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Carboxyatractyloside resistant human Mitochondrial ADP/ATP

carrier, a novel, quick and efficient selection marker with wide rang

application in cell and gene therapy.

Shahram Khaleghi

Department of Medicine

Karolinska Institute Hddinge. Stockholm, Sweden

Supervisor: Evren Alici

1

CONTENTS

1- Introduction

1-1 -Graft versus host disease

1-2 –suicide genes

1-3 –selection markers

2- Mitochondrial AAC, a novel selection marker

2-1-structure

2-2-ADP binding site

3- AAC specific inhibitors

3-1- CATR

3-2- CATR binding site

3-3- CATR binding capacity in mutants

3-4-resistant molecules

4- Aims

4-1- specific aims

5- Material and Methods

5-1- DNA extraction

5-2- PCR amplification

5-3- cloning and transfection

5-4-PCR mutagenesis

5-5-retroviral production and transfection

5-6-analysis of transfected cell's viability in presence of CATR

5-7-primery human mononuclear cells viability assay in presence of CATR

6- References

1-Introduction

2

Overview

The cell and gene therapy center in Karolinska Institute is a part of Hematology

division located in Huddinge this unit consists of a principle investigator, Evren Alici

which is the group leader, a postdoc researcher, 3 GMP certified biomedical

scientists, a phD student and one biomedical engineer.

The center has access to GMP facilities, exclusive cell culture laboratory and GMP

grad multi parameter flowcytometry instrument and other state-of-the-art-molecular

lab instruments.

This unit has collaboration with Vecura which is producing cells in large scale for

clinical applications also vectors for gene therapy purposes.

The group research field is basically cancer immunotherapy by genetically

modification and expansion of NK cells and Tcells to overcome defective immunity

in malignancies

This research group has been involved in first clinical train for genetherapy in

Sweden as well as several other clinical trials in tumor immunotherapy.

What is Gene Therapy Treatment of errors in the genetic make up of cells is the application of gene therapy.

This defect can be congenital or acquired by mutations in a gene which in some cases

ended up in malignancies. Therefore on the one hand gene therapy tries to restore a

genetic defect and on the other hand to abrogate cancerous cells.

Hematopoietic stem cell transplantation (HSCT) is considered as one of the

therapeutic approaches in many patients suffering from hematological malignancies.

Along with progenitor cells, in graft, there are differentiated immunocompetent

Tcells. These cells are key players in therapeutic aspect of HSCT in which

immunocompetent transplanted cells eliminate residual tumor cells (1).

It was shown in 1960 that in animal model, leukemia mice receiving allogenic graft

had better tumor eradication rate. Graft versus Leukemia effect of HSCT has led to a

shift toward an immunotherapy application (2).

Donor lymphocyte infusion (DLI) is an immunotherapeutic strategy which is

employed in treatment of chronic phase chronic myeloid leukemia (3-4), in

myelodysplastic syndromes and acute leukemias (5). Moreover, in Tcell depleted

graft, DLI has important contribution in efficient engraftment (6) and host residual

disease eradication (7).

Donor derived T cells are important from different aspects. Reconstruction of immune

system in immunocompromised patients after HSCT, graft versus infection and

leukemia are beneficiary functions of these cells. Also these immunocompetent cells

can eliminate remaining patient's immune cells, therefore, enhance efficient

engraftment. On the other hand graft versus host disease is a common complication as

a result of reaction of these cells against host tissues (8)

1-1-Graft versus host disease

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This is an outcome of an immune response mediated by alloreactive donor Tcells

against host tissues. It can be categorized according to its manifestation to acute, late

acute, chronic and acute chronic overlapping forms (8). In acute form three organs

(skin, gastrointestinal tract and liver) are involved and patients develop the disease

within 3 months after transplantation (9).

According to billingham speculation, four prerequisites need to be met for GvHD

development;

1-immunocompromised recipient

2-precense of immunocompetent cells in graft

3-HLA incompatibility between donor and recipient

4-migration of effector cells to target tissues (10).

Graft versus host disease (GvHD) related mortality and morbidity is main concern

related to HSCT and DLI.

The mild GvHD (grad I) with little mortality is not considered as big concern. On

contrary, is a sign of better recognition of tumor cells by donor T cells. More

advanced GvHD (grade II to IV) are associated with high mortality rate. In these

patients and in grade IV cases the mortality rise up to more than 90 percent.

Exploiting desired function of transplanted cells in the absence of unwanted GvHD is

a main challenge in cell transplantation.

In a new approach, allogenic mesenchymal stem cells (MSC) were infused into

patients and clinical symptoms of GvHD disappeared (2, 11).

Figure 1: suicide gene therapy in HSCT. (Adapted from Georgoudaki, Sutlu & Alici,

2010). Donor lymphocytes are transduced with suicide genes and selection marker carrying vector.

The desire outcomes are graft versus leukemia or graft versus infection as well as enhanced

engraftment. the prodrug is administered in case of GvHD development. HSCT: Hematopoietic stem cell transplantation; GMC: Genetically modified cells; GvHD: Graft versus

host disease.

1-2- Suicide Genes:

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All toxin genes which can cause cell death are classified as suicide genes. Genes who

their expression cause cell death like diphtheria toxin and genes which are not toxic

but they product can convert nontoxic prodrugs to toxic substances for cells carrying

these genes.

Cancer suicide gene therapy has been widely studied for a variety or tumors. In this

therapeutic approach, cancer calls are transduced by viral vectors carrying HSVtk, the

most commonly used suicide gene (12, 13).

