PEG-MODIFIED ARGININE/LYSINE OXIDOREDUCTASE

Abstract

directed to an arginine/lysine oxidoreductase modified with polyethylene glycol and to a production method thereof and to methods of treating disorders responsive to a modification of amino acid levels reactive oxygen species and/or ammonium.

Description

FIELD OF THE INVENTION

[0001]The present invention is directed to an arginine/lysine oxidoreductase modified with polyethylene glycol.

BACKGROUND OF THE INVENTION

[0002]Since decades, tremendous efforts are made to collect pharmacologically active substances from natural organisms and to test their effects in various disease areas.

[0003]The sea hare Aplysia punctata produces a purple ink to protect itself from predation. This ink was shown to contain an anti-tumor activity (Butzke et al., 2004). Subsequently, studies were conducted to isolate the factor from crude ink, resulting in the discovery of APIT, the Aplysia Punctata Ink Toxin. Recently, the factor was cloned and characterized to be a weakly glycosylated FAD-binding L-amino acid oxidase that catalyzes the oxidative deamination of L-lysine and L-arginine and thereby produces hydrogen peroxide (H₂O₂), ammonium (NH₄.sup.+) and the corresponding alpha-keto acids (Butzke et al., 2005).

[0004]L-amino acid oxidases (LAAOs, EC 1.4.3.2), can be found in secretions and venoms. Members of this family of flavoenzymes catalyze the stereospecific oxidative deamination of L-amino acids and thereby produce H₂O₂, ammonium and the corresponding alpha-keto acids (Du et al., 2002). The individual LAAOs differ in their substrate specificity: Snake venom L-amino acid oxidases (sv-LAAOs) which constitute up to 30% (by weight) of the crude venom (Ponnudurai et al., 1994), possess a clear preference for hydrophobic amino acids. A fish capsule LAAO termed AIP (Apoptosis-Inducing Protein) which is induced by larval nematode infection of Scomber japonicus is highly specific for L-lysine (Jung et al., 2000). Achacin, a mucus LAAO from the African snail Achatina fulica, metabolizes a very broad range of substrates, including hydrophobic amino acids along with L-lysine, L-arginine, L-cysteine, L-asparagine and L-tyrosine (Ehara et al., 2002).

[0005]L-amino acid oxidases have not been investigated for their half-life after administration to mammalians at any routes. With respect to drug development, this is however an important issue and a prerequisite to efficiently exert a therapeutic effect. Data in this respect are only available for a D-amino acid oxidase employing D-proline as substrate (Fang et al., 2002). After intravenous injection, native D-amino acid oxidase was, however, rapidly cleared from the circulation. A pegylated derivative did not exhibit a significantly increased circulation half-life time which was well below 1 h. A product with such a short circulation half life time cannot be expected to have a substantial therapeutic benefit for therapy, e.g. for cancer therapy. Thus, according to the teaching of the prior art it would have been expected that pegylated amino acid oxidases would lack therapeutically efficacy.

[0006]An object of the present invention was to increase the therapeutic efficacy of L-amino acid oxidoreductases in order to provide a therapeutic product with improved characteristics.

SUMMARY OF THE INVENTION

[0007]The present invention addresses for the first time an arginine/lysine oxidoreductase modified with at least one polyethylene glycol moiety and pharmaceutical compositions comprising said modified arginine/lysine oxidoreductase, a production method thereof and methods to treating diseases responsive to modulation of plasma amino acid levels or/and responsive to reactive oxygen species or/and ammonium, for example proliferative diseases, viral infections or/and microbial infections.

[0008]In a preferred embodiment, an arginine/lysine oxidoreductase termed Aplysia punctata ink toxin (APIT) is modified with at least one polyethylene glycol moiety having a weight average molecular weight of from about 1,000 to about 10,000.

[0009]An additional embodiment of the invention describes methods of production of a pegylated arginine/lysine oxidoreductase.

[0010]A further embodiment describes a method of enhancing the enzymatic activity and circulation time of an arginine/lysine oxidoreductase comprising pegylation of an arginine/lysine oxidoreductase.

[0011]An additional embodiment refers to a method of protecting an arginine/lysine oxidoreductase against inactivation by body fluids comprising pegylation of an arginine/lysine oxidoreductase.

[0012]In a further aspect, the present invention refers to a method for depleting the amino acids lysine or/and arginine in a liquid, comprising adding an arginine/lysine oxidoreductase to the liquid.

[0013]Yet another embodiment refers to a kit comprising an arginine/lysine oxidoreductase of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0014]The object of the present invention is achieved by covalently modifying arginine/lysine oxidoreductases, particularly L-arginine/lysine oxidases, with polyethylene glycol (PEG). The present invention is based on the surprising discovery that arginine/lysine oxidoreductases modified with polyethylene glycol are circulating in the plasma of mice for more than 48 h and retain their enzymatic activity. In contrast, unmodified arginine/lysine oxidoreductases are active in circulation for only about 3 h at equivalent activity-based dose.

[0015]Furthermore, it was surprisingly found that dosing of a PEG modified arginine/lysine oxidoreductase can be significantly reduced compared with the unmodified arginine/lysine oxidoreductase.

[0016]Moreover, it was surprisingly found that PEG modified arginine/lysine oxidoreductase has a smaller IC50 value for inhibition of lung cancer cells than unmodified arginine/lysine oxidoreductase.

[0017]Furthermore, arginine/lysine oxidoreductases modified with polyethylene glycol provide a unique means in lowering amino acid levels lysine and arginine thereby producing reactive oxygen species and ammonium. All these effects or parts thereof are sufficient to treat certain types of disorders. When compared to a native arginine/lysine oxidoreductase, a pegylated arginine/lysine oxidoreductase retains most of its enzymatic activity, is able to deplete lysine and arginine in mammalians for 48 h and more, and is much more efficacious in the treatment of diseases responsive to a modification of amino acid levels and/or reactive oxygen species or ammonium. Moreover, the pegylated arginine/lysine oxidoreductase is active even in the presence of specific antibodies directed to the unmodified arginine/lysine oxidoreductase.

[0018]In a first aspect, the present invention provides a conjugate comprising an arginine/lysine oxidoreductase and at least one polyethylene glycol moiety.

[0019]"Conjugate" as used herein refers to a compound which comprises a polypeptide portion preferably including a coenzyme such as FAD to which at least one polyethylene glycol moiety has been coupled by a covalent linkage.

[0020]The conjugate of the present invention may comprise any known arginine/lysine oxidoreductase, particularly L-arginine/lysine oxidoreductase, which catalyzes the conversion of L-arginine or/and L-lysine into the respective alpha imino acid. In this step, the enzyme produces H₂O₂ in stoichiometric amounts. In a second step, the alpha imino acid is converted into an alpha keto acid under release of ammonium. The second step is not dependent upon the arginine/lysine oxidoreductase activity. Details of the reaction catalyzed by arginine/lysine oxidoreductase are described in WO 2004/065415 which is included herein by reference.

[0021]In another preferred embodiment of the present invention, the conjugate comprises an arginine/lysine oxidoreductase which is an L-arginine/L-lysine oxidoreductase.

[0022]In a further preferred embodiment, the conjugate comprises an arginine/lysine oxidoreductase which is specific for arginine or/and lysine. In particular, the enzymatic activity for the processing of lysine or/and arginine, in particular L-lysine or/and L-arginine, is at least a factor of about 3 or about 4 larger than the enzymatic activity for the processing of other amino acids, in particular of alpha-L-amino acids naturally present in organisms.

[0023]A preferred arginine/lysine oxidoreductase may be obtained from an Aplysia species, in particular from Aplysia punctata. For example, the Aplysia Punctata Ink Toxin (APIT), which is an arginine/lysine oxidoreductase of the present invention, can be found in the ink of the sea hare Aplysia punctata. This enzyme and its manufacture is described in WO 2004/065415 which is included herein by reference.

[0024]Further, a suitable L-lysine a-oxidase is described in Lukasheva E V, Berezov T T. L-Lysine alpha-oxidase: physiochemical and biological properties. Biochemistry (Mosc.) 2002 October; 67(10):1152-8.

[0025]The arginine/lysine oxidase may be a native molecule isolated from a natural source or a recombinant molecule obtained from a recombinant, e.g. non-naturally ocurring source. In a preferred embodiment, the conjugate may also comprise a recombinant arginine/lysine oxidoreductase.

[0026]In an especially preferred embodiment, the conjugate of the present invention comprises an arginine/lysine oxidoreductase comprising

(a) the sequence SEQ ID NO: 2, 4, 6, or/and 8,(b) a sequence with at least which is at least 70% identical to the sequence of (a), or/and(c) a fragment of (a) or/and (b).

[0027]SEQ ID NO: 2, 4, 6, and 8 differ in several amino acid positions. SEQ ID NO: 8 describes a preferred arginine/lysine oxidoreductase isolated from Aplysia punctata (see examples of the present invention). SEQ ID NO: 2, 4, and 6 describe further arginine/lysine oxidoreductases isolated from Aplysia punctata. SEQ ID NO: 2, 4, and 6 are described in WO 2004/065415 which is included herein by reference.

[0028]The preferred sequence of (a) is SEQ ID NO: 8.

[0029]The sequence of (b) is a sequence which is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, most preferably at least 99% identical to the sequence of (a). The identity is determined in the region of maximal overlap. The person skilled in the art can determine the region of maximal overlap by commonly known algorithms such as FASTA, BLAST or/and derivatives thereof relating to amino acid sequences.

[0030]The fragment of (c) is any enzymatically active fragment of an arginine/lysine oxidoreductase of (a) or (b) and may have a length of a least about 30 amino acid residues, preferably at least about 50 amino acid residues, more preferably at least about 100 amino acid residues, most preferably at least about 200 amino acid residues.

[0031]The fragment of (c) may have a length of smaller than the full length of SEQ ID NO: 2, 4, 6, or/and 8, preferably at the maximum about 500 amino acid residues, more preferably at the maximum about 400 amino acid residues, most preferably at the maximum about 300 amino acid residues.

[0032]The fragment of (c) may be selected from sequences derived from SEQ ID NO: 2, 4, 6 or/and 8 by removing N-terminal amino acids. The N-terminus may represent signal sequences which are not required for the oxidoreductase function. Preferably, up to about 5, up to about 10, up to about 20, or up to about 50 N-terminal amino acids are removed. More preferably, in SEQ ID NO: 2, 18 N-terminal amino acids are removed, or/and in SEQ ID NO:4, 17 N-terminal amino acids are removed.

[0033]The conjugate of the present invention is preferably active in the circulation for at least 6 h, i.e. the conjugate has preferably a circulation time of at least 6 h. In the context of the present invention, "circulation time" refers to the time the conjugate of the present invention retains its activity, in particular its enzymatic activity, during circulation in a subject. In the present invention, the term "circulation time" is also applied to other compounds for comparative purposes. For instance, "circulation time" is applied to unmodified APIT. The circulation time may also be expressed by the circulation half life time.

[0034]The conjugate of the present invention is more preferably active in the circulation for at least 12 h, even more preferably at least 24 h, most preferably at least 48 h.

[0035]It was surprisingly found that the dose of the conjugate of the present invention can significantly be reduced compared with the corresponding unmodified arginine/lysine oxidoreductase. For instance, an amount of 0.6 Units/kg PEG-5000-APIT is capable of depleting lysine and arginine levels in blood plasma in mice for 6 h. Unmodified APIT requires 1000 Units/kg to achieve the same effect. Thus the dose of PEG-5000-APIT can be reduced by a factor of about 1667 compared with unpegylated APIT. In other words, the activity of PEG-5000-APIT is a factor of about 1667 larger than the activity of unmodified APIT.

