Prokineticin polypeptides, related compositions and methods

Abstract

The invention provides isolated polypeptides that stimulate gastrointestinal smooth muscle contraction, including human prokineticin 1 and human prokineticin 2 polypeptides, and functional fragments and modifications thereof. Also provided are methods of stimulating gastrointestinal smooth muscle contraction in a mammal, by administering to the mammal an effective amount of a prokineticin polypeptide. The invention also provides nucleic acid molecules encoding a prokineticin polypeptide, and antibodies that selectively bind a prokineticin polypeptide. Further provided are methods of identifying a prokineticin receptor ligand, agonist or antagonist.

1 . An isolated polypeptide that stimulates gastrointestinal smooth muscle contraction, comprising an amino acid sequence at least 80% identical to the sequence of human prokineticin 1 (SEQ ID NO:3), said sequence comprising the N-terminal 6 amino acids of SEQ ID NO:3, the 10 conserved cysteine residues of SEQ ID NO:3, and from 0 to 9 of the 9 C-terminal amino acids of SEQ ID NO:3.  
     
     
         2 . The isolated polypeptide of  claim 1 , wherein amino acid residues that differ from residues in SEQ ID NO:3 are conservative substitutions thereof.  
     
     
         3 . The isolated polypeptide of  claim 1 , wherein amino acid residues that differ from residues in SEQ ID NO:3 consist of the corresponding residues from SEQ ID NO:6.  
     
     
         4 . The isolated polypeptide of  claim 3 , comprising SEQ ID NO: 13.  
     
     
         5 . The isolated polypeptide of  claim 1 , comprising amino acids 1-77 of SEQ ID NO:3.  
     
     
         6 . The isolated polypeptide of  claim 1 , comprising SEQ ID NO:3.  
     
     
         7 . The isolated polypeptide of  claim 1 , comprising a 6XHis tag.  
     
     
         8 . The isolated polypeptide of  claim 1 , which is detectably labeled.  
     
     
         9 . An isolated peptide comprising at least 10 contiguous amino acids of SEQ ID NO:3, wherein said peptide is immunogenic.  
     
     
         10 . A pharmaceutical composition, comprising the isolated polypeptide of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         11 . A method of stimulating gastrointestinal smooth muscle contraction in a mammal, comprising administering to said mammal an effective amount of the polypeptide of  claim 1 .  
     
     
         12 . A nucleic acid molecule encoding the polypeptide of  claim 1 .  
     
     
         13 . An expression vector containing the nucleic acid molecule of  claim 12  operatively linked to a promoter of gene expression.  
     
     
         14 . A host cell comprising the expression vector of  claim 13 .  
     
     
         15 . A method of preparing the isolated polypeptide of  claim 1 , comprising culturing the host cell of  claim 14  so as to express said polypeptide, substantially purifying said polypeptide, and refolding said polypeptide.  
     
     
         16 . An antibody that selectively binds the polypeptide of  claim 1 .  
     
     
         17 . An isolated polypeptide that stimulates gastrointestinal smooth muscle contraction, comprising an amino acid sequence at least 80% identical to the sequence of human prokineticin 2 (SEQ ID NO:6), said sequence comprising the N-terminal 6 amino acids of SEQ ID NO:6, the 10 conserved cysteine residues of SEQ ID NO:6, and from 0 to 4 of the 4 C-terminal amino acids of SEQ ID NO:6.  
     
     
         18 . The isolated polypeptide of  claim 17 , wherein amino acid residues that differ from residues in SEQ ID NO:6 are conservative substitutions thereof.  
     
     
         19 . The isolated polypeptide of  claim 17 , wherein amino acid residues that differ from residues in SEQ ID NO:6 consist of the corresponding residues from SEQ ID NO:3.  
     
     
         20 . The isolated polypeptide of  claim 19 , comprising SEQ ID NO: 14.  
     
     
         21 . The isolated polypeptide of  claim 17 , comprising amino acids 1-77 of SEQ ID NO:6.  
     
     
         22 . The isolated polypeptide of  claim 17 , comprising SEQ ID NO:6.  
     
     
         23 . The isolated polypeptide of  claim 17 , comprising a 6XHis tag.  
     
     
         24 . The isolated polypeptide of  claim 17 , which is detectably labeled.  
     
     
         25 . An isolated peptide comprising at least 10 contiguous amino acids of SEQ ID NO:6, wherein said peptide is immunogenic.  
     
     
         26 . A pharmaceutical composition, comprising the isolated polypeptide of  claim 17  and a pharmaceutically acceptable carrier.  
     