Suicide genes can be categorized according to their dependence on cell cycle. One

group members are independent to cell cycle and exert their toxicity to all cells

expressing suicide gene whereas, other genes in other group can only exert their effect

on dividing cells (8).

Table 1: suicide genes (adapted from Georgoudaki, Sutlu & Alici, 2010).

ADCC: antibody depended cell cytotoxicity; AZT: 3’-azido-3’deoxythymidine; CD: Cytosine

deaminase; CID: Chemical inducer of dimerization; DED-FADD: Death effector domain of Fas-

associated death domain-containing protein; GCV: Ganciclovir; GPT: Guanine phosphoribosyl

transferase; HSV-tk: Herpes simplex thymidine kinase; iCasp9: Inducible caspace 9; MeP:

MethylpurineMethylpurine; MePdR: Methylpurine deoxyriboside; MP: Monophosphate; NR: Not

reported; Ntr: Nitroreductase; PNP: Purine nucleoside phosphorylase; tmpk: Thymidylate kinase; TP:

Triphosphate; VZV-tk: Varicella zoster virus thymidine kinase.

Tcell suicide gene therapy is an attractive application of suicide genes. these cells are

readily manipulated and get transfected by Moloney murin leukemia virus

(MoMuLV) which is highly lymphotrophic. Application of genetically modified

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lymphocytes with suicide gene has been studied in clinical trial in setting of HSCT

(13).

Suicide gene modification of transplanted cells allowing safe administration and

elimination of self reactive cells in case of GvHD reaction. Application of suicide

gene therapy for GvHD treatment started in 1990 in clinical setting. A population of

83 patients was monitoring for GvHD and 25 patients developed disease following

modified T-Cell administration. All aloreactive T cells and GvHD symptoms

abrogated after administration of prodrug (8).

Since HSVtk/GCV system is more efficient in dividing cells, administration of

prodrug will not affect non reactive transplanted cell and immunocompetency of

patient is not compromised (15).

This system should be effective even in low dose substrate because of disparity in

biodistribution of substrate. Moreover, only genetically modified cells should be able

to convert prodrug to toxic substances.

Herpes simplex virus-1 thymidine kinase:

Herpes simplex virus 1- thymidine kinase gene is the first gene was used and

feasibility of this approach was confirmed by Moolten and colleague in 1986.

This enzyme has ability to phosphorylate gancyclovir an analog for nucleoside and

convert it to a nucleoside monophosphate, which consequently is converted to a

nucleotide three phosphate by cellular kinas (GCV-TP). This nucleotide cause

genomic instability once integrated to genome in addition, GCV-TP block DNA

synthesis by inhibiting DNA polymerase.

HSVtk is the most commonly used suicide gene and comparing to mammalian tk, is

1000- fold more efficient in GCV phosphorylation (17).

.the prodrug, GCV administration dose is maximum 10 mg/kg/day which is toxic

particularly for hematopoietic cells. In addition its effect to abrogate modified cells

take 5-6 days in case of sever GvHD which has high mortality and morbidity (18, 19).

Cytosine deaminase CD/5-Fluorocytosine (5-fu)

In this system a toxic nucleotide analog is produced. This gene derived from either

bacteria or yeast and Mullen in 1992(14) used this gene as suicide gene. Applicability

of this gene has been tried in many research groups. Despite promising preclinical

results partial response in clinical approach brought some debt about using this gene

alone as suicide gene.

1-3- Selection Markers:

To study the function of transgene, the formation of cell lines containing gene of

interest is required. The efficacy of transfection vary according to cell type but it has

been accepted that the efficacy of stable integration is at its best still very low.

Therefore a selection marker to select those transduced cells is indispensable. A

hallmark of ideal selection marker is an easy selection in a variety of cells.

During transfection process, many cells fail to accept vectors. Therefore, enrichment

and selection of genetically modified cells is needed, especially, when suicide gene

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carrying vectors are used. Cells lacking suicide gene will escape crucial suicide gene

control activation step. Infusion of none transduced Tcells increase the risk of

uncontrollable GvHD. Therefore co expression of suicide gene and a selection marker

secure infusion of allogenic cells. for a selection marker to be considered as optimal,

some important properties must be included. Short selection time, immunogenicity,

genotoxicity, cost efficiency; high sensitivity, simplicity of technique and its safety in

clinical setting are important criteria among them. Riddell et al in 1996 reported Tcell

rejection in genetically modified HIV TCD8+ infusion because of expression of

nonself selection marker (20).

Table 2: some selection markers for mammalian cells(55).

Selection marker Comment

Aminoglycoside

phosphotransferase (neo,

G418, APH)

Cells have different sensitivity to G418 and optimization

is required. It is toxic for cells and need long time for

selection.

Bleomycin (phleo, bleo,

zeocin)

It is a bacterial origin marker therefore has the same

downsides as other bacterial derived antibiotic resistant

markers.

Cytosine deaminase

(CDA, CD)

It can also act as a suicide gene. The de novo synthesis

pathway is blocked and cells are forced to rely on CDA

scavenger pathway.

Dihydrofolate reductase

(DHFR)

A MTX resistant mutant of this enzyme provides a good

selection marker for wide range of cells.

Histidinol dehydrogenase

(hisD)

Histidinol is toxic for cells .

Hygromycin-B-

phosphotransferase

(HPH)

The selection process needs high concentration of

Hygromicine. It is a bacterial selection marker.

