sf21 cells can be used for expression of a recombinant protein, Sf21 cells commonly yield slightly faster protein expression than Sf982 and were chosen for the protein expression in this work. Sf21 cells were infected with the ATP7A baculoviruses (3.8 x
108 copies/plaque forming units; multiplicity of infection (MOI) 5) and protein expression was monitored by taking samples at regular time intervals up to 76 hours post-infection (Figure
3.7, Table 3.2).
As shown in Figure 3.7, The Sf9 cells were successfully infected by His-ATP7A baculovirus constructs. Concentration of ATP7A baculovirus used in the experiments to infect Sf21/Sf9 insect host cells is summarized in Table 3.2.
42
Figure 3.7 Evaluation of expression of His-ATP7A proteins in Sf9/Sf21 cells via WB analysis. Sf21 cells were infected with ATP7A Baculovirus (3.8 x 108 copies/plaque forming units (MOI 5)) and protein expression was monitored by taking samples at intervals up to 76 h post-infection. Expression levels were assessed with the antibodies, as indicated.
Table 3.2 Infection of Sf21 cells under the conditions of Figure 3.7 with each culture volume of 50 ml. Construct Volume of virus, mL Sf21cells number b bYHH1705 1.46 1.03x106 (His-ATP7Awt) (1.8x1012) a 94% bYHH1706 1.35 1.01x106 (His-ATP7AD1044E) (1.8x1012) 93% bYHH1707 1.46 1.02x106 (His-ATP7AM1311V) (1.8x1012) 95% bYHH1708 1.58 1.07x106 (His-ATP7AP1386V) (1.8x1012) 92% bYHH1709 1.46 1.05x106 (ATP7Awt) (1.8x1012) 91% a MOI 5, Multiplicity of infection (MOI) = Plaque forming units (PFU) of virus used for infection/ Cells number∼ copies/ PFU, PFU for BacPAK6 = 7.6x10 7 and copies/ml= 4x109 when copies/PFU = 53 (Clontech user manual) b Viability was evaluated by a hemocytometer or automatic cell counter (Life Technologies, Countess II FL). The suspension was diluted (1:1 ratio) with Trypan Blue dye in both methods.
43 3.8 Time-dependent expression of ATP7A proteins
The expression of protein in Sf9/Sf21 cells depends on infection time. Expression in Sf21 cells infected with the recombinant His-ATP7Awt baculovirus and its variants (bYHH1705-8) was monitored. Samples were taken at regular time intervals up to 76 hours after the infection.
Protein expression level increased proportionally with time, as confirmed by WB analysis
(Figure 3.8). Western blot analyses showed a proportional increase in ATP7A expression levels with time following infection.
For Sf21 cells infected with 3.8 x 108 copies/plaque forming units (MOI 5) of the virus, the expression approached a maximum after 72 hours of post-infection, although most of the cells were dead by then. The decline in Sf21 viability (data not shown) is likely due to decreased biosynthetic machinery efficiency, leading to a higher chance of protein degradation due to release of proteases from cell debris and an increase in post-translational processing heterogeneity. This data indicated that cells should be harvested 64-72 hours after infection to minimize protein degradation.
Figure 3.8 WB analysis of time-dependent expression of ATP7A proteins in Sf21 cells. Infection level was 3.8 x 108 copies/plaque forming. A total of 50 g of protein per sample was separated by SDS-PAGE on 4–12% BIS- TRIS gradient gels, followed by transfer onto nitrocellulose membranes. CT77 was used as a secondary antibody.
44 3.9 Summary and concluding remarks
P1B-ATPases are the subclass of P-type ATPase that are responsible for the transport of heavy metals across cell lipid biolayers. Although many proteins are involved in intracellular distribution of copper, a vital homeostatic role belongs to the ATP7A protein. The mutations in ATP7A can lead to a variety of neurogenerative diseases. Therefore, it is important to study this enzyme either in vivo or in vitro. However, evaluating ATP7A protein at the molecular level requires active purified protein.
In this study, His-ATP7Awt and selected variants were expressed in a baculovirus expression system in Sf9 or Sf21 insect host cells employing two essential steps. Selected genes were cloned into the transfer vector pBacPAK9. The vector was then co-transfected with linearized
BacPAK6 viral DNA into Sf9/Sf21 host cells (Figure 3.2). The intrinsic instability of ATP7A cDNA necessitated insertion of a low copy origin of replication into the vector ATP7A cDNA
(Figure 3.1).
As discussed in section 3.4, ATP7A genes were subcloned into pWSK29, followed by insertion of the N-terminal His-tags. Finally, His-tagged ATP7A proteins were cloned successfully into the transfer vector. All new constructs were evaluated by digestion with appropriate restriction enzymes and sequenced. The plasmids used in this work are listed in Table 2.2. The ATP7A transfer vectors were co-transfected with linearized viral DNA into Sf9/Sf21 host cells. As it is shown in Table 3.1 and Table 3.2, the concentration of ATP7A baculovirus was calculated for each construct and 3.8 x 108 copies/plaque forming units; multiplicity of infection (MOI) 5 was used to infect Sf9/Sf21 cells. Protein expression was confirmed by WB analysis (Figure
3.7). Infected Sf21 cells were harvested at 64-72 hours of after infection in order to minimize protein degradation. Protein purification is presented in chapter 4.
45 Chapter 4: Purification and preliminary characterization
of ATP7A proteins
4.1 Introduction
The role of the P1B-type ATPase ATP7A (a polytopic integral membrane protein) in copper transport and homeostasis was discussed in Chapter 1.67, 83, 84 Mutations in this copper pump and an analogue ATP7B can lead to copper imbalance, resulting in Menkes’ and Wilson diseases along with a variety of related neuro-pathologies.85, 86 In recent years, knowledge of the nature of Cu-ATPases at the cellular level has increased as a result of valuable in vivo cell studies. Moreover, biochemical study of bacterial Cu-ATPases such as CopA, has offered a platform for future investigation of other Cu pumps.35, 87, 88
However, understanding of copper pumps at molecular levels has fallen behind as it requires proteins purified to high levels. As discussed in Chapter 1.11, the main difficulties are associated with expression and handling of membrane proteins plus development of sophisticated approaches to the copper chemistry. Success is dependent upon:-
1. Production of adequate quantities of purified and functionally active enzyme;
2. Quantification of binding of Cu(I) and its link to ATPase activity.
This chapter details strategies used to isolate and purify the His-ATP7Awt protein and a number of variants.
4.2 Semi-purification of ATP7A proteins in microsomes
Microsomes are heterogenous vesicle-like artifacts (20-200 nm in diameter) which can be segregated from other cellular debris using differential centrifugation.89 They form from pieces of the endoplasmic reticulum (ER) with attached ribosomes when eukaryotic cells are
46 fragmented in vitro.90 Consequently, they can contain over-expressed membrane proteins.
Microsomes can be prepared from Sf9 or Sf21 insect cells or from yeast by heterologous expression.91, 92,93 Hence, they can be valuable for study of membrane enzyme activity or inhibition for research in vitro.83, 94-96 This approach was employed in this study to probe the enzymatic activity of the expressed ATP7A proteins.
Three batches of Sf21-His-ATP7A microsomes were prepared (See 2.21). Their presence in microsomes was confirmed by WB analysis using the CT77 antibody (Figure 4.1). The concentration of total protein in the microsomes were calculated using a Pierce™ BCA Protein
Assay Kit (Thermo Fisher) according to the manufacturer’s instructions (section 2.5). The results are summarized in the Table 4.1.
Figure 4.1 Detection of ATP7A proteins in microsomes generated from infected Sf21 cells via Western blot analysis using antibody CT77. Sf9/EE-ATP7A was used as a control. One set of experiments is shown
47 Table 4.1 Concentration of total protein embedded in Sf21/ATP7A microsomes a
Construct concentration
(mg/ml)
Sf21-His-Tag-ATP7Awt (I) 2.44
Sf21-His-Tag-ATP7AD1044E (I) 2.61
Sf21-His-Tag-ATP7AM1311V (I) 2.78
Sf21-His-Tag-ATP7AP1386E (I) 3.83
Sf21-ATP7Awt (I) 3.38
Sf21-His-Tag-ATP7Awt (II) 3.21
Sf21-His-Tag-ATP7AD1044E (II) 3.01
Sf21-His-Tag-ATP7AM1311V (II) 3.41
Sf21-His-Tag-ATP7AP1386E (II) 3.13
Sf21-ATP7Awt (II) 2.42
Sf21-His-Tag-ATP7Awt (III) 2.91
Sf21-His-Tag-ATP7AD1044E (III) 3.21
Sf21-His-Tag-ATP7AM1311V (III) 2.42
Sf21-His-Tag-ATP7AP1386E (III) 2.78
Sf21-ATP7Awt (III) 3.9 a Protein concentration in extracts from whole cells were estimated using a Pierce™ BCA Protein Assay Kit (Thermo Fisher), according to the manufacturer’s instructions (See 2.20).
