Claudin polypeptides, polynucleotides, and methods of making and use thereof

Abstract

This invention relates to new members of the human Claudin polypeptide family, to methods of making such polypeptides, and to methods of using them to treat Claudin-associated conditions and to identify agents that alter Claudin polypeptide activities.

1 . An isolated polypeptide consisting essentially of an amino acid sequence selected from the group consisting of: 
 (a) amino acids 31 through 76 of SEQ ID NO:6;    (b) amino acids 138 through 159 of SEQ ID NO:6;    (c) amino acids 5 through 99 of SEQ ID NO:6;    (d) amino acids 112 through 182 of SEQ ID NO:6;    (e) amino acids 5 through 182 of SEQ ID NO:6; and    (f) an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97.5%, or at least 99% identical across the length of any of the amino acid sequences of (a)-(e) above.    
     
     
         2 . An isolated polypeptide consisting essentially of an amino acid sequence selected from the group consisting of: 
 (a) the amino acid sequence of a first or second extracellular loop of a Claudin-23 polypeptide;    (b) the amino acid sequence of a fragment of a Claudin-23 polypeptide, wherein the fragment consists of, in N-to-C order, a first transmembrane domain, a first extracellular loop, and a second transmembrane domain of a Claudin-23 polypeptide;    (c) the amino acid sequence of a fragment of a Claudin-23 polypeptide, wherein the fragment consists of, in N-to-C order, a third transmembrane domain, a second extracellular loop, and a fourth transmembrane domain of a Claudin-23 polypeptide;    (d) the amino acid sequence of a fragment of a Claudin-23 polypeptide, wherein the fragment consists of, in N-to-C order, a first transmembrane domain, a first extracellular loop, a second transmembrane domain, an intracellular loop, a third transmembrane domain, a second extracellular loop, and a fourth transmembrane domain of a Claudin-23 polypeptide of a Claudin-23 polypeptide;    (e) an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97.5%, or at least 99% identical across the length of any of the amino acid sequences of (a)-(e) above.    
     
     
         3 . The isolated polypeptide of  claim 2 , wherein the amino acid sequence of the first extracellular loop is x1 through 76 of SEQ ID NO:6, and wherein x1 is an amino acid between amino acid 28 and 31 of SEQ ID NO:6.  
     
     
         4 . The isolated polypeptide of  claim 2 , wherein the amino acid sequence of the second extracellular loop is x2 to 159 of SEQ ID NO:6, and wherein x2 is an amino acid between 135 and 138 of SEQ ID NO:6.  
     
     
         5 . An isolated polynucleotide consisting essentially of a nucleotide sequence encoding the polypeptide of  claim 1 .  
     
     
         6 . An expression vector comprising a polynucleotide of  claim 5 .  
     
     
         7 . A recombinant host cell into which the polynucleotide of  claim 5  has been introduced.  
     
     
         8 . The recombinant host cell of  claim 7 , wherein the polynucleotide is integrated into the host cell genome.  
     
     
         9 . A method for expressing a polypeptide of  claim 1 , comprising culturing a recombinant host cell into which a polynucleotide encoding the polypeptide has been introduced.  
     
     
         10 . The method of  claim 9  wherein the recombinant host cell is cultured under conditions promoting expression of the polypeptide from the polynucleotide.  
     
     
         11 . The method of  claim 9  further comprising isolating the polypeptide.  
     
     
         12 . An isolated polypeptide produced by the process of  claim 11 .  
     
     
         13 . An isolated antibody that binds to the polypeptide of  claim 12 , wherein binding of the antibody to the polypeptide agonizes the activity of the polypeptide.  
     
     
         14 . The antibody of  claim 13 , wherein the activity of the polypeptide is tight junction formation activity.  
     
     
         15 . The antibody of  claim 13 , wherein the antibody is a monoclonal antibody.  
     
     
         16 . The antibody of  claim 13 , wherein the antibody is a human antibody.  
     
     
         17 . The antibody of  claim 13 , wherein the antibody is a humanized antibody.  
     
     
         18 . A method for identifying an agent that modulates polypeptide activity comprising mixing a test agent with a polypeptide of  claim 12 , and determining whether there is a change in the activity of the polypeptide in the presence of the test agent relative to the activity of the polypeptide in the absence of the test agent.  
     
     
         19 . The method of  claim 18 , wherein the polypeptide activity is tight junction formation activity.  
     
     
         20 . The method of  claim 18 , wherein determining whether there is a change in polypeptide activity comprises an assessment of transcription and/or translation of skin differentiation markers such as, but not limited to, filaggrin, profilaggrin, involucrin, and keratin markers.  
     
     
         21 . The method of  claim 18 , wherein the test agent is selected from the group consisting of small molecules, peptides, and antibodies.