Puromycin-N-acetyl

transferase (PAC, puro)

Its efficiency is comparable to neo. It has bacterial origin.

Xanthine-guanine

phosphoribosyltransferase

(XGPRT, gpt)

This is a bacterial enzyme which does not have

mammalian homologous.

neomycin

phosphotransferase II

gene(Neo)

Bacterial gene.

Multi drug resistant gene

MDR-1

Transduced cells develop anti cancer drug resistance.

Membrane markers

( FACS sorting system)

It is a slow process and not suitable for clinical setting

when high cell number is required.

Membrane markers

(immunomegnetic beads)

Only high expressing cells are selected and false positive

selection is a big concern.

.

Bacterial derived antibiotic resistant, for example, neomycin phosphotransferase II

gene (NeoR) has been used as selection marker (18). This kind of markers need long

culture time for selection process which negatively affect functional characteristics of

cells and impose major limitation for clinical purpose. Antibiotics are toxic for cells

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and the immunogenicity of these bacterial derived genes is another major limitation.

In biosystronic construct system, the expression of the IRES- linked partner shows

reduction in NeoR system (30).

Antibody based sorting system:

Truncated low affinity grow factor receptor (dLNGFRD) has been used in many

preclinical and clinical studies and no adverse event has been reported (31).

Truncated CD34, which normally is expressed on hematopoietic stem and progenitor

cells have been used as a selection marker. Cytoplasmic region which is recognized

by kinase is missing therefore cytoplasmic signal of binding to ligand is blocked. It

has been shown that this technique can give a high purity of transduced cells (97-

99%) (32, 33).however it has been shown that ecto expression of this molecule affect

cell traffics and cell homing mechanism (34).

Only some of the human Tcells express Thy (CD90) while all Tcells in mice express

this molecule. It has been used as a selection marker without toxicity. Function and

ligand of this molecule has not been identified, So more investigation is required to

study its potential phenotoxicity(35).This molecule is attached to cell membrane by

glycophosphatidylinositol (GPI),therefore can be shed by phospholipase C which

make it unsuitable at least for hematopoietic cells .

CD19 which normally expressed by Bcells and its presence is crucial for their

development, has been used as a selection marker (36). Truncated molecules give

high purity (98%).

These membrane proteins which their cytoplasmic part is missing can be shed into

medium which interfere with detection. High rate of false positive necessitates several

selection procedure which is time consuming also it is harmful for stem cells.

Moreover FACS sorting system is a slow process which is not suitable for clinical

setting when a large number of cells are required.

Magnetic cell separation is more suitable. Selection of cells with Immunomagnetic

beads is used for rapid selection of cells. The beads are activated with antibodies

against specific surface membrane marker however, false positive selection remain a

big challenge .only highly expressing modified cells are selected which significantly

reduce the number of selected modified cells.

Bacterial derived antibiotic resistant genes and other Chemical selections are more

cost effective however they are time consuming and toxic for cells. Recently a new

selection marker has been developed by genetically engineered Na+,K+ ATPase

which ubiquitously is expressed and control basic cellular homeostasis by maintaining

the potassium and sodium gradient in the cell. The Na,K,ATPase/oubain selection

marker has been demonstrated to have a high efficiency(99%). The selection time is

48 hours (2).

Ouabain is a non toxic cardiac medicine which blocks Na+, K+ ATPase.OuaR is

being used as selection marker for human cells. Big gene size is its shortcoming

which reduces packaging efficiency. This marker has rat origin, since this ATPase

shows less sensitivity to Ouabain in rat. Application of rat origin protein in clinical

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setting potentially can trigger immune response which leads to elimination of infused

cells.

2- Mitochondrial ACC, a novel selection marker

Eukaryotic cells need a transport system in their inner mitochondrial membrane to

transfer metabolites from matrix to inner membrane space (IMC) and vice versa. Utter

mitochondrial membrane has more permeability so metabolites can be transferred

between IMC and cytoplasm.

Each of us exchange equivalent to our body mass of ATP every day and this exchange

is achieved by ADP/ATP carrier in inner mitochondrial membrane, so its function is

vital for cells. This carrier is the most studied protein in mitochondrial carrier family

And consists of 298 amino acids in human. The carrier catalysis the exchange of 1mol

ADP for 1 mol ATP and exclude all other nucleotides. The rate of exchange at 30C is

about 30μm/min/g wet wt or 600 μm/min/g mitochondrial proteins. There are 3

isoforms of this protein with different pattern of expression level in different tissues

(ANT1, ANT2 and ANT3)(37, 38, and 39).

The amino acids sequence is highly conserved between species, so that there is 90%

similarity between human and bovine isoforms I which mainly is expressed in skeletal

and cardiac muscle cells. This sequence identity between yeast and bovine carrier is

equal to 50% (39).

However the physiological role of ANT is to exchange ADP with ADP, but it is also

member of mitochondrial permeability transition pore (MPTR) and is a strong

apoptosis inducer. Overexpression of ANT-1 induces apoptosis, however highly

expressed in normal cells but it is inhibited by other proteins. It has been shown that

only NH2 end of carrier is enough for induction of apoptosis.

Surprisingly ANT-2 can not induce apoptosis in spite of 90% similarity with ANT-

1(40).

This protein can trigger apoptosis in two different ways. Mutant carriers which have

lost their transmembrane activity still are able to induce apoptosis. Half of ANT-1 is

dispensable for apoptosis induction and the NH2 terminal half is enough to provoke

apoptosis. Over expressed ANT-1 induces apoptosis in different way from binding of

CATR (40).