[0036]Therefore, in a preferred embodiment, the activity of the conjugate of the present invention is a factor of at least 30, preferably at least 100, more preferably at least 300, even more preferably at least 1000, most preferably at least 1500 larger than the activity of the arginine/lysine oxidoreductase not carrying a polyethylene glycol moiety. The activity is in particular the enzymatic activity. More particularly the activity is the enzymatic activity in the blood plasma after administration to a subject, for instance a mammal such as a rodent or a human. The enzymatic activity may be determined by determination of the dose required to deplete arginine or/and lysine in a liquid, for instance a body fluid such as blood plasma, for a predetermined length of time. The length of time may be selected from ranges of about 3 to about 48 h, about 6 to about 24 h, or about 12 to about 18 h, or may preferably be about 3 h, about 6 h, about 12 h, about 24 h, or about 48 h. Arginine or/and lysine may be depleted to a predetermined level, for instance 0 μM. The predetermined level may be selected from a range of 0 μM to about 10 μM, 0 μM to 20 μM, or 0 μM to about 100 μM.

[0037]The factor of activity improvement can for instance be calculated by dividing the dose of the unpegylated arginine/lysine oxidoreductase by the dose of the conjugate of the present invention, which doses are required for depletion of arginine or/and lysine for a predetermined length of time.

[0038]In a further preferred embodiment, the conjugate of the present invention comprises at least one polyethylene glycol moiety having a weight average molecular weight of about 1,000 Dalton to about 10,000 Dalton, preferably of about 3,000 Dalton to about 8,000 Dalton, more preferably of about 4,000 Dalton to about 6,000 Dalton, even more preferably about 4,500 Dalton to about 5,500 Dalton, most preferably about 5,000 Dalton.

[0039]The polyethylene glycol moiety as employed herein includes unmodified and modified polyethylene glycol moieties suitable for coupling to polypeptides. The modified polyethylene glycol moieties preferably include terminally modified polyethylene glycol moieties, wherein the terminal OH group has been modified, e.g. by alkylation, acylation and/or oxidation. More preferably, the polyethylene glycol moieties have terminal OH groups or modified terminal groups selected from O--C_1-3 alkyl groups and acyl groups or combinations thereof.

[0040]In yet another preferred embodiment, the conjugate comprises at least one polyethylene glycol moiety which is covalently coupled to the arginine/lysine oxidoreductase via a linking group. The linking group may be a succinimide group, preferably a succinimidyl succinate group.

[0041]In yet another preferred embodiment, the conjugate of the present invention comprises from 1 to about 30 polyethylene glycol moieties, preferably from 1 to about 20 polyethylene glycol moieties, more preferably from 1 to about 10 polyethylene glycol moieties.

[0042]In the conjugate of the present invention, the at least one polyethylene glycol group may be coupled to any amino acid with a side chain carrying a reactive functional group, such as a carboxylate group, amino group, thiol group and/or hydroxy group, for example an amino acid selected from aspartate, glutamate or/and lysine. A preferred embodiment refers to a conjugate wherein the at least one polyethylene glycol moiety is coupled via a lysine residue to the arginine/lysine oxidoreductase. Since SEQ ID NO: 8 comprises 23 lysine residues, a conjugate of the present invention comprising a polypeptide of SEQ ID NO: 8 may comprise up to 23 polyethylene glycol moieties, preferably up to about 10 polyethylene glycol moieties coupled via a lysine residue.

[0043]It is more preferred that in the conjugate of the present invention, the at least one polyethylene group is coupled to lysine via a linker group, e.g. a succinimide linker group or any other suitable linker group for coupling to amino groups. It is even more preferred that in the conjugate of the present invention, the at least one polyethylene group has a weight average molecular weight of about 4,500 to about 5,500 Dalton and is coupled to lysine by via a linker group, e.g. a succinimide linker group.

[0044]Most preferred is a conjugate comprising an L-arginine/L-lysine oxireductase comprising SEQ ID NO: 8 and at least one polyethylene glycol group having a weight average molecular weight of about 4,500 to 5,500 Dalton.

[0045]Yet another aspect is a method of producing the conjugate of the present invention, comprising the steps

(a) recombinantly expressing an arginine/lysine oxidoreductase or/and isolating an arginine/lysine oxidoreductase from a natural source,(b) coupling at least one polyethylene glycol moiety to the arginine/lysine oxidoreductase of (a).

[0046]Preferred natural sources of an arginine/lysine oxidoreductase which may be employed in step (a) are as described above.

[0047]Step (a) preferably comprises a recombinant expression of a nucleic acid comprising

(i) the sequence SEQ ID NO: 1, 3, 5, or/and 7,(ii) a sequence complementary to the sequence of (i),(iii) a sequence within the scope of the degeneracy of the genetic code of the sequence of (i) or (ii),(iv) a sequence which is at least 70% identical to the sequence of (i), (ii) or/and (iii),(v) a sequence which hybridizes with any of the sequences (i), (ii), (iii) or/and (iv) under stringent conditions, or/and (vi) a fragment of any of the sequences (i), (ii), (iii), (iv), or/and (v).

[0048]SEQ ID NO: 1, 3, 5, and 7 are nucleic acid sequences encoding polypeptides comprising the amino acid sequences SEQ ID NO: 2, 4, 6, and 8, respectively. SEQ ID NO: 1, 3, 5, and 7 differ in several nucleotide positions. SEQ ID NO: 7 describes the nucleotide sequence encoding an arginine/lysine oxidoreductase isolated from Aplysia punctata (see examples of the present invention). SEQ ID NO:1, 3, and 5 describe further nucleotide sequences encoding arginine/lysine oxidoreductases isolated from Aplysia punctata. SEQ ID NO:1, 3, and 5 are described in WO 2004/065415 which is included herein by reference.

[0049]A preferred sequence of (i) is SEQ ID NO: 7.

[0050]The nucleotide sequence of (iv) is a sequence which is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, most preferably at least 99% identical to the sequence of (i), (ii) or/and (iii). The identity of the nucleotide sequences is determined within the region of maximal overlap. A person skilled in the art can easily determine the region of maximal overlap by commonly known algorithms such as FASTA or BLAST.

[0051]The person skilled in the art knows stringent hybridization conditions (see e.g. Sambrook J. et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.). Hybridization under stringent conditions in step (v) preferably means that after washing for 1 h with 1×SSC and 0.1% SDS at 55° C., preferably at 62° C. and more preferably at 68° C., particularly after washing for 1 h with 0.2×SSC and 0.1% SDS at 55° C., preferably at 62° C. and more preferably at 68° C., a hybridization signal is detected.

[0052]The fragment of (vi) may have a length of a least about 90 nucleotide residues, preferably at least about 150 nucleotide residues, more preferably at least about 300 nucleotide residues, most preferably at least about 600 nucleotide residues.

[0053]The fragment of (vi) may have a length of smaller than the full length of SEQ ID NO: 1, 3, 5, or/and 7, preferably at the maximum about 1500 nucleotide residues, more preferably at the maximum about 1200 nucleotide residues, most preferably at the maximum about 900 nucleotide residues.

[0054]The fragment of (vi) may be selected from sequences derived from SEQ ID NO: 1, 3, 5 and 7 by removing 5' nucleotides encoding the N-terminal amino acids which may represent signal sequences which are not required for the oxidoreductase function. Preferably, up to about 15, up to about 30, up to about 60, or up to about 150 5' nucleotides are removed. More preferably, in SEQ ID NO: 1, 54 5' nucleotides are removed, or/and in SEQ ID NO:3, 51 5' nucleotides are removed.

[0055]The person skilled in the art knows methods for recombinant expression of a protein, isolation, refolding and introduction of prosthetic groups such as FAD. In the examples of the present invention the recombinant expressing of SEQ ID NO: 7 is described in order to obtain a polypeptide comprising SEQ ID NO: 8. Preferred host cells for recombinant expression are prokaryotic or eukaryotic host cells, e.g. yeast cells or bacterial cells, particularly Gram-negative bacterial cells, such as E. coli.

[0056]The method for the preparation of the conjugate of the present invention may further comprise the introduction of a coenzyme such as FAD into the arginine/lysine oxidoreductase.

[0057]Step (b) of the method for the preparation of the conjugate of the present invention refers to coupling of at least one polyethylene glycol moiety to the arginine/lysine oxidoreductase obtained in step (a). Pegylation in step (b) may be performed by standard procedures known by a person skilled in the art.

[0058]In step (b), a polyethylene glycol may be employed having a weight average molecular weight of about 1,000 Dalton to about 10,000 Dalton, preferably of about 3,000 Dalton to about 8,000 Dalton, more preferably of about 4,000 Dalton to about 6,000 Dalton, even more preferably about 4,500 Dalton to about 5,500 Dalton, most preferably about 5,000 Dalton.

[0059]The polyethylene glycol moiety may be covalently bound via a linking group, which may be a succinimide group, preferably a succinimidyl succinate.

[0060]Other linking groups may also be employed. The person skilled in the art knows suitable linking groups.

[0061]It is preferred that the at least one polyethylene glycol moiety is coupled via a lysine residue to the arginine/lysine oxidoreductase. Coupling to lysine can be performed by applying a succinimide linker group. Coupling may also performed to other amino acid side chains carrying a functional group such as a carboxylase group, amino group, thiol group or/and hydroxy group, such as aspartate or glutamate.

[0062]In order to provide a conjugate of the present invention comprising from 1 to about 30 polyethylene glycol moieties, preferably from 1 to about 20 polyethylene glycol moieties, more preferably from 1 to about 10 polyethylene glycol moieties, an excess of polyethylene glycol may be employed in step (b). The polyethylene glycol may be provided in an amount of from about 10 to about 500 equivalents, preferably from about 10 to about 50 equivalents with respect to the number of free residues available for coupling, for instance the number of free lysine residues. In particular, about 10, about 20, about 30, about 40, about 50, about 200, or about 500 equivalents polyethylene glycol may be employed. An excess of 50 equivalents may result in coupling of about 10 PEG molecules per molecule arginine/lysine oxidoreductase.

[0063]Yet another aspect of the present invention is a pharmaceutical composition comprising a conjugate of the present invention optionally together with pharmaceutically acceptable carriers, adjuvants, diluents or/and additives.

[0064]The pharmaceutical composition of the present invention may be suitable for the prevention, alleviation or/and treatment of a disease responsive to reactive oxygen species or/and ammonium, or/and responsive to modulation of plasma amino acid levels, in particular of plasma lysine or/and arginine levels. The pharmaceutical composition of the present invention may also be suitable for the prevention, alleviation or/and treatment of a disease selected from microbial infections, viral infections such as HIV, hepatitis B or/and C virus infections, and proliferative diseases such as cancer.

[0065]The pharmaceutical composition of the present invention is suitable for the treatment of solid tumors and leukemias in general including apoptosis resistant and multi drug resistant cancer forms.

[0066]The proliferative disease to be treated with the pharmaceutical composition of the present invention may be lung cancer, MDR lung cancer, head and neck cancer, breast cancer, prostate cancer, colon cancer, cervix cancer, uterus cancer, larynx cancer, gastric cancer, liver cancer, Ewings sarkoma, acute lymphoid leukemia, acute and chronic myeloid leukemia, apoptosis resistant leukemia, pancreas cancer, kidney cancer, gliomas, melanomas, chronic lympoid leukemia, or/and lymphoma.