48 4.3 Affinity chromatography
The proteins were purified in two steps: affinity chromatography for high resolution protein capture and size exclusion chromatography to enhance homogeneity. Affinity chromatography is a robust and effective method to purify proteins with a specific affinity tag obtained by different expression systems. A high level of protein purification is possible as a particular ligand is covalently conjugated to a solid phase matrix allowing specific binding to proteins tagged with an affinity label.97
The ATP7A proteins were tagged with an 8x His-tag at the N-terminus. An affinity column
(1.5 x 10 cm, max. vol. 18 ml, cross-sectional area 1.77 cm, low-pressure chromatography) preloaded with His GraviTrap TALON Superflow Co2+ resin was used in the first step of purification. After 64-72 h post-infection of Sf21 cells with 3.8 x 108 copies/plaque forming units (MOI 5) of the ATP7A baculovirus for each construct, infected cells were harvested and membrane proteins were extracted with detergent buffer (50 mM MOPS-TRIS, pH 7.5, 10 mM DDM, 150 mM NaCl, 10% glycerol, 2 mM βME, and EDTA-free protease inhibitor) and the extract loaded onto the column. To maximize the protein capture, the flow-through fractions was usually re-loaded onto the column twice after the initial application. The column loaded with cell extract was kept in a cold room at 4C for 2 h to enhance the target protein binding efficiency. A variety of conditions (variation of concentration of eluant imidazole, for instance) were explored to maximize purification efficiency. The eluted fractions were analysed by SDS-page and visualized by Coomassie brilliant blue staining, as shown in Figure
4.2.
49
Figure 4.2 SDS-PAGE analysis of protein fractions from the affinity column loaded with cell extract containing His-ATP7Awt, washed with buffer (50 mM MOPS-TRIS buffer, pH 7.5) and then eluted with the same buffer containing 15 mM imidazole in A or 10 mM imidazole in B.
4.4 Size exclusion chromatography
Size exclusion chromatography (SEC) was employed for further purification of His-ATP7A proteins eluting from the affinity column. Using a calibrated column and standards proteins,
SEC also allows estimation of protein molecular masses.98 Affinity-purified His-ATP7A proteins were concentrated and added to the column (Superose-12 10/30 HR (matrix exclusion
6 limit ~2 x10 Mr, bead diameter (µm) 8–12, prepacked column bed dimensions (mm) 10 x 300–
310, bed volume (ml) 24, bed pressure 430 psi, flow rate (ml/min) 0.5); fractionation range, 1-
300 kDa) equilibrated with MOPS-TRIS buffer (50 mM, pH 7.5, 2 mM DDM, 150 mM NaCl,
10% glycerol, 2 mM ME).
50 A size exclusion chromatogram for His-ATP7Awt is shown in Figure 4.3, as an example. The sample was contaminated with some degraded and other species, as normally expected (Figure
4.3A). WB analysis with different antibodies indicated that fractions 3-6 contained significant quantities of ATP7A proteins (Figure 4.3B). Concentrated fractions were analysed by SDS-
PAGE (Figure 4.3C), confirming that fractions 3-6 contained highly purified His-ATP7Awt.
Figure 4.3 Size-exclusion chromatography (SEC) of affinity purified His-ATP7Awt.
A. SEC elution profile of affinity-purified protein. B. WB analysis of SEC fractions with Red and CT77 antibody C. SDS-PAGE analysis of the SEC fractions (concentrated). Fraction samples were concentrated and resolved on 4–12% BIS-TRIS gradient gels, and proteins were visualised by Coomassie Brilliant Blue staining.
51 Other ATP7A variant proteins behaved in a similar way. In each case, the identity of the His-
ATP7A proteins was confirmed by WB analysis using the antibodies against their N-terminal
(Red) and C-terminal (CT77) domains. The purified proteins were stored in MOPS-TRIS buffer (50 mM, pH7.5, 2 mM DDM, 150 mM NaCl, 10% glycerol, and 2 mM ME) at -80C.
For large scale purification of the His-ATP7A, 100-200 ml of Sf21 cells (approximately 1 million cells per ml) were infected (3.8 x 108 copies/plaque forming units (MOI 5) of the
ATP7A baculovirus). Sf21 membrane vesicles containing ATP7A and its variants proteins were solubilized in buffer containing DDM (10 M). Soluble samples were collected by centrifugation (20000 x g for 30 min) at 4 C. The soluble membrane extract then was loaded to an affinity column (1.5 x 10 cm, glass chromatography column, max. vol. 24 ml, cross- sectional area 1.77 cm, low-pressure chromatography, His GraviTrap TALON Superflow Co2+ resin) followed by size exclusion chromatography as described before. It is apparent that highly purified protein is accessible, as shown in Figure 4.4. The concentration of the purified proteins then calculated with NanoDrop (Thermo Fisher) instrument. 0.03-0.05 M of the purified proteins were used in functional assays (Chapter 5).
52
Figure 4.4 WB (Red, N-terminal antibody) and SDS (4 – 12% Bis-Tris gradient) gels of large-scale ATP7A variants purification. C = concentrated.
A. His-ATP7Awt; B. His-ATP7AD1044E; C. His-ATP7AM1311V; D. His-ATP7AP1386S.
4.5 Acyl-phosphorylation activity of ATP7A proteins
Formation of the signature transient acyl-phosphate intermediate is one of the key steps in the catalytic cycle of P-type ATPases.72, 99 To validate the ability of ATP7A variants expressed in
Sf21 cells to form the transient acyl-phosphate intermediate, Sf21 microsomes containing
ATP7A proteins were assayed semi-quantitatively using BODIPY® FL ATP-γ-S (Life
Technologies), a fluorescent chromophore that binds to the active site of the P-type ATPases.
53 The phosphor-enzyme pixel intensity was measured using ImageJ software (version 2.0.0, http://imagej.net). The copper-dependent kinetics was analysed using GraphPad PrismTM
Software (version 8.0.1, http://www.graphpad.com).
4.5.1 Labelling ATP7A microsomes with BODIPY® FL ATP-γ-S.
The microsomes were labelled with this fluorophore for various time periods as indicated in
Figure 4.5-A. Reactions were initiated by the addition of 5 l fluorophore to 45 l mixture of microsome (10 g total) in MOPS buffer (20 mM, pH 6.8, 150 mM NaCl, 5 mM MgCl2, fresh
50 M DTT, 5M CuCl2) and the samples were incubated in ice for different time periods.
Figure 4.5 Labelling ATP7A proteins in microsomes with BODIPY® FL ATP-γ-S.
A. Images of labelled proteins detected at emission maximum 509 nm. B. Rate of acyl-phosphorylation from three independent experiments. C. Comparison of rate of acyl-phosphorylation.
Vertical lines in B,C represent standard errors of the mean (+/-SEM).
54 gels were incubated in gel fixing buffer and exposed in a Typhoon FLA 9500 imager (Life
Science) at an emission maximum of 509 nm. Longer labelling times led to an increase in acyl- phosphorylation (Figure 4.5-A). As expected, the negative control protein His-ATP7AD1044E were able to bind the probe fluorescent form of ATP at low levels only (probably adventitious)
(Figure 4.5-B,C). The abilities of the other proteins to bind the fluorophore do not differ significantly. The His-tag does not appear to affect the labelling process (Figure 4.5-B,C).
4.5.2 Pulse-chase of ATP7A proteins, labelled with BODIPY® FL ATP-γ-S
A pulse-chase experiment is a two-phase technique used to examine cellular processes that take place over a period of time.100 During the pulse phase, cells are exposed to a compound (which may be labelled) that is incorporated into the molecule or pathway being studied.101
To pulse-chase the acyl-phosphate complex, “cold” ATP (1 mM) was added, at a final volume of 50 l to the labelled samples that had been treated with BODIPY® FL ATP-γ-S for 25 sec in ice, as described in the previous section (4.5.1). Reactions were stopped by adding 20% SDS
(5 L) at various time intervals and the proteins resolved by SDS-PAGE on an acid (5.5%) polyacrylamide gel (Figure 4.6-A). The gels were incubated in gel-fixing buffer and exposed to a Typhoon FLA 9500 imager (Life Science).
As seen in Figure 4.6-B-C-D, there are significant differences in pulse change behaviour among the various ATP7A proteins. Taken together, with the exception of negative control
His-ATP7AD1044E, the ATP7A variants expressed in Sf21 insect cells were active and able to form the transient acyl-phosphate intermediate with this probe form of ATP. An increase in acyl-phosphorylation corresponded with longer labelling times and reached a maximum after
25 sec for each protein, independent of reaction rate.