Manvel et al (1999) suggested that apoptotic activity of ANT-1 is not by forming an

active pore but is achieved by specific protein- protein interaction. Different levels of

ANT-1 isoforms in different tissues cause different sensitivities to apoptosis.

2-1-STRUCTURE

9

X-ray crystallography to a resolution of 2.2 A, of bovine isoforms I has provided an

insight to its transport mechanism and it has been shown that nucleotide transport is

mediated by conformational changes (39).

ANT consists of six transmembrane helices and both carboxyl and amino termini are

located in inner membrane space (42).

As other mitochondrial carrier, ANT has a three fold duplication structure in three

fold repeat of about 100 amino acids with 15% sequence similarity which are tilted

and form a deep cone –shaped barrel. The cavity has depth of 30A and a maximal

diameter of 20A (44).

Figure 2: mutations in the AAC2 from S. cerevisiae. Residues have been signified as for negative

residues, for positive residues and for neutral residues (45).

Sharp kink in odd numbered helixes is due to proline residues which are located in

conserved sequence, which are characteristic of mitochondrial inner membrane

carrier.

The bovine K22 (38 in yeast) is an unusual basic residue in first helix which occurs at

the same position in all ANTs. This residue is the only one which completely

protected by CATR in structural studies. Also it involves in translocation and

substrate binding site (41).

A cationic cluster comprise of two lysine {bovine (K22, K32)} and Five argentine

{bovine (R79, R137.R234, R235, R279)} are toward the cavity and points to the

solvent (45).

Two basic residues located at the entrance of channel have their contribution to

attracting nucleotides towards their binding site. Tyrosine ladder {(Y186, Y190,

Y194) (bovine)} are important in this regard .it has been shown that Y186 and Y190

(Y203 and Y207 in yeast) replacement with alanine reduce or prevent cellular growth.

10

R236 (R254 in yeast) forms a salt bridge with E264, contributing in conformational

changes through interaction with M3 core(42).

M3 matrix loop has a sequence of 8 amino acids including E264, K267 and K271

which are charged .Y203and Y 207(yeast) are implicated in transferring process and

crucial for carrier function but not for substrate binding. Matrix loop M2 participates

in transport activity so any changes preclude its movement can abolish carrier

function (42,43,and 45).

Intrahelically charged residues are of major importance in sustaining electrically

active transport of ATP. Extra helical lysine binds to cardiolipin head groups and thus

explain specificity of cardiolipin for AAC (46).

There are only 6 charged residues {yeast (K38, R96, R204 and R 294)} located in

transmembrane helixes. It has been shown that any mutation in these residues leave

the carrier inactive. Also all three argentine in third domain {yeast (252-254)} are

essential and any modification case inactivity (39, 41).

Also it has been shown that E45 (yeast) is not required for carrier function and D

26and R88 are dispensable alsoK179and K182 (yeast) can be replaced by uncharged

residues without serious consequences. all cysteines{yeast(C73,C244,C271and C288}

has been modified and it has been shown that they are all non- essential .the proline

247 has been mutated to glycine and it did not affect cell growth. this seems unusual

since proline residues are highly conserved in first, third and fifth helices and also in

all mitochondrial carrier(45).

All mutants who have lost their function involve charged residues. Many neutral

residues have been tested by mutation and they seem to be non essential for activity.

Some revertants have been developed either extragenic or intragenic; however

intragenic revertants are more common. Mutants can be same site or second site

revertants. Same site mutation is achieved by double or triple nucleotide changes in

primary mutation place (R152A resulted in A152K which restoring positive charge).

More interesting revertants are second –site revertants which fall into two categories.

In one of them, neutral residues mutation is replaced by another neutral residue. In

other one they lead to loss of complementary charge. The E45 G or E45Q revertants

are gly+ while their parent R294A is not. The homology between domains is an

interesting feature of ANT.R96 in second transmembrane helix is homologous to

R296 in sixth transmembrane helix. In addition D149 is homologous to E45, while

both have negative charge(47, 48).

Some second site revertants are as follow: R 96A gives three revertants,

Y305H,D26V and C288Y,Also R96L gives S33I .in another attempt, in argentine

residues(252-257) which are highly conserved and any mutations leaves the protein

inactive, each residue was mutated to iLe and they gave many revertant which were

able to grow on glycerol . WhileR252I never gave any revertants, R254I gave 11 and

R253I gave 4.these information reveal a strong relevance of sequence position on the

ability to revert. Accordingly R252 are essential while R253 and R254 nearly not.

Interestingly only one revertant, namely N53I, was on the matrix side as triple

arginine. The others were located either on cytoplasmic side or in the transmembrane

region near cytoplasmic side. Since almost all revertant are located distant from triple

arginine, it can be concluded that mutation in these arginines change the geometry of

11

protein on far side by changing the configuration of 5th

helix bottom. And variety of

revertants is able to restore this defect.

Some of revertant mutant for triplet arginine are the same position as revertant for

R96 mutant. Some revertants are as follow:

S33N revertant of R254I.

D26E revertant of R254I

G30V revertant of R96T, D149G (together) (both of these mutant are gly-)

G30C revertant of R254I

Mutation in R96 or triple arginines has similar effect on structure of carrier and both

can be restored by same revertant at G30 ( 47, 49, 50).

Some residues are structurally and some of them functionally important like K38,

K179, and K182. Mutation inD149S delimits the transmembrane helix and is

completely inactive.

