Tryparedoxin, expression plasmid, process of preparation, method of use, test kit and pharmaceutical composition
The present invention provides novel enzymes, tryparedoxins, their isolation from Crithidia fasciculata, a method for the production thereof in genetically transformed bacteria, and their use as molecular targets for the discovery of trypanocidal drugs
This is a continuation of International Application No. PCT/EP97/06983 filed Dec. 12, 1997, the entire disclosure of which is incorporated herein by reference.
INTRODUCTIONFlagellated protozoan parasites of the family Trypanosomatidae are among the most prevalent human pathogens in tropical and subtropical areas. These organisms have complex life cycles and some of them are the causative agents of debilitating or life-threatening diseases, such as American Chagas' disease (Trypanosoma cruzi), African sleeping sickness (T. brucei ganibienise and T. b. rhodesienise), oriental sore (Leishmania tropica), kala azar (L. donovani) and mucocutaneous leishmaniasis (L. brasiliensis). Others infect hosts as diverse as plants (Phytomonas species), insects (Crithidia and Leptomonas species) and livestock (T. congolenise, T. b. brucei, T. evansi). Many of the human pathogens are also endemic in wildlife. Worldwide, more than 30 million people are estimated to suffer from trypanosomal and leishmanial infections (World Health Organisation, 1996). Vaccination strategies have so far failed and most of the chemotherapeutic drugs currently used for treatment are unsatisfactory in terms of both efficacy and toxicity (Risse, 1993). Nifurtimox, for instance, a drug widely used in the treatment of Chagas' disease, is an unspecific redox cycler affecting not only the peroxide sensitive parasites but also the host. Accordingly, the defense system against oxidants in the trypanosomatids, which differs substantially from the analogous host metabolism, has been discussed as a potential target area for the development of more specific trypanocidal agents. (Fairlamb, 1996; Jacoby et al., 1996).
As parasites, the trypanosomatids are inevitably exposed to various reactive oxygen species, such as superoxide radicals, hydrogen peroxide and myeloperoxidase products, generated during the host defense reaction. However, their ability to cope with such oxidative stress appears to be surprisingly weak. Although they possess an iron-containing superoxide dismutase to scavenge phagocyte-derived superoxide (LeTrant et al., 1983), most of them lack both catalase and glutathione peroxidase (Docampo, 1990), the major hydroperoxide metabolising enzymes of the host organisms (Chance et al., 1979; Flohe, 1989). They also contain conspicuously low concentrations of glutathione (GSH), the major antioxidant sulfhydryl compound in mammalian cells. Instead they form a unique GSH derivative known as trypanothione (T(SH)2; N1,N8-bis(glutathionyl)spermidine) (
The present invention is based on the discovery that hydroperoxide metabolism in the trypanosomatids is enzymatic in nature, but distinct from any known metabolic pathway of the host organisms (Nogoceke et al., 1997). Apart from the previously known trypanothione reductase (TR), the parasitic pathway comprises two novel proteins, called tryparedoxin (TXN) and tryparedoxin peroxidase (TXNPx), which together catalyse the reduction of hydroperoxides at the expense of NADPH as depicted in
The uniqueness of this cascade of oxidoreductases offers the possibility to inhibit the parasitic metabolism without causing adverse effects in the host organism.
Thus, one embodiment of the invention concerns proteins, which we called tryparedoxins (trypanothione: peroxiredoxin oxidoreductases) and which are characterized by their capability of transferring reductive equivalents of trypanothione to a peroxiredoxin-type protein such as tryparedoxin peroxidase. As regards peroxi-redoxins and peroxi-redoxin-type proteins, reference is made to Chae et al. in J. Biol. Chem., 269(1994) 27670-24678 and in PNAS USA, 91 (1994) 7017-7021 and to EP 96 120 016.9 or PCT/EP 97/04 990. Tryparedoxins exhibit a catalytic site similar to that of thioredoxin, an ubiquituous redox mediator with pleiotropic functions. Typical thioredoxins have never been found in any trypanosomatid. It therefore appears conceivable that in the trypanosomatids the tryparedoxins substitute for thioredoxin in such diverse metabolic functions as reduction of ribonucleotides, differentiation, regulation of transcription or other regulatory processes depending on the cellular thiol/disulphide equilibrium. The possibility of multiple biological functions of tryparedoxins is further suggested by the coexistence of more than one tryparedoxin in the same species, as described below.