[0067]Yet another aspect of the present invention is a method for the prevention, alleviation or/and treatment of a disease responsive to reactive oxygen species, ammonium or/and responsive to modulation of amino acid levels in body fluids, in particular of plasma lysine or/and arginine levels, which method comprises administering an effective amount of the conjugate of the of the present invention or/and a pharmaceutical composition of the present invention to a subject in need thereof. The disease to be treated by the method is in particular a microbial infection, a viral infection or/and a proliferative disease as defined above.

[0068]Yet another aspect of the present invention is a kit comprising the conjugate of the present invention or/and the pharmaceutical composition of the present invention.

[0069]A further aspect of the present invention refers to a method for enhancing the enzymatic activity or/and circulation time of an arginine/lysine oxidoreductase, which method comprises coupling at least one polyethylene glycol moiety to the arginine/lysine oxidoreductase. In this method, the arginine/lysine oxidoreductase may in particular be the arginine/lysine oxidoreductase as defined herein. The polyethylene glycol moiety may be as defined herein.

[0070]Yet a further aspect of the present invention is a method for protecting an arginine/lysine oxidoreductase against inactivation in body fluids such as blood plasma comprising coupling at least one polyethylene glycol moiety to the arginine/lysine oxidoreductase. In this method, the arginine/lysine oxidoreductase may in particular be the arginine/lysine oxidoreductase as defined herein. The polyethylene glycol moiety may be as defined herein. In particular, inactivation in body fluids may be inactivation by antibodies.

[0071]A PEG group, in particular a PEG group as described herein, may be used for enhancing the enzymatic activity or/and circulation time of an arginine/lysine oxidoreductase, in particular of an arginine/lysine oxidoreductase as defined herein.

[0072]A PEG group, in particular a PEG group as described herein, may be used for protecting an arginine/lysine oxidoreductase, in particular an arginine/lysine oxidoreductase as defined herein against inactivation in body fluids such as blood plasma, in particular by antibodies.

[0073]The conjugate of the present invention as defined herein or/and the pharmaceutical composition of the present invention as defined herein may be used for the depletion of lysine or/and arginine in a liquid or/and for the production of hydrogen peroxide, ammonium or/and metabolites of lysine or/and arginine in a liquid. The liquid may in particular be a body fluid of a mammal.

[0074]The invention is further demonstrated in the following examples and figures, which are for purposes of illustration, and are not intended to limit the scope of the present invention.

[0075]Figure Legends

[0076]FIG. 1: Primary amino acid sequence of arginine/lysine oxidoreductase, APIT (SEQ ID NO: 8) and a nucleotide sequence coding therefor (SEQ ID NO:7).

[0077]FIG. 2: Pegylation of arginine/lysine oxidoreductase, APIT with PEG-5000.

[0078]FIG. 3: Activity of PEG-5000-APIT in the presence of anit-APIT polyclonal antibodies.

[0079]FIG. 4: Effect of single intravenous dose of APIT (250 U/kg) and APIT (1000 U/kg) on plasma lysine levels in mice.

[0080]FIG. 5: Effect of single intravenous dose of APIT (250 U/kg) and APIT (1000 U/kg) on plasma arginine levels in mice.

[0081]FIG. 6: Comparison of single intravenous dose of APIT (250 U/kg) and PEG-5000-APIT at an equivalent dose of 250 U/kg on plasma lysine levels in mice.

[0082]FIG. 7: Comparison of single intravenous dose of APIT (250 U/kg) and PEG-5000-APIT at an equivalent dose of 250 U/kg on plasma arginine levels in mice.

[0083]FIG. 8: Dose-dependent depletion of plasma lysine levels after single intravenous administration of PEG-5000-APIT to mice.

[0084]FIG. 9: Dose-dependent depletion of plasma arginine levels after single intravenous administration of PEG-5000-APIT to mice.

[0085]FIG. 10: Antitumor efficacy of PEG-5000-APIT.

[0086]FIG. 11: Nucleotide sequences of cDNA encoding arginine/lysine oxidoreductases isolated from ink of Aplysia punctata (SEQ ID NO: 1, 3 and 5) and the derived amino acid sequences (SEQ ID NO: 2, 4, and 6). The dinucleotide binding fold (28 amino acid residues) and the GG motif (8 amino acid residues) are indicated by boxes.

EXAMPLES

Example 1

Method of bulk production of recombinant arginine/lysine oxidoreductase, ART in E. coli

[0087]A B-Braun Biostat B10 Fermenter with 5000 ml medium is inoculated with a working cell starter culture containing the APIT-cDNA comprising SEQ ID NO: 7 under control of a suitable promoter (e.g. T7). Cells are grown up to an optical density of 6-8 (at 600 nm) at 37±0.5° C. Subsequently the cells are induced through the addition of IPTG and further fermented for additional 3 h at 37±0.5° C. Thereafter, cells are harvested through centrifugation and stored at ≦-60° C. The resuspended cells are disrupted using a French press and the inclusion bodies are separated from the supernatant by centrifugation. The inclusion bodies are stored up to further processing at ≦-60° C. APIT is isolated with urea from the inclusion bodies of the E. coli biomass. The solubilized protein is then refolded, concentrated and desalted by TFF (Tangential Crossflow Filtration). Subsequently, APIT is purified by anion exchange chromatography and size exclusion chromatography (SEC).

Isolation of APIT from Inclusion Bodies:

[0088]The pellet is resuspended two times in succession in washing-buffer, then NaCl-Washing-buffer and Urea-washing-buffer. The suspension is centrifuged following each resuspension-step, while the remaining supernatant is discarded. Thereafter, the pellet is resuspended in Resuspensions-Buffer following a final centrifugation step. The supernatant is filtrated through 0.2 μm and the (APIT-containing) solution is stored frozen at <-15° C.

Refolding:

[0089]This process step is carried out at room temperature (22-25° C.). 200 mL of the isolated APIT from E. coli (in Resuspension-Buffer) are mixed with 200 mL Guanidine-HCl. This mixture is then injected into 20 L refolding buffer containing L-arginine and FAD, using a pump. The mixing/dilution step is supported by a (maximal active) magnet stirrer. Die dilution is realized via 4 Injection sites.

Purification by TFF:

[0090]The RF-mixture is filtered using a TFF (0.2 μm) to separate unfolded protein. Thereafter, the system is rinsed with 1.6 L Tris-Buffer. APIT is concentrated using a 10 kDa-Membrane. Thereafter, the RF-Buffer is dialyzed against the Tris-Buffer. During this step, the conductivity of the RF-mixture and Tris-buffered mixture is measured. The 10 kDa-Membrane is maintained to change the buffer and to continue the concentration step. The level of conductivity decrease in the concentrated APIT solution indicates the duration of the procedure.

Anion-Exchange Chromatography:

[0091]The APIT containing sample is further purified by anion-exchange chromatography (SOURCE 30Q-column). The elution of the product from the column is done using a salt-gradient (0-500 mM NaCl) at pH 8.0. The elute is collected in fractions.

Size Exclusion Chromatography:

[0092]The APIT-containing sample is desalted using a size exclusion chromatography (Sephadex G25). The purification occurs with D-PBS. The eluted product is collected in fractions.

Filling and Storage:

[0093]The filling of the bulk product is in sterile PE-containers at a volume of 1.000 μl and stored at 2-8° C. The primary amino acid sequence of recombinant APIT is shown in FIG. 1.

Example 2

Pegylation of Arginine/Lysine Oxidoreductase, APIT with PEG-5,000

[0094]Purified arginine/lysine oxidoreductase, APIT from Example 1 was desalted with HiPrep Desalting 26/10 column and brought into a pegylation reaction buffer containing 50 mM sodiumbicarbonat, pH 9.5. The pegylation reactions were carried by adding a linear polydisperse polyethylene-glycol succinimidylester, MW 5000 Da (PEG-5000-SS) at varying ratios of PEG-5000-SS to arginine/lysine oxidoreductase (based on free lysine residues in APIT) from 10 to 50 equivalents (eq) and stirring for 1 hour at 800 rpm and 25° C. The PEG-conjugated arginine/lysine oxidoreductase variants, PEG-5000-APIT (10 eq), PEG-5000-APIT (20 eq), PEG-5000-APIT (40 eq), PEG-5000-APIT (50 eq) were individually purified by gelfiltration from reaction-products and unconjugated PEG-reagent and the buffer was exchanged to sodium phosphate buffered saline PH 7). The final products were analyzed on SDS-page and for enzymatic activity (FIG. 2). An excess of 50 eq PEG resulted in coupling of about 10 molecules PEG to one molecule APIT.

[0095]FIG. 2 shows that the PEG-5000-APIT had an apparent molecular weight ranging from 60 kda to >212 kDa. All PEG-5000-APIT variants were enzymatically active in comparison to unconjugated APIT. The relative enzymatic activities of PEG-5000-APIT (10 eq), PEG-5000-APIT (20 eq), PEG-5000-APIT (40 eq), PEG-5000-APIT (50 eq) ranged from 58% to 72%.

Example 3

Activity in the Presence of Anti-APIT Polyclonal Antibodies

[0096]A high titer (>1:200,000) polyclonal antibody serum against APIT was raised in rabbits. In order to investigate if PEG-5000-APIT is inactivated by antibodies against APIT, pre-immune rabbit serum and anti-APIT rabbit immune serum was incubated with PEG-5000-APIT for 1 h at 37° C. following analysis of amino acid levels for lysine and arginine. FIG. 3 shows that lysine and arginine levels in rabbit pre-immune rabbit serum and anti-APIT rabbit immune serum were normal (lysine: 238 μM and 265 μM respectively; arginine 207 μM and 179 μM respectively). Incubation with PEG-5000-APIT resulted in levels of lysine and arginine of 0 μM in both, pre-immune rabbit serum and anti-APIT rabbit immune serum demonstrating that PEG-5000-APIT is enzymatically active in the presence of anti-APIT polyclonal antibodies and allows for complete depletion of lysine and arginine in liquids.

Example 4

Application to Mice

[0097]Mice received a single intravenous administration (tail vein) of unconjugated APIT (250 U/kg or 1000 U/kg) or PEG-5000-APIT (250 U/kg, 79 U/kg, 11 U/kg, 2 U/kg, 0.6 U/kg) or D-PBS (vehicle control) respectively. Serial blood samples were collected from each animal via orbital sinus under etherization at -1 h pre-dose and at the indicated time-points post-dose and transferred into tubes containing anticoagulant (heparin). Each blood sample was centrifuged at 4° C. to prepare plasma for analysis of plasma amino acid levels.

[0098]For the amino acid analysis, 25 μL heparin plasma samples were mixed with 4 μL precipitation-buffer containing sulfosalicyl acid. Then, 6 μL sample dilution buffer containing the internal standard norleucine (Nle, c: 1 nmol/μL) was added and samples were filled up to a final volume of 60 μL with sample dilution buffer. Thereafter, samples were mixed (Vortex) and centrifuged. The supernatants were passed through a centrifuge filter by centrifugation. The centrifugate was transferred into a sample vial and analyzed with the amino acid analyzer A200 using a physiologic program.

[0099]FIG. 4 and FIG. 5 show the effect of APIT at a single intravenous dose of 250 U/kg and 1000 U/kg to mice on plasma arginine or plasma lysine levels, respectively. A 4-fold increase of the APIT dose from 250 U/kg to 1000 U/kg results in an elongation of depletion of lysine or arginine from 3 h to 6 h post-dose.