The residues E45, D149 and D249, terminate the first helix in each domain on matrix

side and any mutation result in losing their negative charge impair insertion of helices

into phospholipids(45).

Only three mutants out of eight mutants (E45G, K179M, K179I+K182I) were able to

grow on nonfermentable carbon resource. On the other hand other mutants (K38A,

K48I, R152A, D149S and D249S) have impaired oxidative phosphorylation

therefore unable to grow in non fermentable media.

2-2- ADP binding site:

Several biochemical and genetical approaches have been tried to identify the

important residues for nucleotide binding.R96H mutation in S.cervisiae( R76

bovine)reduce ADP binding affinity ,All highly conserved basic residues located at

the bottom of the cavity are putatively involved in binding to negatively charged

ADP. R234 and 235 are possibly implicated in ADP binding and also binding to

CATR molecule(45, 51).

3- AAC specific Inhibitors

Atractyloside (ATR) and bangkorkic acid are two specific inhibitors for carrier. BKA

binds to matrix side of carrier and competitively prevents binding of ATP molecule.

This binding leave the pore in closed state and stabilize it, therefore it is an apoptosis

inhibitor. This antibiotic blocks the oxidative phosphorylation (39, 41).

Atractyloside or its derivative, carboxyatractyloside (CATR), which has ten times

more affinity for carrier, bind to carrier from cytoplasmic side and their binding site

partially overlap with ADP binding site. CATR stabilize the carrier in open state and

lead to loss of inner membrane potential which consequently results in apoptosis.

Functional carrier unit is dimmer and each inhibitor molecule bind to a dimmer

carrier.

3-1-Carboxyatractyloside (CATR):

12

Cocklebur ( Xanthium Strunarium and Pitark in Turkish) has a world wide

distribution and its seeds contain a highly toxic glycoside for animals which is known

as CATR. Drying does not affect its toxicity and 0/30- 0/45% of body weight

consumption from seed is fatal. Poisoning usually occur 24 hours after ingestion (52).

Fig 3:Xanthum Strunarium.

CATR structurally is quite similar to ATR. The only difference is one extra carboxyl

group. First this molecule called as gummiferin, but later on was renamed as

carboxyatractyloside (53). It is a non penetrable inhibitor which binds selectively and

strongly to ACC molecule. The binding of CATR is non competitive, irreversible and

functionally different from ATR which is competitive and reversible. Its dissociation

constant is 5-10nm. CATR bind to mammalian, yeast ant plant mitochondria. In

mammalian mitochondria concentration of 10-7

M effectively inhibits carrier while, in

isolate hepatocytes it is 10-5

(53).

Molecular weight 768.80

Empirical formula C31H44O18S2 .XK+

biological source from Xanthium sibiricum

Assay ≥98% (HPLC)

Form Solid

storage condition Desiccated

Color White

Solubility H2O: ≥10 mg/mL

storage temp. −20°C

Table2: http://www.sigmaaldrich.com

3-2- CATR binding site:

Each CATR binds to functional dimeric form (37. 46) and gets trapped through many

interactions, deeply in cavity. This strong binding at the bottom of the cavity is a

result of many hydrogen bounds between CATR and basic residues and of course

water molecules. Crystal Structure of bovine mitochondrial ADP/ATP carrier in

complex with carboxyatractyloside reveals the identity of residues and kind of

13

interaction between carrier and CATR. Accordingly, ASN 87, LYS91, ARG79,

ASP231 and ARG234 via H_ bond and

ARG79,LEU127,GLY182,TYR186,SER227,ASP231,PHE230,ASP231 via

hydrophobic bond interact with CATR (39).

The two carboxylic groups in CATR form a salt bridge with R79. The other

carboxylic group which is characteristic of CATR interacts with R279 mediated by a

water molecule therefore reinforce CATR interaction compare to ATR. (10 times

higher Kd for CATR). Hydrophobic side chains (L127, V130, and I183) are in Van

der Waals interacts with isovaleric group.L127 (bovine) has 15 times lower affinity

than wild type (39).

3-3-CATR Binding capacity in mutants:

Fig4: Binding of specific inhibitors (CAT &BKA) to ANT. (A) Binding of CAT to

various carrier mutants. (B) Binding of BKA to various carrier mutants (45).

14

Since CATR binding site overlaps with ADP binding site, many of mutations which

can prevent CATR binding, leave the carrier inactive.K22A (K38 in yeast) mutant has

60% binding capacity for CATR, comparing wild type but has only 4% of Oxphos .

K162M (K179yeast) and K162I+K165I (K179I+K182I in yeast) are only two

positively charged mutants which retain their transport activity while CATR binding

value reaches to 30% and 60% respectively. In R137A(R 152 A yeast) this value is

about 20% but this mutant is gly- and lacks transport activity (45, 39).

K48A (yeast) and D149S (yeast) have strong suppression of inhibitor binding and

complete loss of affinity reported for D249S.

All mutants showed that they have measurable respiration except D249S (yeast).

First resistant mutations to CATR and BKA were repotted in three mutants K48A,

D149S and D249S, but they do not have transport activity and their expressions were

decreased. . E 45, D149 and D 249 are negatively charged residues which are located

on the matrix side of first helix in each of three domains and are well preserved.

It is surprising that neutralizing mutations in these three residues have divergent

effects (45).

E45G has 30% of wild type activity of ADP/ATP transport and has little effect on

Oxphos while D149S and D249S are completely suppressed (22-45)

Crop mutation of neutralization of positively charged residues does not exhibit

binding suppression for CATR, only for R96H mutant shows an increase in Kd for

CATR (54-45).

