Polypeptides having phospholipase a2 activity

Abstract

The present invention relates to a novel phospholipase A 2 polypeptide, DNA encoding the polypeptide, a vector comprising the DNA, a transformant transformed with the vector, and a process for producing the phospholipase A 2 polypeptide. The present invention also relates to a method of utilizing the polypeptide, e.g., a method of screening for a compound having agonist or antagonist activity by using the polypeptide or an antibody to the polypeptide, and a pharmaceutical comprising the polypeptide or an antibody to the polypeptide. The present invention further relates to a polypeptide inhibiting the phospholipase A 2 activity of a phospholipase A 2 polypeptide (hereinafter referred to as inhibitor polypeptide), DNA encoding the inhibitor polypeptide, a vector comprising the DNA encoding the inhibitor polypeptide, a transformant transformed with the vector, a pharmaceutical comprising the inhibitor polypeptide, and a process for producing the inhibitor polypeptide.

1 - 3 . (canceled)  
     
     
         4 . An isolated DNA encoding a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity.  
     
     
         5 . An isolated DNA having a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 2, 23, 27 and 39.  
     
     
         6 . A DNA which hybridizes to a DNA consisting of a nucleotide sequence complementary to a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 2, 23, 27 and 39 at 65° C. in the presence of 0.7 to 1.0 mmol/l NaCl followed by washing with 0.1 to 2-fold SSC and which encodes a polypeptide having phospholipase A 2  activity.  
     
     
         7 . A recombinant vector comprising the DNA according to any of  claims 4  to  6 .  
     
     
         8 . A transformant carrying the recombinant vector according to  claim 7 .  
     
     
         9 . The transformant according to  claim 8 , wherein the transformant is selected from the group consisting of a microorganism, an animal cell, a plant cell and an insect cell.  
     
     
         10 . The transformant according to  claim 9 , wherein the microorganism belongs to the genus  Escherichia.    
     
     
         11 . The transformant according to  claim 9 , wherein the microorganism is  Escherichia coli  JM109/p5269+C5 (FERM BP-7281).  
     
     
         12 . A process for producing a polypeptide having phospholipase A 2  activity, which comprises culturing the transformant according to  claim 10  in a medium, allowing the polypeptide to form and accumulate in the culture, and recovering the polypeptide from the culture.  
     
     
         13 . An oligonucleotide selected from the group consisting of a sense oligonucleotide having a nucleotide sequence identical with a sequence of 5 to 60 consecutive nucleotides in the nucleotide sequence of the DNA according to any of  claims 4  to  6 , an antisense oligonucleotide having a nucleotide sequence complementary to that of said sense oligonucleotide, and a derivative of said sense oligonucleotide or antisense oligonucleotide.  
     
     
         14 . An oligonucleotide consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 13, 14, 28, 29, 30, 31, 46 and 47.  
     
     
         15 . The oligonucleotide according to  claim 13 , wherein the oligonucleotide derivative is selected from the group consisting of an oligonucleotide derivative wherein the phosphodiester bond in an oligonucleotide is converted to a phosophorothioate bond, an oligonucleotide derivative wherein the phosphodiester bond in an oligonucleotide is converted to an N3′-P5′ phosphoamidate bond, an oligonucleotide derivative wherein the ribose-phosphodiester bond in an oligonucleotide is converted to a peptide-nucleic acid bond, an oligonucleotide derivative wherein the uracil in an oligonucleotide is substituted by C-5 propynyluracil, an oligonucleotide derivative wherein the uracil in an oligonucleotide is substituted by C-5 thiazolyluracil, an oligonucleotide derivative wherein the cytosine in an oligonucleotide is substituted by C-5 propynylcytosine, an oligonucleotide derivative wherein the cytosine in an oligonucleotide is substituted by phenoxazine-modified cytosine, an oligonucleotide derivative wherein the ribose in DNA is substituted by 2′-O-propylribose, and an oligonucleotide derivative wherein the ribose in an oligonucleotide is substituted by 2′-methoxyethoxyribose.  
     
     
         16 . A method for detecting an mRNA encoding a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, which comprises using the oligonucleotide according to  claim 13 .  
     
     
         17 . A method for inhibiting the expression of a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, which comprises using the oligonucleotide according to  claim 13 .  
     
     
         18 . An antibody recognizing a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity.  
     
