WO1994014950A1

WO1994014950A1 - Human variant manganese superoxide dismutase

Info

Publication number: WO1994014950A1
Application number: PCT/JP1993/001917
Authority: WO
Inventors: Masatoshi Kondo; Kimio Katsuta
Original assignee: Sii Technoresearch, Inc.
Priority date: 1992-12-28
Filing date: 1993-12-28
Publication date: 1994-07-07
Also published as: AU5717094A

Abstract

A human manganese superoxide dismutase (Mn-SOD) having an elevated isoelectric point and an increased capability of tissue penetration is prepared by replacing an amino acid residue present at the site which does not affect the enzymatic activity even when another amino acid residue is present thereat in the amino acid sequence of the dismutase by a more positively charged amino acid residue. Examples thereof include one prepared by replacing the 4th serine residue on the N-terminal side of a recombinant human variant Mn-SOD by a basic amino acid residue and one prepared by replacing, in addition to the 4th amino acid residue, the 42nd glutamic acid residue by a valine residue. These human variant Mn-SODs have an excellent drug activity on various diseases on which the conventional Mn-SOD is insufficiently efficacious, for example, various inflammatory diseases caused by active oxygen, cancer, retinopathy of prematurity, hypertension and diabetes. Further they are useful as a cosmetic ingredient.

Description

Description Name of Invention

Human mutant manganese superoxide disperse

The present invention relates to human manganese superoxide dismutase, and more particularly, to a part of the amino acid constituting the peptide sequence of human manganese superoxide dismutase (human Mn-SOD). Accordingly, the present invention relates to a novel Mn-SOD mutated to another amino acid, a DNA gene encoding the amino acid sequence of the Mn-SOD, a host cell, a method for producing the Mn-SOD and its use .

^ Scenic technology

Oxygen is essential for biological, ultraviolet, under the action of such external factors such as radiation has been known that force ^s becomes a variety of reactive oxygen species in vivo tissue. Under normal conditions, it is said that about 1% of oxygen is present as active oxygen. These reactive oxygen species exert a strong bactericidal effect by directly acting on bacteria and the like by phagocytosis of macrophages and the like in vivo, and play an important role as an in vivo infection defense mechanism it is conceivable that.

However, it is also known that when oxygen is present in a living body at a high concentration, it adversely affects the living body. It is thought that this is also due to the action of various active oxygens generated by the reduction of oxygen force in the biotissue. .

Active oxygen has a longer life span in a hydrophobic environment than in a hydrophilic environment, whereas unsaturated fatty acids in phospholipids are extremely sensitive to active oxygen and can be more susceptible to peroxidation: ^ Are known. Therefore, unsaturated fatty acids in biological membranes become lipid peroxides when subjected to a peroxidation reaction, and this lipid peroxide acts on structural proteins and the like, causing cell damage, causing arteriosclerosis, aging, carcinogenesis, etc. Has harmful effects. This active oxygen is generated by irradiating a living body with ultraviolet rays, radiation, or the like. Therefore, there is sebum containing unsaturated fatty acids such as squalane in biological membranes such as skin exposed to sunlight, particularly ultraviolet rays, and the unsaturated fatty acids are subjected to the action of active oxygen to form peroxyacids. It becomes hylipid and causes inflammation, pigmentation, and aging. It is also assumed that when the amount of lipid peroxide is significantly increased, melamine production is promoted and melamine deposition is caused. Furthermore, when skin cells are damaged by burns or trauma and skin cells come into direct contact with air, active oxygen is generated in the cell membrane, and this active oxygen further acts on unsaturated fatty acids in the skin cells. will be lipid peroxidation force ^s generated. The formation of this lipid peroxide delays the healing of damaged areas of the skin.

As mentioned above, recently, it has been reported that this active oxygen is one of the causes of various inflammatory diseases and carcinogenesis, retinopathy of premature babies, and senile diseases. Furthermore, it is estimated that immunity is deeply involved.

By the way, living organisms that breathe oxygen and live (aerobic organisms) naturally supply the oxygen they take in to the whole body and supply active oxygen at the same time. super one force ^s various active oxygen enzymatic degradation are erased Okishido Jisumu evening is Ichize (hereinafter SOD and referred). Therefore, it can be said that SOD is an essential component for aerobic organisms to survive. In fact, it is known that the difference in the amount of SOD in the body between various organisms has a clear correlation with the life span of the organism.

Numerous documents have reported that the local decrease in the concentration of S 0 D causes the above-mentioned numerous diseases.

For example, in cancer cells have been reduced force ^s Remarkably force ^s report of manganese super one Okishidojisumuta Ichize (Mn- SOD) (Yoshihiko Oyanagi: Tokishikoroji one Forum 1 1 Volume (1), 62- 70, 1988, etc.).

Thus, SOD has the potential to be used for treatment or prevention as a causal therapy for many diseases caused by reduced local SOD levels.

Examples of the superoxide dismutase having the above-mentioned action (hereinafter abbreviated as S〇D) include manganese mono-SOD (Mn-SOD), copper zinc-SOD (CuZn-SOD), and iron- S 0 D (F e -SOD) and the like.

Of these, when administering animal-derived SOD to humans, there are problems such as rejection due to the antigen-antibody reaction, and therefore, many difficulties are expected to be developed as pharmaceuticals. On the other hand, human-derived SOD is preferable for development as a medicinal product because it has no problem.

In this sense, Fe-S0D is inappropriate because it is derived from bacteria and plants.

However, human-derived SOD also has extremely serious problems in production and supply, and at the same time, there is concern about infection due to contamination with viruses. Therefore, attempts have been made to produce human SOD by genetic engineering means as a means for solving the above-mentioned problems. In other words, manganese-SOD (Mn—SOD) and copper-zinc— Since SOD (CuZn-SOD) has the potential to be manufactured by genetic engineering techniques, the feasibility of applying these SODs to drugs, cosmetics, etc. is being investigated. In addition, copper-zinc-one SOD (CuZn-SOD) has a shorter life span, so it is more preferable to use Mn-SOD, which has a longer half-life in the body, when it is applied to pharmaceuticals, cosmetics, etc. .

By the way, Mn-SOD is mainly contained in mitochondria in cells in animals including humans, and forms a tetramer composed of a peptide having a molecular weight of about 20,000. It is a metalloprotein containing one per peptide. This Mn ion is essential for enzyme activity.

Mn-S0D has a half-life of about 10 times longer in the body than CuZn-SOD, and is more resistant to hydrogen peroxide, a metabolite of S0D. Mn-SOD is considered to be more useful as a drug.

By the way, regarding the amino acid sequence of Mn-SOD, it was reported that the amino acid sequence consisted of 196 out of 198 total amino acids of human Mn-S0D [Barra et al., J. Biol. Chem259, 12595- 12601, 1984)].