55
Figure 4.6 Pulse chase of fluorescent labelled ATP7A proteins forming transient acyl-phosphate intermediate;
A. Image of Fluorescent labelled ATP7A and ATP7A variants using BODIPY® FL ATP-γ-S followed by pulse chase with unlabelled ATP (one set of experiments is shown). B. percentage of pixel density of bands in A calculated by ImageJ software (version 2.0.0). C. Acyl-Phosphorylation of ATP7A and ATP7A variants is modelled using dissociation kinetics, model: Y=(Y0-NS) *exp (-K*X) + NS. Y0 is the binding at time zero, NS is the binding (nonspecific) at infinite time, both in units of the Y axis. K is the rate constant in inverse units of the X axis. D. Comparison of turnover points ATP7A proteins. The half-life equals ln2 D1044E divided by K. t1/2 for ATP7A is forced to zero since no activity was observed.
56 It is interesting that ATP7Awt and the disease proteins ATP7AM1311V and ATP7AP1386S all show significant ability to form acyl-phosphate complexes, a necessary property for ATPase activity.
It is possible that the disease proteins may be viable ATPases but not effective in either copper transport or protein translocation.
4.6 Estimation of ATP7A protein concentration in prepared microsomes using WB analysis. As discussed in previous sections (4.5.1 and 4.5.2), semi-purified His-ATP7A proteins suspended in microsomes display acyl-phosphorylation activity. There remains an important question: what is the actual concentration of ATP7A versus total proteins embedded in Sf21 microsomes?
To address that question, the ATP7A protein concentrations in various microsome preparations were estimated using WB analysis in the presence and absence of detergent DDM with control of a purified ATP7A protein of known concentration as a calibration standard. Known concentrations of purified ATP7A (10-100 ng) were loaded into an SDS-page gel (4-12% BIS-
TRIS Plus Gels), followed by transfer to a polyvinylidene fluoride (PVDF) membrane using a dry transfer system (iBlot® 2 Transfer Stacks, PVDF, mini) for WB detection. The intensity of each WB band was calculated in units of pixels per square inch (PPI) using ImageJ software
(version 2.0.0, http://imagej.net). A standard curve based on %PPI versus ATP7A concentration was devised (Figure 4.7). The curve is based upon ATP7A concentration and
%PPI (after normalization to the highest band intensity and forcing to zero by removing the background). Also, different concentrations of microsomes of His-ATP7A proteins
(with/without DDM) were loaded onto the same SDS-page gel and blotted, respectively.
Concentrations of ATP7A protein then was calculated and are listed in Table 4.2.
57 These experiments estimated the concentrations of various ATP7A proteins in microsomes quite consistently in the presence and absence of DDM (Table 4.2).
Figure 4.7 Estimation of ATP7A concentration in microsome constructs using WB analysis in the presence and absence of DDM. One set of experiments only is shown in each case). A. WB images and standard curve for purified His-ATP7Awt plus DDM (2 mM, 0.02%), His-tag antibody. B. WB images and standard curve for purified His-ATP7Awt without DDM
C. WB image of Sf21/His-ATP7Awt its variants in the presence of DDM (2 mM, 0.02%)
1. 1 l of Sf21/His-ATP7Awt microsome (3.21 mg/ml) 2. 2 l of Sf21/His-ATP7Awt microsome (3.21 mg/ml) 3. 1 l of Sf21/His-ATP7AD1044E microsome (3.01 mg/ml) 4. 2 l of Sf21/His-ATP7AD1044E microsome (3.01 mg/ml) 5. 1 l of Sf21/His-ATP7AM1133V microsome (3.41 mg/ml) 6. 2 l of Sf21/His-ATP7AM1133V microsome (3.41 mg/ml) 7. 1 l of Sf21/His-ATP7AP1386S microsome (3.13 mg/ml) 8. 2 l of Sf21/His-ATP7AP1386S microsome (3.13 mg/ml)
58 D. WB image of Sf21/His-ATP7Awt and its variants in the absence of DDM.
1. 1 l of Sf21/His-ATP7Awt (3.21mg/ml) 2. 2 l of Sf21/His-ATP7Awt microsome (3.21mg/ml) 3. 1 l of Sf21/His-ATP7AD1044E microsome (3.01mg/ml) 4. 2 l of Sf21/His-ATP7AD1044E microsome (3.01mg/ml) 5. 1 l of Sf21/His-ATP7AM1133V microsome (3.41mg/ml) 6. 2 l of Sf21/His-ATP7AM1133V microsome (3.41mg/ml) 7. 1 l of Sf21/His-ATP7AP1386S microsome (3.13mg/ml) 8. 2 l of Sf21/His-ATP7AP1386S microsome (3.13mg/ml)
Table 4.2 Quantification of ATP7A protein in Sf21/His-ATP7Awt and its variants a
PPI% PPI% [His-ATP7A]t His-ATP7A]t (2.0l (2.0l + DDM g/ml ATP7A in isolated microsome+ microsome) g/ml Sf21 microsomes DDM) His-ATP7Awt (I) 43.1 ± 4 40.5 ± 3 2.4 ± 0.2 2.1 ± 0.1 His-ATP7AD1044E (I) 38.2 ± 5 42.9 ± 4 2.3 ± 0.1 2.4 ± 0.2 His-ATP7AM1113V (I) 48.4 ± 4 49.7 ± 7 2.6 ± 0.5 2.3 ± 0.4 His-ATP7AP1386S (I) 41 ± 2 39.8 ± 5 2.1 ± 0.2 2.2 ± 0.1 His-ATP7Awt (II) 25.5 ± 5 27.9± 3 1.5 ± 0.5 2.0 ± 0.2 His-ATP7AD1044E (II) 33.3 ± 4 31.0 ± 3 1.8 ± 0.1 1.5 ± 0.3 His-ATP7AM1113V (II) 26.7± 5 27.9 ± 5 1.5 ± 0.2 1.9 ± 0.1 His-ATP7AP1386S (II) 31.3 ± 4 37.2 ± 4 2.0 ± 0.2 2.2 ± 0.1 His-ATP7Awt (III) 36.2 ± 3 40.0± 3 2.4 ± 0.3 2.1 ± 0.2 His-ATP7AD1044E (III) 41.3 ± 5 40.8 ± 3 2.1 ± 0.1 2.5 ± 0.2 His-ATP7AM1113V (III) 40.0 ± 5 43.8 ± 5 2.3 ± 0.3 2.4 ± 0.3 His-ATP7AP1386S (III) 38.9 ± 4 40.3 ± 5 2.1 ± 0.2 2.0 ± 0. 3 a The ATP7A concentration in each Sf21 microsome preparation was calculated using the standard curve built, under the same conditions, from the known concentrations of purified ATP7A samples.
(I), (II), (III) different batches of prepared microsomes in Sf21 cells
59 4.7 Summary and concluding remarks
Molecular characterisation (in vitro) of copper transporters requires purified proteins. Two approaches to purification of His-ATP7A proteins were explored in this work. Initially, the expression was undertaken in Sf21 cells and Sf21 microsomes (three different batches) were prepared as described in section 4.2. The presence of His-ATP7A proteins in the membranes was confirmed by WB analysis (Figure 4.1). The concentration of total protein for each batch of microsome preparation was determined using the BCA assay and is summarized in Table
4.1.
These microsomes were then subjected to an acyl-phosphorylation assay including labelling of the constructs (4.5.1) and pulse-chase experiments (4.5.2) to evaluate the ability of the ATP7A proteins in the microsome to bind and exchange ATP, a necessary property of an ATPase.
It is interesting that ATP7Awt and the disease proteins ATP7AM1311V and ATP7AP1386S all show a significant ability to form acyl-phosphate complexes. These preliminary experiments suggest that the disease proteins may be effective ATPases but not effective in copper transport or in protein translocation. In addition, His-ATP7Awt protein and four variants were successfully extracted from Sf21 cells with detergent DDM and purified using His-tag affinity chromatography and size exclusion chromatography.
Since the expressed ATP7A protein can only be a small fraction of the total proteins present in the isolated microsome preparations, the concentration of His-ATP7Awt and its variants were estimated using WB analysis with control of a calibration curve of a purified ATP7A of known concentration (summarized in Table 4.2) as it was described in section 4.6. It is necessary to further quantify the observed activity of ATP7A proteins. This is addressed in the next chapter where the ATPase activities for His-ATP7Awt and its variants (either dissolved in DDM detergent or embedded in microsomes) are evaluated.
