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

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

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

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:

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-3deoxythymidine; CD: Cytosine deaminase; CID: Chemical inducer of dimerization; DED-FADD: Death effector domain of Fasassociated 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 4

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 5

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 Cells have different sensitivity to G418 and optimization phosphotransferase (neo, is required. It is toxic for cells and need long time for G418, APH) selection. Bleomycin (phleo, bleo, It is a bacterial origin marker therefore has the same zeocin) downsides as other bacterial derived antibiotic resistant markers. Cytosine deaminase It can also act as a suicide gene. The de novo synthesis (CDA, CD) pathway is blocked and cells are forced to rely on CDA scavenger pathway. Dihydrofolate reductase A MTX resistant mutant of this enzyme provides a good (DHFR) selection marker for wide range of cells. Histidinol dehydrogenase Histidinol is toxic for cells . (hisD) Hygromycin-BThe selection process needs high concentration of phosphotransferase Hygromicine. It is a bacterial selection marker. (HPH) Puromycin-N-acetyl Its efficiency is comparable to neo. It has bacterial origin. transferase (PAC, puro) Xanthine-guanine This is a bacterial enzyme which does not have phosphoribosyltransferase mammalian homologous. (XGPRT, gpt) neomycin Bacterial gene. phosphotransferase II gene(Neo) Multi drug resistant gene Transduced cells develop anti cancer drug resistance. MDR-1 Membrane markers It is a slow process and not suitable for clinical setting ( FACS sorting system) when high cell number is required. Membrane markers Only high expressing cells are selected and false positive (immunomegnetic beads) 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 6

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 (9799%) (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

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 30m/min/g wet wt or 600 m/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 ANT1(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

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

for negative

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.

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

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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):

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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 Form 98% (HPLC) Solid

storage condition Desiccated Color Solubility storage temp. White H2O: 10 mg/mL 20C

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

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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).

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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.

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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 l of red blood cell lysis buffer to 500 l 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 l nucleic lysis buffer ( pipetting) 6- Add 600 l chloroform and 100 l 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 l). 9- Add 800 l 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 l TE , vortex it and store it in 4C . The expecting yield is between 100-300 ng/ l DNA and 1l 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/l) 1 l

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PCR products (purified by gel electrophoresis) 10x universal KGB buffer dNTPs (2-2.5 mM each) ATP 1 m M Restriction enzyme T4 DNA polymerase T4 DNA ligase H20

0.05-0.5 pmol 2 l 2 l 1 l 2 units 1 unit 3 units to total 20 l 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 l 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 l 10m M dNTPs 2- 0,2 l of primers (1 and2) 3- 1 l plasmid template (10 ng) 4- 37.6 l H2O 5- 2 l 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 30 seconds 55C 12 12 minutes 68C

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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 CMVCATRR 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 g plasmid into ependorf tube and add TE ( 0.1%) till final volume of 450 l. 4- Vortex it after adding 50 l CaCL2, leave it in upright position for5 min under hood. 5- Add 500 l 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

18

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.

6-References
1-Horowitz MM, Gale RP, Sondel PM et al.: Graft-versus-leukemia reactions after bone marrow transplantation. Blood,1990. 75(3),p: 555562. 2-Alexandera Treschow., Optimization of suicide gene therapy in stem cell transplantation with special reference to the selection marker OuaSelect. thesis for doctorate degree,2009:p7-30 3-Kolb HJ, Schattenberg A, Goldman JM et al.: Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood,1995. 86(5),p: 20412050. 4-Sehn LH, Alyea EP, Weller E et al.: Comparative outcomes of T-cell-depleted and non-T-cell-depleted allogeneic bone marrow transplantation for chronic myelogenous leukemia: Impact of donor lymphocyte infusion. J. Clin. Oncol,1999.17(2),p: 561 568. 5-Helg C, Starobinski M, Jeannet M, Chapuis B: Donor lymphocyte infusion for the treatment of relapse after allogeneic hematopoietic stem cell transplantation. Leuk. Lymphoma, 1998. 29(34),p: 301313. 19

6-Nakamura K, Inaba M, Sugiura K et al.: Enhancement of allogeneic hematopoietic stem cell engraftment and prevention of GVHD by intra-bone marrow bone marrow transplantation plus donor lymphocyte infusion. Stem Cells 22(2), 125134 (2004). 7-Aversa F, Tabilio A, Velardi A et al.: Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. N. Engl. J. Med, 1998. 339(17),p:11861193. 8- Anna MG.,Tolga S., Evren A.: Suicide gene therapy for graft-versus-host disease, Immunotherapy ,2010. 2(4),p: 521537.,..4444h 9- Martin PJ, Schoch G, Fisher L et al.: A retrospective analysis of therapy for acute graft-versus-host disease: initial treatment. Blood,1990. 76(8),p: 14641472. 10- Billingham RE: The biology of graft-versus host reactions. Harvey Lect,1966.62,p: 2178.