The proteins according to the invention can be characterized in that they can be prepared by means of and/or isolated from a species of the family Trypanosomatidae.
Further, the proteins according to the invention can be characterized in that their preparation and/or isolation can be carried out by genetic engineering, especially by means of an oligonucleotide as probe having the oligonucleotide sequence and encoding the amino acid sequence of SEQ ID NO 1 (
Further, the proteins according to the invention can be characterized by a molecular weight of 15-19 kDa.
Further, a protein according to the invention can be characterized by a WCPPC motif and catalyzing the reduction of protein disulphide bonds by means of trypanothione.
Further, a protein according to the invention can be a protein
-
- (a) having the amino acid sequence SEQ ID NO 2 (
FIG. 3 , positions 1 to 150) or - (b) having an amino acid sequence which is homologous to said according to (a), has the same number or a smaller or slightly smaller or larger number of amino acids than SEQ ID NO 2 and is encoded by an oligonucleotide which is hybridizable with an oligonucleotide which encodes a protein comprising or having the amino acid sequence SEQ ID NO 1 or SEQ ID NO 2.
- (a) having the amino acid sequence SEQ ID NO 2 (
Further, the protein according to (b) can be a protein having an amino acid sequence which is homologous to SEQ ID NO 1 or SEQ ID NO 2 by at least 70% and especially at least 75%.
Another embodiment of the invention concerns plasmids for the expression of proteins according any of the preceding claims and comprising a nucleic acid sequence encoding said proteins.
The plasmids according to the invention may comprise DNA sequences encoding tryparedoxin especially of Crithidia fasciculata.
Further, a plasmid according to the invention may comprise a DNA sequence encoding functionally active derivatives of tryparedoxin designed for the isolation in a manner known per se.
Further, a plasmid according to the invention may comprise a DNA sequence encoding functionally active derivatives of tryparedoxin wherein the tryparedoxin is derivatised by a His tag.
Still another embodiment of the invention concerns a process for the production of a protein according to the invention characterized in that it is produced by means of a DNA sequence encoding the amino acid sequence of SEQ ID NO 2 by genetic engineering in a manner known per se.
The process according to the invention can be characterized in that the production is carried out by means of a plasmid according to the invention.
Further, the process according to the invention can be characterized in that the host is selected from the group consisting of bacteria, fungi, yeast, plant cells, insect cells, mammalian cells and cell cultures (heterologous expression).
Further, the process according to the invention can be characterized in that Escherichia coli is used as host.
Still another embodiment of the invention concerns the use of a protein according to the invention for testing and recovering inhibitory substances which inhibit activities of said protein.
Still another embodiment of the invention concerns a test system for testing the catalytic activity of a protein according to the invention or obtained according to the process according to the invention, wherein the testing system contains or comprises trypanothione, trypanothione reductase, a tryparedoxin peroxidase, a tryparedoxin and, in addition, a hydroperoxide as indicator enzyme, mediator and substrate, respectively.
Finally, another embodiment of the invention concerns a pharmaceutical preparation having a trypanocidal activity and comprising an inhibitory substance inhibiting the catalytic activity of a protein according to the invention or of a protein which can be obtained according to the process according to the invention.
The pharmaceutical composition according to the invention can be characterized in that it can be obtained by a use according to the invention and by using a test system according to the invention.
The invention is now described in greater detail by means of figures and examples.