     
         27 . A method of stimulating gastrointestinal smooth muscle contraction in a mammal, comprising administering to said mammal an effective amount of the polypeptide of  claim 17 .  
     
     
         28 . A nucleic acid molecule encoding the polypeptide of  claim 17 .  
     
     
         29 . An expression vector containing the nucleic acid molecule of  claim 17  operatively linked to a promoter of gene expression.  
     
     
         30 . A host cell comprising the expression vector of  claim 29 .  
     
     
         31 . A method of preparing the isolated polypeptide of  claim 17 , comprising culturing the host cell of  claim 30  so as to express said polypeptide, substantially purifying said polypeptide, and refolding said polypeptide.  
     
     
         32 . An antibody that selectively binds the polypeptide of  claim 17 .  
     
     
         33 . A method of identifying a prokineticin receptor ligand, comprising contacting a preparation comprising prokineticin receptor with one or more candidate compounds, and identifying a compound that specifically binds to said receptor, said compound being characterized as a prokineticin receptor ligand.  
     
     
         34 . The method of  claim 33 , wherein said preparation is an intestinal smooth muscle preparation or membrane preparation thereof.  
     
     
         35 . The method of  claim 33 , wherein said preparation is a cell line or membrane preparation thereof.  
     
     
         36 . The method of  claim 35 , wherein said cell line is M2A7 (ATCC CRL-2500).  
     
     
         37 . The method of  claim 33 , wherein the ability of said ligand to selectively agonize or antagonize prokineticin receptor signaling is further determined.  
     
     
         38 . The method of  claim 37 , wherein said signaling is determined in a cell line.  
     
     
         39 . The method of  claim 38 , wherein said cell line is M2A7 (ATCC CRL-2500).  
     
     
         40 . The method of  claim 37 , wherein said signaling is determined by monitoring calcium mobilization.  
     
     
         41 . The method of  claim 33 , wherein the ability of said ligand to modulate smooth muscle contractility is further determined.  
     
     
         42 . A method of identifying a prokineticin receptor agonist, comprising contacting a preparation comprising a prokineticin receptor with one or more candidate compounds, and identifying a compound that selectively promotes production of a prokineticin receptor signal, said compound being characterized as a prokineticin receptor agonist.  
     
     
         43 . The method of  claim 42 , wherein said preparation is a cell line.  
     
     
         44 . The method of  claim 43 , wherein said cell line is M2A7 (ATCC CRL-2500).  
     
     
         45 . The method of  claim 42 , wherein said signaling is determined by monitoring calcium mobilization.  
     
     
         46 . The method of  claim 42 , wherein the ability of said agonist to modulate smooth muscle contractility is further determined.  
     
     
         47 . A method of identifying a prokineticin receptor antagonist, comprising contacting a preparation comprising a prokineticin receptor with one or more candidate compounds in the presence of a prokineticin, and identifying a compound that selectively inhibits production of a prokineticin receptor signal, said compound being characterized as a prokineticin receptor antagonist.  
     
     
         48 . The method of  claim 47 , wherein said prokineticin comprises an amino acid sequence selected from the group consisting of amino acids 1-77 of SEQ ID NOS:3 and amino acids 1-77 of SEQ ID NO:6.  
     
     
         49 . The method of  claim 47 , wherein said preparation is a cell line.  
     
     
         50 . The method of  claim 49 , wherein said cell line is M2A7 (ATCC CRL-2500).  
     
     
         51 . The method of  claim 47 , wherein said signaling is determined by monitoring calcium mobilization.  
     
     
         52 . The method of  claim 47 , wherein the ability of said antagonist to modulate smooth muscle contractility is further determined.  
     
     
         53 . The isolated polypeptide of  claim 17 , comprising SEQ ID NO:5.  
     
     
         54 . The isolated polypeptide of  claim 17 , consisting of SEQ ID NO:5.  
     
     
         55 . A composition, comprising the isolated polypeptide of  claim 17  and a carrier, wherein the polypeptide comprises amino acids 23-108 of SEQ ID NO:5.  
     
     
         56 . The composition of  claim 55 , wherein the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.  
     
     
         57 . A composition, comprising the isolated polypeptide of  claim 17  and a carrier, wherein the polypeptide comprises SEQ ID NO:5.  
     
     
         58 . The composition of  claim 57 , wherein the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.  
     
     
         59 . The isolated polypeptide of  claim 17 , comprising amino acids 23-108 of SEQ ID NO:5.  
     
     
         60 . The isolated polypeptide of  claim 17 , consisting of amino acids 23-108 of SEQ ID NO:5.  
     
     
         61 . The isolated polypeptide of  claim 17 , comprising amino acids 23-108 of SEQ ID NO:5 and an affinity tag.  
     