11-Soui ane Ghannam et al., Immunosuppression by mesenchymal stem cells: mechanisms and clinical applications.stem cell research & therapy ,2010,1:2 12-Fillat, C., et al., Suicide gene therapy mediated by the Herpes Simplex Virus thymidine kinase gene/ Canciclovir system: fifteen years of application. Curr Gene Ther,2003. 3(1):p.13-26 13-Link,C.J.,Jr.,et al.,Adoptiveimmunotherapyfor leukemia: donor lymphocytes transduced with the herpes simplex thymidin kinase gene for remission induction. HGTRI 0103.Hum Gene Ther,1998.9(1):p.115-34 14-Portsmouth, D., J. Hlavaty and M. Renner , Suicide gene for cancer therapy. Mol Aspects Med, 2007. 28(1):p.4-41. 15-Baum, C., et al., Novel retroviral vectors for efficient expression of the multidrug resistant (mdr-1) gene in early hematopoitic cells. J viral, 1995. 69(12):p.7541-7. 16-Moolten, F. L., Tumor chemosensitivity conferred by inserted herpes thymidine kinase gene: paradigm for a prospective cancer control strategy. Cancer Res, 1986.46(10):p.5276-81.

17- Jennifer H.,H., et al., Functional expression of thymidine kinase in human leukaemic and colorectal cells, delivered as EGFP fusion protein by herpesvirus saimiri-based vector. Cancer Gene Therap,2004.11:p613-624. 18-Bomini,C.,et al.,HSV-TK gene transfer into donor lymphocyte for control of allogenic graft- versus- leukemia. Science, 1997.276(5319):p1719-24. 19-Tiberghien, p., Use of suicide gene- expression donor T-cell to control alloreactivity after heamatopoitic stem cell transplantation. J Intern Med, 2001.249

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94):p.369-77. 20-Riddell SR, Elliott M, Lewinsohn DA et al,. : T-cell mediated rejection of genemodified HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nat. Med, 1996. 2(2).p:216223. 21-Alar, A, Vector development for suicide gene therapy. Karolinska University Press,2002.p:31 22-Dilber,M.S., and Gahrton, G. Suicide gene therapy: possible application in hematopoietic disorders. J intern Med, 2001. 249:p 359-367. 23-Medin, J.A and Karlsson, S. Viral vectorsfor gene therapy for hematopoitic cells. Immunothechnology, 1997.3.p:3-19. 24-Miller, A.D., and Rosman, G.J. Improved retroviral vectors for gene transfer and expression.Biotechniques,1998. 7.p:980-982. 25-Licht, T., Herrmann, F., Gottesman, M.M., and Pastan, I. In vivo drug- selectable genes: a new concept in gene therapy. Stem Cells,1997.15.p:104-111. 26-Nolan, G. P ., Fiering, S., Nicolas, J.F.Fluorescence- activated cell analysis and sorting of viable mammalian cells based on beta D- galactosidase activity after transduction of Escherichia coli lac Z.Proc Natl Acad Sci USA,1998.85.p:2603-2607. 27- Grigani, F ., et al .,High efficiency gene transfer and selection of human hematopoitic progenitor cells with a hybrid EBV/retroviral vector expression the green fluorescence protein. Cancer Res,1998.58.p:14-19. 28-Mavilo, F., et al., Peripheral blood lymphocytes as target cells of retroviral vectormediated gene transfer.Blood 2001 Apr 1;97(7):1951-9.83,1988-1997. 29-Pawliuk, R., et al., Selection of retrovirally transduced hematopoietic cell using CD24 as a marker of gene transfer. Blood 98,2002-2007. 30-2Byun, J. M., Robbins, P.D. The selectable marker neo gene down regulates gene expression from retroviral vectors containing an internal ribosom entry site. Gene Ther,1998.5.p:1441-1444. 31-Bonini,C.,et al.,The suicide gene therapy challenges: how to improve a successful gene therapy approach .Mol Ter,2007.15(7):p.1248-52. 32-Fackeler,M.J.,et al., Full length but not truncated CD34 inhibits hematopoitic cell differentiation of M1 cells. Blood ,1995.85(11):p.3040-7. 33-Fehes, B., et al., A novel sort-suicide fusion gene vector for T cell manipulation. Gene Ther, 2002.9(23):p 1633-8. 34-Lange,C,. et al.,CD34 modulates the trafficking behavior of hematopoitic cells in vivo. Stem Cell Dev, 2007.16(2):p.297-304.