[0100]FIGS. 6 and 7 show a direct comparison of the effect of unconjugated APIT and PEG-5000-APIT at a single intravenous dose of 250 U/kg. While unconjugated APIT mediates depletion of lysine (FIG. 6) and arginine (FIG. 7) only for 3 h post-dose, PEG-5000-APIT depletes both amino acids for at least 48 h post-dose.

[0101]Lysine and arginine plasma levels after single intravenous administration of different doses of PEG-5000-APIT to mice are shown in FIG. 8 and FIG. 9, respectively.

[0102]FIG. 8 demonstrates that a single intravenous dose of 11 Units/kg or 25 U/kg or 79 U/kg PEG-5000-APIT completely depletes plasma lysine levels in mice. A dose of 0.6 U/kg or 2 U/kg still mediates a complete depletion of Lysine in mice for 6 h, which is the effect of 1000 U/kg unconjugated APIT. FIG. 9 demonstrates that a single intravenous dose of 79 Units/kg PEG-5000-APIT completely depletes plasma arginine levels in mice. A dose of 25 U/kg or 11 U/kg mediates a complete depletion of arginine in mice for 24 h.

[0103]A dose of 0.6 U/kg or 2 U/kg still mediates a complete depletion of arginine in mice for 6 h, which is the effect of 1000 U/kg unconjugated APIT.

[0104]Taken together, this unexpected findings demonstrated in FIGS. 8 and 9 mean that less than 0.1% PEG-5000-APIT relating to unconjugated APIT dose mediates equivalent effects on lysine and arginine levels in mammalians.

Example 5

Antitumor Efficacy of PEG-5000-APIT

[0105]The antitumor efficacy of PEG-5000-APIT was assessed in comparison to unconjugated APIT on A549 lung cancer cells by the MIT assay. The MTT assay is based upon the cleavage of the yellow tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to purple formazan crystals by metabolically active cells. The tumor cells were seeded into 96-well culture plates and maintained in culture for 24 hours before adding the APIT or PEG-5000-APIT for another 96 h. After treatment, 20 μl of MTT labeling reagent were added to each well and plates were incubated at 37° C. for 4 h. Following MTT incubation, the cultures were incubated with DMSO and the spectrophotometric absorbance of the samples was detected by using a microtiter plate reader at a wavelength of 550 nm. The result was then plotted against the test substance concentration to obtain a dose-response curve. The test substance concentration that leads to 50% inhibition (IC50) of the metabolic activity was graphically determined and is shown for unconjugated APIT or PEG-5000-APIT in FIG. 10. Unexpectedly, the IC 50 of PEG-5000-APIT was lower (633 pU/mL) than the IC50 with unconjugated APIT (717 pU/mL) on A549 cells, indicating a higher antitumor effect of PEG-5000-APIT compared to that of unconjugated APIT.

REFERENCES

[0106]Butzke, D., Machuy, N., Thiede, B., Hurwitz, R., Goedert, S., & Rudel, T. (2004) Hydrogen peroxide produced by Aplysia ink toxin kills tumor cells independent of apoptosis via peroxiredoxin I sensitive pathways. Cell Death. Differ., 11, 608-617. [0107]Butzke, D. Hurwitz, R., Thiede, B., Goedert, S., and Rudel, T. (2005) Cloning and biochemical characterization of APIT, a new L-amino acid oxidase from Aplysia punctata. Toxicon, 46, 479-489. [0108]Du, X. Y. & Clemetson, K. J. (2002) Snake venom L-amino acid oxidases. Toxicon, 40, 659-665. [0109]Ponnudurai, G., Chung, M. C., & Tan, N. H. (1994) Purification and properties of the L-amino acid oxidase from Malayan pit viper (Calloselasma rhodostoma) venom. Arch. Biochem. Biophys., 313, 373-378. [0110]Jung, S. K., Mai, A., Iwamoto, M., Arizono, N., Fujimoto, D., Sakamaki, K., & Yonehara, S. (2000) Purification and cloning of an apoptosis-inducing protein derived from fish infected with Anisakis simplex, a causative nematode of human anisakiasis. J. Immunol., 165, 1491-1497. [0111]Ehara, T., Kitajima, S., Kanzawa, N., Tamiya, T., & Tsuchiya, T. (2002) Antimicrobial action of achacin is mediated by L-amino acid oxidase activity. FEBS Lett., 531, 509-512. [0112]Fang J, Sawa T, Akaike T, Maeda H. Tumor-targeted delivery of polyethylene glycol-conjugated D-amino acid oxidase for antitumor therapy via enzymatic generation of hydrogen peroxide. Cancer Res. 2002 Jun. 1; 62 (11):3138-43.