Then, a DNA probe was prepared by citing the amino acid sequence of the above-mentioned article, and it was disclosed that a clone encoding 198 amino acids, which is the entire amino acid sequence of Mn-SOD, was identified from human T cells (Japanese Patent Application Laid-Open No. H10-163,878). (Showa 64-27470). Furthermore, Japanese Patent Application Laid-Open No. 63383/1988 describes a human Mn—S0D force ^, of which the isoelectric point of the polypeptide corresponding to IVa (wild type) is P It is stated that i was 8.15. The hMn-SOD described in the publication does not have any other amino acid residue substitution with respect to the wild-type native Mn-S0D. The DNA sequence described in the publication is significantly different from the human mutant Mn-SOD according to the present invention described later. In addition, the power of this isoelectric point measured by any method ^ "is indeterminate, and the literature [B.A.

Omar & JM McCord: J. Mol Cell Cardiol 23, 149-159 (1991)], the isoelectric point of this peptide is 7.4. Considering that the isoelectric point of human Mn-SOD is usually between 7.0 and 7.4, the isoelectric point mentioned above is too high compared to the usual and suddenly I have to say that it is too much.

In addition, it has been reported that the third amino acid Ser from the N-terminal of the amino acid sequence of human SOD was mutated to Arg (SL Church: Biochimica et Biophysica Acta, 1087, 250-252, 1990). . However, then the author himself This amino acid sequence has been corrected as a misinformation (S. Church: BBA, 1171, 341, 1993).

By the way, since Mn—S 0 D is a polymer, it cannot be said that tissue permeability is good. On the other hand, oxygen radicals are generated in any tissue or organ because oxygen is distributed without gaps in a living body. Therefore, Mn-SOD force administered from outside the body requires good tissue permeability to effectively exert a therapeutic or preventive action in the body.

However, when conventional Mn-S0D is administered to various inflammatory diseases caused by S0D deficiency, cancer, retinopathy of prematurity, hypertension, diabetes mellitus, etc. Unlike sex S0D, it did not penetrate the required local area, and the therapeutic effect was unsatisfactory. Therefore, there is a strong demand for the development of human-type Mn-SOD with good tissue permeability. ,

As mentioned above, even though Mn-SOD is not good in tissue permeability, compared to SOD containing CuZn, Mn-SOD has a better ability to penetrate into the heron myocardium. [BA Omar & JM McCord: J. Mol Cell Cardiol. 23, 149-159 (1991)]. It is said that this is related to the fact that the isoelectric point indicated by Mn-S0D is higher than CuZn-S0D. Molecules having a high affinity for the synovium are more effective. In the case of humans, the isoelectric point of CuZn-SOD is Pi about 5.1, whereas that of Mn-SOD is about 7.0-7.4. Thus, Mn-SOD with a higher isoelectric point is expected to increase the pharmacological effect because of its permeability into tissues.

Therefore, the present invention provides a human mutant Mn-SOD obtained after performing various mutation operations using a recombinant Mn-SOD (rMn-SOD) gene. Another object of the present invention is to create a recombinant mutant Mn-SOD having an isoelectric point higher than that of a natural type Mn-SOD.

Further, another object of the present invention is to create a novel Mn-SOD which does not reduce the enzyme activity and has a sustainability equal to or higher than that of the natural type by reducing the isoelectric point of the Mn-SOD. I do.

Furthermore, another object of the present invention is to provide various inflammatory diseases, cancers, immaturities, etc. as compared with the conventionally known Mn-SOD due to the low isoelectric point and the high tissue permeability. It is an object of the present invention to provide a new recombinant human mutant Mn-SOD that exerts a stronger pharmacological effect against retinopathy of the infant, hypertension, diabetes, and the like.

In addition to these, the present invention provides a gene encoding a human mutant Mn-SOD, It is an object of the present invention to provide an expression plasmid containing the gene sequence of the present invention, a host cell containing the expression plasmid, a method for producing a human mutant Mn-S0D, and a method for preventing or treating a disease using the same.

Until now, if even one amino acid in the amino acid sequence of Mn-SOD was substituted and mutated with another basic amino acid, its enzymatic activity was reduced, and it was not possible to achieve its intended purpose.

However, surprisingly, it was surprising that the human manganese superoxide dismutation in which the serine residue (Ser), the third amino acid residue from the N-terminal of the amino acid sequence of human Mn-SOD, was changed to an arginine residue (Arg). In Yuze, its isoelectric point increased significantly from about 7.1 to 7.4 pi to 8.1 to 8.4 pi, while enzyme activity, half-life in the body, and peroxidation No change was seen in the resistance to hydrogen, and it was found that the above-mentioned problem was satisfied. When human Mn-SOD is recombined into Escherichia coli, a methionine residue (Met) is added to the N-terminus of the recombinant Mn-SOD. The residue (Ser) will be converted to an arginine residue (Arg).

Similarly, the serine residue (Ser), which is the third amino acid residue from the N-terminus of the amino acid sequence of human Mn-SOD, is changed to an arginine residue (Arg), and the amino acid residue at position 42 is replaced with an amino acid residue. Human variant manganese superoxide dismutase, in which a certain glutamic acid residue (Glu) is replaced with a palin residue (Val), also has an isoelectric point of about 7.1 to 7. 4 pi was found to rise significantly from pi 8.5 to 9.4.

Based on the results, the present inventors have made intensive efforts to further solve the above problems, and as a result, in the case of Mn-SOD, the total power of polar amino acid residues is reflected in the isoelectric point. I found it. In other words, for example, the total number of acidic amino acids in Mn-SOD is 19, and the number of basic amino acids is 29. The total number of basic amino acids is 10 more. It was found that the isoelectric point decreased when the number was reduced, while the isoelectric point increased when the number was increased.

In other words, in the peptide sequence of human mutated manganese superoxide dismutase (hereinafter sometimes abbreviated as "hMn-S0D"), a site that does not affect enzyme activity even if it is replaced with another amino acid residue. In addition, it was found that by substituting with a more positively charged amino acid, it was possible to obtain a human mutant Mn-SOD having an isoelectric point higher than a desired value, thereby completing the present invention. . Disclosure of the invention

The human mutant manganese superoxide dismutase (hereinafter sometimes abbreviated as "hMn-S0DJ") according to the present invention has an enzyme even if it is substituted with another amino acid residue in its peptide sequence. It is a human mutant Mn-SOD in which a site that does not affect the activity is substituted with a more positively charged amino acid.

In the present invention, each of a plurality of sites in the peptide sequence of the human mutant manganese superoxide dismutase that does not affect the enzyme activity even when substituted with other amino acid residues is more positively added. Also provided is a human variant Mn-SOD having an increased isoelectric point by substituting a charged amino acid.