     
         19 . A method for immunological detection of a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, which comprises using the antibody according to  claim 18 .  
     
     
         20 . A method for immunohistochemical staining of a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, which comprises using the antibody according to  claim 18 .  
     
     
         21 . An immunohistochemical staining agent comprising the antibody according to  claim 18 .  
     
     
         22 . A method for screening for a compound varying the phospholipase A 2  activity of a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, which comprises contacting said polypeptide with a test sample, and measuring the phospholipase A 2  activity of said polypeptide.  
     
     
         23 . A method for screening for a compound varying the expression level of a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, which comprises contacting cells expressing said polypeptide with a test sample, and detecting the expression level of said polypeptide.  
     
     
         24 . The method according to  claim 23 , wherein said detection of the expression level of said polypeptide is detection of an mRNA encoding a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity using an oligonucleotide selected from the group consisting of a sense oligonucleotide having a nucleotide sequence identical with a sequence of 5 to 60 consecutive nucleotides in the nucleotide sequence of the DNA according to any of  claims 4  to  6 , an antisense oligonucleotide having a nucleotide sequence complementary to that of said sense oligonucleotide, and a derivative of said sense oligonucleotide or antisense oligonucleotide.  
     
     
         25 . The method according to  claim 23 , wherein said detection of the expression level of said polypeptide utilizes an antibody recognizing said polypeptide.  
     
     
         26 . The method according to  claim 22 , wherein said variation of the phospholipase A 2  activity of a polypeptide is an increase in the phospholipase A 2  activity of said polypeptide.  
     
     
         27 . The method according to  claim 22 , wherein said variation of the phospholipase A 2  activity of the polypeptide is a decrease in the phospholipase A 2  activity of said polypeptide.  
     
     
         28 . The method according to any of  claims 23  to  25 , wherein said variation of the expression of the polypeptide is an increase in the expression level of said polypeptide.  
     
     
         29 . The method according to any of  claims 23  to  25 , wherein said variation of the expression of the polypeptide is a decrease in the expression level of said polypeptide.  
     
     
         30 . A compound which is identified by the method according to any of  claims 22  to  27 .  
     
     
         31 . A promoter DNA regulating the transcription of a DNA encoding a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity.  
     
     
         32 . A method for screening for a compound varying the efficiency of transcription of a DNA encoding a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, which comprises contacting a transformant carrying a plasmid containing the promoter DNA according to  claim 31  and a reporter gene ligated downstream of said promoter DNA with a test sample, and measuring the content of the translation product of said reporter gene.  
     
     
         33 . The method according to  claim 32 , wherein the reporter gene is a gene selected from the group consisting of a chloramphenicol acetyltransferase gene, a β-galactosidase gene, a luciferase gene, a β-glucuronidase gene, an aequorin gene and a green fluorescent protein gene.  
     
     
         34 . The method according to  claim 32  or  33 , wherein said variation of the efficiency of transcription of a DNA encoding the polypeptide is an increase in the efficiency of transcription of said DNA.  
     
     
         35 . The method according to  claim 32  or  33 , wherein said variation of the efficiency of transcription of a DNA encoding the polypeptide is a decrease in the efficiency of transcription of said DNA.  
     
     
         36 . A compound which is identified by the method according to claims  32  or  33 .  
     
     
         37 . (canceled)  
     
     
         38 . An isolated polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.  
     
     
         39 . An isolated polypeptide consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 3 and having the activity of inhibiting phospholipase A 2  activity.  
     
     
         40 . An isolated polypeptide consisting of an amino acid sequence which has 60% or more homology to SEQ ID NO: 3 and having the activity of inhibiting phospholipase A 2  activity.  
     
     
         41 . An isolated DNA encoding the polypeptide according to any of  claims 38  to  40 .  
     
     
         42 . An isolated DNA comprising the nucleotide sequence of SEQ ID NO: 4.  
     
     
         43 . An isolated DNA which hybridizes to a DNA consisting of a nucleotide sequence complementary to SEQ ID NO: 4 at 65° C. in the presence of 0.7 to 1.0 mol/l NaCl followed by washing with 0.1 to 2-fold SSC and which encodes a polypeptide having the activity of inhibiting phospholipase A 2  activity.  
     
     
         44 . A recombinant vector comprising the DNA according to  claim 41 .  
     
