US2004191887A1PendingUtilityA1

Botulinum neurotoxin B receptors and use thereof

49
Priority: Oct 31, 2002Filed: Oct 28, 2003Published: Sep 30, 2004
Est. expiryOct 31, 2022(expired)· nominal 20-yr term from priority
Inventors:Edwin R. ChapmanMin Dong
A61P 39/02A61P 25/00C07H 21/02A61P 3/04C07K 14/70571C07H 21/04
49
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Claims

Abstract

It is disclosed here that synaptotagmin I (syt I) and synaptotagmin II (syt II) are the cellular receptors for botulinum neurotoxin B (BoNT/B) that mediate the cellular entry and toxicity of BoNT/B. The BoNT/B binding domains of syt I and II are also disclosed. While syt I needs gangliosides for BoNT/B binding, syt II can bind to BoNT/B in the absence of gangliosides. Various nucleic acids and polypeptides that relate to the BoNT/B binding domain of syt I or II are disclosed. Further disclosed are methods of reducing BoNT/B toxicity, methods of identifying agents that can block the binding between BoNT/B and syt I or II, methods of identifying agents that can bind to the BoNT/B binding domain of syt I or II, and methods of detecting BoNT/B or Clostridium botulinum.

Claims

exact text as granted — not AI-modified
We claim:  
     
         1 . An isolated nucleic acid comprising a polynucleotide selected from the group consisting of: 
 (1) a first polynucleotide that encodes an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10;    (2) a second polynucleotide that is at least 80% identical to the first polynucleotide over the entire length of the first polynucleotide;    (3) a third polynucleotide that hybridizes to the first polynucleotide under stringent or moderately stringent hybridization conditions; and    (4) a fourth polynucleotide that is a complement of the first, second or third polynucleotide,    with the proviso that a nucleic acid comprising a polynucleotide that encodes a full length synaptotagmin I or II is excluded.    
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein the nucleic acid comprises the first polynucleotide or a complement of the first polynucleotide wherein the first polynucleotide encodes an amino acid sequence that is at least 80% identical to to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         3 . The isolated nucleic acid of  claim 1 , wherein the nucleic acid comprises the first polynucleotide or a complement of the first polynucleotide wherein the first polynucleotide encodes an amino acid sequence that is at least 90% identical to to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         4 . The isolated nucleic acid of  claim 1 , wherein the nucleic acid comprises the first polynucleotide or a complement of the first polynucleotide wherein the first polynucleotide encodes an amino acid sequence that is at least 95% identical to to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         5 . The isolated nucleic acid of  claim 1 , wherein the nucleic acid comprises the first polynucleotide or a complement of the first polynucleotide wherein the first polynucleotide encodes an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         6 . A vector comprising the polynucleotide of  claim 1  operably linked to a non-native promoter.  
     
     
         7 . A vector comprising the polynucleotide of  claim 5  operably linked to a non-native promoter.  
     
     
         8 . A host cell comprising the polynucledtide of  claim 1  operably linked to a non-native promoter.  
     
     
         9 . A host cell comprising the polynucleotide of  claim 5  operably linked to a non-native promoter.  
     
     
         10 . An isolated polypeptide comprising an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10, with the proviso that a polypeptide comprising a full length synaptotagmin I or II is excluded.  
     
     
         11 . The isolated polypeptide of  claim 10 , wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         12 . The isolated polypeptide of  claim 10 , wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         13 . The isolated polypeptide of  claim 10 , wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         14 . The isolated polypeptide of  claim 10 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         15 . An antibody that binds specifically to an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         16 . A method for reducing BoNT/B toxicity in a human or non-human animal subject comprising the step of: 
 administering to the subject an agent that reduces binding between BoNT/B and an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.    
     
     
         17 . The method of  claim 16 , wherein the subject is a human subject.  
     
     
         18 . The method of  claim 16 , wherein the agent can compete for binding to BoNT/B with an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         19 . The method of  claim 18 , wherein the agent is a polypeptide that comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-79 of SEQ ID NO:2, amino acids 32-79 of SEQ ID NO:4, amino acids 33-80 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         20 . The method of  claim 19 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of amino acids 1-61 of SEQ ID NO:7, amino acids 1-87 of SEQ ID NO:7, amino acids 40-87 of SEQ ID NO:7, amino acids 40-267 of SEQ ID NO:7, amino acids 1-267 of SEQ ID NO:7, amino acids 1-422 of SEQ ID NO:7, amino acids 1-61 of SEQ ID NO:9, amino acids 1-87 of SEQ ID NO:9, amino acids 40-87 of SEQ ID NO:9, amino acids 40-267 of SEQ ID NO:9, amino acids 1-267 of SEQ ID NO:9, amino acids 1-422 of SEQ ID NO:9, amino acids 1-57 of SEQ ID NO:10, amino acids 1-84 of SEQ ID NO:10, amino acids 37-84 of SEQ ID NO:10, amino acids 37-264 of SEQ ID NO:10, amino acids 1-264 of SEQ ID NO:10, and amino acids 1-419 of SEQ ID NO:10.  
     
     
         21 . The method of  claim 19 , wherein the agent further comprises a ganglioside.  
     