C. fasciculata was cultivated in a 100 I fermenter as described (Shim and Fairlamb, 1988). The cells were harvested in the late log phase, suspended in 50 mM sodium phosphate pH 5.8 (buffer B) containing 0.1 mM PMSF, then frozen and thawed twice to complete cell disruption. Cell debris was removed by centrifugation at 25,000×g for 30 min and the supernatant was applied on an S-Sepharose column pre-equilibrated with buffer B. Tryparedoxin peroxidase eluted at 150 mM NaCl in buffer B and was directly loaded on a hydroxyapatite (BioRad, USA) column pre-equilibrated with 10 mM sodium phosphate pH 6.8. Tryparedoxin peroxidase was eluted stepwise with 0.4 M potassium phosphate pH 6.8. The protein was extensively dialyzed against 20 mM Tris pH 7.6 (buffer C) and purified to homogeneity on a Resource Q column, eluting at 0.1 M NaCl in buffer C. The flow-through of the S-Sepharose column containing trypanothione reductase and tryparedoxin can be used to measure the enzymatic activity of tryparedoxin peroxidase (see example 2). The flow-through of the S-Sepharose column, containing trypanothione reductase and tryparedoxin, was adjusted to pH 7.2 with 1 M NaOH. The extract was adjusted to 3% (w/v) streptomycin sulphate, brought to 50%. ammonium sulphate saturation, and centrifuged for 10 min at 11,000×g. The supernatant was adjusted to 80% ammonium sulphate saturation and recentrifuged. The pellet was dissolved in, then dialyzed extensively, against 20 mM bis-Tris propane pH 7.2 containing 1 mM EDTA and 1 mM DTT (buffer D). The enzyme extract was loaded on a DEAE-Sepharose column and eluted with a linear gradient of 0.4 M KCl in buffer D. The sample eluting at 80-120 mM KCl was concentrated by ultrafiltration (Omegacell, Filtron, Germany), washed with 20 mM potassium phosphate pH 7.2 containing 1 mM EDTA and 1 mM DTT (buffer E) and loaded on a 2′5′ADP-Sepharose 4B column. Trypanothione reductase was eluted with 5 mM NADP in buffer E and purified to homogeneity on a Sephacryl S-200 column. The unbound fraction was concentrated by ultrafiltration and fractionated on an Ultrogel AcA54 (LKB, Sweden) gel filtration column in 50 mM Hepes pH 7.6 containing 150 mM NaCl, 1 mM EDTA and 1 mM DTT to yield homogeneous tryparedoxin. The authentic tryparedoxin, thus isolated, is termed tryparedoxin I (TXN I). The overall yields of the final purification scheme are shown in Table 1.
Based on the purification factors yielding homogeneous products the minimum concentrations of tryparedoxin and tryparedoxin peroxidase in the starting material were estimated to amount to 5% and 6% of the total soluble protein, respectively. The homogeneity and approximate molecular masses of the purified proteins are shown in
Analyses of the spectral properties of the two proteins confirmed the absence of any chromophoric cofactors absorbing in the visible region.
EXAMPLE 2 Determination of Tryparedoxin ActivityIn essence, the activity of tryparedoxin activity is measured by coupling the catalytic reduction of hydroperoxide mediated by tryparedoxin peroxidase to NADPH consumption by means of trypanothione and trypanothione reductase. For example, an assay sample may contain 0.1 mM NADPH in 50 mM Hepes pH 7.6, 1 mM EDTA, 50 M H2O2 or t-butyl hydroperoxide (t-bOOH), 45 M T(SH)2, 16.5 μg/ml tryparedoxin peroxidase and 0.34 U trypanothione reductase and an unknown amount of tryparedoxin. Unless otherwise stated, the reaction is started with the addition of the hydroperoxide. Dihydro-trypanothione is obtained by chemical reduction of TS2 (Bachem, Switzerland) as described (Fairlamb et al., 1986). t-BOOH may be replaced by other hydroperoxides, such as H2O2, linoleic acid hydroperoxide or phosphatidylcholine hydroperoxide.
Since the N-terminus of tryparedoxin I was blocked, the protein was digested with either bovine trypsin or endoproteinase Glu-C from Staphylococcus aureus (both sequencing grade, Promega) according to Stone and Williams (1993). The peptides were separated by HPLC (Applied Biosystems 172A) on an Aquapore OD-300 RP-18 column. Automated Edman degradation was performed with an Applied Biosystems, Inc. sequencer with an on-line C-18 reverse phase HPLC. Database searches were performed with the BLAST and FASTA programs. Peptides were aligned with the Bestfit program, Genetics Computer Group (GCG), Madison, Wis., USA.
Seven fragments could be sequenced and could be aligned to a thioredoxin-like protein of C. elegans (
Cells culture and DNA extraction: C. fasciculata (HS6) was grown as described by Shim and Fairlamb (1988). The cells were harvested by centrifugation for 15 min at 7000 rpm, washed twice with saline solution (0.9% NaCl) and resuspended in 5 ml buffer (50 mM TrisHCl, 100 mM EDTA, 15 mM NaCl, 0.5% SDS, 100 μg ml−1 Proteinase K, pH 8.0). Resuspended cells were preincubated at 50° C. for 40 min. The genomic DNA was extracted twice with equivalent volumes of phenol (incubation: 60° C. for 45 min; centrifugation: 20 min, 4500 rpm) followed by phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol extraction (24:1). Genomic DNA was precipitated with sodium acetate and ethanol.