Sequence CWU 1

811608DNAAplysia punctataCDS(1)..(1608) 1atg tcg tct gct gtg ctt ctc ctg gct tgt gcg ttg gtc atc tct gtc 48Met Ser Ser Ala Val Leu Leu Leu Ala Cys Ala Leu Val Ile Ser Val1 5 10 15cac gcc gac ggt atc tgc aga aac aga cgt caa tgt aac aga gag gtg 96His Ala Asp Gly Ile Cys Arg Asn Arg Arg Gln Cys Asn Arg Glu Val 20 25 30tgc ggt tct acc tac gat gtg gcc gtc gtg ggg gcg ggg cct ggg gga 144Cys Gly Ser Thr Tyr Asp Val Ala Val Val Gly Ala Gly Pro Gly Gly 35 40 45gct aac tcc gcc tac atg ctg agg gac tcc ggc ctg gac atc gct gtg 192Ala Asn Ser Ala Tyr Met Leu Arg Asp Ser Gly Leu Asp Ile Ala Val 50 55 60ttc gag tac tcg gac cga gtg ggc ggc cgg ctg ttc acc tac cag ctg 240Phe Glu Tyr Ser Asp Arg Val Gly Gly Arg Leu Phe Thr Tyr Gln Leu65 70 75 80ccc aac aca ccc gac gtt aac ctg gag att ggc ggc atg agg ttc atc 288Pro Asn Thr Pro Asp Val Asn Leu Glu Ile Gly Gly Met Arg Phe Ile 85 90 95gaa ggc gcc atg cac agg ctc tgg agg gtc att tca gaa ctc ggc cta 336Glu Gly Ala Met His Arg Leu Trp Arg Val Ile Ser Glu Leu Gly Leu 100 105 110acc ccc aag gtg ttc aag gaa ggt ttc ggc aag gag ggc aga caa aga 384Thr Pro Lys Val Phe Lys Glu Gly Phe Gly Lys Glu Gly Arg Gln Arg 115 120 125ttt tac ctg cgg gga cag agc ctg acc aag aaa cag gtc aag agt ggg 432Phe Tyr Leu Arg Gly Gln Ser Leu Thr Lys Lys Gln Val Lys Ser Gly 130 135 140gac gta ccc tat gac ctc agc ccg gag gag aaa gaa aac cag gga aat 480Asp Val Pro Tyr Asp Leu Ser Pro Glu Glu Lys Glu Asn Gln Gly Asn145 150 155 160ctg gtc gaa tac tac ctg gag aaa ctg aca ggt cta caa ctc aac ggc 528Leu Val Glu Tyr Tyr Leu Glu Lys Leu Thr Gly Leu Gln Leu Asn Gly 165 170 175gag ccg ctc aaa cgt gag gtt gcg ctt aaa cta acc gtg ccg gac ggc 576Glu Pro Leu Lys Arg Glu Val Ala Leu Lys Leu Thr Val Pro Asp Gly 180 185 190aga ttc ctc tat gac ctc tcg ttt gac gaa gcc atg gat ctg gtt gcc 624Arg Phe Leu Tyr Asp Leu Ser Phe Asp Glu Ala Met Asp Leu Val Ala 195 200 205tcc cct gag ggc aaa gag ttc acc cga gac acg cac gtc ttc aca gga 672Ser Pro Glu Gly Lys Glu Phe Thr Arg Asp Thr His Val Phe Thr Gly 210 215 220gag gtc acc ctg gac gcg tcg gct gtc tcc ctc ttc gac gac cac ctg 720Glu Val Thr Leu Asp Ala Ser Ala Val Ser Leu Phe Asp Asp His Leu225 230 235 240gga gag gac tac tat ggc agt gag atc tac acc cta aag gaa gga ctg 768Gly Glu Asp Tyr Tyr Gly Ser Glu Ile Tyr Thr Leu Lys Glu Gly Leu 245 250 255tct tcc gtc cca caa ggg ctc cta cag gct ttt ctg gac gcc gca gac 816Ser Ser Val Pro Gln Gly Leu Leu Gln Ala Phe Leu Asp Ala Ala Asp 260 265 270tcc aac gag ttc tat ccc aac agc cac ctg aag gcc ctg aga cgt aag 864Ser Asn Glu Phe Tyr Pro Asn Ser His Leu Lys Ala Leu Arg Arg Lys 275 280 285acc aac ggt cag tat gtt ctt tac ttt gag ccc acc acc tcc aag gat 912Thr Asn Gly Gln Tyr Val Leu Tyr Phe Glu Pro Thr Thr Ser Lys Asp 290 295 300gga caa acc aca atc aac tat ctg gaa ccc ctg cag gtt gtg tgt gca 960Gly Gln Thr Thr Ile Asn Tyr Leu Glu Pro Leu Gln Val Val Cys Ala305 310 315 320caa aga gtc atc ctg gcc atg ccg gta tac gct ctg aac caa cta gac 1008Gln Arg Val Ile Leu Ala Met Pro Val Tyr Ala Leu Asn Gln Leu Asp 325 330 335tgg aat cag ctc aga aat gac cga gcc acc caa gcg tac gct gcc gtt 1056Trp Asn Gln Leu Arg Asn Asp Arg Ala Thr Gln Ala Tyr Ala Ala Val 340 345 350cgc ccg att cct gca agt aag gtg ttc atg tcc ttt gat cag ccc tgg 1104Arg Pro Ile Pro Ala Ser Lys Val Phe Met Ser Phe Asp Gln Pro Trp 355 360 365tgg ttg gag aac gag agg aaa tcc tgg gtc acc aag tcg gac gcg ctt 1152Trp Leu Glu Asn Glu Arg Lys Ser Trp Val Thr Lys Ser Asp Ala Leu 370 375 380ttc agc caa atg tac gac tgg cag aag tct gag gcg tcc gga gac tac 1200Phe Ser Gln Met Tyr Asp Trp Gln Lys Ser Glu Ala Ser Gly Asp Tyr385 390 395 400atc ctg atc gcc agc tac gcc gac ggc ctc aaa gcc cag tac ctg cgg 1248Ile Leu Ile Ala Ser Tyr Ala Asp Gly Leu Lys Ala Gln Tyr Leu Arg 405 410 415gag ctg aag aat cag gga gag gac atc cca ggc tct gac cca ggc tac 1296Glu Leu Lys Asn Gln Gly Glu Asp Ile Pro Gly Ser Asp Pro Gly Tyr 420 425 430aac cag gtt acc gaa ccc ctc aag gac acc att ctt gac cac ctc act 1344Asn Gln Val Thr Glu Pro Leu Lys Asp Thr Ile Leu Asp His Leu Thr 435 440 445gag gct tat ggc gtg gag cga gac tcg atc ccg gaa ccc gtg acc gcc 1392Glu Ala Tyr Gly Val Glu Arg Asp Ser Ile Pro Glu Pro Val Thr Ala 450 455 460gct tcc cag ttc tgg aca gac tac ccg ttt ggc tgt gga tgg atc acc 1440Ala Ser Gln Phe Trp Thr Asp Tyr Pro Phe Gly Cys Gly Trp Ile Thr465 470 475 480tgg agg gcc ggc ttc cat ttc gat gac gtc atc agc acc atg cgt cgc 1488Trp Arg Ala Gly Phe His Phe Asp Asp Val Ile Ser Thr Met Arg Arg 485 490 495ccg tca ctg aaa gat gag gta tac gtg gtg gga gcc gac tac tcc tgg 1536Pro Ser Leu Lys Asp Glu Val Tyr Val Val Gly Ala Asp Tyr Ser Trp 500 505 510gga ctt atc tcc tcc tgg ata gag ggc gct ctg gag acc tcg gaa aac 1584Gly Leu Ile Ser Ser Trp Ile Glu Gly Ala Leu Glu Thr Ser Glu Asn 515 520 525gtc atc aac gac tac ttc ctc taa 1608Val Ile Asn Asp Tyr Phe Leu 530 5352535PRTAplysia punctata 2Met Ser Ser Ala Val Leu Leu Leu Ala Cys Ala Leu Val Ile Ser Val1 5 10 15His Ala Asp Gly Ile Cys Arg Asn Arg Arg Gln Cys Asn Arg Glu Val 20 25 30Cys Gly Ser Thr Tyr Asp Val Ala Val Val Gly Ala Gly Pro Gly Gly 35 40 45Ala Asn Ser Ala Tyr Met Leu Arg Asp Ser Gly Leu Asp Ile Ala Val 50 55 60Phe Glu Tyr Ser Asp Arg Val Gly Gly Arg Leu Phe Thr Tyr Gln Leu65 70 75 80Pro Asn Thr Pro Asp Val Asn Leu Glu Ile Gly Gly Met Arg Phe Ile 85 90 95Glu Gly Ala Met His Arg Leu Trp Arg Val Ile Ser Glu Leu Gly Leu 100 105 110Thr Pro Lys Val Phe Lys Glu Gly Phe Gly Lys Glu Gly Arg Gln Arg 115 120 125Phe Tyr Leu Arg Gly Gln Ser Leu Thr Lys Lys Gln Val Lys Ser Gly 130 135 140Asp Val Pro Tyr Asp Leu Ser Pro Glu Glu Lys Glu Asn Gln Gly Asn145 150 155 160Leu Val Glu Tyr Tyr Leu Glu Lys Leu Thr Gly Leu Gln Leu Asn Gly 165 170 175Glu Pro Leu Lys Arg Glu Val Ala Leu Lys Leu Thr Val Pro Asp Gly 180 185 190Arg Phe Leu Tyr Asp Leu Ser Phe Asp Glu Ala Met Asp Leu Val Ala 195 200 205Ser Pro Glu Gly Lys Glu Phe Thr Arg Asp Thr His Val Phe Thr Gly 210 215 220Glu Val Thr Leu Asp Ala Ser Ala Val Ser Leu Phe Asp Asp His Leu225 230 235 240Gly Glu Asp Tyr Tyr Gly Ser Glu Ile Tyr Thr Leu Lys Glu Gly Leu 245 250 255Ser Ser Val Pro Gln Gly Leu Leu Gln Ala Phe Leu Asp Ala Ala Asp 260 265 270Ser Asn Glu Phe Tyr Pro Asn Ser His Leu Lys Ala Leu Arg Arg Lys 275 280 285Thr Asn Gly Gln Tyr Val Leu Tyr Phe Glu Pro Thr Thr Ser Lys Asp 290 295 300Gly Gln Thr Thr Ile Asn Tyr Leu Glu Pro Leu Gln Val Val Cys Ala305 310 315 320Gln Arg Val Ile Leu Ala Met Pro Val Tyr Ala Leu Asn Gln Leu Asp 325 330 335Trp Asn Gln Leu Arg Asn Asp Arg Ala Thr Gln Ala Tyr Ala Ala Val 340 345 350Arg Pro Ile Pro Ala Ser Lys Val Phe Met Ser Phe Asp Gln Pro Trp 355 360 365Trp Leu Glu Asn Glu Arg Lys Ser Trp Val Thr Lys Ser Asp Ala Leu 370 375 380Phe Ser Gln Met Tyr Asp Trp Gln Lys Ser Glu Ala Ser Gly Asp Tyr385 390 395 400Ile Leu Ile Ala Ser Tyr Ala Asp Gly Leu Lys Ala Gln Tyr Leu Arg 405 410 415Glu Leu Lys Asn Gln Gly Glu Asp Ile Pro Gly Ser Asp Pro Gly Tyr 420 425 430Asn Gln Val Thr Glu Pro Leu Lys Asp Thr Ile Leu Asp His Leu Thr 435 440 445Glu Ala Tyr Gly Val Glu Arg Asp Ser Ile Pro Glu Pro Val Thr Ala 450 455 460Ala Ser Gln Phe Trp Thr Asp Tyr Pro Phe Gly Cys Gly Trp Ile Thr465 470 475 480Trp Arg Ala Gly Phe His Phe Asp Asp Val Ile Ser Thr Met Arg Arg 485 490 495Pro Ser Leu Lys Asp Glu Val Tyr Val Val Gly Ala Asp Tyr Ser Trp 500 505 510Gly Leu Ile Ser Ser Trp Ile Glu Gly Ala Leu Glu Thr Ser Glu Asn 515 520 525Val Ile Asn Asp Tyr Phe Leu 530 53531605DNAAplysia punctataCDS(1)..(1605) 3tcg tct gct gtg ctt ctc ctg gct tgt gcg ttg gtc atc tct gtc cac 48Ser Ser Ala Val Leu Leu Leu Ala Cys Ala Leu Val Ile Ser Val His1 5 10 15gcc gac ggt gtc tgc aga aac aga cgt caa tgt aac aga gag gtg tgc 96Ala Asp Gly Val Cys Arg Asn Arg Arg Gln Cys Asn Arg Glu Val Cys 20 25 30ggt tct acc tac gat gtg gcc gtc gtg ggg gcg ggg cct ggg gga gct 144Gly Ser Thr Tyr Asp Val Ala Val Val Gly Ala Gly Pro Gly Gly Ala 35 40 45aac tcc gcc tac atg ctg agg gac tcc ggc ctg gac atc gct gtg ttc 192Asn Ser Ala Tyr Met Leu Arg Asp Ser Gly Leu Asp Ile Ala Val Phe 50 55 60gag tac tca gac cga gtg ggc ggc cgg ctg ttc acc tac cag ctg ccc 240Glu Tyr Ser Asp Arg Val Gly Gly Arg Leu Phe Thr Tyr Gln Leu Pro65 70 75 80aac aca ccc gac gtt aat ctc gag att ggc ggc atg agg ttc atc gag 288Asn Thr Pro Asp Val Asn Leu Glu Ile Gly Gly Met Arg Phe Ile Glu 85 90 95ggc gcc atg cac agg ctc tgg agg gtc att tca gaa ctc ggc cta acc 336Gly Ala Met His Arg Leu Trp Arg Val Ile Ser Glu Leu Gly Leu Thr 100 105 110ccc aag gtg ttc aag gaa ggt ttc gga aag gag ggc aga cag aga ttt 384Pro Lys Val Phe Lys Glu Gly Phe Gly Lys Glu Gly Arg Gln Arg Phe 115 120 125tac ctg cgg gga cag agc ctg acc aag aaa cag gtc aag agt ggg gac 432Tyr Leu Arg Gly Gln Ser Leu Thr Lys Lys Gln Val Lys Ser Gly Asp 130 135 140gta ccc tat gac ctc agc ccg gag gag aaa gaa aac cag gga aat ctg 480Val Pro Tyr Asp Leu Ser Pro Glu Glu Lys Glu Asn Gln Gly Asn Leu145 150 155 160gtc gaa tac tac ctg gag aaa ctg aca ggt cta caa ctc aat ggt gaa 528Val Glu Tyr Tyr Leu Glu Lys Leu Thr Gly Leu Gln Leu Asn Gly Glu 165 170 175ccg ctc aaa cgt gag gtt gcg ctt aaa cta acc gtg ccg gac ggc aga 576Pro Leu Lys Arg Glu Val Ala Leu Lys Leu Thr Val Pro Asp Gly Arg 180 185 190ttc ctc tat gac ctc tcg ttt gac gaa gcc atg gat ctg gtt gcc tcc 624Phe Leu Tyr Asp Leu Ser Phe Asp Glu Ala Met Asp Leu Val Ala Ser 195 200 205cct gag ggc aaa gag ttc acc cga gac acg cac gtc ttc acc gga gag 672Pro Glu Gly Lys Glu Phe Thr Arg Asp Thr His Val Phe Thr Gly Glu 210 215 220gtc acc ctg ggc gcg tcg gct gtc tcc ctc ttc gac gac cac ctg gga 720Val Thr Leu Gly Ala Ser Ala Val Ser Leu Phe Asp Asp His Leu Gly225 230 235 240gag gac tac tac ggc agt gag atc tac acc ctc aag gaa gga ctg tct 768Glu Asp Tyr Tyr Gly Ser Glu Ile Tyr Thr Leu Lys Glu Gly Leu Ser 245 250 255tcc gtc cct caa ggg ctc cta cag gct ttt ctg gac gcc gca gac tcc 816Ser Val Pro Gln Gly Leu Leu Gln Ala Phe Leu Asp Ala Ala Asp Ser 260 265 270aac gag ttc tat ccc aac agc cac ctg aag gcc ctg aga cgt aag acc 864Asn Glu Phe Tyr Pro Asn Ser His Leu Lys Ala Leu Arg Arg Lys Thr 275 280 285aac ggt cag tat gtt ctt tac ttt gag ccc acc acc tcc aag gat gga 912Asn Gly Gln Tyr Val Leu Tyr Phe Glu Pro Thr Thr Ser Lys Asp Gly 290 295 300caa acc aca atc aac tat ctg gaa ccc ctg cag gtt gtg tgt gca cag 960Gln Thr Thr Ile Asn Tyr Leu Glu Pro Leu Gln Val Val Cys Ala Gln305 310 315 320aga gtc att ctg gcc atg ccg gtc tac gct ctc aac cag ttg gat tgg 1008Arg Val Ile Leu Ala Met Pro Val Tyr Ala Leu Asn Gln Leu Asp Trp 325 330 335aat cag ctc aga aat gac cga gcc acc caa gcg tac gct gcc gtg cgc 1056Asn Gln Leu Arg Asn Asp Arg Ala Thr Gln Ala Tyr Ala Ala Val Arg 340 345 350ccg att cct gca agt aag gtg ttc atg acc ttt gat cag ccc tgg tgg 1104Pro Ile Pro Ala Ser Lys Val Phe Met Thr Phe Asp Gln Pro Trp Trp 355 360 365ttg gag aac gag agg aaa tcc tgg gtc acc aag tcg gac gcg ctt ttc 1152Leu Glu Asn Glu Arg Lys Ser Trp Val Thr Lys Ser Asp Ala Leu Phe 370 375 380agt caa atg tac gac tgg cag aag tct gag gcg tcc gga gac tac atc 1200Ser Gln Met Tyr Asp Trp Gln Lys Ser Glu Ala Ser Gly Asp Tyr Ile385 390 395 400ctg atc gcc agc tac gcc gac ggc ctc aaa gcc cag tac ctg cgg gag 1248Leu Ile Ala Ser Tyr Ala Asp Gly Leu Lys Ala Gln Tyr Leu Arg Glu 405 410 415ctg aag aat cag gga gag gac atc cca ggc tct gac cca ggc tac aac 1296Leu Lys Asn Gln Gly Glu Asp Ile Pro Gly Ser Asp Pro Gly Tyr Asn 420 425 430cag gtc acc gaa ccc ctc aag gac acc att ctt gac cac ctc act gag 1344Gln Val Thr Glu Pro Leu Lys Asp Thr Ile Leu Asp His Leu Thr Glu 435 440 445gcc tat ggc gtg gag cga gac tcg atc cgg gaa ccc gtg acc gcc gct 1392Ala Tyr Gly Val Glu Arg Asp Ser Ile Arg Glu Pro Val Thr Ala Ala 450 455 460tcc cag ttc tgg aca gac tac ccg ttt ggc tgt gga tgg atc acc tgg 1440Ser Gln Phe Trp Thr Asp Tyr Pro Phe Gly Cys Gly Trp Ile Thr Trp465 470 475 480agg gcc ggc ttc cat ttc gat gac gtc atc agc acc atg cgt cgc ccg 1488Arg Ala Gly Phe His Phe Asp Asp Val Ile Ser Thr Met Arg Arg Pro 485 490 495tca ctg aaa gat gag gtc tac gtg gtg gga gcc gat tac tcc tgg gga 1536Ser Leu Lys Asp Glu Val Tyr Val Val Gly Ala Asp Tyr Ser Trp Gly 500 505 510ctt atc tcc tcc tgg ata gag ggc gct ctg gag acc tca gaa aac gtc 1584Leu Ile Ser Ser Trp Ile Glu Gly Ala Leu Glu Thr Ser Glu Asn Val 515 520 525atc aac gac tac ttc ctc taa 1605Ile Asn Asp Tyr Phe Leu 5304534PRTAplysia punctata 4Ser Ser Ala Val Leu Leu Leu Ala Cys Ala Leu Val Ile Ser Val His1 5 10 15Ala Asp Gly Val Cys Arg Asn Arg Arg Gln Cys Asn Arg Glu Val Cys 20 25 30Gly Ser Thr Tyr Asp Val Ala Val Val Gly Ala Gly Pro Gly Gly Ala 35 40 45Asn Ser Ala Tyr Met Leu Arg Asp Ser Gly Leu Asp Ile Ala Val Phe 50 55 60Glu Tyr Ser Asp Arg Val Gly Gly Arg Leu Phe Thr Tyr Gln Leu Pro65 70 75 80Asn Thr Pro Asp Val Asn Leu Glu Ile Gly Gly Met Arg Phe Ile Glu 85 90 95Gly Ala Met His Arg Leu Trp Arg Val Ile Ser Glu Leu Gly Leu Thr 100 105 110Pro Lys Val Phe Lys Glu Gly Phe Gly Lys Glu Gly Arg Gln Arg Phe 115 120 125Tyr Leu Arg Gly Gln Ser Leu Thr Lys Lys Gln Val Lys Ser Gly Asp 130 135 140Val Pro Tyr Asp Leu Ser Pro Glu Glu Lys Glu Asn Gln Gly Asn Leu145 150 155 160Val Glu Tyr Tyr Leu Glu Lys Leu Thr Gly Leu Gln Leu Asn Gly Glu 165 170 175Pro Leu Lys Arg Glu Val Ala Leu Lys Leu Thr Val Pro Asp Gly Arg 180 185 190Phe Leu Tyr Asp Leu

