WO1994014950A1 - Dismutase de superoxyde de manganese humaine de type variant - Google Patents

Dismutase de superoxyde de manganese humaine de type variant Download PDF

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WO1994014950A1
WO1994014950A1 PCT/JP1993/001917 JP9301917W WO9414950A1 WO 1994014950 A1 WO1994014950 A1 WO 1994014950A1 JP 9301917 W JP9301917 W JP 9301917W WO 9414950 A1 WO9414950 A1 WO 9414950A1
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amino acid
sod
human
human mutant
superoxide dismutase
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PCT/JP1993/001917
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Japanese (ja)
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Masatoshi Kondo
Kimio Katsuta
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Sii Technoresearch, Inc.
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Priority to AU57170/94A priority Critical patent/AU5717094A/en
Publication of WO1994014950A1 publication Critical patent/WO1994014950A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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 .
  • 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.
  • 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.
  • SOD is an essential component for aerobic organisms to survive.
  • the difference in the amount of SOD in the body between various organisms has a clear correlation with the life span of the organism.
  • SOD has the potential to be used for treatment or prevention as a causal therapy for many diseases caused by reduced local SOD levels.
  • S ⁇ D superoxide dismutase having the above-mentioned action
  • Mn-SOD manganese mono-SOD
  • CuZn-SOD copper zinc-SOD
  • F e -SOD iron- S 0 D
  • Fe-S0D is inappropriate because it is derived from bacteria and plants.
  • CuZn-SOD copper-zinc-one SOD
  • Mn-SOD which has a longer half-life in the body, when it is applied to pharmaceuticals, cosmetics, etc. .
  • 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.
  • 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.
  • the isoelectric point of this peptide is 7.4.
  • 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.
  • Mn—S 0 D is a polymer, it cannot be said that tissue permeability is good.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • serine residue 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.
  • 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.
  • hMn-S0D human mutated manganese superoxide dismutase
  • 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.
  • 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.
  • 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.
  • Ser serine residue
  • Arg arginine residue
  • 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,
  • 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.
  • 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
  • a neutral amino acid residue such as palin, leucine, isoleucine, glycine, or alanine residue or a basic amino acid residue
  • a basic amino acid residue such as lysine, arginine, or histidine residue
  • 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.
  • 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.
  • the site to be replaced with another amino acid in the peptide sequence of hMn-SOD is 1 Pcs to 4 pcs preferred.
  • 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.
  • another amino acid residue such as arginine residue.
  • basic amino acid residues such as lysine, histidine, glutamine, and asparagine, or Examples include amino acid residues such as syn, isoleucine, alanine, and glycine.
  • 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.
  • 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.
  • 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.
  • the acidic amino acid for example, glutamic acid
  • a basic amino acid such as glutamine
  • 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. .
  • 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.
  • N-terminal of Mn-SOD N-terminal of Mn-SOD
  • 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'
  • examples of the primer at the C-terminal side of Mn—S0D include the following.
  • HMS-6 3 'GCTAGC AATACGACGT CTACMTTC 5'
  • the serine residue of the Met Lys His Ser Leu Pro Asp Leu... sequence includes all of the mutant sequences of the N-terminal bimer which change other basic amino acid sequences into gene sequences to be instructed.
  • 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.
  • the corresponding gene sequence that directs the native N-terminal Mn—S 0 D peptide sequence is as follows.