Primers, hybridization probes and sequence analysis: Based on the peptide sequences of tryparedoxin I (Nogoceke et al., 1997) degenerate oligodeoxyribonucleotides were synthesized. Polymerase chain reaction (PCR) amplification was performed using the GeneAmp PCR Core kit (Perkin Elmer) using 0.2 μg of C. fasciculata genomic DNA as template, 5 μl of 10× reaction buffer, 3 μl 25 mM MgCl2, 1 μl of each 10 μM dNTP, 100 pmol of each primer and 0.25 U Taq polymerase. An annealing temperature of 52° C. was used. The PCR product was analysed by agarose gels and purified using the QIAquick PCR purification kit (QIAGEN Inc.). Sequencing was performed on a 373A DNA Sequencer (Applied Biosystems) using the PRISM Ready Reaction DyeDeoxy Terminator Sequencing Kit (1550V, 19 mA, 30 W, 42° C.). When used as a hybridization probe the PCR product was labelled with digoxigenin using the DIG DNA Labeling Kit (Boehringer Mannheim) according to the instructions provided by the supplier.
Library construction and screening procedure: The genomic DNA was partially digested for 5-30 min with a ratio unit Sau3A /μg DNA of 0.005. The efficiency of the digestion was monitored by electrophoresis on agarose gels. Proteins were removed from the DNA using StrataClean Resin (Stratagene). The Sau3A sites were partially refilled with dATP and dGTP and Klenow fragment. The genomic DNA was ligated into Lambda GEM-11 Xho I half site arms (Promega) at a molar ratio of DNA to genomic DNA (average size 15 kb) of 1:0.7. The ligated DNA was packaged using the Packagene Lambda DNA Packaging System (Promega) according to the suppliers' instructions. The phages were used to infect the E. coli host strain LE392 (Promega) according to the standard protocol. 5.1×103 pfu of the genomic library were plated on agar. The plaques were transferred to 9 cm diameter Biodyne-A nylon membranes and screened with the DIG-labelled PCR probes following the instructions provided by the supplier but using a hybridization temperature of 54 C. DIG labelled nucleic acids were detected calorimetrically with the DIG Nucleic Acid Detection Kit (Boehringer Mannheim). Positive clones were rescreened, amplified and suspended in SM buffer. The phages were precipitated by PEG 8000 and purified in CsCl gradients. The isolated DNA was used for restriction analyses (Sac I, EcoR I, BamH I, Xho I, Nco I) or as template for PCR reactions. The digestion products were eluted from agarose gels and ligated into pBluescript II KS (±) phagemids (Stratagene) or pET24d(+) vector (Novagen). The ligated DNA was used to transform E. coli LE392. Transformed cells were selected by ampicillin (pBluescript II KS (±) phagemid) or kanamycin (pET24d(+) vector) resistance, plasmids were purified using QIAprep Spin Plasmid Kit (Qiagen Inc.) and analyzed by restriction enzyme digestion and sequencing.