Ser Phe Asp Glu Ala Met Asp Leu Val Ala Ser 195 200 205Pro Glu Gly Lys Glu Phe Thr Arg Asp Thr His Val Phe Thr Gly Glu 210 215 220Val Thr Leu Gly Ala Ser Ala Val Ser Leu Phe Asp Asp His Leu Gly225 230 235 240Glu Asp Tyr Tyr Gly Ser Glu Ile Tyr Thr Leu Lys Glu Gly Leu Ser 245 250 255Ser Val Pro Gln Gly Leu Leu Gln Ala Phe Leu Asp Ala Ala Asp Ser 260 265 270Asn Glu Phe Tyr Pro Asn Ser His Leu Lys Ala Leu Arg Arg Lys Thr 275 280 285Asn Gly Gln Tyr Val Leu Tyr Phe Glu Pro Thr Thr Ser Lys Asp Gly 290 295 300Gln Thr Thr Ile Asn Tyr Leu Glu Pro Leu Gln Val Val Cys Ala Gln305 310 315 320Arg Val Ile Leu Ala Met Pro Val Tyr Ala Leu Asn Gln Leu Asp Trp 325 330 335Asn Gln Leu Arg Asn Asp Arg Ala Thr Gln Ala Tyr Ala Ala Val Arg 340 345 350Pro Ile Pro Ala Ser Lys Val Phe Met Thr Phe Asp Gln Pro Trp Trp 355 360 365Leu Glu Asn Glu Arg Lys Ser Trp Val Thr Lys Ser Asp Ala Leu Phe 370 375 380Ser Gln Met Tyr Asp Trp Gln Lys Ser Glu Ala Ser Gly Asp Tyr Ile385 390 395 400Leu Ile Ala Ser Tyr Ala Asp Gly Leu Lys Ala Gln Tyr Leu Arg Glu 405 410 415Leu Lys Asn Gln Gly Glu Asp Ile Pro Gly Ser Asp Pro Gly Tyr Asn 420 425 430Gln Val Thr Glu Pro Leu Lys Asp Thr Ile Leu Asp His Leu Thr Glu 435 440 445Ala Tyr Gly Val Glu Arg Asp Ser Ile Arg Glu Pro Val Thr Ala Ala 450 455 460Ser Gln Phe Trp Thr Asp Tyr Pro Phe Gly Cys Gly Trp Ile Thr Trp465 470 475 480Arg Ala Gly Phe His Phe Asp Asp Val Ile Ser Thr Met Arg Arg Pro 485 490 495Ser Leu Lys Asp Glu Val Tyr Val Val Gly Ala Asp Tyr Ser Trp Gly 500 505 510Leu Ile Ser Ser Trp Ile Glu Gly Ala Leu Glu Thr Ser Glu Asn Val 515 520 525Ile Asn Asp Tyr Phe Leu 53051554DNAAplysia punctataCDS(1)..(1554) 5gac ggt atc tgc aga aac aga cgt caa tgt aac aga gag gtg tgc ggt 48Asp Gly Ile Cys Arg Asn Arg Arg Gln Cys Asn Arg Glu Val Cys Gly1 5 10 15tct acc tac gat gtg gct gtc gtg ggg gcg ggg cct ggg gga gct aac 96Ser Thr Tyr Asp Val Ala Val Val Gly Ala Gly Pro Gly Gly Ala Asn 20 25 30tcc gcc tac atg ctg agg gac tcc ggc ctg gac atc gct gtg ttc gag 144Ser Ala Tyr Met Leu Arg Asp Ser Gly Leu Asp Ile Ala Val Phe Glu 35 40 45tac tca gac cga gtg ggc ggc cgg ctg ttc acc tac cag ctg ccc aac 192Tyr Ser Asp Arg Val Gly Gly Arg Leu Phe Thr Tyr Gln Leu Pro Asn 50 55 60aca ccc gac gtt aat ctc gag att ggc ggc atg agg ttc atc gag ggc 240Thr Pro Asp Val Asn Leu Glu Ile Gly Gly Met Arg Phe Ile Glu Gly65 70 75 80gcc atg cac agg ctc tgg agg gtc att tca gaa ctc ggc cta acc ccc 288Ala Met His Arg Leu Trp Arg Val Ile Ser Glu Leu Gly Leu Thr Pro 85 90 95aag gtg ttc aag gaa ggt ttc gga aag gag ggc aga cag aga ttt tac 336Lys Val Phe Lys Glu Gly Phe Gly Lys Glu Gly Arg Gln Arg Phe Tyr 100 105 110ctg cgg gga cag agc ctg acc aag aaa cag gtc aag agt ggg gac gta 384Leu Arg Gly Gln Ser Leu Thr Lys Lys Gln Val Lys Ser Gly Asp Val 115 120 125ccc tat gac ctc agc ccg gag gag aaa gaa aac cag gga aat ctg gtc 432Pro Tyr Asp Leu Ser Pro Glu Glu Lys Glu Asn Gln Gly Asn Leu Val 130 135 140gaa tac tac ctg gag aaa ctg aca ggt cta aaa ctc aac ggc gga ccg 480Glu Tyr Tyr Leu Glu Lys Leu Thr Gly Leu Lys Leu Asn Gly Gly Pro145 150 155 160ctc aaa cgt gag gtt gcg ctt aaa cta acc gtg ccg gac ggc aga ttc 528Leu Lys Arg Glu Val Ala Leu Lys Leu Thr Val Pro Asp Gly Arg Phe 165 170 175ctc tat gac ctc tcg ttt gac gaa gcc atg gac ctg gtt gcc tcc cct 576Leu Tyr Asp Leu Ser Phe Asp Glu Ala Met Asp Leu Val Ala Ser Pro 180 185 190gag ggc aaa gag ttc acc cga gac acg cac gtg ttc acc gga gaa gtc 624Glu Gly Lys Glu Phe Thr Arg Asp Thr His Val Phe Thr Gly Glu Val 195 200 205acc ctg gac gcg tcg gct gtc tcc ctc ttc gac gac cac ctg gga gag 672Thr Leu Asp Ala Ser Ala Val Ser Leu Phe Asp Asp His Leu Gly Glu 210 215 220gac tac tat ggc agt gag atc tac acc cta aag gaa gga ctg tct tcc 720Asp Tyr Tyr Gly Ser Glu Ile Tyr Thr Leu Lys Glu Gly Leu Ser Ser225 230 235 240gtc cca caa ggg ctc cta cag act ttt ctg gac gcc gca gac tcc aac 768Val Pro Gln Gly Leu Leu Gln Thr Phe Leu Asp Ala Ala Asp Ser Asn 245 250 255gag ttc tat ccc aac agc cac ctg aag gcc ctg aga cgt aag acc aac 816Glu Phe Tyr Pro Asn Ser His Leu Lys Ala Leu Arg Arg Lys Thr Asn 260 265 270ggt cag tat gtt ctt tac ttt gag ccc acc acc tcc aag gat gga caa 864Gly Gln Tyr Val Leu Tyr Phe Glu Pro Thr Thr Ser Lys Asp Gly Gln 275 280 285acc aca atc aac tat ctg gaa ccc ctg cag gtt gtg tgt gca cag aga 912Thr Thr Ile Asn Tyr Leu Glu Pro Leu Gln Val Val Cys Ala Gln Arg 290 295 300gtc atc ctg gcc atg ccg gtc tac gct ctc aac caa ctg gac tgg aat 960Val Ile Leu Ala Met Pro Val Tyr Ala Leu Asn Gln Leu Asp Trp Asn305 310 315 320cag ctc aga aat gac cga gcc acc caa gcg tac gct gcc gtg cgc ccg 1008Gln Leu Arg Asn Asp Arg Ala Thr Gln Ala Tyr Ala Ala Val Arg Pro 325 330 335att cct gca agt aaa gtg ttc atg acc ttt gat cag ccc tgg tgg ttg 1056Ile Pro Ala Ser Lys Val Phe Met Thr Phe Asp Gln Pro Trp Trp Leu 340 345 350gag aac gag agg aaa tcc tgg gtc acc aag tcg gac gcg ctt ttc agc 1104Glu Asn Glu Arg Lys Ser Trp Val Thr Lys Ser Asp Ala Leu Phe Ser 355 360 365caa atg tac gac tgg cag aag tct gag gcg tcc gga gac tac atc ctg 1152Gln Met Tyr Asp Trp Gln Lys Ser Glu Ala Ser Gly Asp Tyr Ile Leu 370 375 380atc gcc agc tac gcc gac ggc ctc aaa gcc cag tac ctg cgg gag ctg 1200Ile Ala Ser Tyr Ala Asp Gly Leu Lys Ala Gln Tyr Leu Arg Glu Leu385 390 395 400aag aat cag gga gag gac atc cca ggc tct gac cca ggc tac aac cag 1248Lys Asn Gln Gly Glu Asp Ile Pro Gly Ser Asp Pro Gly Tyr Asn Gln 405 410 415gtc acc gaa ccc ctc aag gac acc att ctt gac cac ctc act gag gct 1296Val Thr Glu Pro Leu Lys Asp Thr Ile Leu Asp His Leu Thr Glu Ala 420 425 430tat ggc gtg gaa cga gac tcg atc ccg gaa ccc gtg acc gcc gct tcc 1344Tyr Gly Val Glu Arg Asp Ser Ile Pro Glu Pro Val Thr Ala Ala Ser 435 440 445cag ttc tgg acc gac tac ccg ttc ggc tgt gga tgg atc acc tgg agg 1392Gln Phe Trp Thr Asp Tyr Pro Phe Gly Cys Gly Trp Ile Thr Trp Arg 450 455 460gca ggc ttc cat ttt gat gac gtc atc agc acc atg cgt cgc ccg tca 1440Ala Gly Phe His Phe Asp Asp Val Ile Ser Thr Met Arg Arg Pro Ser465 470 475 480ctg aaa gat gag gtc tac gtg gtg gga gcc gat tac tcc tgg gga ctt 1488Leu Lys Asp Glu Val Tyr Val Val Gly Ala Asp Tyr Ser Trp Gly Leu 485 490 495atc tcc tcc tgg ata gag ggc gct ctg gag acc tcg gaa aac gtc atc 1536Ile Ser Ser Trp Ile Glu Gly Ala Leu Glu Thr Ser Glu Asn Val Ile 500 505 510aac gac tac ttc ctc taa 1554Asn Asp Tyr Phe Leu 5156517PRTAplysia punctata 6Asp Gly Ile Cys Arg Asn Arg Arg Gln Cys Asn Arg Glu Val Cys Gly1 5 10 15Ser Thr Tyr Asp Val Ala Val Val Gly Ala Gly Pro Gly Gly Ala Asn 20 25 30Ser Ala Tyr Met Leu Arg Asp Ser Gly Leu Asp Ile Ala Val Phe Glu 35 40 45Tyr Ser Asp Arg Val Gly Gly Arg Leu Phe Thr Tyr Gln Leu Pro Asn 50 55 60Thr Pro Asp Val Asn Leu Glu Ile Gly Gly Met Arg Phe Ile Glu Gly65 70 75 80Ala Met His Arg Leu Trp Arg Val Ile Ser Glu Leu Gly Leu Thr Pro 85 90 95Lys Val Phe Lys Glu Gly Phe Gly Lys Glu Gly Arg Gln Arg Phe Tyr 100 105 110Leu Arg Gly Gln Ser Leu Thr Lys Lys Gln Val Lys Ser Gly Asp Val 115 120 125Pro Tyr Asp Leu Ser Pro Glu Glu Lys Glu Asn Gln Gly Asn Leu Val 130 135 140Glu Tyr Tyr Leu Glu Lys Leu Thr Gly Leu Lys Leu Asn Gly Gly Pro145 150 155 160Leu Lys Arg Glu Val Ala Leu Lys Leu Thr Val Pro Asp Gly Arg Phe 165 170 175Leu Tyr Asp Leu Ser Phe Asp Glu Ala Met Asp Leu Val Ala Ser Pro 180 185 190Glu Gly Lys Glu Phe Thr Arg Asp Thr His Val Phe Thr Gly Glu Val 195 200 205Thr Leu Asp Ala Ser Ala Val Ser Leu Phe Asp Asp His Leu Gly Glu 210 215 220Asp Tyr Tyr Gly Ser Glu Ile Tyr Thr Leu Lys Glu Gly Leu Ser Ser225 230 235 240Val Pro Gln Gly Leu Leu Gln Thr Phe Leu Asp Ala Ala Asp Ser Asn 245 250 255Glu Phe Tyr Pro Asn Ser His Leu Lys Ala Leu Arg Arg Lys Thr Asn 260 265 270Gly Gln Tyr Val Leu Tyr Phe Glu Pro Thr Thr Ser Lys Asp Gly Gln 275 280 285Thr Thr Ile Asn Tyr Leu Glu Pro Leu Gln Val Val Cys Ala Gln Arg 290 295 300Val Ile Leu Ala Met Pro Val Tyr Ala Leu Asn Gln Leu Asp Trp Asn305 310 315 320Gln Leu Arg Asn Asp Arg Ala Thr Gln Ala Tyr Ala Ala Val Arg Pro 325 330 335Ile Pro Ala Ser Lys Val Phe Met Thr Phe Asp Gln Pro Trp Trp Leu 340 345 350Glu Asn Glu Arg Lys Ser Trp Val Thr Lys Ser Asp Ala Leu Phe Ser 355 360 365Gln Met Tyr Asp Trp Gln Lys Ser Glu Ala Ser Gly Asp Tyr Ile Leu 370 375 380Ile Ala Ser Tyr Ala Asp Gly Leu Lys Ala Gln Tyr Leu Arg Glu Leu385 390 395 400Lys Asn Gln Gly Glu Asp Ile Pro Gly Ser Asp Pro Gly Tyr Asn Gln 405 410 415Val Thr Glu Pro Leu Lys Asp Thr Ile Leu Asp His Leu Thr Glu Ala 420 425 430Tyr Gly Val Glu Arg Asp Ser Ile Pro Glu Pro Val Thr Ala Ala Ser 435 440 445Gln Phe Trp Thr Asp Tyr Pro Phe Gly Cys Gly Trp Ile Thr Trp Arg 450 455 460Ala Gly Phe His Phe Asp Asp Val Ile Ser Thr Met Arg Arg Pro Ser465 470 475 480Leu Lys Asp Glu Val Tyr Val Val Gly Ala Asp Tyr Ser Trp Gly Leu 485 490 495Ile Ser Ser Trp Ile Glu Gly Ala Leu Glu Thr Ser Glu Asn Val Ile 500 505 510Asn Asp Tyr Phe Leu 51571554DNAAplysia punctataCDS(1)..(1554) 7atg gac ggt gtc agc aga aac aga cgt caa tgt aac aga gag gtg tgc 48Met Asp Gly Val Ser Arg Asn Arg Arg Gln Cys Asn Arg Glu Val Cys1 5 10 15ggt tct acc tac gat gtg gcc gtc gtg ggg gcg ggg cct ggg gga gct 96Gly Ser Thr Tyr Asp Val Ala Val Val Gly Ala Gly Pro Gly Gly Ala 20 25 30aac tcc gcc tac atg ctg agg gac tcc ggc ctg gac atc gct gtg ttc 144Asn Ser Ala Tyr Met Leu Arg Asp Ser Gly Leu Asp Ile Ala Val Phe 35 40 45gag tac tca gac cga gtg ggc ggc cgg ctg ttc acc tac cag ctg ccc 192Glu Tyr Ser Asp Arg Val Gly Gly Arg Leu Phe Thr Tyr Gln Leu Pro 50 55 60aac aca ccc gac gtt aac ctg gag att gga ggc atg agg ttc atc gaa 240Asn Thr Pro Asp Val Asn Leu Glu Ile Gly Gly Met Arg Phe Ile Glu65 70 75 80ggc gcc atg cac agg ctc tgg agg gtc att tca gaa ctc ggc cta acc 288Gly Ala Met His Arg Leu Trp Arg Val Ile Ser Glu Leu Gly Leu Thr 85 90 95ccc aag gtg ttc aag gaa ggt ttc ggc aag gag ggc aga caa aga ttc 336Pro Lys Val Phe Lys Glu Gly Phe Gly Lys Glu Gly Arg Gln Arg Phe 100 105 110tac ctg cgg gga cag agc ctg acc aag aaa cag gtc aag agt ggg gac 384Tyr Leu Arg Gly Gln Ser Leu Thr Lys Lys Gln Val Lys Ser Gly Asp 115 120 125gta ccc tat gac ctc agc ccg gag gag aaa gaa aac cag gga aat ctg 432Val Pro Tyr Asp Leu Ser Pro Glu Glu Lys Glu Asn Gln Gly Asn Leu 130 135 140gtc gaa tac tac ctg gag aaa ctg aca ggt cta caa ctc aac ggc gag 480Val Glu Tyr Tyr Leu Glu Lys Leu Thr Gly Leu Gln Leu Asn Gly Glu145 150 155 160ccg ctc aaa cgt gag gtt gcg ctt aaa cta acc gtg ccg gac ggc aga 528Pro Leu Lys Arg Glu Val Ala Leu Lys Leu Thr Val Pro Asp Gly Arg 165 170 175ttc ctc tat gac ctc tcg ttt gac gaa gcc atg gat ctg gtt gcc tcc 576Phe Leu Tyr Asp Leu Ser Phe Asp Glu Ala Met Asp Leu Val Ala Ser 180 185 190cct gag ggc aaa gag ttc acc cga gac acg cac gtc ttc aca gga gag 624Pro Glu Gly Lys Glu Phe Thr Arg Asp Thr His Val Phe Thr Gly Glu 195 200 205gtc acc ctg gac gcg tcg gct gtc tcc ctc ttc gac gac cac ctg gga 672Val Thr Leu Asp Ala Ser Ala Val Ser Leu Phe Asp Asp His Leu Gly 210 215 220gag gac tac tat ggc agt gag atc tac acc cta aag gaa gga ctg tct 720Glu Asp Tyr Tyr Gly Ser Glu Ile Tyr Thr Leu Lys Glu Gly Leu Ser225 230 235 240tcc gtc cca caa ggg ctc cta cag gct ttt ctg gac gcc gca gac tcc 768Ser Val Pro Gln Gly Leu Leu Gln Ala Phe Leu Asp Ala Ala Asp Ser 245 250 255aac gag ttc tat ccc aac agc cac ctg aag gcc ctg aga cgt aag acc 816Asn Glu Phe Tyr Pro Asn Ser His Leu Lys Ala Leu Arg Arg Lys Thr 260 265 270aac ggt cag tat gtt ctt tac ttt gag ccc acc acc tcc aag gat gga 864Asn Gly Gln Tyr Val Leu Tyr Phe Glu Pro Thr Thr Ser Lys Asp Gly 275 280 285caa acc aca atc aac tat ctg gaa ccc ctg cag gtt gtg tgt gca cag 912Gln Thr Thr Ile Asn Tyr Leu Glu Pro Leu Gln Val Val Cys Ala Gln 290 295 300aga gtc atc ctg gcc atg ccg gta tac gct ctg aac caa cta gac tgg 960Arg Val Ile Leu Ala Met Pro Val Tyr Ala Leu Asn Gln Leu Asp Trp305 310 315 320aat cag ctc aga aat gac cga gcc acc caa gcg tac gct gcc gtt cgc 1008Asn Gln Leu Arg Asn Asp Arg Ala Thr Gln Ala Tyr Ala Ala Val Arg 325 330 335ccg att cct gca agt aag gtg ttc atg acc ttt gat cag ccc tgg tgg 1056Pro Ile Pro Ala Ser Lys Val Phe Met Thr Phe Asp Gln Pro Trp Trp 340 345 350ttg gag aac gag agg aaa tcc tgg gtc acc aag tcg gac gcg ctt ttc 1104Leu Glu Asn Glu Arg Lys Ser Trp Val Thr Lys Ser Asp Ala Leu Phe 355 360 365agc caa atg tac gac tgg cag aag tct gag gcg tcc gga gac tac atc 1152Ser Gln Met Tyr Asp Trp Gln Lys Ser Glu Ala Ser Gly Asp Tyr Ile 370 375 380ctg atc gcc agc tac gcc gac ggc ctc aaa gcc cag tac ctg cgg gag 1200Leu Ile Ala Ser Tyr Ala Asp Gly Leu Lys Ala Gln Tyr Leu Arg Glu385 390 395 400ctg aag aat cag gga gag gac atc cca ggc tct gac cca ggc tac aac 1248Leu Lys Asn Gln Gly Glu Asp Ile Pro Gly Ser Asp Pro Gly Tyr Asn 405 410 415cag gtc acc gaa ccc ctc aag gac acc att ctt gac cac ctc act gag 1296Gln Val Thr Glu Pro Leu Lys Asp Thr Ile Leu Asp His Leu Thr Glu 420 425 430gct tat ggc gtg gaa cga gac tcg atc ccg gaa ccc gtg acc gcc gct 1344Ala Tyr Gly Val Glu Arg Asp Ser Ile Pro Glu Pro Val Thr Ala Ala 435 440 445tcc cag ttc tgg aca gac tac ccg ttt ggc tgt gga tgg atc acc tgg 1392Ser Gln Phe Trp Thr Asp Tyr Pro Phe Gly Cys Gly Trp Ile Thr Trp 450 455 460agg gcc ggc ttc cat ttc gat gac gtc atc agc acc atg cgt cgc ccg 1440Arg Ala Gly Phe His Phe Asp Asp Val Ile Ser Thr Met Arg Arg Pro465 470 475 480tca ctg aaa gat