Furthermore, in the present invention, in the peptide sequence of the human mutant manganese superoxide dismutase, an amino acid residue present at a site that does not affect the enzyme activity even if it is substituted with another amino acid residue is included. Substitution with a basic amino acid residue or a basic amino acid residue provides a human mutant M n -SOD force having an increased isoelectric point.

Other objects, features and advantages will be apparent from the following detailed description of the invention which refers to the accompanying drawings.

By the way, when the amino acid sequence of human Mn-SOD is systematically compared, only the conserved amino acid sequence centering on the amino acid sequence of human Mn-SOD is shown below. Is a conserved amino acid, and the numerical value above the amino acid abbreviation indicates the position of the amino acid from the N-terminus in the amino acid sequence of human recombinant Mn-SOD.

Ten

Met Lys His Ser (Leu Pro) Asp (Leu) Pro Tyr Asp Tyr Gly (Ala Leu) Glu

20 30

Pro His He Asn Ala Glu lie Met Glu Leu (His) His Ser Lys (His His) Ala

40 50 Ala Tyr Val Asn Asn Leu (Asn) Val Tyr Glu Glu Lys Tyr Gin Glu Ala Leu

60

Ala Lys Gly Asp Val Thr Ala Gin lie Ala Leu Gin Pro Ala Leu Lys Phe Asn 70 80

Gly Gl Gly His lie Asn (His) Ser lie Phe (Trp) Thr Asn Leu Ser (Pro)

90 100

Asn Gly Gly Gly Glu Pro Lys (Gly) Glu Leu Leu Glu Ala lie Lys Arg Asp. 110

Phe Gly Ser Phe Asp Lys Phe Lys Glu Lys Leu Thr Ala Ala Ser Val Gly

120 130

Val Gin (Gly Ser) Gly (Trp) Gly Trp Leu Gly Phe Asn Lys Gin Arg Gly

140 150

His Leu Gin lie Ala Ala Cys Pro Asn Gin Asp (Pro) Leu Gin Gly Thr Thr

160

Gly Leu lie (Pro) Leu Leu Gly lie (Asp) Val (Trp) (Glu His) Ala (Tyr

170 180 Tyr) Leu Gin Tyr Lys Asn Val Arg Pro Asp Tyr Leu Lys Ala lie Trp Asn

190 199

Val He Asn Trp Glu Asn Val Thr Glu Arg Tyr Met Ala Cys Lys Lys In this amino acid sequence of human Mn-SOD, a recombinant human mutant Mn-SOD in which amino acid residues are mutated to different sequences by deletion, duplication, substitution, etc. Obtainable. For example, by changing the fourth serine residue from the N-terminus to an arginine residue (the third in natural Mn-SOD purified from human-derived material because the methionine residue is missing at the N-terminus), pi is changed. 8. The ability to increase to 1 to 8.4.

More specifically, for example, a human in which a serine residue (Ser), which is the third amino acid residue from the N-terminus of the amino acid sequence of a native (wild-type) human Mn-SOD, is substituted with an arginine residue (Arg) When the mutant Mn-SOD is recombined into Escherichia coli, a methionine residue (Met) is added to the N-terminus of human Mn-SOD. The amino acid sequence at the N-terminus is shown below. It is. That is,

N-terminal-Met Lys his Ser Leu Pro Asp Leu—

1

N-end Kyu-Met Lys His Arg Leu Pro Asp Leu—

In this case, in the recombinant human mutant Mn-SOD, the fourth serine residue (Ser) from the N-terminus is mutated to an arginine residue (Arg). This serine residue can be replaced with a basic amino acid residue such as a lysine or histidine residue in addition to an arginine residue.

Furthermore, in the amino acid sequence of the human recombinant Mn-SOD, the four serine residues from the N-terminal were replaced with an arginine residue or another amino acid residue, and Has an amino acid sequence in which the glutamic acid residue at position 43 is replaced with a neutral amino acid residue such as palin, leucine, isoleucine, glycine, or alanine residue or a basic amino acid residue such as lysine, arginine, or histidine residue Although the human mutant MnS OD does not affect the enzyme activity, half-life in the body, or resistance to hydrogen peroxide, it only increases its isoelectric point to about PI 8.5-9.4. Was found.

Furthermore, only the glutamic acid residue at position 43 from the N-terminus in the amino acid sequence of the human and recombinant Mn-SOD is replaced with a neutral amino acid residue such as valine, leucine, isoleucine, glycine, or alanine residue or lysine. The human mutant Mn-SOD having an amino acid sequence substituted with a basic amino acid residue such as an arginine or histidine residue is also slightly different from the human mutant Mn-SOD having an amino acid sequence in which the above two sites are mutated. It was found to exhibit an inferiorly elevated isoelectric point.

In the above-described amino acid sequence of human Mn-SOD, all amino acids other than the conserved amino acids are mutable amino acids unless they cause a significant structural change. In other words, theoretically, even if it is substituted with another amino acid, the enzyme activity of the polypeptide is not lost, and all or many of the amino acid residues present at the site are more positively charged. It is also possible to substitute a substituted amino acid residue, or a neutral amino acid residue or a basic amino acid residue. For example, the substitution of too many positions with another amino acid residue may result in a completely different protein in some cases. In order to develop it, it must be treated as a completely new substance, and the advantages of developing it as human Mn-SOD are diminished and unrealistic. In addition, the novel human Mn-SOD obtained by substituting amino acids at sites greater than the above-mentioned number with other amino acid residues also has problems such as rejection reactions that are difficult to overcome such as antigen-antibody reactions. The development of such Mn-SOD as a medicinal product is expected to be quite difficult because of the potential for the development of Mn-SOD.

Therefore, in consideration of problems such as rejection in the development of pharmaceuticals, eh cosmetics, etc., in the present invention, the site to be replaced with another amino acid in the peptide sequence of hMn-SOD is 1 Pcs to 4 pcs preferred.

In the peptide sequence of hMn-SOD, an amino acid residue at a position where enzyme activity does not change even if it is substituted with another amino acid residue is replaced with another amino acid residue such as arginine residue. In addition to basic amino acid residues such as lysine, histidine, glutamine, and asparagine, or Examples include amino acid residues such as syn, isoleucine, alanine, and glycine.

In the present invention, the isoelectric point achieved by substituting an amino acid residue existing at a site substituted with another amino acid in the peptide sequence of hMn-SOD with another amino acid residue is about PI It is 7.6 or more, preferably about pI 8.0 or more, and more preferably about P18.1 to 9.4. By increasing the isoelectric point in this way, the tissue permeability of human Mn-SOD can be increased.