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35-Lemoine, F.M., Efficient transduction and selection of human T lymphocytes with bicistronic Thy 1/HSV1-TK retroviral vector produced by a human cell line. J Gene Med,2004.6(4):p.374-86. 36-Del Nagro,C.J., CD 19 function in central and peripheral B cell development. Immuno Res, 2005.31(2):p.119-31. 37-Martin, K., David, R., Structure function relationship of the ADP/ATP carrier. Biochimica et Biophysica Acta, 1994.1187.p:241-244. 38- Claudine, D., Bertrand A., et al., Two Residues of a Conserved Aromatic Ladder of the Mitochondrial ADP/ATP Carrier Are Crucial to Nucleotide Transport. Biochemistry, 2008.47.p: 1322313231.

39-Eva P., P, Structure of mitochondrial ADP/AT carrier in complex with carboxyatractyloside , Nature,2003.426. 40-Manuel, K.A., et al., Adenine Nucleotide Translocase-1, a Component of the Permeability Transition Pore, Can Dominantly Induce Apoptosis. The Journal of Cell Biology, 1999.147. 41-Eva, P.P., Ge rard, B. Nucleotide exchange in mitochondria: insight at a molecular level. Current Opinion in Structural Biology, 2004.14:p.420425. 42-Cecile, D.G., Conformation-Dependent Swinging of the Matrix Loop m2 of the Mitochondrial Saccharomyces cereVisiae ADP/ATP Carrier. Biochemistry, 2005,.44,p: 16310-16320. 43-Pierre, V.V. Molecular and physiological aspects of adenine nucleotide transport in mitochondria. Biochimica et Biophisica Acta,1976. 456.p:1_38. 44-Julius, S., Jordan, K., Ladislav, K., Obligatory requirement of intramitochondrial ATP for normal functioning of the eukaryotic cells.Biochemical and Biophysical research communications,1972. 49(1). 45-Veronika, M,. et al., Mutagenesis of Some Positive and Negative Residues Occurring in Repeat Triad Residues in the ADP/ATP Carrier from Yeast.Biochemistry,1997.36.p:16008-16018. 46-Steven, M., et al., Cardiolipin, a critical determinant of mitochondrial carrier protein assembly and function. Biochimica et Biophysica Acta , 2009.1788.p: 2059 2068. 47- David R. Nelson. The yeast ADP/ATP Carrier. Mutagenesis and second site revertants. Biochimica et Biophysica Acta, 1996.(1275).p:133-137. 48- Klingenberg,M,. and Nelson, D.R. In biochemistry of cell membrans,. Birkhauser, Basal, Switzarland, 1995. pp. 191-219.

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49- Nelson, D.R., Lawson, J.E, Klingenberg, M. And Douglas, M.G. J. Molec. Biol,1993. 230.p:1159-1170. 50- Nelson, D.R. and Douglas, M.G. J. Molec. Biol. 1993. 230. P: 1171-1182. 51-Pierre, R., et al., Conformational Changes of the Yeast Mitochondrial Adenosine Diphosphate/Adenosine Triphosphate Carrier Studied through Its Intrinsic Fluorescence. 2.Assignment of Tryptophanyl Residues of the Carrier to the Responses to Specific ligands. Biochemistry, 1996.(35).p:16125-16531. 52-Mehmet. T., et al., Carboxyatractyloside poisoning in humans. Annals of Tropical Paediaincs, 2005.(25).p:125-134. 53- Marion, S., Inhibitors of Adenine nucleotide translocase. Pharma Ther, 1997.(7).p:329-349. 54- G. SCHAFER., et al., 8- Azido-ADP, A covalent binding inhibitor of mitochondrial adenine nucleotide translocation. Febs Letters,1976.64(1). 55-Richard M. Mortensen1 and Robert E. Kingston2.Selection of Transfected Mammalian Cells. Current Protocols in Molecular Biology, 2009.

23

Transmembrane helix Matrix side helix turns Different residues between yeast and bovine carrier.

REVERTANTS

Yeast
AAC2

Bovine ANT1

Human ANT2 S D Q A L S F L K D F L A G T A V

S S K E S N

20 21 22

I DV rev of R96A DE rev of R254I dispensable 26

1 2 3 4 5 6 7 8 9 11 11 12 13 14

M GV rev of R96T &D146G **** GC rev of R254I SI rev of R96L, SN rev of R254I

29 30

33 34 35 36 38

15 G 16 V 17 A 18 19 21 21 22 A A I S K

V A It is involved in nucleotide binding KA

60% affinity to CATR but only 4% Oxphos.

This residue is the only one which is completely protected by CATR.

EG or Q rev of R294A 30% translocation

activity and normal

Dispensable for function EG gly+,

45

23 24 25 26 27 28 29

T A V A P I E

24

Oxphos.