     
         45 . An isolated transformant carrying the recombinant vector according to  claim 44 .  
     
     
         46 . The transformant according to  claim 45 , wherein the transformant is selected from the group consisting of a microorganism, an animal cell, a plant cell and an insect cell.  
     
     
         47 . A process for producing a polypeptide having the activity of inhibiting phospholipase A 2  activity, which comprises culturing the transformant according to  claim 46  in a medium, allowing the polypeptide having the activity of inhibiting phospholipase A 2  activity to form and accumulate in the culture, and recovering the polypeptide from the culture.  
     
     
         48 . A pharmaceutical for the diagnosis, prevention or treatment of a disease in which a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity is concerned, which comprises, as an active ingredient, a compound varying the phospholipase A 2  activity of said polypeptide, wherein said disease is asthma, ischemic diseases, arthritis, rheumatism, sepsis, dermatitis, arteriosclerosis, pain, Parkinson disease, Alzheimer disease, malignant tumor, nephritis, diabetes or ischemic reperfusion injury.  
     
     
         49 . (canceled)  
     
     
         50 . A pharmaceutical for the diagnosis, prevention or treatment of a disease in which a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity is concerned, which comprises, as an active ingredient, the DNA according to any of  claims 4  to  6 , wherein said disease is asthma, ischemic diseases, arthritis, rheumatism, sepsis, dermatitis, arteriosclerosis, pain, Parkinson disease, Alzheimer disease, malignant tumor, nephritis, diabetes or ischemic reperfusion injury.  
     
     
         51 . (canceled)  
     
     
         52 . A pharmaceutical for the diagnosis, prevention or treatment of a disease in which a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity is concerned, which comprises, as an active ingredient, the DNA according to  claim 41 , wherein said disease is asthma, ischemic diseases, arthritis, rheumatism, sepsis, dermatitis, arteriosclerosis, pain, Parkinson disease, Alzheimer disease, malignant tumor, nephritis, diabetes or ischemic reperfusion injury.  
     
     
         53 . A pharmaceutical for the diagnosis, prevention or treatment of a disease in which a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity is concerned, which comprises, as an active ingredient, the oligonucleotide according to  claim 13 , wherein said disease is asthma, ischemic diseases, arthritis, rheumatism, sepsis, dermatitis, arteriosclerosis, pain, Parkinson disease, Alzheimer disease, malignant tumor, nephritis, diabetes or ischemic reperfusion injury.  
     
     
         54 . A pharmaceutical for the diagnosis, prevention or treatment of a disease in which a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity is concerned, which comprises, as an active ingredient, the antibody according to  claim 18 , wherein said disease is asthma, ischemic diseases, arthritis, rheumatism, sepsis, dermatitis, arteriosclerosis, pain, Parkinson disease, Alzheimer disease, malignant tumor, nephritis, diabetes or ischemic reperfusion injury.  
     
     
         55 . A pharmaceutical for the diagnosis, prevention or treatment of a disease in which a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity is concerned, which comprises, as an active ingredient, the compound according to  claim 30 , wherein said disease is asthma, ischemic diseases, arthritis, rheumatism, sepsis, dermatitis, arteriosclerosis, pain, Parkinson disease, Alzheimer disease, malignant tumor, nephritis, diabetes or ischemic reperfusion injury.  
     
     
         56 . (canceled)  
     
     
         57 . A pharmaceutical for the diagnosis, prevention or treatment of diabetes, which comprises, as an active ingredient, a compound obtainable by the method according to  claim 28 .  
     
     
         58 . A pharmaceutical for the diagnosis, prevention or treatment of a disease in which a polypeptide (i) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38, (ii) consisting of an amino acid sequence wherein one to twenty amino acid residues are deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity, or (iii) consisting of an amino acid sequence which has 60% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 26 and 38 and having phospholipase A 2  activity is concerned, which comprises, as an active ingredient, the compound according to  claim 36 , wherein said disease is asthma, ischemic diseases, arthritis, rheumatism, sepsis, dermatitis, arteriosclerosis, pain, Parkinson disease, Alzheimer disease, malignant tumor, nephritis, diabetes or ischemic reperfusion injury.  
     
     
         59 . A pharmaceutical for the diagnosis, prevention or treatment of diabetes, which comprises, as an active ingredient, a compound obtainable by the method according to  claim 34.