Fig 5: Oxidative phosphorylation rate in various mutants by isolated

mitochondria before and after correction with CAT (45).

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3-4-Resistant molecule:

Our ultimate goal is creating a CATR resistant functional carrier. Overlapping of

CATR binding site with ADP binding site and also numerous interactions between

structurally and functionally important residues with CATR make it more complex.

Based on previous mutations and other information, following residues are potential

target for new mutants and revertants with strong CATR binding resistancy, while

they are functional and cell are able to grow in non fermentable media and in presence

of CATR.

1-D134D (D149 in yeast) mutation in this residue leaves the carrier inactive however,

this mutation cause CATR binding inhibition. David, R., (1996) showed that in yeast,

G30V is a revertant of this mutant (G14V bovine).

2-R79: this residue interacts with CATR molecule through 2 H-bond and 3

hydrophobic interactions. Moreover two carboxylic groups from CATR molecule

make a salt bridge with this residue. Due to importance of this residue Any mutation

lead to inactivity of carrier but it has been shown that there are some revertants for

mutants of this amino acid. R79A which is gly- has three revertants(Y305H, D26V

and C288Y).S33I is a revertant of R79L and G3OV is a revertant of R79T.

3- K162M which is the only positive residues that its mutation does not affect carrier

transport function. Mutant has lower affinity for CATR. This information has been

gathered by yeast mutant.

4-K162I+K165 I: T this mutant has low affinity for CATR and is able to grow (yeast

mutation).

5-R235: this residue possibly interacts with CATR therefore can be considered as

target. Y290H is a revertant of R235I (Y305H in yeast).

6-R279: this residue interact with CATR via water molecule and this interaction cause

10 times higher affinity for CATR compare to ATR .E29Q and E29G are revertants of

R279A(yeast mutations).

7-L127 interacts with CATR via hydrophobic interactions and it has been shown that

mutant has 15 times lower affinity to CATR.

8- F230 interacts with CATR via hydrophobic interaction.

4-Aims The overall aim of this study is to develop a selection system based on human gene

. In particular the quick selection of genetically modified suicide gene carrying cells is

our main goal. The efficacy of gene delivery for genetic modification is not 100%

therefore elimination of nonmodified cells compensates this shortcoming.

4-1-The specific aims:

1- Study of sensitivity of different cell lines to CATR.

2- Study of different ANT mutant to identify CATR resistant mutants.

3- Study of efficacy of CATR-resistant mutant in cell selection.

16

5-MATERIAL AND METHODS:

5-1-DNA extraction:

Materials:

1-EDTA (0.5M) ,PH 8.0

2-1M Tris –hcl, Ph 7.6

3- Red blood cell lysis buffer

4-nucleic lysis buffer

5-TE buffer( pH 8.0)

6- chloroform( 4C)

7- Absolute ethanol (-20C)

Method:

1- add 1000 μl of red blood cell lysis buffer to 500 μl blood sample into ependorf

tube and shake it gently(homogenized)

2- spin the sample at 7000 rpm for 2 min and discard supernatant.

3- Repeat steps 1 and 2 for 3 times to get rid of all hemoglobin. (pellet must be

vortexed and rinsed to wash out any hemoglobin residual from white blood

cells)

4- While taking enough care from contamination, put the tube downward on

tissue paper for few seconds.

5- Resuspend the pellet in 400 μl nucleic lysis buffer ( pipetting)

6- Add 600 μl chloroform and 100 μl of saturated NACL (5M) and mix it at

room temperature ( on a rotator blood mixer)

7- Spin it at 7000 rpm for 2 min.

8- Transfer supernatant to a new ependorf (400 μl).

9- Add 800 μl absolute ethanol (-20C), DNA should be visible after gentle

vortex.

10- Centrifuge it at 12000 rpm for 1 min. discard supernatant and put the tube

downward on tissue paper till it is completely dried.

11- After adding 50 μl TE , vortex it and store it in 4C .

The expecting yield is between 100-300 ng/ μl DNA and 1μl is enough for a PCR

reaction.

Also Blood cells from Buffy coat can be achieved. DNA is extracted by Blood ready

Multiplex PCR kit.

5-2- PCR amplification

Forward and reverse primers are designed for ANT gene with binding site for EcoRI

and BamH1 endonuclease binding sites. Gene of interest is amplified by PCR.

5-3-Cloning and transformation

PCR products are cloned in pCMVcloning vector.

1-add these solution together;

Plasmid (50ng/μl) 1 μl

17

PCR products (purified by gel electrophoresis) 0.05-0.5 pmol

10x universal KGB buffer 2 μl

dNTPs (2-2.5 mM each) 2 μl

ATP 1 m M 1 μl

Restriction enzyme 2 units

T4 DNA polymerase 1 unit

T4 DNA ligase 3 units

H20 to total 20 μl volume

*10x universal KGB= 1Mpotassium acetate, 0.25M tris acetate, 5m M

2mercaptoethanol, 0.1 M magnesium acetate.

2-overnight incubation at 16C or 4 hours at 22C.

3-Incubate for 0.5-16 hours after adding 2 units restriction enzymes

4-take 2 μl of reaction mixture to transform Ecoli.

5-culture on agar plate with antibiotic.

Ref: molecular cloning,3rd

edition ( sambrook and Russell) CSHL press 2001)

5-4-PCR mutagenesis:

Design the primers:

For any mutation we need two primers which are complementary to each other. In any

of primers mutant sequence is flanked by 20 bases on each side. must of primers will

be annealing to each other and will not be extended( avoid normal taq).