Isolation and sequencing of tryparedoxin genes from C. fasciculata: Sequenced peptide fragments obtained from isolated tryparedoxin I of C. fasciculata (Nogoceke et al., 1997) could be aligned along the established deduced amino acid sequence of a thioredoxin-like protein of Caenorhabditis elegans. This enabled appropriate degenerate PCR primers to be designed for the generation of a PCR product from the C. fasciculata genomic DNA. This PCR product, which coded for approximately the 50% of tryparedoxin I (
New primers were designed using the information obtained in example 4 and the complete gene was sequenced directly from the 6 kb fragment. The previously obtained PCR product is about 60% identical in its amino acid sequence to the correponding region of the isolated gene. The full length encoding DNA and the deduced amino acid sequence are shown in
The tryparedoxin gene contained in the cloned 6 kb fragment was amplified by PCR with a forward primer A (5′-TCGTGATTCCGTTCCGCATATGTCAGGGC-3′) that contains an Nde I site and overlaps the 5′ end of the coding sequence, and a reverse primer B. (5′-GCAACTCAATCGCTCCCCTCGAGCTTCTTGGCCTCC-3′) which overlaps the 3′ end of the coding sequence and contains an Xho I site. Consequently a leucine and a glutamate residue are added, the stop codon is deleted and the protein will contain 6 histidine residues at its carboxyl-terminal end. Amplification was performed as above but using the Expand High Fidelity polymerase mixture and buffer (Boehringer Mannheim) at an annealing temperature of 50° C. with the extension temperature being increased in 10 sec increments per cycle during cycles 10-20. The amplified coding region was digested with Nde I and Xho I and ligated to a pET24a(+) vector (Novagen) treated with the same enzymes and dephosphorylated. The resulting plasmid (pET/TXN II H6) was used to transform E. coli BL21(DE3). Transformed cells were selected by kanamycin resistance, the plasmids purified and sequenced.
The same procedure, but using a reverse primer C (5′- CAGCAACTCAATGGATCC TCATTACTTCTTGGCC-3′) instead of reverse primer B, was used to express tryparedoxin II with no changes at the carboxyl-terminal end. In this case the reverse primer contained an extra stop codon and a BamH I site at the 5′-end of the extra stop codon, with the digestions for the cloning step being performed with Nde I and BamH I. The resulting plasmid was called pET/17 II and was used to transform E. coli BL21(DE3). Transformed cells were selected by kanamycin resistance, the plasmids purified and sequenced.
E. coli BL21(DE3) pET/TXN IIH6 were grown to A600 of 0.9-1.0 at 25° C. and 180 rpm in LB medium with 30 μg kanamycin/ml, then expression of the tryparedoxin II gene was induced with 1 mM isopropyl-D-thiogalactopyranoside. E. coli BL21(DE3) containing the pET24a plasmid was grown in the same way. Samples taken at, different times were centrifuged, resuspended in 50 mM Tris-HCl pH 8.0, 1 mM EDTA buffer, sonicated and centrifuged. Enzyme activity was determined as in Nogoceke et al. (1997); protein concentration was determined using Coomassie Brilliant Blue-G reagent (BioRad) with bovine serum albumin as standard. After induction of the transformed bacteria, a marked increase in tryparedoxin activity was detected in supernatants of sonicated cells. Activity increased to a maximum 6 hours after induction and no activity was found in the control (
E. coli BL21(DE3) pET/TXN II were grown to A600 of 0.9-1.0 at 25° C. and 180 rpm in LB medium with 30 μg kanamycin/ml, then expression of the tryparedoxin II gene was induced with 1 mM isopropyl-D-thiogalactopyranoside. E. coli BL21(DE3) containing the pET24a plasmid was grown in the same way. Samples taken at different times were centrifuged, resuspended in 50 mM Tris-HCl pH 8.0, 1 mM EDTA buffer, sonicated and centrifuged. Enzyme activity was determined as in Nogoceke et al. (1997); protein concentration was determined using Coomassie Brilliant Blue-G reagent (BioRad) with bovine serum albumin as standard. After induction of the transformed bacteria, a marked increase in tryparedoxin activity was detected in supernatants of sonicated cells. Activity increased to a maximum 6 hours after induction and no activity was found in the control (
E. coli BL21(DE3) pET/TXN II H6 was grown at 25° C. and 180 rpm in LB medium with 30 μg kanamycin/ml to A600 of 0.9-1.0, then expression of the tryparedoxin II gene was induced with 1 mM isopropyl-D-thiogalactopyranoside. After 6 h the culture was centrifuged and either stored at −20° C. or the cells were resuspended in 0.05 culture volumes of binding buffer (5 mM imidazole, 500 mM NaCl and 20 mM Tris-HCl pH 7.9). The cell suspension was sonicated on ice and centrifuged for 40 min at 4° C., 13000 rpm. The supernatant was applied to a His Bind resin (Novagen) column charged with Ni2+ and equilibrated with binding buffer, at a flow rate of about 10 column volumes per hour. The column was washed with 10 volumes of binding buffer and 6 volumes of 500 mM NaCl, 20 mM Tris-HCl pH 7.9 buffer containing 100 mM imidazole. Tryparedoxin eluted in the buffer containing 500 mM imidazole. Active fractions were pooled and immediately dialysed against 50 mM Tris-HCl pH 7.6 buffer containing 1 mM DTT and 1 mM EDTA. Tryparedoxin II eluted at 500 mM imidazole and was shown to be pure by SDS-PAGE and subsequent silver staining (
The purified recombinant enzyme had a specific activity of 7.7 U/mg compared to 2.3 U/mg for the authentic enzyme.