     
         22 . The method of  claim 18 , wherein the polypeptide comprises an amino acid sequence selected from amino acids 40-87 of SEQ ID NO:7, amino acids 1-87 of SEQ ID NO:7, amino acids 40-267 of SEQ ID NO:7, amino acids 1-267 of SEQ ID NO:7, amino acids 40-87, amino acids 1-87 of SEQ ID NO:9, amino acids 40-267 of SEQ ID NO:9, amino acids 1-267 of SEQ ID NO:9, amino acids 37-84 of SEQ ID NO:10, amino acids 1-84 of SEQ ID NO:10, amino acids 40-264 of SEQ ID NO:10, and amino acids 1-264 of SEQ ID NO:10.  
     
     
         23 . The method of  claim 16 , wherein the agent can compete with BoNT/B for binding to an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         24 . The method of  claim 23 , wherein the agent is an antibody specific to an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         25 . The method of  claim 16 , wherein the agent can reduce the expression of at least of one of synaptotagmin I and II in the subject.  
     
     
         26 . The method of  claim 16 , wherein the agent can reduce the binding between gangliosides and an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 53-79 of SEQ ID NO:2, amino acids 53-79 of SEQ ID NO:4, amino acids 54-80 of SEQ ID NO:5, amino acids 61-87 of SEQ ID NO:7, amino acids 61-87 of SEQ ID NO:9, amino acids 58-84 of SEQ ID NO:10.  
     
     
         27 . The method of  claim 26 , wherein the agent can reduce the amount of gangliosides available for binding to a ganglioside domain of at least of one of synaptotagmin I and II in the subject.  
     
     
         28 . The method of  claim 26 , wherein the agent can compete with gangliosides for binding to an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 53-79 of SEQ ID NO:2, amino acids 53-79 of SEQ ID NO:4, amino acids 54-80 of SEQ ID NO:5, amino acids 61-87 of SEQ ID NO:7, amino acids 61-87 of SEQ ID NO:9, amino acids 58-84 of SEQ ID NO:10.  
     
     
         29 . The method of  claim 28 , wherein the agent is an antibody specific to an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 53-79 of SEQ ID NO:2, amino acids 53-79 of SEQ ID NO:4, amino acids 54-80 of SEQ ID NO:5, amino acids 61-87 of SEQ ID NO:7, amino acids 61-87 of SEQ ID NO:9, amino acids 58-84 of SEQ ID NO:10.  
     
     
         30 . The method of  claim 16 , wherein the agent is a dominant negative synaptotagmin I or II.  
     
     
         31 . A method for identifying an agent that can block binding between BoNT/B and synaptotagmin I or II, the method comprising the steps of: 
 measuring binding between BoNT/B and a polypeptide in the presence of a test agent wherein the polypeptide comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-79 of SEQ ID NO:2, amino acids 32-79 of SEQ ID NO:4, amino acids 33-80 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10, with the proviso that a polypeptide comprising a full length synaptotagmin I or II is excluded; and    comparing the binding to that of a control measured under the same conditions but in the absence of the test agent, wherein a lower than control binding indicates that the agent can block binding between BoNT/B and synaptotagmin I or II.    
     
     
         32 . The method of  claim 31 , wherein the polypeptide consists of an amino acid sequence selected from the group consisting of amino acids 32-79 of SEQ ID NO:2, amino acids 32-79 of SEQ ID NO:4, amino acids 33-80 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         33 . The method of  claim 31 , wherein all three steps are performed in vitro.  
     
     
         34 . The method of  claim 31 , wherein the polypeptide is provided on a cell surface and the cell is exposed to the test agent.  
     
     
         35 . The method of  claim 34 , wherein the binding between BoNT/B and the polypeptide is measured indirectly by monitoring the entry of BoNT/B into the cell.  
     
     
         36 . A method for identifying an agent that can bind to a BoNT/B binding domain of synaptotagmin I or II, the method comprising the steps of: 
 exposing a polypeptide to a test agent wherein the polypepetide comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10, with the proviso that a polypeptide comprising a full length synaptotagmin I or II is excluded; and    determining whether the agent binds to the polypeptide.    
     
     
         37 . The method of  claim 36 , wherein the polypeptide consists of an amino acid sequence selected from the group consisting of amino acids 32-52 of SEQ ID NO:2, amino acids 33-53 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10.  
     
     
         38 . The method of  claim 36 , wherein all three steps are carried out in vitro.  
     
     
         39 . The method of  claim 36 , where the polypeptide is provided and exposed to a test agent in a cell.  
     
     
         40 . A method for detecting BoNT/B or  Clostridium botulinum  comprising the steps of: 
 exposing a sample suspected of containing BoNT/B to a polypeptide that comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 32-79 of SEQ ID NO:2, amino acids 32-79 of SEQ ID NO:4, amino acids 33-80 of SEQ ID NO:5, amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10, with the proviso that a polypeptide comprising a full length synaptotagmin I or II is excluded;    exposing the sample to a ganglioside wherein this step is optional when the polypeptide used comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of amino acids 40-60 of SEQ ID NO:7, amino acids 40-60 of SEQ ID NO:9, and amino acids 37-57 of SEQ ID NO:10; and    detecting binding of the polypeptide to BoNT/B.

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