  • N-terminal variant Mn-S0D peptide sequence portion corresponding to the above-mentioned native terminal Mn-S0D peptide partial sequence is as follows.
  • the gene sequence that directs the N-terminal mutant M n-SOD peptide sequence corresponding to the above is as follows.
  • N-terminal side Mn-S0D peptide sequence corresponding to the above-mentioned native terminal Mn-S0D peptide partial sequence is as follows.
  • 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.
  • Partial gene sequence 7 -AAG CAC AAG CTC CCC GAC...
  • the gene sequence that directs the N-terminal mutant M n-SOD peptide sequence corresponding to the above is as follows.
  • Partial gene sequence 8 ⁇ MG CAC CM CTC CCC GAC...
  • Partial gene sequence 9 —AAG CAC CAG CTC CCC GAC
  • 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.
  • the corresponding gene sequence that directs the native N-terminal Mn—S 0 D peptide sequence is as follows.
  • 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.
  • the gene sequence that directs the N-terminal mutant M n-SOD peptide sequence corresponding to the above is as follows.
  • 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. .
  • 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.
  • 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,
  • 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.
  • 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.
  • FIG. 1 is a diagram showing an expression vector.
  • Figure 2 is a graph showing the persistence of MHS2: Mn-SOD in rats.
  • ⁇ indicates MHS: ⁇ -SOD
  • Hata indicates recombinant human Mn-SOD
  • mouth indicates CuZn-SOD.
  • FIG. 3 shows the isoelectric focusing of MHS2: Mn-SOD (ampholyte, pH
  • FIG. 4 is a graph showing the anti-inflammatory effect (rat) of MHS: Mn—SOD on adjuvant arthritis.
  • ⁇ indicates MHS: Mn—SOD
  • 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.
  • (a) shows recombinant human Mn-SOD
  • (b) shows MHS: Mn-SOD.
  • FIG. 6 is a graph showing the myocardial protective action in a cardiac ischemia reperfusion model.
  • (a) shows control
  • (b) shows recombinant human Mn-SOD
  • (c) shows MHS: Mn-SOD.
  • 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
  • C-terminal primer HMS-6 was chemically synthesized, and both HMS-5 and HMS-6 primers were synthesized.
  • HMS-5 primer (Sequence Table 1) was chemically synthesized as an N-terminal primer
  • C-terminal primer HMS-6 was chemically synthesized, and both HMS-5 and HMS-6 primers were synthesized.
  • HMS-5 primer (Sequence Table 1) was chemically synthesized as an N-terminal primer
  • C-terminal primer HMS-6 (Sequence Table 2) was chemically synthesized, and both HMS-5 and HMS-6 primers were synthesized.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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
  • 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.
  • MH S Mn—SOD showed a pI of about 8.2, which was clearly different from the native Mn—SOD (FIG. 2).
  • MHS Mn-SOD
  • Mn-S0D 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. .
  • 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.
  • 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).
  • 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.
  • Example 9 Plasma creatinine kinase release inhibitory action
  • MHS Mn-SOD
  • Mn-SOD like recombinant human Mn-S0D
  • Mn-SOD 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.
  • 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
  • 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.
  • 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.
  • 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.
  • Mn-SOD 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.
  • the recombinant ⁇ 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.
  • 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
  • Gly Asp Val Thr Ala Gin lie Ala Leu Gin Pro Ala Leu Lys Phe Asn Gly Gly 220 230 240 250 260 270
  • Lys Ala lie Tr Asn Val lie Asn Trp Glu Asn Val Thr Glu Arg Tyr Met Ala
  • 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.
  • MH S Mn-SOD showed a pI of about 8.5 to 9.4, which was clearly different from that of native Mn-SOD.
  • 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.
  • Xan resistance was no different from M n -SOD.
  • Mn—S ⁇ D 10 mg / kg
  • blood was sampled from the carotid artery over time and the persistence was measured. Minutes, 76 minutes and 88 minutes.
  • 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.
  • 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.