60 Chapter 5: ATPase activity of ATP7A proteins using
1H-NMR as a detection probe
5.1 Introduction
P1B-type ATPase enzymes use energy derived from ATP hydrolysis to pump heavy metal ions across cell membranes against thermodynamic gradients.102, 103 Copper ATPases such as
EcCopA, ATP7A and ATP7B are selective for Cu(I) and their catalytic cycles are commonly presumed to follow the Albers-Post model (Figure 1.2).57, 102 ATP hydrolysis provides the driving force for conformational changes of the enzymes between E1 and E2 states of high and low affinity for Cu(I), respectively, and facilitates Cu(I) transport. The hydrolysis yields adenosine diphosphate (ADP) and inorganic phosphate (Pi). Monitoring the evolution of these products or the loss of reactant ATP allows evaluation of ATPase activity of the enzymes.63,
104-106 Many assays are based on detection of inorganic phosphate either by formation of a chromophoric phosphomolybdate anion or by coupled enzyme reactions which are indirect and complex to perform.107 The formation of the phosphomolybdate complex requires acid conditions which may lead to spontaneous hydrolysis of ATP, imposing a high non-enzymic background.108 On the other hand, the coupled enzyme assays require many reagents (for two coupled enzyme reactions) and the ATPase activity may not be the rate-determining step. On top of this, background phosphate is present in many biological reaction conditions. Hence, to reduce these uncertainties, detailed evaluations are essential.109
Recently, it was shown that ATP hydrolysis can be monitored by simultaneous detection of both reactant ATP and product ADP via ion-exchange chromatography or 1H-NMR spectroscopy.74, 110 These approaches are not only insensitive to background levels of phosphate and other contaminations, but also present the possibility of using ubiquitous and
61 convenient phosphate buffers as they do not interfere with the detection of either ATP or ADP.
Anion exchange chromatography, for instance, is an easy and versatile method that separates the three nucleotides ATP, ADP and adenosine monophosphate (AMP) based upon ion exchange using a shallow salt gradient elution under conditions of high performance liquid chromatography (HPLC).110 This Chapter details application of the 1H-NMR approach to study of ATP7A activity. The approach has enabled monitoring of the ATPase activity of His-
ATP7Awt and its variants in real time with improved sensitivity. It was adapted and extended from recent reports (see section 2.25).35, 74
5.2 The principle of the ATPase activity assay by 1H NMR
It is possible to quantify the conversion of ATP to ADP and determine the rates of hydrolysis in real time by comparison of the relative intensities of the H8 proton chemical shifts of ATP and ADP (Figure 5.1).35, 74
Figure 5.1 Schematic illustration of detection of ATP and ADP using 1H-NMR. A. Location of the H8 proton in ATP and ADP. B. The difference in chemical shift positions of H8 in ATP and ADP and the quantitative correlation of their relative intensities to their relative concentrations. In the absence of an ATPase enzyme, the percentage of ATP in solutions containing different ratios of ATP (50 M) and ADP (50 M) in Na-MOPS buffer (50 mM, pH 7.5) remained unchanged, i.e., there is no background hydrolysis under these conditions.
62 The relative concentrations of ATP and ADP in the same assay solution are seen to correlate proportionally to the intensities of their H8 chemical shift peaks, obtained by integration of the respective peak (Figure 5.1B).74 Consequently, the percentage decrease in ATP concentration over time can be obtained by plotting the percentage of the ATP intensity over the sum of the
∫ 푨푻푷 푯ퟖ 풑풆풂풌 ATP and ADP intensities (i.e.: ATP % = x 100) relative to the ∫ 푨푻푷 푯ퟖ 풑풆풂풌 +∫ 푨푫푷 푯ퟖ 풑풆풂풌 reaction time. The gradient of the linear plot can be converted to the ATPase activity of the enzyme under specific conditions. The activity is presented as molar turnover number of ATP by His-ATP7A proteins per min. As seen in Figure 5.1B, the percentage of ATP was monitored in different samples including 100% ATP, 50% ATP + 50% ADP and 100% ADP without any enzyme to confirm that the 1H-NMR instrument was able to detect the H8 protons of both the
ATP and ADP molecules and that ATP is stable under the experimental conditions specified in the Figure legend.
The following general protocol was adapted from recent reports and used for each experiment.35, 74 The reaction mixture (0.8 mL) contained: ATP (50-400 M, depending on the assay), MgCl2 (in 1:1 ratio to ATP, 50-400 M), NaCl (50 mM), D2O (10 % v/v), CuSO4 ( 10
μM), BCA (100 μM), GSH (25 M), ascorbate (500 μM, as a reductant), Na-MOPS buffer (50 mM, pH 7.5). Within experimental error, no hydrolysis of ATP was observed under these conditions. Upon addition of a specific ATP7A enzyme (to a final concentration in the range of 0.03-0.05 M) to initiate the catalysis, the reaction mixture was transferred immediately into an NMR tube and the reaction monitored by the NMR spectroscopy. To improve the signal to noise (S/N) ratio, 8 and 16 consecutive scans were taken for each reading at a total scan time of approximately 1.03-1.57 min for each complete spectrum. Using TopSpin 4.0.6 App, the peak areas corresponding to ATP and ADP were integrated to calculate the percentage of ATP hydrolysis.
63 5.3 Optimization of reaction conditions for ATP7A enzymes
ATPase activity can be affected by several factors including temperature, assay components and the concentration of each component. The enzymes may exhibit higher activity under a particular set of conditions, while other suboptimal conditions may lead to a decease or even a complete loss of the enzyme activity. For instance, increasing temperature may accelerate the reaction rate, while decreasing temperature may slow it down.111 However, too high temperatures can lead to enzyme denaturation.112 Therefore, conditions were optimized to obtain the physiologically relevant and, importantly, reproducible activities of ATP7A and its variants (either dissolved in DDM detergent or embedded in microsomes) for a reliable comparative structure-function study.
5.3.1 Effect of enzyme concentration on the catalytic rate of ATP hydrolysis
Enzyme concentration will affect the enzyme activity.90 While there is sufficient substrate to bind to the enzyme, increasing the concentration of enzyme will accelerate the reaction rate.113
In order to determine the enzyme activity under a set of specific assay conditions, the amount of enzyme used for the assay must fall within a certain concentration range to ensure that the catalytic rate varies with the enzyme concentration linearly and this can be satisfied only when the reaction is zero order on substrate, i.e., under substrate saturation conditions.
Since expression and purification of membrane proteins is challenging, it is important to use the purified enzyme efficiently. Different concentrations of the ATP7A enzyme (either dissolved in DDM detergent or embedded in microsomes) were used to find the optimum working concentration under a given set of conditions. As shown in Figure 5.2, under the assay conditions specified in the figure legend with ATP at 200 µM, the NMR approach can monitor
64 the catalytic reaction sensitively. The catalytic rate increased linearly with the enzyme concentrations in the range 0 – 0.05 µM.
Figure 5.2 Linearity of the catalysis of purified His-ATP7A variants and Sf21/His-ATP7A microsomes with different concentrations of enzyme. A. Catalytic time course for purified His-ATP7Awt. B. Catalytic time course for Sf21/His-ATP7Awt microsomes. C. Linear correlation of reaction rate vs enzyme concentration for purified His-ATP7Awt. D. Linear correlation of reaction rate vs enzyme concentration for Sf21/His-ATP7Awt microsomes.
Other components: ATP (200 M), MgCl2 (200 M), NaCl (100 mM), D2O (10 %; v/v), GSH (100 µM), ascorbate
(100 M), Na-MOPS buffer (50 mM, pH 7.5), Cu(I) (10 μM as CuSO4).
65 5.3.2 Effect of ATP concentration on the rate of ATP hydrolysis
The ATPase activity of ATP7Awt and its variants (either dissolved in DDM detergent or embedded in microsomes) at a concentration range of 0 – 0.05 µM increased with ATP concentration and approached a maximum at [ATP] > 100 µM (Figures 5.3, 4). This is consistent with classic Michaelis-Menten enzyme kinetics: the catalytic rate of an enzyme will increase with substrate concentration until all the enzyme reaction sites are saturated by the substrate. For the ATP7Awt enzyme, a set of kinetic constants were derived from these
-1 experiments: Km = 25.8 M, Kcat = 8.7 min for the purified form in DDM and Km = 16.3 M,
-1 Kcat = 6.8 min for the form embedded in microsomes.
Figure 5.3 Catalytic time course for purified His-ATP7Awt with different ATP concentrations. A. Catalytic progress with different concentrations of substrate ATP (M) B. Michaelis-Menten kinetics for substrate ATP -1 (Km = 25.8 M, Kcat = 8.7 min ).
wt Other components: MgCl2 (200 M), His-ATP7A enzyme (0.03 M), GSH (100 µM), NaCl (100 mM), D2O
(10 %; v/v), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5), Cu (I) (10 μM as CuSO4).
66
Figure 5.4 Catalytic time course for Sf21/His-ATP7Awt microsomes with different ATP concentrations. A. Catalytic progress with different concentrations of substrate ATP (M) for Sf21/His-ATP7Awt microsomes. B. -1 Michaelis-Menten kinetics for substrate ATP (Km = 16.31 M, Kcat = 6.8 min ).
wt Other components: MgCl2 (200 M), Sf21/M/His-ATP7A enzyme (0.03 M), GSH (100
µM), NaCl (100 mM), D2O (10 %; v/v), ascorbate (100 M), Na-MOPS buffer (50 mM, pH
7.5), Cu (I) (10 μM as CuSO4).