gag gta tac gtg gtg gga gcc gac tac tcc tgg gga 1488Ser Leu Lys Asp Glu Val Tyr Val Val Gly Ala Asp Tyr Ser Trp Gly 485 490 495ctt atc tcc tcc tgg ata gag ggc gct ctt gag acc tcg gaa aac gtc 1536Leu Ile Ser Ser Trp Ile Glu Gly Ala Leu Glu Thr Ser Glu Asn Val 500 505 510atc aac gac tac ttc ctc 1554Ile Asn Asp Tyr Phe Leu 5158518PRTAplysia punctata 8Met Asp Gly Val Ser Arg Asn Arg Arg Gln Cys Asn Arg Glu Val Cys1 5 10 15Gly Ser Thr Tyr Asp Val Ala Val Val Gly Ala Gly Pro Gly Gly Ala 20 25 30Asn Ser Ala Tyr Met Leu Arg Asp Ser Gly Leu Asp Ile Ala Val Phe 35 40 45Glu Tyr Ser Asp Arg Val Gly Gly Arg Leu Phe Thr Tyr Gln Leu Pro 50 55 60Asn Thr Pro Asp Val Asn Leu Glu Ile Gly Gly Met Arg Phe Ile Glu65 70 75 80Gly Ala Met His Arg Leu Trp Arg Val Ile Ser Glu Leu Gly Leu Thr 85 90 95Pro Lys Val Phe Lys Glu Gly Phe Gly Lys Glu Gly Arg Gln Arg Phe 100 105 110Tyr Leu Arg Gly Gln Ser Leu Thr Lys Lys Gln Val Lys Ser Gly Asp 115 120 125Val Pro Tyr Asp Leu Ser Pro Glu Glu Lys Glu Asn Gln Gly Asn Leu 130 135 140Val Glu Tyr Tyr Leu Glu Lys Leu Thr Gly Leu Gln Leu Asn Gly Glu145 150 155 160Pro Leu Lys Arg Glu Val Ala Leu Lys Leu Thr Val Pro Asp Gly Arg 165 170 175Phe Leu Tyr Asp Leu Ser Phe Asp Glu Ala Met Asp Leu Val Ala Ser 180 185 190Pro Glu Gly Lys Glu Phe Thr Arg Asp Thr His Val Phe Thr Gly Glu 195 200 205Val Thr Leu Asp Ala Ser Ala Val Ser Leu Phe Asp Asp His Leu Gly 210 215 220Glu Asp Tyr Tyr Gly Ser Glu Ile Tyr Thr Leu Lys Glu Gly Leu Ser225 230 235 240Ser Val Pro Gln Gly Leu Leu Gln Ala Phe Leu Asp Ala Ala Asp Ser 245 250 255Asn Glu Phe Tyr Pro Asn Ser His Leu Lys Ala Leu Arg Arg Lys Thr 260 265 270Asn Gly Gln Tyr Val Leu Tyr Phe Glu Pro Thr Thr Ser Lys Asp Gly 275 280 285Gln Thr Thr Ile Asn Tyr Leu Glu Pro Leu Gln Val Val Cys Ala Gln 290 295 300Arg Val Ile Leu Ala Met Pro Val Tyr Ala Leu Asn Gln Leu Asp Trp305 310 315 320Asn Gln Leu Arg Asn Asp Arg Ala Thr Gln Ala Tyr Ala Ala Val Arg 325 330 335Pro Ile Pro Ala Ser Lys Val Phe Met Thr Phe Asp Gln Pro Trp Trp 340 345 350Leu Glu Asn Glu Arg Lys Ser Trp Val Thr Lys Ser Asp Ala Leu Phe 355 360 365Ser Gln Met Tyr Asp Trp Gln Lys Ser Glu Ala Ser Gly Asp Tyr Ile 370 375 380Leu Ile Ala Ser Tyr Ala Asp Gly Leu Lys Ala Gln Tyr Leu Arg Glu385 390 395 400Leu Lys Asn Gln Gly Glu Asp Ile Pro Gly Ser Asp Pro Gly Tyr Asn 405 410 415Gln Val Thr Glu Pro Leu Lys Asp Thr Ile Leu Asp His Leu Thr Glu 420 425 430Ala Tyr Gly Val Glu Arg Asp Ser Ile Pro Glu Pro Val Thr Ala Ala 435 440 445Ser Gln Phe Trp Thr Asp Tyr Pro Phe Gly Cys Gly Trp Ile Thr Trp 450 455 460Arg Ala Gly Phe His Phe Asp Asp Val Ile Ser Thr Met Arg Arg Pro465 470 475 480Ser Leu Lys Asp Glu Val Tyr Val Val Gly Ala Asp Tyr Ser Trp Gly 485 490 495Leu Ile Ser Ser Trp Ile Glu Gly Ala Leu Glu Thr Ser Glu Asn Val 500 505 510Ile Asn Asp Tyr Phe Leu 515