PCR set up:

1- 4 μl 10m M dNTPs

2- 0,2 μl of primers (1 and2)

3- 1 μl plasmid template (10 ng)

4- 37.6 μl H2O

5- 2 μl Pfu polymerase (inc Mg)

Note: Pfu polymerase has 3 →5 exonuclease activity therefore can start chewing up

primers (SSDNA), Thus make them less specific. To avoid this assemble the reaction

on ice and keep on cold till putting them in PCR machine which is already heated to

94C.

PCR program for PCR mutagenesis

Duration Temperature Cycles

60 seconds 94C 1

30 seconds 94C

12 30 seconds 55C

12 minutes 68C

18

6-After cooling down the PCR reaction to the room temperature add Dpnl

(restriction enzyme) and incubate it at 37C for 1 hours. This enzyme cuts parents

DNA which are methylated.

7- Transform the reaction into Ecoli

8- Miniprep some colonies and verify by sequencing

P CMV-CATR is mutated by PCR mutagenesis and resulting plasmid would be called

p CMV- CATRR. The fragment between endonuclease binding sites from p CMV-

CATRR inserted to p EGFP and p EGFP-CATRr is produced.

5-5-Retroviral production and Transfection:

FuGENE 6 is used for transfection according to manufacturer's introduction. Phoenix

Gp cells are transiently transduced by vector constructs. Filtered supernatant is used

for transduction of packing cell line (pG13) for stable production.

-transfection (CaCL2 method).

1- culture cells in pettri dish in 10 ml DME 0.5% CS ( 1x106 cells per 10 cm)

2- incubate cells and 24 hours later prepare fresh following solutions;

280 m M NaCL

50m M Hepes

1.5 m M Na2 HPO4 (PH 7)

pH to 7.1

3- Add 20 μg plasmid into ependorf tube and add TE ( 0.1%) till final volume

of 450 μl.

4- Vortex it after adding 50 μl CaCL2, leave it in upright position for5 min

under hood.

5- Add 500 μl Hepes buffered saline to a 15 ml plastic centrifuge.

6- in less than 30 second add drop by drop plasmid solution to 2X Hepes

solution while vortexing.

7- Leave it for 30 min under hood.

8- Drop by drop add the mixture (I ml final volume)

9- Incubate it for 4 hours ( CO2 incubator)

10- Replace the media after 24 hours.

Trypsinaze the cells and transfer 1/5 or 1/10 of entire cells in a new pettri

dish

5-6-Analysis of transfected cell's viability in presence of CATR

Transfected cells are cultured in different concentration of CATR to determine

maximum cytotoxicity and also to identify any non specific effect of CATR on cells.

The viability of CATR resistant mutant cells in presence of CATR is measured by

19

WST-1 reagent. Viable cells are able to reduce WST-1 and produce a soluble salt

which is followed by spectrometry assay.

5-7- Primary human mononuclear cells viability assay in presence of CATR. For this purpose CATRr gene is cloned in SF91 which gives stable expression in

hematopoietic cells (Hildinger et al).

The mononuclear blood cells are separated on lymphoprep.

Lymphoprep TM

Protocol:

1- collect sample blood into a tube ( containing anticoagulant)

2- 2-add equal volume of 0.9% NACL.

3- Take 12-15 mm centrifuge tube and layer 6 ml of diluted sample over 3 ml

lymphoprepTM . To prevent the formation of aerosols, tube must be caped.

4- Centrifuge at 800g for 20 min (room temperature and in swing out rotor)

5- Remove the cells from formed band at the sample/medium interface by a

Pasteur pipette.

6- To reduce the density, dilute it with NACL (0.9%).

7- Spin it for 10 min at 250g

Harvested cells are transfected and their viability in presence of CATR is measure by

WST-1 reagent.

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24

Transmembrane helix

Matrix side helix

turns

Different residues between yeast and

bovine carrier.

Human

ANT2

Bovine

ANT1

Yeast AAC2

REVERTANTS

S T 1 S

D 2 S

Q A 3 K

A 4 20 E

L V 5 21 S

S 6 22 N

F 7

L A 8

K 9 I

D 11 26 dispensable D→V rev of R96A

D→E rev of R254I

F 11

L 12

A 13 29 M

G 14 30 G→V rev of R96T

&D146G ****

G→C rev of R254I

G 15

V 16

A 17 33 S S→I rev of R96L,

S→N rev of R254I

A 18 34

A 19 35

I 21 36 V

S 21 A

This residue is the

only one which is

completely

protected by CATR.

K 22 38 K→A 60% affinity to

CATR but only 4%

Oxphos.

It is involved in

nucleotide binding

T 23

A 24

V 25

A 26

P 27

I 28

E 29 45 Dispensable

for function

E→G or Q rev of

R294A

E→G gly+, 30% translocation

activity and normal

25

Oxphos.

R 31

V 31

K 32 48 K→I gly-

K→A strong

suppression of

CATR binding but

15% Oxphos and

inactive transport

activity

L 33 49

L 34 51

L 35 51 I

Q 36 52

V 37 53 N

Q 38 54#

H 39 56 E

A 41 57 M

S 41 58 L

K 42 59

Q 43 61

I 44 61 G

S T 45 62 T

A 46 L

E D 47 D

K 48 R

Q 49 K

Y 51

K 51 A

G 52

I 53

I 54 L

D 55

C 56 Non essential

V 57 F

V 58 L

R 59

I 61 T

P 61 A

K 62 T

E 63 Q

Q 64 E

G 65

F V 66 V

L 67 I

S 68

F 69

W 71

R 71 Dispensable for

26

function

G 72

N 73

L 74 T

A 75

N 76

V 77

I 78

Interact with CATR

via 3 H bond, and 2

hydrophobic bond. two carboxyl groups

from CATR make salt

bridge with this residue.