For any meaningful comparison of the catalytic activities of different enzyme forms, the assay must be conducted under substrate saturation conditions, i.e., zero order reaction rate for the substrate concentration. Then, the catalytic rate depends on the enzyme concentration only (see
Section 5.3.1). Consequently, an ATP concentration of 200 µM was chosen for all activity assays in this work to ensure the catalysis is conducted under ATP saturation conditions.
5.3.3 Effect of MgCl2 concentrations on the rate of ATP hydrolysis
Mg2+ is essentional for the biological activity of ATPase enzymes as it is needed to stabilize
ATP polyphosphates for optimal reaction.114, 115 Hence, the Mg2+ concentration has impact on the activity of the ATP7A enzyme. For this reason, it is also important that the concentrations of metal-chelating ligands such as EDTA are carefully controlled to ensure a sufficient supply of Mg2+ to the ATP substrate. As shown in Figure 5.5, the catalysis rate was affected at ratios
116 D1044E of MgCl2 to ATP of less than 1, but not at higher ratios, as seen previously. His-ATP7A
67 variant and Sf21 microsomes were included in the experiment as controls. As expected, the rate of ATPase reaction for those controls was negligible. All assays in this work were carried out with the MgCl2 concentration equal to the ATP concentration.
Figure 5.5. Optimization of MgCl2 concentration for ATP7A enzymes. A. Catalytic time course for purified His- wt D1044E ATP7A and its inactive variant His-ATP7A with different concentrations of MgCl2. B. Similar plot for
Sf21/His-ATP7A microsomes.
Other components; ATP (200 M), NaCl (150 mM), D2O (10 %; v/v), GSH (100 µM), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5) ), Cu(I) (10 μM; added as CuSO4), purified His-ATP7A variants and ATP7A microsome variants (0.03M).
5.3.4 Effect of temperature on ATPase activity
Previous studies have shown that catalytic ATP hydrolysis is temperature dependent.117-119 This is confirmed in this work for ATP7A enzymes (Figure 5.6). Therefore, it is important to conduct the activity assay under a constant known temperature for the activity comparison. All the experiments in this work were conducted at 37 C since, at this temperature, the enzymes exhibit high activity without denaturing within the experimental time frame.
68
Figure 5.6 Optimization of temperature for ATP7A enzyme purified His-ATP7A variant and Sf21/His-ATP7A microsomes. A. Catalytic time course for purified His-ATP7Awt at various temperature. B. Catalytic time course for Sf21/His-ATP7Awt microsomes at various temperature.
Other components; ATP (200 M), NaCl (150 mM), D2O (10 %; v/v), GSH (100 µM), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5) ), Cu(I) (10 μM; added as CuSO4), purified His-ATP7A variants and ATP7A microsome variants (0.03M).
5.3.5 Effect of Cu(I)-stabilising ligand GSH on ATPase activity
In previous in vitro assays, the required copper was added to the assay solution as Cu(II), together with reductants such as ascorbic acid120 or tris (2-carboxyethyl) phosphine (TCEP)121 to reduce Cu(II) to Cu(I). However, Cu(I) is easily oxidized back to Cu(II) since it is thermodynamically unstable.122 Hence, to prevent this oxidation, it is essential to add a ligand that can stabilize Cu(I) in aqueous solution under aerobic conditions.123 Glutathione (GSH)
- was used in this study as a background Cu(I)-stabilising ligand. It binds Cu(I) as [Cu4(GH)6]
-15 124 with Kd ~ 10 M. It is present in many cellular compartments such as the cytosol and nucleus. It is the most ubiquitous light-meromyosin thiol found in eukaryotes.125 Among its diverse functions126, it can be an effective reductant for Cu(II), although the primary reductant used in this work was ascorbate.
69 As shown in Figure 5.7, the presence of GSH increases the rate of ATPase of the ATP7A protein considerably, relative to a control lacking GSH or any other Cu(I)-stabilising ligand.
Hence, GSH (100 µM) is included in all assays as a background Cu(I) ligand. The ATPase activities of ATP7AWT and its three protein variants in media containing 100 µM GSH will be analysed systematically in section 5.4.1.
Figure 5.7 Catalytic time course for purified His-ATP7Awt and Sf21/His-ATP7Awt microsomes with and without GSH. A. Rate of ATPase reaction for purified His- ATP7Awt with (100 M) and without GSH. B. Rate of ATPase reaction for Sf21/His-ATP7Awt microsomes with (100 M) and without GSH.
Other components : ATP (200 M), MgCl2 (200 M), NaCl (100 mM), D 2O (10 %; v/v), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5), Cu(I) (10 μM; wt wt added as CuSO4) and purified His-ATP7A and Sf21-His-ATP7A microsome variants (0.03M).
5.3.6 Effects of Cu(I) concentration on the rate of ATP hydrolysis
The impact of Cu(I) concentration on the rate of ATPase reaction was evaluated for the ATP7A enzyme (either dissolved in DDM detergent or embedded in microsomes), as shown in Figure
5.8. The ATP7A enzyme was active even in the presence of a small amount of Cu(I).
70
Figure 5.8 Catalytic time course for purified His-ATP7Awt and Sf21/His-ATP7Awt microsomes with and without copper, with [HVO4]2- (0.2 mM) as inhibitor. A. Rate of ATPase reaction for purified His-ATP7Awt and its variant with and without of Cu(I). B. Rate of ATPase reaction for Sf21/His-ATP7Awt and its variant microsomes with and without of Cu(I).
Other components : ATP (200 M), MgCl2 (200 M), NaCl (100 mM), D2O (10 %; v/v), GSH (100 µM), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5), Cu(I) (10 μM; added as
CuSO4) and purified His-ATP7A variants and ATP7A microsome variants (0.03M).
Experiments were conducted by adding 10 M Cu (as CuSO4) in the presence of 100 µM GSH as Cu(I) ligand. Notably, ATP7A enzyme was inactive in the presence of vanadate that inhibits
ATP binding to D1044.
5.4 Analysis of ATPase activity of ATP7A proteins under different levels of Cu availability Although many recent studies (in vivo) have shown that ATPase activity of copper ATPase can change in the presence of different metals,56, 127-130 there are few studies that evaluate the
ATPase activity of copper pumps in vitro.35, 131 This work aimed to evaluate the ATPase activity of ATP7A and its variants .in the presence of various Cu(I) binding ligands. The latter included the background Cu(I) ligand GSH (discussed above in section 5.3.5) and other Cu(I)
71 ligands of varying affinity that will be presented and discussed next. These include
-12 132 -18 bicinchoninic acid (BCA; Kd ~ 10 M) , bathocuproine disulfonate anion (BCS; Kd ~ 10
132 -19 133 M) , the N-terminal metal binding domain 1 of EcCopA (MBD1; Kd ~ 10 M) , metal
-17 134 biding domain 6 of human Wilson disease protein (WLN6; Kd ~ 10 M) and the copper
-18 134 chaperone ATOX1 (Kd ~ 10 M).
The ATPase activity was calculated by dividing the rate of the ATP hydrolysis reaction for each sample by the concentration of enzyme. The activities of ATP7Awt and the M1311V,
P1386S and D1044E mutations (either purified ATP7A dissolved in DDM detergent or the one embedded in Sf21 microsomes) were then compared. The experimental results are summarised in Table 5.1 and 5.2 and presented systematically below.
5.4.1 ATPase activity in the presence of GSH with/without copper
ATPase activity of His-ATP7Awt and its variants (either purified ATP7A dissolved in DDM detergent or the one embedded in Sf21 microsomes) were investigated in the presence of GSH
(as Cu(I) supporting ligand) under different conditions (Table 5.1 and Table 5.2, Expt No 1-4).
As shown in section 5.3.5, adding 100 l of GSH increase the rate of ATPase reaction for His-
ATP7Awt. However, His-ATP7AD1044E variant (negative control) was not active in the presence or the absence of copper as expected. On the other hand, His-ATP7AM1311V and His-
ATP7AP1386S presented higher ATPase activity in the presence GSH and Cu(I), as expected
(Table 5.1 and Table 5.2, Expt No. 2 Vs Expt No. 4). Figure 5.9 demonstrates the ATPase
wt activity of His-ATP7A and its variants in the presence of GSH with Cu(I) (added as CuSO4)
(Table 5.1 and Table 5.2 Expt No. 2) and without copper (Table 5.1 and Table 5.2, Expt No.
4).