     
         62 . The isolated polypeptide of  claim 17 , comprising amino acids 23-108 of SEQ ID NO:5 and an affinity tag selected from the group consisting of polyhistidine, protein A, glutathione S transferase, Glu-Gln affinity tag, substance P, Flag peptide, streptavidin binding peptide and maltose-binding protein.  
     
     
         63 . A composition comprising a carrier and an isolated polypeptide of  claim 22 .  
     
     
         64 . The composition of  claim 63 , wherein the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.  
     
     
         65 . An isolated polypeptide, comprising amino acid residues 23 to 108 of SEQ ID NO:5.  
     
     
         66 . The isolated polypeptide of  claim 65 , comprising the amino acid sequence of SEQ ID NO:5.  
     
     
         67 . The isolated polypeptide of  claim 65 , wherein the polypeptide has an amino acid sequence consisting of the amino acid sequence of SEQ ID NO:5.  
     
     
         68 . The isolated polypeptide of  claim 65 , and an affinity tag.  
     
     
         69 . The isolated polypeptide of  claim 68 , wherein the affinity tag is selected from the group consisting of: polyhistidine tract, protein A, glutathione S transferase, Glu-Glu affinity tag, substance P, Flag peptide, streptavidin binding peptide, and maltose-binding protein.  
     
     
         70 . The composition, comprising a carrier and a polypeptide that comprises amino acid residues 23 to 108 of SEQ ID NO:5.  
     
     
         71 . The composition of  claim 70 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:5.  
     
     
         72 . An isolated polypeptide comprising amino acid residues 1 to 108 of SEQ ID NO:5.  
     
     
         73 . The isolated polypeptide of  claim 72 , and an affinity tag.  
     
     
         74 . The isolated polypeptide of  claim 73 , wherein the affinity tag is selected from the group consisting of: poly-histidine tract, protein A, glutathione S transferase, Glu-Glu affinity tag, substance P, Flag peptides, streptavidin binding peptide, and maltose-binding protein.  
     
     
         75 . An isolated polypeptide consisting of amino acid residues 28 to 108 of SEQ ID NO:5.  
     
     
         76 . A composition, comprising a carrier and a polypeptide that comprises amino acid residues 28 to 108 of SEQ ID NO:5.  
     
     
         77 . An isolated polypeptide comprising an amino acid sequence that is at least 70%, identical to amino acid residues 23 to 108 of SEQ ID NO:5, wherein the isolated polypeptide can specifically bind with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:5.  
     
     
         78 . The isolated polypeptide of  claim 72 , wherein the polypeptide comprises an amino acid sequence that is at least 80%, identical to amino acid residues 23 to 108 of SEQ ID NO:5.  
     
     
         79 . The isolated polypeptide of  claim 77 , wherein the polypeptide comprises an amino acid sequence that is at least 90%, identical to amino acid residues 23 to 108 of SEQ ID NO:5.  
     
     
         80 . The isolated polypeptide of  claim 77 , wherein the polypeptide comprises the amino acid sequence of amino acid residues 23 to 108 of SEQ ID NO:5.  
     
     
         81 . An isolated nucleic acid molecule selected from the group consisting of (a) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:5, or (b) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule consisting of the nucleotide sequence of nucleotides 66 to 161 of SEQ ID NO:4, the nucleotide sequence of nucleotides 288 to 389 of SEQ ID NO:4, or to the complement of the nucleotide sequence of either nucleotides 66 to 161 of SEQ ID NO:4 or nucleotides 288 to 389 of SEQ ID NO:4.  
     
     
         82 . The isolated nucleic acid molecule of  claim 81 , wherein any difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of SEQ ID NO:5 is due to a conservative amino acid substitution.  
     
     
         83 . The isolated nucleic acid molecule of  claim 81 , wherein the nucleic acid molecule comprises the nucleotide sequence of nucleotides 132 to 389 of SEQ ID NO:4.  
     
     
         84 . A vector, comprising the isolated nucleic acid molecule of  claim 83 .  
     
     
         85 . An expression vector, comprising the isolated nucleic acid molecule of  claim 78 , a transcription promoter, and a transcription terminator, wherein the promoter is operably linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator.  
     
     
         86 . A recombinant host cell comprising the expression vector of  claim 80 , wherein the host cell is selected from the group consisting of bacterium, yeast cell, fungal cell, avian cell, insect cell, mammalian cell, and plant cell.  
     
     
         87 . A method of producing Prokineticin protein, the method comprising the step of culturing recombinant host cells that comprise the expression vector of  claim 80 , and that produce the Prokineticin protein.  
     