Claims

1. A conjugate comprising an arginine/lysine oxidoreductase and at least one polyethylene glycol moiety.

2. The conjugate of claim 1, wherein the at least one polyethylene glycol moiety has a weight average molecular weight of from about 1,000 Daltons to about 10,000 Daltons.

3. The conjugate of claim 1, wherein the at least one polyethylene glycol moiety is covalently coupled to the arginine/lysine oxidoreductase via a linking group.

4. The conjugate of claim 3, wherein the linking group is a succinimide group.

5. The conjugate of claim 3, wherein the at least one polyethylene glycol moiety is coupled via a lysine residue to the arginine/lysine oxidoreductase.

6. The conjugate of claim 1 comprising from 1 to about 30 polyethylene glycol moieties, preferably from 1 to about 10 polyethylene glycol moieties.

7. The conjugate of claim 1, wherein the arginine/lysine oxidoreductase is an L-arginine/L-lysine oxidoreductase.

8. The conjugate of claim 1, wherein the arginine/lysine oxidoreductase is isolated from a natural source.

9. The conjugate of claim 1, wherein the arginine/lysine oxidoreductase is a recombinant arginine/lysine oxidoreductase.

10. The conjugate of claim 1, wherein the arginine/lysine oxidoreductase is selected from the group consisting of:(a) an arginine/lysine oxidoreductase having the sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, and/or SEQ ID NO: 8,(b) a sequence which is at least 70% identical to the sequence of (a), or/and(c) a fragment of (a) or (b).

11. The conjugate of claim 10, wherein the fragment has a length of at least about 30 amino acid residues.

12. The conjugate of claim 1, wherein the conjugate is active in the circulation of a subject for at least 6 hours.

13. The conjugate of claim 1, wherein the conjugate has an activity which is a factor of at least 30 larger than the activity of an arginine/lysine oxidoreductase not carrying a polyethylene glycol moiety.

14. A pharmaceutical composition comprising the conjugate of claim 1 in combination with one or more pharmaceutically acceptable carriers, adjuvants, diluents and/or additives.

15-16. (canceled)

17. A method for the prevention, alleviation and/or treatment of a disease responsive to reactive oxygen species and/or to ammonium, and/or which is responsive to modulation of amino acid levels in body fluids, comprising administering an effective amount of the conjugate of claim 1 to a subject in need thereof.

18. A method of producing the conjugate of claim 1, comprising the steps of:(a) recombinantly expressing the arginine/lysine oxidoreductase and/or isolating the arginine/lysine oxidoreductase from a natural source, and(b) coupling at least one polyethylene glycol moiety to the arginine/lysine oxidoreductase of (a).

19. The method of claim 18, wherein step (a) comprises recombinantly expressing a nucleic acid selected from the group consisting of:(i) a nucleic acid having the sequence SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, and/or SEQ ID NO:7,(ii) a nucleic acid having a sequence complementary to the sequence of (i),(iii) a nucleic acid having a sequence within the scope of the degeneracy of the genetic code of the sequence of (i) or (ii),(iv) a nucleic acid having a sequence which is at least 70% identical to the sequence of (i), (ii) and/or (iii),(v) a nucleic acid having a sequence which hybridizes with any of the sequences (i), (ii), (iii) and/or (iv) under stringent conditions, and(vi) a nucleic acid comprising a fragment of any of the sequences of (i), (ii), (iii), (iv), and/or (v).

20. The method of claim 19, wherein the fragment has a length of at least 90 nucleotide residues.

21. A kit comprising the conjugate of claim 1.

22. A method for enhancing the enzymatic activity and/or circulation time of an arginine/lysine oxidoreductase, comprising coupling at least one polyethylene glycol moiety to the arginine/lysine oxidoreductase.

23. A method for protecting an arginine/lysine oxidoreductase against inactivation in body fluids comprising coupling at least one polyethylene glycol moiety to the arginine/lysine oxidoreductase.

24. (canceled)

25. Use of the conjugate of claim 1 for the depletion of lysine and/or arginine in a liquid and/or for the production of hydrogen peroxide, ammonium and/or metabolites of lysine and/or arginine in a liquid.

26. Use of claim 25, wherein the liquid is a body fluid of a mammal.

27. Use of at least one polyethylene glycol group for increasing the activity and/or circulation time of an arginine/lysine oxidoreductase.

28. Use of at least one polyethylene glycol group for protecting an arginine/lysine oxidoreductase against inactivation in body fluids.

29. The method of claim 17, wherein the disease is selected from the group consisting of microbial infections, viral infections, and proliferative diseases, and wherein the proliferative disease can be cancer.

30. The method of claim 17, wherein the amino acid levels in body fluids are lysine or/and arginine levels in plasma.

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