72 wt 2- Moreover, none of the His-ATP7A and its variants were active in the presence of [HVO4]
(0.2 mM) as an inhibitor (Table 5.1 and Table 5.2 Expt No. 3). This again confirms the vital role of copper in the ATP7A enzyme ATPase activity. Also, these experiments highlight the role of GSH in stabilizing the Cu(I).
Table 5.1 ATPase activities of purified ATP7A and its variants dissolved in detergent DDM using 1H-NMR as the detection probe a
-1 b,c Expt Cu(I) ligand [L]tot [Cu]tot ATPase (min )
No. (L) (M) (M) wt D1044E M1311V P1386S 1 GSH 100 10 0 d 0 d 0 d 0 d 2 GSH 100 10 19 ± 2.5 0.2 ± 0.1 18 ± 2.5 15 ± 1.5 3 GSH 100 10 0.6 ± 0.2e 0.4 ± 0.2e 0.3 ± 0.1e 0.3 ± 0.4e 4 GSH 100 0 3 ± 0.5 0.6 ± 0.1 2 ± 1 1 ± 2.5 5 GSH/BCS 100/100 0 0.4 ± 0.02 0.5 ± 0.2 0.1 ± 0.2 0.2 ± 0.2 6 - 0 10 8 ± 1.5 0.3 ± 0.1 7 ± 2.5 5 ± 1 7 GSH/BCA 100/25 10 20 ± 3.5 0.9 ± 0.2 18 ± 1.5 15 ± 2.5 8 GSH/ATOX 100/25 10 24 ± 3 0.7 ± 0.2 21 ± 2.5 18 ± 1.5 9 GSH/BCS 100/25 10 11 ± 1 0.3 ± 0.1 10 ± 0.5 8 ± 1.5 10 GSH/WLN6 100/25 10 12 ± 2 0.4 ± 0.1 11 ± 2.5 9 ± 1 11 GSH/MBD1 100/25 10 11 + 2.5 0.7 ± 0.2 9 ± 1.5 8 ± 2.5
a Other components in the assay mixture: ATP (200 M), MgCl2 (200 M), NaCl (100 mM),
D2O (10%; v/v), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5), Cu(I) (10 μM; added as CuSO4); b Unless indicated, the enzyme concentrations used were in the range 0.03 – 0.05 µM; c ATPase activities were analysed using GraphPad PrismTM Software (version 8.0.1, http://www.graphpad.com); d no enzyme control; e 2- ATP7A and its variants were pre-mixed with [HVO4] (0.2 mM).
73 Table 5.2 ATPase activities of ATP7A and its variants isolated in microsomes using 1H-NMR as the detection probe a
-1 b,c Expt Cu(I) ligand [L]tot [Cu]tot ATPase (min )
No. (L) (M) (M) wt D1044E M1311V P1386S 1 GSH 100 10 0 d 0 d 0 d 0 d 2 GSH 100 10 13 ± 4 1 ± 1.5 12 ± 2.5 10 ± 1.5 3 GSH 100 10 0.1± 0.04 e 0.1± 0.05 e 0.1± 0.2 e 0.1± 0.3 e 4 GSH 100 0 2 ± 1.5 1 ± 0.5 1 ± 0.5 0.5 ± 0.1 5 GSH/Bcs 100/100 0 0.1± 0.05 0.1 ± 0.4 0.1± 0.5 0.1 ± 0.3 6 - 0 10 5 ± 3 1 ± 0.5 5 ± 1.5 4 ± 1.5 7 GSH/Bca 100/25 10 12 ± 4 1 ± 0.5 11 ± 2.5 10 ± 1.5 8 GSH/Atox1 100/25 10 17 ± 5.5 1 ± 0.4 16 ± 2.5 14 ± 2 9 GSH/Bcs 100/25 10 8 ± 5 1 ± 0.3 8 ± 3.5 7 ± 1 10 GSH/WLN6 100/25 10 7 ± 4 1 ± 0.5 6.5 ± 3.5 6 ± 1.5 11 GSH/MBD1 100/25 10 6 ± 3.5 1 ± 0.5 6 ± 1.5 6 ± 2.5
a Other components in the assay mixture: ATP (200 M), MgCl2 (200 M), NaCl (100 mM),
D2O (10%; v/v), ascorbate (100 M), Na-Mops buffer (50 mM, pH 7.5), Cu(I) (10 μM; added as CuSO4); b Unless indicated, the enzyme concentrations used were in the range 0.03 – 0.05 µM; c ATPase activities were analysed using GraphPad PrismTM Software (version 8.0.1, http://www.graphpad.com); d no enzyme control; e 2- ATP7A and its variants were pre-mixed with [HVO4] (0.2 mM).
74
Figure 5.9 ATPase activity of ATP7A and its variants in presence of GSH with/without copper. A. ATPase activity of ATP7A and its variants in presence of GSH (100 M) with CuSO4 (10 M). B. A. ATPase activity of ATP7A and its variants in presence of GSH (100 M) without copper.
Other components : ATP (200 M), MgCl2 (200 M), NaCl (100 mM), D2O (10 %; v/v), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5) and purified His-ATP7A variants and ATP7A microsome variants (0.03M).
5.4.2 ATPase activity in the presence of GSH with the addition of BCA
The activity of the wild type ATP7A enzyme was evaluated in the presence of BCA (see Table
5.1 and 5.2, Expt No. 7). As shown in Figure 5.10, there is a little difference in rate in the presence and absence of BCA. It is probable that the Cu(I) affinities of GSH and BCA are comparable under the conditions and that, in the presence of ascorbate reductant, the functions of GSH and BCA are redundant. Indeed, the catalytic rate remained little changed upon increasing the BCA concentration to 100 µM or upon a complete removal of GSH in the presence of Bca. These experiments suggest that while both GSH and BCA can provide effect protection for Cu(I) and that their Cu(I) affinities are not high enough to restrict Cu(I) availability to the enzymes.
75
Figure 5.10 Catalytic time course for purified His-ATP7Awt with (100 M) and without BCA in presence of (GSH 100 M). A. Rate of ATPase reaction for purified His-ATP7Awt with (25 M) and without BCA. B. ATPase activity of ATP7A and its variants with BCA (25 M)
Other components : ATP (200 M), MgCl2 (200 M), NaCl (100 mM), D2O (10 %; v/v), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5), Cu(I) (10 μM; added as CuSO4) and purified His-ATP7Awt and Sf21-His-ATP7Awt microsome variants (0.03M).
5.4.3 ATPase activity in the presence of GSH with addition of BCS
-17 The ATPase activity of ATP7A enzyme was evaluated in the presence of BCS (Kd ~ 10 M)
(see Figure 5.11; Expts 2 versus 9 in Table 5.1, 5.2,). The ATPase activity of ATP7A enzyme and its variants decrease in presence of BCS, suggesting that Bcs possesses a sufficiently high affinity for Cu(I) that it restricts Cu(I) availability for the ATP7A enzyme.30
Notably, the relative ATPase activities between ATP7Awt and its three protein variants remained essentially unchanged, consistent with minimal disruption of these mutations to the
Cu(I) binding and transport pathways. Indeed, these mutation sites (D1044, M1311, P1386) are all remote from the potential Cu(I) binding and transport sites (see Figure 5.11B134).
76
Figure 5.11 Evaluation of His-ATP7Awt and its variants (dissolved in DDM and embedded in microsomes) in the presence and absence of BCS. A. Catalytic time course for purified His-ATP7Awt with BCS (25 M). B. ATPase activity of ATP7A and its variants with BCS (25 M)
Other components : ATP (200 M), MgCl2 (200 M), NaCl (100 mM), D2O (10 %; v/v), GSH (100 µM), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5), Cu(I) (10 μM; added as CuSO4), purified His-ATP7A variants and ATP7A microsome variants (0.03M).
5.4.4 ATPase activity in the presence of GSH with addition of WLN6
Human Wilson protein (ATP7B) has six N terminal cytosolic copper binding domains (WLN1-
6). The first four domains are involved in acquiring of copper from copper chaperone ATOX1, while WLN5-6 are vital for copper transport across the cell membrane.135, 136 As shown in
Figure 5.12 that compared experimental results of Expts No. 2 versus 10 in Table 5.1 and 5.2, the ATPase activities of ATP7A enzyme and its variant in the presence of WLN6 were comparable to those in the presence of Bcs but were lower than those in the presence of Bca, under the same general assay conditions. This again can be attributed to the high affinity of
-17 134 WLN6 for Cu(I) (KD = 10 M) that restricts the Cu(I) availability for the enzyme. Again, the relative ATPase activities of ATP7Awt and its three variants remained essentially unchanged, consistent with the modulation of the enzyme activities by WLN6 by restriction of
Cu(I) availability to the enzymes only.
77
Figure 5.12 Evaluation of His-ATP7Awt and its variants ATPase (dissolved in DDM and embedded in microsomes) in presence of WLN6. A. Catalytic time course for purified His-ATP7Awt with WLN6 (25 M). B. ATPase activity of ATP7A and its variants with WLN6 (25 M).