     
         88 . The method of  claim 87 , further comprising the step of isolating the Prokineticin protein from the cultured recombinant host cells.  
     
     
         89 . An antibody or antibody fragment that specifically binds with the polypeptide of  claim 80 .  
     
     
         90 . A method of detecting the presence of Prokineticin 2 RNA in a biological sample, comprising the steps of: (a) contacting a Prokineticin 2 nucleic acid probe under hybridizing conditions with either (i) test RNA molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of the nucleic acid molecule of  claim 83 , or the complement of the nucleotide sequence of the nucleic acid molecule of  claim 83 , and (b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicates the presence of Prokineticin 2 RNA in the biological sample.  
     
     
         91 . A method of detecting the presence of Prokineticin 2 in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody, or an antibody fragment, of  claim 89 , wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.  
     
     
         92 . An anti-idiotype antibody, or anti-idiotype antibody fragment, that specifically binds with the antibody or antibody fragment of  claim 89 .  
     
     
         93 . A composition, comprising a carrier and at least one of the expression vector of  claim 85  or a recombinant virus that comprises the expression vector of  claim 80 .  
     
     
         94 . A fusion protein, comprising the polypeptide of  claim 80 .  
     
     
         95 . The fusion protein of  claim 94 , further comprising an immunoglobulin moiety.  
     
     
         96 . An isolated polypeptide, comprising an amino acid sequence that shares an identity with the amino acid sequence of SEQ ID NO:5 selected from the group consisting of at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or greater than 95% identity, and wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NO:5 is due to one or more conservative amino acid substitutions.  
     
     
         97 . An isolated polypeptide, comprising an amino acid sequence selected from the group consisting of: amino acid residues 82 to 108 of SEQ ID NO:5, amino acid residues 28 to 79 of SEQ ID NO:5, amino acid residues 28 to 72 of SEQ ID NO:5, amino acid residues 75 to 78 (amide) of SEQ ID NO:5, and amino acid residues 28 to 79 (amide) of SEQ ID NO:5.  
     
     
         98 . The isolated polypeptide of  claim 97 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: amino acid residues 75 to 108 of SEQ ID NO:5, amino acid residues 28 to 108 of SEQ ID NO:5, amino acid residues 23 to 108 of SEQ ID NO:5, amino acid residues 22 to 108 of SEQ ID NO:5, amino acid residues 21 to 108 of SEQ ID NO:5, and amino acid residues 20 to 108 of SEQ ID NO:5.  
     
     
         99 . The isolated polypeptide of  claim 97 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: amino acid residues 23 to 79 of SEQ ID NO:5, amino acid residues 22 to 79 of SEQ ID NO:5, amino acid residues 21 to 79 of SEQ ID NO:5, amino acid residues 20 to 79 of SEQ ID NO:5, and amino acid residues 1 to 79 of SEQ ID NO:5.  
     
     
         100 . The isolated polypeptide of  claim 97 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: amino acid residues 20 to 72 of SEQ ID NO:5, amino acid residues 21 to 72 of SEQ ID NO:5, amino acid residues 22 to 72 of SEQ ID NO:5, and amino acid residues 23 to 72 of SEQ ID NO:5.  
     
     
         101 . The isolated polypeptide of  claim 97 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: amino acid residues 23 to 78 (amide) of SEQ ID NO:5, amino acid residues 22 to 78 (amide) of SEQ ID NO:5, amino acid residues 21 to 78 (amide) of SEQ ID NO:5, and amino acid residues 1 to 78 (amide) of SEQ ID NO:5.  
     
     
         102 . The isolated polypeptide of  claim 97 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: amino acid residues 23 to 79 (amide) of SEQ ID NO:5, amino acid residues 22 to 79 (amide) of SEQ ID NO:5, amino acid residues 21 to 79 (amide) of SEQ ID NO:5, and amino acid residues 20 to 79 (amide) of SEQ ID NO:5.  
     
     
         103 . An isolated polypeptide, comprising an amino acid sequence selected from the group consisting of: (a) amino acid residues 20 to 105 of SEQ ID NO:5, (b) amino acid residues 18 to 105 of SEQ ID NO:5, (c) amino acid residues 1 to 70 of SEQ ID NO:5, (d) amino acid residues 20 to 70 of SEQ ID NO:5, (e) amino acid residues 18 to 70 of SEQ ID NO:5, (f) amino acid residues 76 to 105 of SEQ ID NO:5, (g) amino acid residues 66 to 105 of SEQ ID NO:5, and (h) amino acid residues 82 to 105 of SEQ ID NO:5.