Isolated GRP94 ligand binding domain polypeptide and nucleic acid encoding same, and screening methods employing same

Abstract

An isolated GRP94 ligand binding domain polypeptide. In some embodiments, the isolated GRP94 ligand binding domain polypeptide includes (a) an amino acid sequence encoded by a nucleic acid molecule that includes a nucleotide sequence selected from among: (i) nucleotides 310-1116 of SEQ ID NO: 1; (ii) nucleotides 142-948 of SEQ ID NO: 3; (iii) nucleotides 142-948 of SEQ ID NO: 5; and (iv) an amino acid sequence encoded by a nucleotide sequence at least 95% identical to any of (i)-(iii); (b) an amino acid sequence selected from among (v) amino acids 69-337 of SEQ ID NO: 2; (vi) amino acids 48-316 of SEQ ID NO: 4; (vii) amino acids 48-316 of SEQ ID NO: 6; and (viii) an amino acid sequence at least 95% identical to any of (iv)-(vi); or (c) an amino acid sequence that is immunologically cross-reactive with an antibody that specifically binds to a polypeptide consisting of an amino acid sequence as set forth in any one of (a) and (b), wherein the GRP94 ligand binding domain (LBD) polypeptide binds a GRP94 ligand selected from the group consisting of 4,4′-dianilino-1,1-binaphthyl-5,5-disulfonic acid (bis-ANS) and N-ethylcarboxamidoadenosine (NECA). Also disclosed are isolated polynucleotides encoding the disclosed polypeptides and methods of using the same to identify modulators of Hsp90 proteins.

1 . An isolated GRP94 ligand binding domain (LBD) polypeptide comprising:
 (a) an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
 (i) nucleotides 310-1116 of SEQ ID NO: 1; 
 (ii) nucleotides 142-948 of SEQ ID NO: 3; 
 (iii) nucleotides 142-948 of SEQ ID NO: 5; and 
 (iv) an amino acid sequence encoded by a nucleotide sequence at least 95% identical to any of (i)-(iii); 
   (b) an amino acid sequence selected from the group consisting of:
 (v) amino acids 69-337 of SEQ ID NO: 2; 
 (vi) amino acids 48-316 of SEQ ID NO: 4; 
 (vii) amino acids 48-316 of SEQ ID NO: 6; and 
 (viii) an amino acid sequence at least 95% identical to any of (iv)-(vi); or 
   (c) an amino acid sequence that is immunologically cross-reactive with an antibody that specifically binds to a polypeptide consisting of an amino acid sequence as set forth in any one of (a) and (b),   wherein the GRP94 ligand binding domain (LBD) polypeptide binds a GRP94 ligand selected from the group consisting of 4,4′-dianilino-1,1-binaphthyl-5,5-disulfonic acid (bis-ANS) and N-ethylcarboxamidoadenosine (NECA).   
     
     
         2 . An isolated GRP94 ligand binding domain (LBD) polypeptide comprising:
 (a) an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
 (i) nucleotides 169-1116 of SEQ ID NO: 1; 
 (ii) nucleotides 1-948 of SEQ ID NO: 3; 
 (iii) nucleotides 1-948 of SEQ ID NO: 5; and 
 (iv) an amino acid sequence encoded by a nucleotide sequence at least 95% identical to any of (i)-(iii); 
   (b) an amino acid sequence selected from the group consisting of:
 (v) amino acids 22-337 of SEQ ID NO: 2; 
 (vi) amino acids 1-316 of SEQ ID NO: 4; 
 (vii) amino acids 1-316 of SEQ ID NO: 6; and 
 (viii) an amino acid sequence at least 95% identical to any of (iv)-(vi); or 
   (c) an amino acid sequence that is immunologically cross-reactive with an antibody that specifically binds to a polypeptide consisting of an amino acid sequence as set forth in any one of (a) and (b),   wherein the GRP94 ligand binding domain (LBD) polypeptide binds a GRP94 ligand selected from the group consisting of 4,4′-dianilino-1,1-binaphthyl-5,5-disulfonic acid (bis-ANS) and N-ethylcarboxamidoadenosine (NECA).   
     
     
         3 . An antibody or a fragment thereof that specifically binds to a GRP94 polypeptide of  claim 1  or  claim 2 . 
     
     
         4 . The antibody or fragment thereof of  claim 3 , wherein the antibody is a monoclonal antibody. 
     
     
         5 . The antibody or fragment thereof of  claim 4 , wherein antibody or fragment thereof is selected from the group consisting of a humanized antibody, a chimeric antibody, a single chain antibody, a single chain fragment variable (scFv), antibody, and a Fab fragment. 
     
     
         6 . The antibody or fragment thereof of  claim 3 , wherein the antibody or fragment thereof specifically binds to the GRP94 polypeptide in a Western blot. 
     
     
         7 . The antibody or fragment thereof of  claim 3 , wherein the antibody or fragment thereof specifically binds to the GRP94 polypeptide in an ELISA. 
     
     
         8 . A method for producing an antibody that specifically recognizes a GRP94 polypeptide, the method comprising:
 (a) recombinantly or synthetically producing a GRP94 polypeptide of  claim 1  or  claim 2 , or a fragment thereof;   (b) formulating the GRP94 polypeptide of (a), or the fragment thereof, whereby it is an effective immunogen;   (c) administering to an animal the formulation of (b) to generate an immune response in the animal comprising production of antibodies, wherein antibodies are present in the animal; and   (d) collecting from the animal of (c) the antibodies that specifically recognize a GRP94 polypeptide.   
     
     
         9 . A method for detecting a GRP94 polypeptide, the method comprising:
 (a) obtaining a biological sample comprising peptidic material; and   (b) detecting a GRP94 polypeptide in the biological sample of (a) by immunochemical reaction with the antibody of  claim 3 .   
     
     
         10 . The method of  claim 9 , further comprising determining an amount of the GRP94 polypeptide in the biological sample.