Other components : ATP (200 M), MgCl2 (200 M), NaCl (100 mM), D 2O (10 %; v/v), GSH (100 µM), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5),
Cu(I) (10 μM; added as CuSO4), purified His-ATP7A variants and ATP7A microsome variants (0.03M).
5.4.3 ATPase activity in the presence of GSH with addition of EcCopA MBD1
There are two metal binding domains in EcCopA (MBD1-2) which can bind Cu(I) with high
−19 133 affinity (Kd ~ 10 M). As shown in Figure 5.13, the catalytic time course and ATPase activity of wild type ATP7A was evaluated in the presence of MBD1. The ATPase activity of
ATP7A enzyme and its variant reduced by adding the MBS1. Although the Cu(I) affinity of
MBD1 is comparable to that of WLN6, the Cu(I) affinity (considering Kd) of MBD1 is much higher than that of GSH (Figure 5.13, Table 5.1 and Table 5.2, Expt No. 11).
78
Figure 5.13 Evaluation of His-ATP7Awt and its variants ATPase (dissolved in DDM and embedded in microsomes) in presence of MSD1. A. Catalytic time course for purified His-ATP7Awt with MSD1 (25 M). B. ATPase activity of ATP7A and its variants with MSD1 (25 M).
Other components : ATP (200 M), MgCl2 (200 M), NaCl (100 mM), D 2O (10 %; v/v), GSH (100 µM), ascorbate (100 M), Na-Mops buffer (50 mM, pH 7.5),
Cu(I) (10 μM; added as CuSO4), purified His-ATP7A variants and ATP7A microsome variants (0.03M).
5.4.4 ATPase activity in the presence of GSH with addition of ATOX1
ATOX1 is the natural Cu(I) chaperone for ATP7A and ATP7B enzymes. It is known that this chaperone can exchange Cu(I) with the six N-terminal MBDs of ATP7A or ATP7B by a series of Cu(I)-ligand exchange intermediates.42, 137 Interaction between Atox1 and ATP7A/ATP7B has been investigated both in vivo and in vitro.138-140
In this work, the ATPase activities of ATP7A and its variants were evaluated in the presence of ATOX1. The experimental data (see Table 5.1 and Table 5.2, Expt No. 8) demonstrated that the ATPase activity was significantly higher in the presence of ATOX1 than in the presence of any of the other Cu(I) ligands tested (Figure 5.14). Although the Cu(I) affinity of ATOX1 is higher than that of GSH and is comparable to those of WLN6 and EcCopA MBD1, ATOX1 is positively charged at biological pH values and is the native copper chaperone for ATP7A. It is
79 optimised for interaction with the six negatively charged MBDs of ATP7A and for efficient donation of Cu(I). Negatively charged WLN6 and EcCopA MND1 are not able to interact as efficiently.
Figure 5.14 Evaluation of His-ATP7Awt and its variants ATPase (dissolved in DDM and embedded in microsomes) in presence of ATOX1 A. Catalytic time course for purified His-ATP7Awt with ATOX1(25 M). B. ATPase activity of ATP7A and its variants with ATOX1(25 M).
Other components : ATP (200 M), MgCl2 (200 M), NaCl (100 mM), D 2O (10 %; v/v), GSH (100 µM), ascorbate (100 M), Na-MOPS buffer (50 mM, pH 7.5),
Cu(I) (10 μM; added as CuSO4), purified His-ATP7A variants and ATP7A microsome variants (0.03M).
80 5.5 Summary and concluding remarks
P1B-type ATPase enzymes (including copper transporting membrane pumps) use energy derived from ATP hydrolysis to transfer heavy metals across the cell membrane against a thermodynamic gradient. ATP hydrolysis yields ADP and inorganic phosphate. Hence, it is possible to investigate the ATPase activity of the enzyme by monitoring the evolution of the products or the reduction of the reactants. ATPase activity assays are commonly based on detection of inorganic phosphate (formation of phosphomolybdate complex or coupled enzyme cascade reaction). However, ATPase activity may not be the total rate determining step using these methods because of either high non-enzymic reaction background or two coupled enzyme reactions.
Ion-exchange chromatography and 1H-NMR are two new approaches that have less background problem. Using these approaches, it is possible to detect and quantify the reactant
ATP and the product ADP simultaneously. This Chapter details improvements carried out to the 1H-NMR (Bruker 600 MHz spectrometer) approach to Cu(I) ATPases that have enabled monitoring of the ATP hydrolysis reaction rate and ATPase activity of His-ATP7Awt and its variants in real time. By using 1H-NMR, it is possible to evaluate the intensities of the H8 proton chemical shift on adenine heterocycle of ATP and ADP (Figure 5.1-A). The prepared
ATP and ADP compositions correlate satisfactorily with their respective NMR peaks’ integration intensity (Figure 5.1-B). The decrease in ATP concentration (percentage, ATP %
∫ 푨푻푷 푯ퟖ 풑풆풂풌 = x100) can be plotted over time where the gradient of the linear ∫ 푨푻푷 푯ퟖ 풑풆풂풌 +∫ 푨푫푷 푯ퟖ 풑풆풂풌 plot can be converted to the ATPase activity of the enzyme.
To obtain the most physiologically relevant and reproducible ATPase activity of ATP7A and its variants (purified or in microsomes), reaction conditions were optimized before conducting the assays.
81 These conditions included:-
1. Enzyme concentration: The catalytic rate of ATP hydrolysis increased linearly with the
enzyme concentration with the certain range (Figure 5.2). A concentration of 0.03 µM
ATP7A enzyme was selected for all assays in this work.
2. ATP concentration: The ATP hydrolysis catalysed by ATP7Awt and its variants follows
the classic Michaelis-Menten kinetics (Figure 5.3 and Figure 5.4). In order to compare
the enzyme activities under different conditions, an ATP substrate concentration at 200
µM was demonstrated to approach the enzyme maximum activity and thus was selected
for all assays in this work.
3. Temperature plus MgCl2, and BCA concentrations (Figure 5.5, 5.6, 5.7): Optimum
conditions were defined as 200 M MgCl2, 25 uM BCA at 37 C.
4. Cu(I) concentration: ATP7A enzymes were able to catalyse ATP hydrolysis in the
presence of 10 M Cu(I). There was slight ATP hydrolysis in the absence of added
Cu(I) which may be related to the buffers that containing some background copper.
However, no activity was observed in the presence of the inhibitor vanadate (Figure
5.8).
ATPase activities of purified ATP7A and its variants (also ATP7A and its variants embedded in microsomes) were investigated under the different conditions summarized in Tables 5.1 and
5.2. The presence of GSH increased the ATPase activity of the ATP7A protein (Figure 5.9) and it was included in the assay mixture as a stabilising ligand for Cu(I) under aerobic conditions. Figure 5.10 demonstrates there is little difference in the rate of the ATPase reaction in the presence and absence of BCA, attributed to the Cu(I) affinities of GSH and Bca being comparable, under the conditions. As shown in Figure 5.11, the ATPase activities decreased in the presence of BCS since this ligand possesses a very high affinity for Cu(I) and restricts the availability of Cu(I) for the enzyme.
82 The ATPase activities in the presence of Cu(I) binding domains ATP7B WLN6 and EcCopA
MBD1were comparable to those in the presence of Bcs but were lower than with Bca, under otherwise identical assay conditions (Figure 5.10-13). This again can be attributed to the high affinities of these domains for Cu(I) that restricts Cu(I) availability for the enzyme. The
ATPase activity of ATP7A was significantly higher in the presence of its native metallo- chaperone ATOX1 than in the presence of the other Cu(I) ligands discussed above (Figure
5.14). Although the affinity of ATOX1 for Cu(I) is comparable to those of WLN6 and MBD1,
ATOX1 is positively charged and presumably interacts with the negative charged MBDs of
ATP7A, enabling more efficient donation of bound Cu(I). It appears that negatively charged
WLN6 and MBD1 interact less efficiently. These conclusions are consistent with very recent findings that copper exchange in related systems is driven by both the relative affinities of the partners for Cu(I) but also the affinities of the specific interactions between the protein pairs.141
The ATPase activity assays evaluated by 1H-NMR demonstrated that:
• ATPase activity is Cu-dependent;
• ATP7A exhibits its highest activity in the presence of its native metallo-
chaperonATOX1;
• The mutation M1113V only marginally affects ATPase activity of the enzyme;
• The mutation P1386S decreases the ATP7A activity significantly, as expected.