Further, the present invention provides a human mutant Mn-SQD protein obtained by recombining the amino acid sequence of the human mutant Mn-SOD, a gene sequence encoding the amino acid sequence of the human mutant Mn-SOD, and a microorganism transformed therewith. Are also included.

Also, the present invention relates to a method for detecting the activity of CuZn-SOD, as reported by Elizabeth D. Getz off et al; Nature: 358, 23 July, NQ. 6384, 1992, for example. After increasing the activity several times by changing the acidic amino acid (for example, glutamic acid) residue to a basic amino acid such as glutamine, for example, the amino acid residue at a predetermined position from the N-terminal is replaced with another amino acid residue. The amino acid sequence of the human variant Mn-SOD and the amino acid sequence of the human variant Mn-SOD which have increased permeability by substitution with the Microorganisms.

The recombinant human mutant Mn-SOD according to the present invention is, for example, substituted by adding a mutation to a gene sequence portion that directs an amino acid residue corresponding to the amino acid sequence of the recombinant amino acid residue in the human Mn-SOD gene sequence. It can be produced by a method of performing amplification by PCR using a primer having a gene sequence that directs the specified amino acid residue. .

That is, for example, an arginine residue or a arginine residue substituted by adding a mutation to a portion of the gene sequence that directs the amino acid residue corresponding to the amino acid sequence of the fourth serine residue from the N-terminal in the human Mn-SOD gene sequence Using a primer having a gene sequence that directs other amino acid residues, and / or mutating the gene sequence portion that directs the amino acid residue corresponding to the amino acid sequence of the glutamic acid residue at position 43 from the N-terminus Can be produced by multiplying by a PCR method using a primer having a gene sequence that directs a substituted valine residue or other amino acid residue.

Therefore, among the primers that can be used in the present invention, N-terminal of Mn-SOD The following are examples of the primer on the end side.

HMS-5: 5 'AT CTG GGC GAA TTC ATG AAG CAC CGC CTC CCC GA 3' or

HMS-7: 5 'C ATG AAG CAC CGC CTC CCC GAC CTG CCC T 3'

Further, examples of the primer at the C-terminal side of Mn—S0D include the following.

HMS-6: 3 'GCTAGC AATACGACGT CTACMTTC 5'

Also, among the N-terminal primers having a gene sequence that directs a part of the N-terminal peptide sequence of the human Mn—S0D, the serine residue of the Met Lys His Ser Leu Pro Asp Leu… sequence The gene sequence to be instructed includes all of the mutant sequences of the N-terminal bimer which change other basic amino acid sequences into gene sequences to be instructed. -For example, a part of the peptide sequence of the first to seventh Mn-SOD from the N-terminal side of the natural type is as follows.

—Lys His Ser Leu Pro Asp Leu

The corresponding gene sequence that directs the native N-terminal Mn—S 0 D peptide sequence is as follows.

—AAG CAC AGC CTC CCC GAC…

One example of the N-terminal variant Mn-S0D peptide sequence portion corresponding to the above-mentioned native terminal Mn-S0D peptide partial sequence is as follows.

— Lys His Arg Leu Pro Asp Leu ···

The gene sequence that directs the N-terminal mutant M n-SOD peptide sequence corresponding to the above is as follows.

Partial gene sequence 1 —AAG CAC CGC CTC CCC GAC….

Partial gene sequence 2 AAG CAC AGG CTC CCC GAC…

Partial gene sequence 3 AAG CAC AGA CTC CCC GAC…

Another example of the N-terminal side Mn-S0D peptide sequence corresponding to the above-mentioned native terminal Mn-S0D peptide partial sequence is as follows.

— Lys His Asn Leu Pro Asp Leu…

The corresponding N-terminal mutant Mn-S0D peptide sequence-directed gene sequence is as follows.

Partial gene sequence 4: ~ MG CAC AAC CTC CCC GAC

Partial gene sequence 5: ~ MG CAC AAT CTC CCC GAC…

N-terminal modification corresponding to the above-mentioned native terminal Mn—S 0 D peptide partial sequence Still another example of the peptide sequence portion of the different Mn—S0D is as follows.

— Lys His Lys Leu Pro Asp Leu ···

The gene sequences that direct the N-terminal mutant Mn—S 0 D peptide sequence corresponding to the above are as follows.

Partial gene sequence 6: ~ MG CAC AAA CTC CCC GAC…

Partial gene sequence 7: -AAG CAC AAG CTC CCC GAC…

Further examples of the peptide sequence portion of the N-terminal mutant Mn-S0D corresponding to the above-mentioned native terminal Mn-S0D peptide partial sequence are as follows.

—Lys His Gin Leu Pro Asp Leu

Partial gene sequence 8: ~ MG CAC CM CTC CCC GAC…

Partial gene sequence 9: —AAG CAC CAG CTC CCC GAC

Also, for example, a part of the peptide sequence of the Mn-SOD at positions 41 to 43 from the N-terminal side of the natural type is as follows.

— Arg Glu lie "·

-CGC GAG ATA…

One example of the peptide sequence portion of the N-terminal mutant Mn—S0D corresponding to the above-mentioned peptide sequence of the native terminal Mn-SOD is as follows.

—Arg Val lie ···

Partial gene sequence 10: "'CGC GTG ATA…

In addition, sequences of genes containing various mutated amino acids and various codons encoding the same can be mentioned. Therefore, the present invention naturally includes, for example, a primer containing a gene sequence corresponding to the amino acid sequence of the substituted amino acid residue as described above. Furthermore, a recombinant human mutant Mn-SOD gene DNA sequence IJ prepared using the above-mentioned Bramer, a cDNA prepared thereby, and a microorganism such as Escherichia coli transformed with the cDNA are also included. .

After culturing the transformed microorganism, the active fractions are collected by column chromatography using DEAE Sepharose (Pharmacia), CM Sepharose, or the like, and endotoxin is collected from the collected active fractions according to a conventional method. By removing such pyrogens, a human mutant Mn-SOD having an increased isoelectric point can be produced.

Various stabilizers and excipients such as albumin, lactalbumin, poly ^ ethylene glycol, amino acids, monosaccharides, disaccharides, trisaccharides, etc. may be added to the recombinant human mutant Mn-SOD of the present invention. Can be. For example, a more preferred example of the above-mentioned disaccharide is a solution containing 0.02 to 1% concentration of trehalose (a- [D-glucopyranosyl] a-D-glucoviranose).

Formulation examples of the human mutant Mn-SOD according to the present invention include, for example, a recombinant human mutant Mn-SOD in which the serine residue at the fourth position from the N-terminus of recombinant human Mn-SOD is substituted with arginine. And the like formulated in an injection solution such as That is,

MHS: SOD 10 Omg

Trehalose 0.23M (final concentration) Sodium glutamate 10mM

After adjusting the pH of the diluted sodium hydroxide to pH 7.2 to 7.4, add distilled water (pyrozen free) to make the total volume 1 Oml.