KI glyKA strong
suppression of CATR binding but

48

31 R 31 V 32 K

15% Oxphos and inactive transport activity

I N E M L

G T L D R K A

49 51 51 52 53 #54 56 57 58 59 61 61 62

L Non essential F L T A T Q E V I

Dispensable for

33 34 35 36 37 38 39 41 41 42 43 44 45 46 47 48 49 51 51 52 53 54 55 56 57 58 59 61 61 62 63 64 65 66 67 68 69 71 71

L L L Q V Q H A S K Q I S A E K Q Y K G I I D C V V R I P K E Q G F L S F W R

T D

25

function 72 73 74 75 76 77 78 79 G N L A N V I ARG,ARG

Y305H, D26V,C288Y S33I G30V

A glyL glyT glyH partially

CATR binding inhibitor

96

Interact with CATR via 3 H bond, and 2 hydrophobic bond.

two carboxyl groups from CATR make salt bridge with this residue.

81 81 82 83 84 85 86 87 88 89 91 91 92 93 94 95 96 97 98 99 111 111 112 113 114 115 116 117 118 119 111

Y F P T Q A L ASN F A F LYS D K Y K Q I F L G G V D R H K Q F W R

Interact with CATR via 2 H bond

Interact with CATR via 2 H bond

I A M G F _ _ K K E E G Y A K

K R T

26

L Mutant has 15 times lower affinity to CATR L

111 112 113 114 115 116 117 118 119 121 121 122 123 124 125 126 127

Y F A G N L A S G G A A G A T S LEU

Interact with CATR via 2 hydrophobic interactions.

S G30V DG glyDS completely inactive. Strong

CATR binding inhibition

128 129 131 131 132 133 149 134

C F V Y P L D

Y RA gly20% affinity to CATR but 8% Oxphos and not nucleotide transport activity

135 F 136 A 152 137 R

Involved in transport activity.

S S Involved in

138 139 141 141 142 143 144 145 146

T R L A A D V G K

27

transport K G G A Q N 147 148 149 151 151 152 153 154 155 156 157 158 159 177 161 178 161 179 162 G A A Q R E F T G L G N C I T K A G A E

I D V Y K The only positively charged residue which its mutation does not affect nucleotide tranport Transport activity decreases but cells are able to grow KM gly+ 30% affinity to
CATR and 120% Oxphos.

D L V Important for translocation activity

KI + K182I gly+
60% affinity to CATR and 50% Oxphos

T L Can be neutralized(refer
to K179)

180 163 I 181 164 F 182 165 K 166 167 168 169 171 171 172 173 174 175 176 177 178 179 181 181 S D G L R G L Y Q G F N V S V Q

V A

L P

28

182 GLY 183 ILE 184 I 185 I 213 186 Y F YA

Interact with CATR via 3 hydrophobic interaction. Interact with CATR via 1 hydrophobic interaction.

V V Implicated in transferring but not in substrate binding A glyG L Implicated in transferring but not in substrate binding

Grow on gly Impaired growth

187 ARG 188 A 189 A 217 191 YF YA

Interact with CATR via 1 H bond

Grow on gly Impaired growth

S L P L L T

191 192 193 194 195 196 197 198 199 211 211 212 213 214 215 216 217 218

F G V Y D T A K G M L P D

Important for transaction not for substrate binding#

G S E G S

P K N V H

T Important for transaction but not for substrate binding# V

F L

219 I 211 I

29

A F L L G W V

T G S T C

Non essential G normal growth

211 212 213 214 215 216 217 218 219 221 221 222 223 224 225 244 226 227 228 247 229

V S W M I A Q T V T A V A G L V S Y P

L DS gly-

231 PHE 231 ASP,ASP 232 T 233 V 252 234 ARG 253 235 R

Interact with CATR via 2 Hydrophobic interactions. Interact with CATR via 1 H bond and 5 hydrophobic bond

No revertant at all Y305H for RI S33N,D26E,G30C

Essential

RI Nearly not essential

254 236 R 237 238 239 241 241 242 243 244 245 246 247 248 249

Interact with CATR via 1 H bond, binding to ADP Possibly bind to CATR and ADP Salt bridge with E264

Q A _ V K 30

M M M Q S G R K G A D I M

D A F Non essential L

V A A

251 251 252 253 254 255 271 256 257 258 259 261 261

Y T G T V D C W R K I A

262 K 263 D 264 E 265 266 267 268 269 271 271 272 288 273 274 275 276 277 278 279 281 281 282 283 284 285 286 287 288 289 315 291 G P K A F F K G A W S N V L ARG G M G G A F V L V L Y

R Salt bridge with E 236 G

V G S L

CY rev of R96A

Non essential

C G A I

E45G, E45Q gly+

A gly-

Interact with CATR mediated by water

V A

G I S M YH rev of R96A and R253I Q L

291 D 292 E 293 I

31

294 295 296 297 298 299 311

K K F V

Y T

32