Tryparedoxin, expression plasmid, process of production,...

Details Tryparedoxin, expression plasmid, process of production,... Tryparedoxin, expression plasmid, process of production,...

1999-06-11

2002-08-13

Navarro, Mark (Department: 1645)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S006120, C435S007220, C435S024000, C435S024000, C435S320100, C435S252300, C435S255300, C536S023200, C536S024300, C536S024320, C424S093100, C424S093200, C424S093210, C424S184100, C424S265100, C424S269100

Reexamination Certificate

active

06432671

ABSTRACT:

INTRODUCTION
Flagellated 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 gambiense
and
T. b. rhodesiense
), 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. congolense,
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 leislmanial 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; Flohé, 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
; N
1
,N
8
-bis(glutathionyl)spermidine) (FIG.
1
), which is believed to play a central role in their antioxidant defense system (Fairlamb et al., 1985; Fairlamb and Cerami, 1992). T(SH)
2
can be oxidized by H
2
O
2
to the corresponding cyclic disulphide (TS
2
) (
FIG. 1
) and is regenerated at the expense of NADPH by trypanothione reductase (TR; Bailey et al., 1993; Jacoby et al., 1996) (FIG.
1
). Whether the reaction of T(SH)
2
with H
2
O
2
is enzymatically catalyzed has, however, been the subject of debate. A T(SH)
2
-dependent peroxidase activity was repeatedly reported for crude extracts of the trypanosomatids (Penketh and Klein, 1986; Henderson et al., 1987; Penketh et al., 1987). However, a pertinent enzymatic entity could never be purified (Henderson et al., 1987; Penketh et al., 1987) and doubts about its existence were raised (Penketh and Klein, 1986). A recent systematic investigation of the various developmental stages of
T. cruzi
even concluded that non-enzymatic oxidation of T(SH)
2
by H
2
O
2
may fully account for the slow hydroperoxide metabolism in this species (Carnieri et al., 1993).
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 FIG.
1
. Isoforms of tryparedoxin exist in one species.
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 of SEQ ID NO: 5 and encoding the amino acid sequence of SEQ ID NO: 4 (
FIG. 2
) or any useful part thereof.
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: 6 (
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: 6 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: 4 or SEQ ID NO: 6.
Further, the protein according to (b) can be a protein having an amino acid sequence which is homologous to SEQ ID NO: 4 or SEQ ID NO: 6 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: 6 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

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