83 Chapter 6: Concluding remarks
6.1 Main principles
Why is it important to study the ATP7A protein at molecular level? ATP7A plays a critical role in copper homeostasis in the human body. Mutations in the ATP7A gene lead to many neurodegenerative diseases such as Menkes' disease (MD), Occipital Horn Syndrome (OHS),
X-linked Distal Hereditary Motor Neuropathy (dHMNx), and Brachial amyotrophic diplegia
(BAD).49 However, the understanding of copper pumps at the molecular level has fallen behind that at the cellular level as considerable challenges remained for isolation of this purified membrane protein. Understanding the molecular mechanisms of the ATP7A enzyme can help improve the current understanding of the aforementioned neurodegenerative diseases and may help development of disease treatments.
Expression of functional ATP7A enzyme remains challenging. E. coli protein expression systems are not a suitable for ATP7A expression since E. coli cannot achieve the necessary post-transitional modifications. Certain studies used a yeast protein expression system, but the final protein size did not match the ATP7A expressed in humans. On the other hand, mammalian protein expression systems are costly and time-consuming. Moreover, despite
(theoretically) high expression levels of membrane proteins expressed using insect or mammalian expression systems, the yields of purified ATP7A enzymes were low. In this work
ATP7A and certain variants were expressed successfully in a baculovirus system using insect
Sf9/Sf21 as host cells.
84 Expression and initial characterisation of ATP7A and its variants. An ATP7A baculovirus expression vector was generated by cloning His-tagged ATP7A into a modified pBacPAK9 transfer vector. A number of variants were included: His-ATP7AWT (positive control), His-
ATP7AD1044E (negative control), His-ATP7AM1311V (BAD mutation) and His-ATP7AP1386S
(dHMNX mutation). DNA sequencing of all constructs confirmed that these genes were successfully cloned into the pBacPAK9low bacterial transfer vector. The vectors were co- transfected with linearized BacPAK6viral DNA into Sf9/Sf21 host cells to generate the ATP7A baculoviruses bYHH1705-9.
Expression of His-ATP7A (180 kDa) and its variants were confirmed with WB analysis using three different antibodies. Approximate concentrations of ATP7A baculovirus constructs
(copies/ml) were estimated (Tables 3.1 and Table 3.2). For rest of the study, the ATP7A protein and its variants were expressed in Sf9/Sf21 insect host cells (50 ml, 100 ml and 200 ml cells cultures with 1x 106 cell per ml, 80% viability) by infection of the cells with the ATP7A baculoviruses bYHH1705-9 (3.8 x 108 copies/plaque forming units (MOI 5)).
Isolation and purification His-ATP7Awt and its variants. They were initially produced in semi-purified form by preparation of Sf21-His-ATP7A microsomes, confirmed by WB analysis. Protein concentrations were estimated by the BCA assay. The proteins were isolated in highly purified form by a two-step procedure: His-tag affinity chromatography followed by size exclusion chromatography. The concentration of purified enzymes was estimated using the Nanodrop instrument and 0.03-0.05 M of proteins were used for functional assays.
Functional assays on ATP7A microsomes and the purified ATP7A proteins. Transient acyl phosphorylation formation in the semi-purified microsomes were investigated using a
85 fluorescent ATP-γ-S probe. All the ATP7A variants were active and able to form transient acyl-phosphate intermediates. An increase in acyl-phosphorylation corresponded with longer labelling times and reached a maximum after 25 sec for each protein, independent of reaction rate. Sf21-His-ATP7Awt and the disease proteins Sf21-His-ATP7AM1311V and Sf21-His-
ATP7AP1386S all show significant ability to form acyl-phosphate complexes, a necessary property for ATPase activity. It is possible that the disease proteins may be effective ATPases but not effective in either copper transport or protein translocation.
ATPase activities of ATP7A and its variants, (purified to homogeneity and embedded in microsomes) were investigated under the different conditions summarized in Tables 5.1 and
5.2. Addition of GSH induced an increase the ATPase activity. There was little difference in the reaction rates of ATP7A proteins in the presence and absence of BCA. This may be due to the Cu(I) affinities of GSH and BCA being comparable.
The ATPase activities decrease in the presence of the Cu(I) high affinity ligand Bcs. It has been observed that Bcs restricts the availability of Cu(I) for the EcCopA ATPase35 and this appears to also be true for ATP7A. The activities in the presence of domains WLN6 and MBD1 were comparable to those in the presence of BCS and lower than those with BCA. This again can be attributed to the high affinity of these domains for Cu(I) that restricts the Cu(I) availability for the enzyme.
The activities were significantly higher in the presence of ATP7A’s natural Cu(I) chaperone
ATOX1 than in the presence of the other Cu(I) ligands. The affinity of ATOX1 for Cu(I) is much higher than that of GSH and is comparable to those of WLN6 and EcCopA MBD1. Atox1 is positively charged and is the native copper chaperone for ATP7A. It appears to interact
86 efficiently with the negative charged MBDs of ATP7A, allowing optimal donation of its bound
Cu(I). In contrast, negatively charged WLN6 and EcCopA MND1 do not interact as efficiently.
6.2 Final conclusion and suggestion for future research
The primary conclusion of this work is that the ATPase activities for binding of Cu(I) by the disease-related variants ATP7AM1311V and ATP7AP1386S are significant. The two mutations are associated with, respectively, X-linked Distal Hereditary Motor Neuropathy (dHMNx), and
Brachial amyotrophic diplegia (BAD). Hence, the problems with these variants would appear to be associated with other aspects of the transport of Cu(I) across the Golgi or plasma membranes or in ATP7A progression thorough the secretary pathway between those membranes.
It is desirable to know which of the above weaknesses is the primary problem. Exploration of the secretary pathway requires expertise in molecular biology. At the present moment in time, molecular aspects of Cu(I) transport can be best be studied in vectorial systems such as giant unilamellar vesicles (GUVs), pioneered recently.35 Attention to both aspects will lead to a better understanding of the mechanisms of the ATP7A enzyme.
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96 Appendix
Figure A. Partial gene synthesis to introduce N-terminal His8-TEV tag
97 Table A . Primers used in this work Primer name Primer sequence (5'->3') #-mer GC content, % Tm ATP7A_SeqF1 CAG CTA TGA CCA TGA TTA CGC 21 47.6 60.7 ATP7A_SeqF2 GCT GAA GAC CAA GGG TGT 18 55.6 61.4 ATP7A_SeqF3 GGT CAC AGC AAA GGA GTC 18 55.6 59.6 ATP7A_SeqF4 CCA GGT GTA AAA TCC ATA CGA G 22 45.5 60.4 ATP7A_SeqF5 CCA ATG ATA GCA GAG TTC ATC C 22 45.5 60.5 ATP7A_SeqF6 CTG TAT TCC TGT AAT GGG GC 20 50 59.8 ATP7A_SeqF7 CGA TGG CTG GAA CAT ATA GC 20 50 60.1 ATP7A_SeqF8 GCA GAC AAA CTC AGT GGC 18 55.6 60.5 ATP7A_SeqF9 GCT CCT ATC CAG CAG TTT G 19 52.6 59.9 ATP7A_SeqF10 CAT TGT GGG AAC TGC TGA AAG 21 47.6 61.6 ATP7A_SeqF11 GAG AGA AAA GGT CGG ACT GC 20 55 62 ATP7A_SeqF12 CTG GAT GTA GTG GCA AGT ATT G 22 45.5 60.7 ATP7A_SeqF13 CGC TCC CTA AAC AGT GTT G 19 52.6 60.3 ATP7A_SeqR1 CAA GGC GAT TAA GTT GGG TAA C 22 45.5 61.4 ATP7A_SeqR2 CAG CAA ACC CAG TTT AGG AG 20 50 60.4 ATP7A_SeqR3 CTT GCC ACT ACA TCC AGA AG 20 50 59.9 ATP7A_SeqR4 GAC CAT TTC TAA TCA TCC ACT CC 23 43.5 60.6 ATP7A_SeqR5 CTC TCC ACC TTT TAT TAG TAT GCC 24 41.7 60.7 ATP7A_SeqR6 GAA TGT CCT TCA ATA ACA CGA C 22 40.9 59.3 ATP7A_SeqR7 CCA GGA ACA TAG AAG AAT GAA GG 23 43.5 60.6 ATP7A_SeqR8 CAC CAT CTC CTT CAT CAG C 19 52.6 59.6 ATP7A_SeqR9 GTT TCT GGA GAG GTT AGT AGA GG 23 47.8 61.1 ATP7A_SeqR10 GGC TGT TGA ATC ATT GGT ATA CG 23 43.5 61.5 ATP7A_SeqR11 CCA GCT CTT TTA TCT GAT TGG C 22 45.5 61.3 ATP7A_SeqR12 CAA GTC ATA CCC TCA ACA GAA ATG 24 41.7 61.5
98
Minerva Access is the Institutional Repository of The University of Melbourne
Author/s: Tondfekr, Reza
Title: Molecular characterization of the ATP7A protein and selected mutants
Date: 2020
Persistent Link: http://hdl.handle.net/11343/258506
File Description: Final thesis file
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