This solution is dispensed into vials and stored.

The dose of the recombinant human mutant Mn-SOD of the present invention is, for example, 0.01 mg to 1 g / kg / day, preferably 0.1 mg to 5 Omg / kg / day, and is used for infusion, intravenous injection, or intramuscular injection. It can also be administered subcutaneously, as a suppository or nasal drop, or into the joint cavity. The administration fee and administration method can be appropriately changed depending on the type and degree of the disease, the condition of the patient, and the like.

The toxicity of the recombinant human mutant Mn-SOD according to the present invention is extremely low. For example, the recombinant human mutant Mn-SOD in which the fourth serine residue from the N-terminal of the recombinant human Mn-SOD is changed to arginine is SD. 5 Omg / kg tail vein to male rat No toxicity was observed with these bolus doses.

The thus obtained recombinant human mutant Mn-SOD in which the fourth amino acid from the N-terminus of the amino acid sequence of human Mn-S0D according to the present invention is mutated to an arginine residue has the effect of arthritis whose effect was not clear until now. A significant anti-inflammatory effect was exhibited on rat models and the like. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing an expression vector. In an E. coli host, a recombinant human mutant Mn-SOD (= MHS: Mn-SOD) gene was linked downstream of the Ptac promoter.

In the figure, (a) shows incorporation into Ptac-hMnSOD, and (b) shows incorporation into Ptac * -hMnSOD.

Figure 2 is a graph showing the persistence of MHS2: Mn-SOD in rats. In the graph, 〇 indicates MHS: ΐνΐη-SOD, Hata indicates recombinant human Mn-SOD, and mouth indicates CuZn-SOD.

Figure 3 shows the isoelectric focusing of MHS2: Mn-SOD (ampholyte, pH

3-9, manufactured by Pharmacia Co., Ltd.). In the graph, (a) shows the recombinant human Mn-SOD, (b) shows MHS2: Mn-SOD, and (c) shows the protein standard.

FIG. 4 is a graph showing the anti-inflammatory effect (rat) of MHS: Mn—SOD on adjuvant arthritis. In the graph, 〇 indicates MHS: Mn—SOD, and reference indicates recombinant human Mn—SOD.

FIG. 5 is a graph showing the effects of MHS _: Mn—S0D on the frequency of occurrence of arrhythmias after coronary artery recanalization and their duration. In the graph, (a) shows recombinant human Mn-SOD, and (b) shows MHS: Mn-SOD.

FIG. 6 is a graph showing the myocardial protective action in a cardiac ischemia reperfusion model. In the graph, (a) shows control, (b) shows recombinant human Mn-SOD, and (c) shows MHS: Mn-SOD. BEST MODE FOR CARRYING OUT THE INVENTION

Example

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples. The present invention is not limited to the following Examples.

Experiments on genetic engineering are basically performed by Sunbrook et al. (Sambrook et al, Cold Spring Harbor Lab., 1989) "Molecular Cloning, A Laboratory Manual J." For protein chemistry, purification and other methods, see Packer Edition [( Lester P acker, ed) Methodin Enzymo logy Vo l. 105, 1984 Ac ademic Press].

Example 1-1: Preparation of cDNA

The mRNA extracted from human cultured cells according to a conventional method has a sequence in which a known phMnS0D4c DNA is used as type II, and then the fourth amino acid residue serine from the N-terminal is mutated to an arginine residue. HMS-5 primer (Sequence Table 1) was chemically synthesized as an N-terminal primer, and C-terminal primer HMS-6 (Sequence Table 2) was chemically synthesized, and both HMS-5 and HMS-6 primers were synthesized. Was used to amplify the human mutant Mn-SOD gene by the gene amplification method.

HMS-5

EcoRI

5ΆΤ CTG GGC GAA TTC ATG AAG CAC CGC CTC CCC GA 3 '

HMS— 6

3'GCTAGC AATACGACGT CTACAATTC 5 '

Pstl

Subsequently, the obtained human mutated SOD gene was digested with the restriction enzymes PstI and EcoRI, and the expression vector digested with the restriction enzymes PstI and EcoRI was added to the human mutated SOD gene. Was linked. The expression vector (A) is used for overexpression of the protein, has a strong tac promoter, is regulated by a 1 ac ribosser in a suitable host (JM105), and It is induced by the addition of galactoside (IPTG) (Fig. 11-A).

Example 1-2:

Using an mRNA extracted from human cultured cells or a known phMnS0D4c DNA according to a conventional method as type III, and then an N-terminal primer having a sequence in which the fourth amino acid residue serine from the N-terminal is mutated to an arginine residue HMS-7 primer was chemically synthesized, and the C-terminal primer HMS-6 was chemically synthesized therewith. Using both HMS-7 and HMS-6 primers, the human mutant S0D gene was amplified by the gene amplification method I let it.

HMS-7

5'C ATG AAG CAC CGC CTC CCC GA 3 ' HMS-6

3 'GCTAGC AATACGACGT CTACMTTC 5'

Pst I

Subsequently, the obtained human Mn-SOD gene was digested with mung-bean nuclease and restriction enzyme PstI, and digested with restriction enzymes NcoI and Mungbean nuclease. Thereafter, the human mutant SOD gene was ligated to an expression vector treated with PstI (FIG. 11B).

Example 2: Selection of promoter and vector

The desired expression plasmid was prepared by ligating cDNA containing the human mutant Mn-SOD gene downstream of Pac and its improved Pt ac * as a promoter for E. coli. EcoR I or Nco I and Pst I sites were placed at both ends of the Mn-SOD gene, respectively, and connected to the promoter. The resulting human mutant Mn—

An expression plasmid containing the S0D gene was introduced into host Escherichia coli, and transformants were selected as ampicillin-resistant bacteria (FIG. 1).

Example 3: Culture and separation and purification

Escherichia coli strain transformed with an expression plasmid containing the human mutant Mn-S0D gene

E.co1iJM105 was cultured with stirring at 37 ° C. for 15 hours in a medium containing ampicillin 4 OmgZl and manganese chloride 15 mM. After the culture, the cells were crushed in a blender and the supernatant was obtained by low-speed centrifugation.

The supernatant thus obtained is loaded on a DEAE sepharose and chromatographed using a phosphate buffer. Subsequently, the active fraction is subjected to CM Sepharose and eluted with a high-concentration phosphate buffer to collect the active fraction. Pyrogens such as endotoxin were removed from the collected active fractions according to a conventional method.