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Abstract

L'invention porte sur une dismutase de superoxyde de manganèse (Mn-SOD) humaine présentant un point isoélectrique élevé et une capacité accrue de pénétration dans les tissus, que l'on prépare en remplaçant un résidu d'acide aminé présent dans le site et n'affectant pas l'activité enzymatique, même lorsqu'un autre résidu d'acide aminé est également présent dans la séquence d'acides aminés de la dismutase, par un résidu d'acide aminé à charge positive supérieure. Dans un mode de réalisation, on remplace le 4ème résidu de sérine du côté N-terminal d'une Mn-SOD humaine de type variant par un résidu d'acide aminé de base. Dans un autre mode de réalisation, on remplace, outre le 4ème résidu d'acide aminé, le 42ème résidu d'acide glutamique par un résidu de valine. Ces Mn-SOD présentent une excellente activité médicamenteuse sur diverses maladies telles que diverses affections inflammatoires dues à l'oxygène actif, le cancer, la rétinopathie de prématurité, l'hypertension et les diabètes sur lesquelles la Mn-SOD classique n'est pas suffisamment efficace. Ces Mn-SOD sont également utiles en tant qu'ingrédients cosmétiques.
PCT/JP1993/001917 1992-12-28 1993-12-28 Dismutase de superoxyde de manganese humaine de type variant WO1994014950A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU57170/94A AU5717094A (en) 1992-12-28 1993-12-28 Human variant manganese superoxide dismutase

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP35995992 1992-12-28
JP4/359959 1992-12-28

Publications (1)

Publication Number Publication Date
WO1994014950A1 true WO1994014950A1 (fr) 1994-07-07

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Family Applications (1)

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PCT/JP1993/001917 WO1994014950A1 (fr) 1992-12-28 1993-12-28 Dismutase de superoxyde de manganese humaine de type variant

Country Status (2)

Country Link
AU (1) AU5717094A (fr)
WO (1) WO1994014950A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006059A3 (fr) * 1997-07-30 1999-08-05 Univ Texas Procedes et compositions relatifs a la cytotoxicite induite par l'oxyde nitrique
US6171856B1 (en) 1997-07-30 2001-01-09 Board Of Regents, The University Of Texas System Methods and compositions relating to no-mediated cytotoxicity
WO2002030453A1 (fr) * 2000-10-12 2002-04-18 Beth Israel Deaconess Medical Center, Inc. Procedes d'inhibition de l'angiogenese utilisant des inhibiteurs de la nadph-oxydase
US8569374B2 (en) 2004-09-16 2013-10-29 The Trustees Of The University Of Pennsylvania NADPH oxidase inhibition pharmacotherapies for obstructive sleep apnea syndrome and its associated morbidities

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6427470A (en) * 1987-03-27 1989-01-30 Bio Technology General Corp Human manganese superoxide dismutase dna, its development, recovery of human maganese superoxide dismutase, human manganese superoxide dismutase analogue or human manganese superoxide dismutase mutant, use and composition thereof and treatment method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6427470A (en) * 1987-03-27 1989-01-30 Bio Technology General Corp Human manganese superoxide dismutase dna, its development, recovery of human maganese superoxide dismutase, human manganese superoxide dismutase analogue or human manganese superoxide dismutase mutant, use and composition thereof and treatment method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J MOL CELL CARDIOL, Vol. 23, (1991), BASSAM A OMAR et al., "Intrestitial Equilibration of Superoxide Dismutase Correlates with its Protective Effect in the Isolated Rabbit Heart", p. 149-159. *
JAPAN BIOCHEMISTRY SOCIETY, "Protein I-Separation.Purification.Quality-", 26 February 1990, TOKYO KAGAKU DOJIN, p. 336-338. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006059A3 (fr) * 1997-07-30 1999-08-05 Univ Texas Procedes et compositions relatifs a la cytotoxicite induite par l'oxyde nitrique
US6171856B1 (en) 1997-07-30 2001-01-09 Board Of Regents, The University Of Texas System Methods and compositions relating to no-mediated cytotoxicity
WO2002030453A1 (fr) * 2000-10-12 2002-04-18 Beth Israel Deaconess Medical Center, Inc. Procedes d'inhibition de l'angiogenese utilisant des inhibiteurs de la nadph-oxydase
US8569374B2 (en) 2004-09-16 2013-10-29 The Trustees Of The University Of Pennsylvania NADPH oxidase inhibition pharmacotherapies for obstructive sleep apnea syndrome and its associated morbidities

Also Published As

Publication number Publication date
AU5717094A (en) 1994-07-19

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