EXAMPLE 8 Inhibition Studies The test system described in example 2 is easily adapted to screen compounds for specific inhibition of tryparedoxin I. As an example the inhibition of tryparedoxin peroxidase by S-modifying agents such as N-ethylmaleimide (NEM), iodoacetamide (IAM) and phenylarsine oxide (PAO) is described (Table 2). Tryparedoxin was preincubated in 50 mM Hepes, 1 mM EDTA, pH 7.6 with or without presumed reducing substrate (T(SH)2), then reacted with inhibitors and activity was checked at 22° C. essentially as described in example 2. Changes in molecular mass were determined by MALDI-TOF-MS (
astored under non-reducing conditions
bvalues in brackets represent predicted mass increments
cone molecule of derivatising agent
dtwo molecules of derivatising agent
einhibition reversible; activity regained within the timescale of the test
Tryparedoxin was preincubated in 50 mM Hepes, 1 mM EDTA, pH 7.6 with presumed reducing substrates, then reacted with iodoacetamide (IAM), NEM or phenylarsine oxide (PAO). Changes in molecular mass were determined my MALDI-TOF-MS. Residual activity was measured at 22° C. using 1 mM T(SH)2 with 1.0 μM tryparedoxin peroxidase and 0.6 μM tryparedoxin.
The disclosure comprises also that of EP 96 120 015.1, the entire disclosure of which is incorporated herein by reference.
References
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Claims
1-28. (canceled)
29. An isolated tryparedoxin (TXN) protein, said TXN protein having an activity which transfers reductive equivalents of trypanothione (T(SH)2) to a peroxiredoxin, said TXN protein having a molecular weight of between 15 kDa to 19 kDa as determined by mass spectrometry, and said protein having a WCPPC motif.
30. The protein of claim 29, wherein said TXN protein is prepared by genetic engineering.
31. The protein of claim 30, wherein said TXN protein is prepared by expression of a plasmid comprising a nucleic acid molecule encoding said protein.
32. The protein of claim 29, wherein said TXN protein is prepared or isolated from a species of the family Trypanosomatidae.
33. The protein of claim 29, wherein said TXN protein catalyzes the reduction of a protein disulfide by T(SH)2.
34. An isolated polypeptide comprising an amino acid sequence that is at least 70 percent homologous to the amino acid sequence of SEQ ID NO: 4 or 6, wherein the polypeptide has tryparedoxin (TXN) activity.
35. The polypeptide of claim 34 comprising the amino acid sequence set forth in SEQ ID NO: 4.
36. The polypeptide of claim 34 comprising the amino acid sequence set forth in SEQ ID NO: 6.
37. The polypeptide of claim 34 comprising a portion of the amino acid sequence of SEQ ID NO: 4, wherein said portion retains a WCPPC motif and retains TXN activity.
38. The polypeptide of claim 34 comprising a portion of the amino acid sequence of SEQ ID NO: 6, wherein said portion retains a WCPPC motif and retains TXN activity.
39. The polypeptide of claim 34 comprising at least 120 amino acids of SEQ ID NO: 6.
40. The polypeptide of any of claims 34-39, wherein said polypeptide is prepared or isolated from a species of the family Trypanosomatidae.
41. The polypeptide of any of claims 34-39, wherein said polypeptide catalyzes the reduction of a protein disulfide by T(SH)2.
42. The polypeptide of claim 31 encoded by the nucleic acid molecule of SEQ ID NO: 5.
43. The polypeptide of claim 31 encoded by the nucleic acid molecule of SEQ ID NO: 7.
Type: Application
Filed: Oct 12, 2004
Publication Date: Jun 30, 2005
Inventors: Leopold Flohe (Braunschweig), Everson Nogoceke (Braunschweig), Henryk Kalisz (Braunschweig), Marisa Montemartini (Braunschweig)
Application Number: 10/962,760