Methods and materials relating to novel CD39-like polypeptides

Abstract

The invention provides novel polynucleotides isolated from cDNA libraries of human fetal liver-spleen and macrophage as well as polypeptides encoded by these polynucleotides and mutants or variants thereof. The polypeptides correspond to a novel human CD39-like protein. Other aspects of the invention include vectors containing polynucleotides of the invention, and related host cells as well a processes for producing novel CD39-like polypeptides, and antibodies specific for such polypeptides.

What is claimed is:  
     
         1 . An isolated polynucleotide encoding an apyrase and/or NDPase and comprising a nucleotide sequence having at least about 80% sequence identity to a human polynucleotide selected from the group consisting of: 
 (a) a polynucleotide having the nucleotide sequence of SEQ ID NO. 2; and    (b) a polynucleotide having the protein coding nucleotide sequence of the polynucleotide sequence of (a).    
     
     
         2 . An isolated polynucleotide encoding an apyrase and comprising a nucleotide sequence having at least about 90% sequence identity to a polynucleotide selected from the group consisting of: 
 (a) a polynucleotide having the nucleotide sequence of SEQ ID NO. 2; and    (b) a polynucleotide having the protein coding nucleotide sequence of the polynucleotide sequence of (a).    
     
     
         3 . An isolated polynucleotide encoding a polypeptide with apyrase and/or NDPase activity, comprising a polynucleotide selected from the group consisting of: 
 (a) polynucleotides that encode the mature protein coding amino acid sequence of SEQ ID NO. 3.    
     
     
         4 . An isolated polynucleotide encoding a polypeptide with apyrase and/or NDPase activity that hybridizes under stringent conditions to the complement of a polynucleotide of SEQ ID NO. 2.  
     
     
         5 . The polynucleotide of any one of claims  1  through  4  which is a DNA.  
     
     
         6 . The DNA of  claim 5  which is a wholly or partially chemically synthesized DNA molecule.  
     
     
         7 . An anti-sense polynucleotide which specifically hybridizes with the complement of the polynucleotide of  claim 4 .  
     
     
         8 . The polynucleotide of  claim 1  which comprises the nucleotide sequence of SEQ ID NO. 2 or 24 or the mature protein coding portions thereof.  
     
     
         9 . An isolated polynucleotide which comprises a complement of the polynucleotide of  claim 1 .  
     
     
         10 . An expression vector comprising the DNA of  claim 5 .  
     
     
         11 . A host cell comprising the DNA of  claim 5 .  
     
     
         12 . A host cell genetically engineered to express the DNA of  claim 5 .  
     
     
         13 . An isolated human polypeptide with apyrase and/or NDPase activity comprising: 
 (a) the mature protein coding sequence of SEQ ID NO. 3; or    (b) an amino acid sequence having at least about 80% sequence identity to SEQ ID NO. 3.    
     
     
         14 . An isolated polypeptide with apyrase and/or NDPase activity comprising: 
 (a) the CD39-like protein coding sequence of SEQ ID NO. 3; or    (b) an amino acid sequence having at least about 90% sequence identity to SEQ ID NO. 3.    
     
     
         15 . The polypeptide of  claim 13  or  14  which comprises the amino acid sequence of SEQ ID NO. 3 or 25 or the mature protein portions thereof.  
     
     
         16 . The polypeptide of  claim 13  or  14  wherein the polypeptide comprises at least one amino acid substitution selected from the group consisting of: D168→T, S170→Q and L175→F.  
     
     
         17 . The polypeptide of  claim 16  comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO. 7.  
     
     
         18 . A method for producing a CD39-like polypeptide comprising the steps of: 
 (a) growing a culture of cells according to  claim 11  under conditions permitting expression of a CD39-like polypeptide; and    (b) isolating the CD39-like polypeptide from the host cell or its growth medium.    
     
     
         19 . A composition comprising the polypeptide of  claim 13 ,  14  or  15  and a pharmaceutically acceptable carrier.  
     
     
         20 . An antibody specifically immunoreactive with a polypeptide encoded by the polynucleotide according to  claim 1 .  
     
     
         21 . The antibody according to  claim 20  which is a monoclonal antibody.  
     
     
         22 . A hybridoma which secretes the antibody according to  claim 21 .  
     
     
         23 . An anti-idiotype antibody specifically immunoreactive with the antibody according to  claim 21 .  
     
     
         24 . A method for detecting a polynucleotide of  claim 1 ,  2  or  3  in a sample comprising the steps of: 
 (a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to detect the complex; and  
 (b) detecting the complex so that if a complex is detected, a polynucleotide of  claim 1 ,  2  or  3  is detected.  
 
     
     
         25 . A method for detecting a polynucleotide of  claim 1 ,  2  or  3  in a sample comprising the steps of: 
 (a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of  claim 1 ,  2  or  3  under such conditions; and  
 (b) amplifying the polynucleotides of  claim 1 ,  2  or  3  so that if a polynucleotide is amplified, a polynucleotide of  claim 1 ,  2  or  3  is detected.  
 