When the obtained purified recombinant human mutant Mn-SOD was measured according to the method of pine code or the like (McCord, J., Friedovich, I., J. Biol. Chem., 244, 6049-6055, 1969), about 4900 units were obtained. It showed the activity of Zmg protein. On the other hand, the recombinant human Mn-S0D used as a control exhibited an activity of about 4800 units / mg, and no difference was observed between the two.

Thus, the mutant Mn-SOD obtained in this example was abbreviated as MHS: Mn-SOD.

The amino acid sequence and gene sequence of MHS _: MnS0D are as follows. EcoRI 10 20 30 40 50

GAA TTC ATG AAG CAC CGC CTC CCC GAC CTG CCC TAC GAC TAC GGC GCC CTG

Met Lys His Arg Leu Pro Asp Leu Pro Tyr Asp Tyr Gly Ala Leu 60 70 80 90 100

5 GAA CCT CAC ATC AAC GCG CAG ATC ATG CAG CTG CAC CAC AGC AAG CAC CAC GCG Glu Pro His lie Asn Ala Gin lie Met Gin Leu His His Ser Lys His His Ala

110 120 130 140 150

GCC TAC GTG AAC AAC CTG AAC GTC ACC GAG GAG AAG TAC CAG GAG GCG GCC

Ala Tyr Val Asn Asn Leu Asn Val Thr Glu Glu Lys Tyr Gin Glu Ala Leu Ala O 160 170 180 190 200 210

AAG GGA GAT ACA GCC CAG ATA GCT CTT CAG CCT GCA CTG AAG TTC AAT GGT

s Gly Asp Val Thr Ala Gin lie Al? Leu Gin Pro Ala Leu Lys Phe Asn Gly

220 230 240 250 260

GGT GGT CAT ATC AAT CAT AGC ΚΠ TTC TGG ACA AAC CTC AGC CCT AAC GGT GGT

5 Gly Gly His lie Asn His Ser lie Phe Trp Thr Asn Leu Ser Pro Asn Gly Gly 270 280 290 300 310 310 320

GGA GAA CCC AAA GGG GAG HG CTG GAA GCC ATC AAA CGT GAC ΊΤΓ GGT TCC ΉΤ

Gly Glu Pro Lys Gly Glu Leu Leu Glu Ala lie Lys Arg Asp Phe Gly Ser Phe

330 340 350 360 370 O GAC AAG ΉΤ AAG GAG AAG CTG ACG GCT GCA TCT GU GGT GTC C GGC TCA GGT

Asp Lys Phe Lys Glu Lys Leu Thr Ala Ala Ser Val Gly Val Gin Gly Ser Gly

380 390 400 410 420

TGG GGT TGG ΖΉ GGT TTC AAT AAG GAA CGG GCA CAC ΉΑ CM ΚΠ GCT GCT TGT

Trp Gly Trp Leu Gly Phe Asn Lys Glu Arg Gly His Leu Gin lie Ala Ala Cys 5 430 440 450 460 470 470 480

CCA AAT CAG GAT CCA CTG C GGA ACA ACA GGC CTT ΚΠ CCA CTG CTG GGG ΚΠ

Pro Asn Gin Asp Pro Leu Gin Gly Thr Thr Gly Leu lie Pro Leu Leu Gly lie

490 500 510 520 530

GAT GTG TGG GAG CAC GCT TAC TAC CTT CAG TAT AAA AAT GTC AGG CCT GAT TAT O Asp Val Trp Glu His Ala Tyr Tyr Leu Gin Tyr Lys Asn Val Arg Pro Asp Tyr

540 550 560 570 580 590

CTA AAA GCT ΚΉ TGG AAT GTA ATC AAC TGG GAG AAT GTA ACT GAA AGA TAC ATG

Leu Lys Ala lie Tr Asn Val lie Asn Trp Glu Asn Val Thr Glu Arg Tyr Met 600 610 620 Pstl

GCT TGC AAA AAGTM ACC ACG ATC GTT ATG CTG CAG

Ala Cys Lys Lys End Example 4: Purified MHS: Enzymatic properties of Mn-SOD

SDS polyacrylamide gel electrophoresis showed a single band with a molecular weight of about 20000. Native and MHS: Isoelectric focusing of PI 3.0 to 9. ◦ containing a slab-type polyacrylamide gel containing 10 wg each of Mn-SOD showed that natural Mn-SOD had a pi of about 7.2. showed that. On the other hand, MH S: Mn—SOD showed a pI of about 8.2, which was clearly different from the native Mn—SOD (FIG. 2).

Investigation of the stability of MHS: Mn-SOD in the presence of various concentrations of hydrogen peroxide and cyanide showed that MHS: Mn-S0D was not deactivated at the concentration of hydrogen peroxide at which CuZn-SOD was deactivated. Cyan resistance was not different from Mn-SOD.

The amino acid sequence of the obtained MHS: Mn-SOD of the present invention is shown above, and it was confirmed by N-terminal analysis that the serine at the fourth position from the N-terminal was a human mutant Mn-SOD in which arginine was mutated. .

Example 5: Substitution of serine residue in other human mutant Mn-SOD gene

The isoleucine residue at position 59 from the N-terminus is replaced by a threonine residue, which reduces the enzymatic activity to about 50%. The N-terminal Met Lys His Ser Leu Pro Asp Leu ... sequence In the case of the recombinant human mutant Mn-SOD in which the serine residue (Ser) of the moiety was changed to an arginine residue (Arg) according to the method of Examples 1-4, the enzyme activity was higher than that of the native form. It was confirmed that the isoelectric point increased from about 7.0 pi to Pl 8.1 to 8.3 with about 50%. As a result, it was confirmed that the thread permeability increased.

Example 6: Persistence of MHS: Mn-S0D in the body

After administration of CuZn-SOD, Mn-SOD and MHS: Mn-SOD: 10 mg / Kg each from the rat tail vein, blood was collected over time from the carotid artery and the persistence was measured. It showed 74 minutes and 82 minutes (Figure 3).

Example 7: Therapeutic effect of MHS: Mn—SOD on adjuvant arthritis

Lewis male rats (6-7 weeks old) Use rats weighing 250-300 g. After ether anesthesia, a 0.1 ml tuber (5 mg gZm 1 liquid paraffin) solution is administered by using a tuberculin needle under the tail subcutaneously.

MHS: Mn—1000 units of SOD Rats were administered to the hind foot joint from day 3 The administration was started once every two days and three times a week. In the MHS administration group, a significant inhibitory effect was seen from week 5 (Fig. 4).