ARG,ARG 79 96 →A gly- Y305H,

D26V,C288Y

→L gly- S33I

→T gly- G30V

→H partially

CATR binding

inhibitor

Y 81

F 81

P 82

T 83

Q 84

A 85

L 86

Interact with CATR

via 2 H bond ASN 87

F 88

A 89

F 91

Interact with CATR

via 2 H bond LYS 91

D 92

K 93

Y 94 I

K 95

Q 96 A

I 97 M

F 98

L 99 G

G 111 F

G 111 _

V 112 _

D 113 K

R K 114 K

H R 115 E

K T 116 E

Q 117 G

F 118 Y

W 119 A

R 111 K

27

Y 111 W

F 112

A 113

G 114

N 115

L 116

A 117

S 118

G 119

G 121

A 121

A 122

G 123

A 124

T 125 L

S 126

Interact with CATR

via 2 hydrophobic

interactions.

LEU 127 Mutant has 15

times lower

affinity to

CATR

C 128 L

F 129

V 131

Y 131

P 132 S

L 133

D 134 149 D→G gly- G30V

D→S

completely

inactive. Strong

CATR binding

inhibition

F 135 Y

A 136

Involved in transport

activity. R 137 152 R→A gly-

20% affinity to

CATR but 8%

Oxphos and not

nucleotide

transport activity

T 138

R 139

L 141

A 141

A 142

D 143

V 144 S

G 145 S

K 146 Involved in

28

transport

G A 147 K

A G 148 G

A A 149 G

Q E 151 A

R 151

E 152 Q

F 153

T R 154 N

G 155

L 156

G 157 I

N D 158 D

C 159 V

I L 161 177 Y

T V 161 178 K

Important for

translocation

activity

K 162 179 K→M gly+

30% affinity to

CATR and 120%

Oxphos.

The only positively

charged residue

which its mutation

does not affect

nucleotide tranport

K→I

+

K182→I gly+ 60% affinity to

CATR and 50%

Oxphos

Transport activity

decreases but cells

are able to grow

I 163 180 T

F 164 181 L

K 165 182 Can be

neutralized(refer

to K179)

S 166

D 167

G 168

L 169 V

R 171 A

G 171

L 172

Y F 173

Q 174 R

G 175

F 176

N 177 L

V 178 P

S 179

V 181

Q 181 V

29

Interact with CATR

via 3 hydrophobic

interaction.

GLY 182

Interact with CATR

via 1 hydrophobic

interaction.

ILE 183

I 184 V

I 185 V

Grow on gly Y→ F 186 213 Implicated in

transferring but

not in substrate

binding

Impaired growth Y→A

Interact with CATR

via 1 H bond ARG 187 →A gly-

A 188 G

A 189 L

Grow on gly Y→F 191 217 Implicated in

transferring but

not in substrate

binding

Impaired growth Y→A

F 191

G 192

V I 193 M

Y 194

D 195

T 196 S

A 197 L

K 198

G 199 P

M 211 L

L 211

P 212 L

Important for

transaction not for

substrate binding#

D 213 T

P 214 G

K 215 S

N 216 E

V T 217 G

Important for

transaction but not

for substrate

binding#

H 218 S

I 219 F

I V 211 L

30

V I 211 A

S 212

W 213 F

M 214 L

I 215 L

A 216 G

Q 217 W

T 218 V

V 219

T 221

A 221 T

V 222 G

A 223

G 224 S

L 225 T

V T 226 244 C Non essential

S 227

Y 228

P 229 247 →G normal

growth

Interact with CATR

via 2 Hydrophobic

interactions.

PHE 231 L

Interact with CATR

via 1 H bond and 5

hydrophobic bond

ASP,ASP 231 D→S gly-

T 232

V 233

Interact with CATR

via 1 H bond,

binding to ADP

ARG 234 252 Essential No revertant at all

Possibly bind to

CATR and ADP R 235 253 Y305H for R→I

Salt bridge with

E264 R 236 254 R→I

Nearly not

essential

S33N,D26E,G30C

M 237

M 238

M 239

Q 241 T

S 241

G 242

R 243 Q

K 244 A

G 245 _

A 246 -

D 247 -

I 248 V

M 249 K

31

Y 251

T 251 D

G 252

T 253 A

V L 254 F

D 255

C 256 271 Non essential

W 257 L

R 258

K 259

I 261

A

261 V

K R 262 A

D 263 A

Salt bridge with E

236 E 264

G 265

P G 266 V

K 267 G

A 268 S

F 269 L

F 271

K 271

G 272

A 273 288 C Non essential C→Y rev of R96A

W 274 G

S 275 A

N 276

V 277 I

L 278

Interact with CATR

mediated by water ARG 279 →A gly- E45G, E45Q gly+

G 281

M 281 V

G 282 A

G 283

A 284

F 285 G

V 286

L 287 I

V 288 S

L 289 M

Y 291 315 Y→H rev of R96A

and R253I

D 291

E 292 Q

I 293 L

32

K 294

K 295

F Y 296

V T 297

298

299

311

.