     
     
         26 . The method of  claim 25  wherein the polynucleotide is an RNA molecule that encodes a polypeptide of  claim 13  or  14 , and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.  
     
     
         27 . A method for detecting a polypeptide of  claim 13  or  14  in a sample comprising: 
 (a) contacting the sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to detect the complex; and  
 (b) detecting the complex so that if a complex is detected, a polypeptide of  claim 13  or  14  is detected.  
 
     
     
         28 . A method for identifying a compound that binds to a polypeptide of  claim 13  or  14  comprising: 
 (a) contacting a compound with a polypeptide of  claim 13  or  14  for a time sufficient to form a polypeptide/compound complex; and  
 (b) detecting the complex so that if a polypeptide/compound complex is detected, a compound that binds to a polypeptide of  claim 13  or  14  is detected.  
 
     
     
         29 . A method for identifying a compound that binds to a polypeptide of  claim 13  or  14  comprising: 
 (a) contacting a compound with a polypeptide of  claim 13  or  14 , in a cell, for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell, and  
 (b) detecting the complex by detecting reporter gene sequence expression so that if a polypeptide/compound complex is detected, a compound that binds to a polypeptide of  claim 13  or  14  is identified.  
 
     
     
         30 . A method of identifying a modulator compound of a CD39-like polypeptide with apyrase activity comprising the steps of: 
 (a) contacting the CD39-like polypeptide encoded by the polynucleotide of  claim 1 ,  2  or  3  with a substrate in the presence and absence of a test compound;    (b) comparing apyrase activity of the CD39-like polypeptide in the presence and absence of the test compound; and    (c) identifying the test compound as a modulator compound when biological activity of the CD39-like polypeptide is increased or decreased in the presence of the test compound.    
     
     
         31 . A method of identifying a modulator compound of a CD39-like polypeptide with NDPase activity comprising the steps of: 
 (a) contacting the CD39-like polypeptide encoded by the polynucleotide of  claim 1 ,  2  or  3  with a substrate in the presence and absence of a test compound;    (b) comparing NDPase activity of the CD39-like polypeptide in the presence and absence of the test compound; and    (c) identifying the test compound as a modulator compound when biological activity of the CD39-like polypeptide is increased or decreased in the presence of the test compound.    
     
     
         32 . A chimeric polypeptide comprising one or more domains of a CD39-like polypeptide fused to one or more domains of heterologous peptide or polypeptide, e.g., an immunoglobulin constant region.  
     
     
         33 . A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of  claim 13  or  14  and a pharmaceutically acceptable carrier.  
     
     
         34 . A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of  claim 13  or  14  and a pharmaceutically acceptable carrier.  
     
     
         35 . A method of inhibiting platelet function comprising administering the polypeptide of  claim 13  or  14  to a medium comprising platelets.  
     
     
         36 . A method of treating thrombotic diseases comprising administering a therapeutic amount of the polypeptide of  claim 13  or  14  to a mammalian subject in need thereof.  
     
     
         37 . A method of hydrolyzing nucleotidediphosphates comprising administering the polypeptide of  claim 13  or  14  to a medium comprising nucleotidediphospates.  
     
     
         38 . A method of inhibiting platelet aggregation in a mammalian subject comprising the step of reducing the ratio of ADP:ATP in said mammalian subject to a less than normal ratio.  
     
     
         39 . The method of  claim 38  wherein said ratio is reduced by administration of CD39-L4 having the sequence set forth in SEQ ID NO: 3 or a polypeptide having NDPase activity and at least about 90% sequence identity to SEQ ID NO: 3.  
     
     
         40 . The method of  claim 38  wherein said ratio is reduced by administration of CD39-L66 having the sequence set forth in SEQ ID NO: 25 or a polypeptide having NDPase activity and at least about 90% sequence identity to SEQ ID NO: 25.  
     
     
         41 . The method of  claim 38  wherein said ratio is reduced by administration of CD39-L2 having the sequence set forth in SEQ ID NO: 27 or a polypeptide having NDPase activity and at least about 90% sequence identity to SEQ ID NO: 27.  
     
     
         42 . The method of  claim 38  -  41  wherein the ratio of ADP:ATP is reduced systemically in circulation.  
     
     
         43 . The method of  claim 38  -  41  wherein the ratio of ADP:ATP is reduced locally within an area selected from the group consisting of heart, brain, kidney, lung and limbs.  
     
     
         44 . The method of claim  38 - 41  wherein the ratio of ADP:ATP is reduced without significantly affecting ATP levels.  
     
     
         45 . A method of identifying a compound capable of reducing the ratio of ADP:ATP to a less than normal ratio comprising the steps of: 
 (a) determining apyrase activity of said compound on ATP;    (b) determining apyrase activity of said compounds on ADP; and    (c) selecting a compound that has greater activity with respect to ADP compared to ATP.