Example 8: Therapeutic effect of MHS: Mn-SOD on reperfusion arrhythmia

The heart of a male SD rat (300-350 g) was excised and subjected to port flow by the Langendorff method. After administration of 100 units / ml of Mn-SOD or MHS: Mn-SOD 5 minutes before ischemia, ischemia was performed for 30 minutes, and the occurrence frequency and duration of arrhythmia after reperfusion were measured.

As a result, as shown in FIG. 5, a significant frequency-reducing effect and a duration-reducing effect were observed as compared with Mn-S〇D.

Example 9: Plasma creatinine kinase release inhibitory action

Finke et al. Have observed that the use of Mn-S0D together with perokinase during coronary thrombolysis protects the release of creatinine kinase [Fincke, U. Arzne im Forcsch 38 Nr. 1: 138-142, 1988)]. 10 mgZkg of the recombinant human mutant Mn-SOD and 3 mgZkg of the high-molecular-weight protein kinase prepared in Examples 1-4 of the present invention were subjected to ischemia in an experimental system using a canine heart ischemia reperfusion model such as Finke. After 6 hours, blood was collected and plasma creatine kinase was measured. As a result, a significant inhibitory effect on plasma creatinine kinase release was observed, and a protective effect on the myocardium was observed (Fig. 6).

Example 10: MHS: Effect of stabilizer on Mn-SOD

MHS: Mn-SOD, like recombinant human Mn-S0D, is unstable when purified to a high degree of purity and its activity may be reduced by freeze-thawing or long-term storage. Therefore, 0.2M trehalose or 12OmgZm1 maltose was added to a 5mg / m1 solution (0.06M phosphate buffer, pH 6.8) of MHS: Mn-SOD, freeze-dried and stored at 55 ° C for 3 weeks. The decrease in activity was examined. As a result, the activity was reduced to about 70% in the group without added sugar, but was not decreased in the group added with sugar.

Example 11: MHS2: cDNA preparation for MnSOD adjustment

The human mutant Mn-SOD gene was amplified according to the method described in Example 1-1, and the human mutant SOD gene was ligated to an expression vector.

Subsequently, mRNA extracted from human cultured cells or known phMnS0D4 cDNA was used as type III, and then the N-terminal side having a sequence in which the fourth amino acid residue serine from the N-terminal was mutated to an arginine residue. The HMS-7 primer was chemically synthesized as a primer, the C-terminal primer HMS-6 was chemically synthesized with the primer, and the HMS-7 and HMS-6 primers were used for gene amplification by the gene amplification method. Mutant S0D gene Amplified. Subsequently, the glutamic acid residue at position 43 from the N-terminus was replaced with a valine residue using a primer in which the base at position 134 in the gene code of FIG. 8 was replaced with thymine.

Subsequently, the obtained human Mn-SOD gene was digested with mung-bean nuclease and restriction enzyme PstI, and then digested with restriction enzymes NcoI and Mungbean nuclease. The above-mentioned human mutant S0D gene was ligated to the expression vector treated with PstI.

Example 12: Selection of promoter and vector of MHS2: Mn-SOD cDNA containing human mutant Mn-SOD gene downstream of Pt ac or its improved Pt ac * was used as a promoter for E. coli. The desired expression plasmid was prepared by ligation. EcoRI or NcoI and PstI sites were placed at both ends of the Mn-SOD gene, respectively, and connected to the promoter. The expression plasmid containing the human mutant Mn-SOD gene thus obtained was introduced into host Escherichia coli, and transformants were selected as ampicillin-resistant bacteria.

Example 13: Culture and separation and purification of MHS2: Mn-SOD

Escherichia coli strain E.co1iJM105 transformed with an expression plasmid containing the MHS2: Mn-SOD gene was cultured according to the method of Example 3, and the obtained MHS2: Mn-SOD was isolated by a conventional method. Purified.

The resulting purified MHS2: Mn-SOD was measured according to the method of McCord, M., Friedovich, I., J. Biol. Chem., 244, 6049-6055, 1969. It showed an activity of about 4950 units / mg protein. On the other hand, the recombinant ぇ human Mn—S0D used as a control exhibited an activity of about 4830 units Zmg, and no difference was observed between the two. An appropriate amount of trehalose was added, lyophilized and stored.

Thus, the mutant Mn-SOD obtained in this example was abbreviated as MHS2: Mn-SOD.

The amino acid sequence and gene sequence of MHS2: MnSOD are as follows. EcoRI 10 20 30 40 50

GAA TTC ATG AAG CAC CGC CTC CCC GAC CTG CCC TAC GAC TAC GGC GCC CTG GAA

Met Lys His Arg Leu Pro Asp Leu Pro Tyr Asp Tyr Gly Ala Leu Glu 60 70 80 90 100

CCT CAC ATC AAC GCG CAG ATC ATG CAG CTG CAC CAC AGC AAG CAC CAC GCG GCC

Pro His lie Asn Ala Gin lie Met Gin Leu His His Ser Lys His His Ala Ala 110 120 130 140 150 160

TAC GTG AAC AAC CTG AAC GTC ACC GTG GAG AAG TAC CAG GAG GCG TTG GCC AAG

Tyr Val Asn Asn Leu Asn Val Thr Val Glu Lys Tyr Gin Glu Ala Leu Ala Lys

170 180 190 200 210 GGA GAT GH ACA GCC CAG ATA GCT CH CAG CCT GCA CTG AAG TTC AAT GGT GGT

Gly Asp Val Thr Ala Gin lie Ala Leu Gin Pro Ala Leu Lys Phe Asn Gly Gly 220 230 240 250 260 270

GGT CAT ATC AAT CAT AGC ΑΠ TTC TGG ACA AAC CTC AGC CCT AAC GGT GGT GGA

Gly His lie Asn His Ser lie Phe Trp Thr Asn Leu Ser Pro Asn Gly Gly Gly

280 290 300 310 320

GAA CCC AAA GGG GAG HG CTG GAA GCC ATC AAA CGT GAC TIT GGT TCC ΉΤ GAC

Glu Pro Lys Gly Glu Leu Leu Glu Al ^ lie Lys Arg Asp Phe Gly Ser Phe Asp

330 340 350 360 370

AAG ΊΤΓ AAG GAG AAG CTG ACG GCT GCA TCT GH GGT GTC CAA GGC TCA GGT TGG

Lys Phe Lys Glu Lys Leu Thr Ala Ala Ser Val Gly Val Gin Gly Ser Gly Trp 380 390 400 410 420 430

GGT TGG CTT GGT TTC AAT AAG GAA CGG GCA CAC ΠΑ CAA Απ GCT GCT TGT CCA Gly Trp Leu Gly Phe Asn Lys Glu Arg Gly His Leu Gin lie Ala Ala Cys Pro 440 450 460 470 480

