Novel proteins within the type e botulinum neurotoxin complex

Abstract

The invention features a polypeptide complex synthesized by bacteria of the genus Clostridia that contains the serotype E botulinum neurotoxin and five neurotoxin associated polypeptides having molecular weights of about 118, 80, 65, 40, and 18 kDa, respectively. The complex is useful in the treatment of diseases or conditions that are caused by excessive release of acetylcholine from presynaptic nerve terminals.

1 . A substantially pure polypeptide complex comprising a  Clostridium botulinum  neurotoxin and more than one  Clostridium botulinum  type E neurotoxin associated polypeptide. 
     
     
         2 . A complex of  claim 1 , wherein the neurotoxin associated polypeptide has a molecular weight of approximately 80 kDa and comprises the amino acid sequence TNLKPYIIYD (SEQ ID NO:4). 
     
     
         3 . A complex of  claim 1 , wherein the neurotoxin associated polypeptide has a molecular weight of approximately 65 kDa and comprises the amino acid sequence MQTTTLNWDT (SEQ ID NO:3). 
     
     
         4 . A complex of  claim 1 , wherein the neurotoxin associated polypeptide has a molecular weight of approximately 40 kDa and comprises the amino acid sequence MRINTNINSM (SEQ ID NO:2). 
     
     
         5 . A complex of  claim 1 , wherein the neurotoxin associated polypeptide has a molecular weight of approximately 18 kDa and comprises the amino acid sequence MKQAFVFEFD (SEQ ID NO:1). 
     
     
         6 . A complex of  claim 1 , wherein the neurotoxin associated polypeptide has a molecular weight of approximately 18 kDa and comprises the amino acid sequence shown in  FIG. 8  (SEQ ID NO:5). 
     
     
         7 . A substantially pure  Clostridium botulinum  serotype E neurotoxin associated polypeptide. 
     
     
         8 . The polypeptide of  claim 7 , wherein the neurotoxin associated polypeptide has a molecular weight of about 80 kDa. 
     
     
         9 . The polypeptide of  claim 8 , wherein the neurotoxin associated polypeptide comprises the amino acid sequence TNLKPYIIYD (SEQ ID NO:4). 
     
     
         10 . The polypeptide of  claim 7 , wherein the neurotoxin associated polypeptide has a molecular weight of about 65 kDa. 
     
     
         11 . The polypeptide of  claim 10 , wherein the neurotoxin associated polypeptide comprises the amino acid sequence MQTTTLNWDT (SEQ ID NO:3). 
     
     
         12 . The polypeptide of  claim 7 , wherein the neurotoxin associated polypeptide has a molecular weight of about 40 kDa. 
     
     
         13 . The polypeptide of  claim 12 , wherein the neurotoxin associated polypeptide comprises the amino acid sequence MRINTNINSM (SEQ ID NO:2). 
     
     
         14 . The polypeptide of  claim 7 , wherein the neurotoxin associated polypeptide has a molecular weight of about 18 kDa. 
     
     
         15 . The polypeptide of  claim 14 , wherein the neurotoxin associated polypeptide comprises the amino acid sequence MKQAFVFEFD (SEQ ID NO:1). 
     
     
         16 . The polypeptide of  claim 14 , wherein the neurotoxin associated polypeptide comprises the amino acid sequence shown in  FIG. 8  (SEQ ID NO:5). 
     
     
         17 . A substantially pure antibody that specifically binds to a  Clostridium botulinum  type E neurotoxin associated polypeptide having a molecular weight of approximately 80, 60, 45, or 18 kDa, or to a complex of any two or more of said neurotoxin associated polypeptides. 
     
     
         18 . A substantially pure antibody that specifically binds to a polypeptide complex of  claim 1 . 
     
     
         19 . A method of detecting a serotype E neurotoxin complex in a sample, the method comprising:
 (a) contacting the sample with an antibody of  claim 17 , and   (b) detecting antibody-bound polypeptide, if any, in the sample, the presence of antibody-bound polypeptide indicating the presence of serotype E neurotoxin in the sample.   
     
     
         20 . The method of  claim 19 , wherein the sample is a foodstuff. 
     
     
         21 . The method of  claim 19 , wherein the sample is a gastrointestinal, blood, or tissue sample obtained from a vertebrate animal. 
     
     
         22 . A method of treating a patient who is suffering from a disease or condition associated with excessive release of acetylcholine from presynaptic nerve terminals, the method comprising administering to the patient a therapeutically effective amount of a polypeptide complex of  claim 1 . 
     
     
         23 . The method of  claim 22 , wherein the excessive acetylcholine release causes undesirable contraction of smooth or skeletal muscle cells. 
     
     
         24 . The method of  claim 22 , wherein the excessive release of acetylcholine causes profuse sweating, lacrimation, or mucous secretion. 
     
     
         25 . A method of treating a patient who is suffering from spasticity occurring secondary to brain ischemia, or traumatic injury of the brain or spinal cord, the method comprising administering to the patient a therapeutically effective amount of a polypeptide complex of  claim 1 . 
     
     
         26 . A method of treating a patient who is suffering from tension headache or pain, the method comprising administering to the patient a therapeutically effective amount of a polypeptide complex of  claim 1 . 
     
     
         27 . A vaccine comprising a polypeptide complex of  claim 1 . 
     
     
         28 . A method of vaccinating an animal against serotype E neurotoxin, the method comprising administering to the animal an effective amount of the vaccine of  claim 27 . 
     
     
         29 . A vaccine comprising a polypeptide of  claim 7 . 
     
     
         30 . A method of detecting a  Clostridium botulinum  serotype E neurotoxin in a sample, the method comprising:
 (a) contacting the sample with a  Clostridium botulinum  type E neurotoxin associated polypeptide (NAP) of  claim 7  that specifically binds a serotype E botulinum neurotoxin and thereby forms a NAP-neurotoxin complex, and   (b) detecting the NAP-neurotoxin complex, if any, in the sample, the presence of a complex indicating the presence of serotype E neurotoxin in the sample.   
     
     
         31 . A complex of  claim 1 , comprising the neurotoxin and neurotoxin associated polypeptides having molecular weights of about 80 kDa, 65 kDa, 40 kDa, and 18 kDa. 
     
     
         32 . The complex of  claim 1 , comprising the neurotoxin and neurotoxin associated polypeptides having molecular weights of about 118 kDa, 80 kDa, 65 kDa, 40 kDa, and 18 kDa.