AAT CAG GAT CCA CTG CAA GGA ACA ACA GGC Ή ΚΠ CCA CTG CTG GGG ΑΉ GAT

Asn Gin Asp Pro Leu Gin Gly Thr Thr Gly Leu lie Pro Leu Leu Gly He Asp 490 500 510 520 530 530 540

GTG TGG GAG CAC GCT TAC TAC CTT CAG TAT AAA AAT GTC AGG CCT GAT TAT CTA

Val Trp Glu His Ala Tyr Tyr Leu Gin Tyr Lys Asn Val Arg Pro Asp Tyr Leu

550 560 570 580 590

AAA GCT ATT TGG AAT GTA ATC AAC TGG GAG AAT GTA ACT GAA AGA TAC ATG GCT

Lys Ala lie Tr Asn Val lie Asn Trp Glu Asn Val Thr Glu Arg Tyr Met Ala

600 610 620 Pstl

Ding GC AAA AAG TAA ACC ACG ATC Gn ATG CTG CAG

Cys Lys Lys End

Example 14: Enzymatic Properties of Purified MHS2: Mn-SOD

SDS polyacrylamide gel electrophoresis showed a single band with a molecular weight of about 20000. Natural type and MHS: Slab type polyac containing Mn—SOD of 1 Og each Isoelectric focusing of PI 3.0-9.0 • containing rilamide gel showed that the native Mn-SOD had a pI of about 7.2. On the other hand, MH S _: Mn-SOD showed a pI of about 8.5 to 9.4, which was clearly different from that of native Mn-SOD.

The stability of MHS2: Mn-SOD in the presence of various concentrations of hydrogen peroxide and cyanide was examined. MHS2: Mn-SOD was not deactivated at the concentration of hydrogen peroxide at which CuZn-SOD was deactivated. However, Xan resistance was no different from M n -SOD.

The amino acid sequence of the obtained MHS2: Mn—SOD of the present invention is shown above, but the human s gene in which the fourth serine from the N-terminus is mutated to arginine is Mn—SOD and the 42nd is valine Confirmed by structural analysis. Example 15: Biopersistence of MHS2: Mn-SOD

After administration of CuZn—S0D, Mn—SOD, and MHS2: Mn—S〇D (10 mg / kg) from the rat tail vein, blood was sampled from the carotid artery over time and the persistence was measured. Minutes, 76 minutes and 88 minutes.

Example 16: Effect of stabilizer on MHS 2: Mn-SOD

MHS 2: Mn-SOD, like recombinant human Mn-S0D, is unstable when purified to a high degree of purity and loses its activity due to freezing and thawing and long-term storage.

Therefore, after adding trehalose 0.2M or maltose 12 OmgZm1 to a 5mg / ml solution of MHS2: Mn-SOD (0.06M phosphate buffer pH6.8), freeze-dry and store for 55 weeks. The decrease in activity was examined. As a result, the activity s was reduced to about 70% in the group without added sugar, whereas the activity s was not reduced in the group added with sugar. Industrial applicability

According to the present invention, it is possible to obtain Mn—S0D having a significantly increased isoelectric point, thereby increasing tissue permeability and various inflammatory diseases, cancer, retinopathy of prematurity, hypertension, diabetes, etc. A novel Mn-SOD which can exert a more powerful pharmacological effect on the skin and can be used as a cosmetic for cosmetics can be provided.

Claims

The scope of the claims

1. In the peptide sequence of human manganese superoxide dismutase, the amino acid residue existing at the site that does not affect the enzyme activity even if it is replaced with another amino acid is replaced with a more positively charged amino acid residue. A human mutant manganese superoxide dismutase characterized by having an increased isoelectric point.

2. In the peptide sequence of human manganese superoxide dismutase, a neutral amino acid residue or a basic amino acid is substituted for an amino acid present at a site that does not affect enzyme activity even if it is substituted with another amino acid. A human mutant manganese superoxide dismutase characterized by increasing the isoelectric point by substitution with a residue.

3. The human mutant manganese superoxide dismutase according to claim 1 or 2, characterized in that there are ^{five or} more site forces substituted with other amino acids.

4. The human mutant manganese peroxide dismutase according to claim 3 is characterized in that there are 1 to 4 sites substituted with another amino acid.

5. The human manganese superoxide dismutase according to claim 1 or 2, wherein the site substituted with another amino acid is the third and / or the second from the N-terminal. I do.

6. The human mutant manganese superoxide xydismumase according to any one of claims 1 to 5, characterized in that its isoelectric point is a pi force of about 7.6 or more.

7. The human mutant manganese superoxide dismutase according to claim 6, wherein the isoelectric point is pI of 8.1 or more.

8. In the peptide sequence of human manganese superoxide dismutase, amino acid residues at sites that do not affect enzyme activity even when substituted with other amino acids are replaced with more positively charged amino acid residues. A gene sequence encoding a human mutant manganese peroxide dismutase characterized by encoding a human mutant manganese superoxide dismutase peptide whose isoelectric point has been increased by substitution. Column.

9. In the peptide sequence of human manganese superoxide dismutase, amino acids present at sites that do not affect enzyme activity even when substituted with other amino acids are replaced with neutral amino acid residues or basic amino acids. A human mutant manganese superoxide dismutase encoding a peptide sequence having an increased isoelectric point by substitution with a residue. A gene sequence encoding a mutated manganese super one year old xydodimutase.

10. The gene sequence encoding the human mutant manganese superoxide dismutase according to claim 8 or 9, which includes a partial gene sequence including a site substituted with another amino acid. Characterized by the fact that a human mutant manganese super one-year-old xydosis

5 Mutase Primer.

11. An expression plasmid characterized by containing the gene sequence of 範 in the so-called sought term, section 9 or section 10.

12. A host cell comprising the expression plasmid according to claim 11.

0 13. A human mutant manganese mutant comprising culturing a host cell containing an expression plasmid containing the gene sequence of the human mutant manganese superoxide dismutase according to item 8 or 9. A method for producing baroxide dismutase.

14.Amplification of .it gene 5 of human mutant manganese super-lactose xidodismutase using the primer of the human mutant manganese super-lactate xidodismutase described in section 10. A method for producing a human mutant manganese superoxide dismutse, which is described as follows.

15. A pharmaceutical or cosmetic composition comprising as an active ingredient the human mutant manganese-baxoxide dismutase according to any one of claims 1 to 7.

0 16. The pharmaceutical composition containing the human mutant manganese super-one-year-old xydismutase described in paragraph 15, paragraph 15 of the request should be applied to human [IV!

7. Scope of claim 15 A medical composition containing the human mutant manganese super-one year old xydismutase described in section 15 above shall be used for the prevention and treatment of diseases caused by active oxygen. The purpose of the human mutant manganese superoxide disposition, which is characterized by the following. o

0