Conotoxins I

Abstract

Substantially pure conotoxins are provided which inhibit synaptic transmissions at the neuromuscular junctions and which are useful both in vivo and in assays because they specifically target particular receptors, such as the acetyl-choline receptor, and ion channels. The peptides are of such length that they can be made by chemical synthesis. They also may be made using recombinant DNA techniques, and the DNA encoding such conotoxins having pesticidal properties can be incorporated as plant defense genes into plant species of interest.

1. A substantially pure conotoxin which is highly selective for a specific human receptor, which conotoxin is selected from the group consisting of:
 Gly-Xaa-Ser-Phe-Cys-Lys-Ala-Asp-Glu-Lys-Xaa-Cys-Glu-Tyr-His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-Ala-Xaa-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cys-Ser-Thr-Ser-Ser-Phe-Phe-Lys-Ile (SEQ ID NO:7) wherein Xaa is 4Hyp;  
 Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln-Xaa-Tyr-Cys-Arg-Xaa-Ser (SEQ ID NO:8) wherein Xaa is Gla and the C-terminus is amidated;  
 His-Xaa-Xaa-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-Xaa-Gly-Cys-Ser-Ser-Ala-Ser-Cys-Cys-Gln (SEQ ID NO:9) wherein Xaa is 4Hyp;  
 Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa-Ala-Asp-Ser-Xaa-Cys-Cys-Thr-Xaa-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe (SEQ ID NO:10) wherein Xaa is Gla and the C-terminus is amidated;  
 Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-Tyr-Cys-Gly-Ser-Thr-Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-Lys-Cys-Ile-Asp-Tyr-Pro-Ser-Asn (SEQ ID NO:11);  
 Gly-Glu-Xaa-Xaa-Val-Ala-Lys-Met-Ala-Ala-Xaa-Leu-Ala-Arg-Xaa-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro (SEQ ID NO:12) wherein Xaa is Gla; and  
 Glu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ ID NO:13), wherein Glu in the 1-position is pGlu and the C-terminus is amidated.  
 
     
     
       2. A conotoxin according to  claim 1  having the formula:
 Gly-Xaa-Ser-Phe-Cys-Lys-Ala-Asp-Glu-Lys-Xaa-Cys-Glu-Tyr-His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-Ala-Xaa-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cys-Ser-Thr-Ser-Ser-Phe-Phe-Lys-Ile (SEQ ID NO:7) wherein Xaa is 4Hyp.  
 
     
     
       3. A conotoxin according to  claim 1  having the formula:
 Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln-Xaa-Tyr-Cys-Arg-Xaa-Ser (SEQ ID NO:8) wherein Xaa is Gla and the C-terminus is amidated.  
 
     
     
       4. A conotoxin according to  claim 1  having the formula:
 His-Xaa-Xaa-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-Xaa-Gly-Cys-Ser-Ser-Ala-Ser-Cys-Cys-Gln (SEQ ID NO:9) wherein Xaa is 4Hyp.  
 
     
     
       5. A conotoxin according to  claim 1  having the formula:
 Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa-Ala-Asp-Ser-Xaa-Cys-Cys-Thr-Xaa-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe (SEQ ID NO:10) wherein Xaa is Gla and the C-terminus is amidated.  
 
     
     
       6. A conotoxin according to  claim 1  having the formula:
 Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-Tyr-Cys-Gly-Ser-Thr-Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-Lys-Cys-Ile-Asp-Tyr-Pro-Ser-Asn (SEQ ID NO:11).  
 
     
     
       7. A conotoxin according to  claim 1  having the formula:
 Gly-Glu-Xaa-Xaa-Val-Ala-Lys-Met-Ala-Ala-Xaa-Leu-Ala-Arg-Xaa-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro (SEQ ID NO:12) wherein Xaa is Gla.  
 
     
     
       8. A conotoxin according to  claim 1  having the formula:
 Glu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ ID NO:13), wherein Glu in the 1-position is pGlu and the C-terminus is amidated.  
 
     
     
       9. The conotoxin according to  claim 8  wherein the Thr residue is glycosylated. 
     
     
       10. A substantially pure conotoxin having the amino acid sequence Glu- Ser - Glu - Glu - Gly - Gly - Ser - Asn - Ala - Thr - Lys - Lys - Pro - Tyr - Ile - Leu  ( SEQ ID NO: 13   ) , wherein Glu in the  1   - position is pGlu.   
     
     
       11. The substantially pure conotoxin of  claim 10 , wherein the Thr residue is glycosylated. 
     
     
       12. An isolated conotoxin which is highly selective for a specific human receptor, comprising a conotoxin selected from the group consisting of:
   Gly - Xaa - Ser - Phe - Cys - Lys - Ala - Asp - Glu - Lys - Xaa - Cys - Glu - Tyr - His - Ala - Asp - Cys - Cys - Asn - Cys - Cys - Leu - Ser - Gly - Ile - Cys - Ala - Xaa - Ser - Thr - Asn - Trp - Ile - Leu - Pro - Gly - Cys - Ser - Thr - Ser - Ser - Phe - Phe - Lys - Ile  ( SEQ ID NO: 7   )  wherein Xaa is  4 Hyp;        Gly - Cys - Cys - Ser - His - Pro - Ala - Cys - Ser - Gly - Lys - Tyr - Gln - Xaa - Tyr - Cys - Arg - Xaa - Ser  ( SEQ ID NO: 8   )  wherein Xaa is Gla and the C - terminus is amidated;        His - Xaa - Xaa - Cys - Cys - Leu - Tyr - Gly - Lys - Cys - Arg - Arg - Tyr - Xaa - Gly - Cys - Ser - Ser - Ala - Ser - Cys - Cys - Gln  ( SEQ ID NO: 9   )  wherein Xaa is  4 Hyp;        Cys - Lys - Thr - Tyr - Ser - Lys - Tyr - Cys - Xaa - Ala - Asp - Ser - Xaa - Cys - Cys - Thr - Xaa - Gln - Cys - Val - Arg - Ser - Tyr - Cys - Thr - Leu - Phe  ( SEQ ID NO: 10   )  wherein Xaa is Gla and the C - terminus is amidated;        Ser - Thr - Ser - Cys - Met - Glu - Ala - Gly - Ser - Tyr - Cys - Gly - Ser - Thr - Thr - Arg - Ile - Cys - Cys - Gly - Tyr - Cys - Ala - Tyr - Phe - Gly - Lys - Lys - Cys - Ile - Asp - Tyr - Pro - Ser - Asn  ( SEQ ID NO: 11   ) ;        Gly - Glu - Xaa - Xaa - Val - Ala - Lys - Met - Ala - Ala - Xaa - Leu - Ala - Arg - Xaa - Asn - Ile - Ala - Lys - Gly - Cys - Lys - Val - Asn - Cys - Tyr - Pro  ( SEQ ID NO: 12   )  wherein Xaa is Gla; and        Glu - Ser - Glu - Glu - Gly - Gly - Ser - Asn - Ala - Thr - Lys - Lys - Pro - Tyr - Ile - Leu  ( SEQ ID NO: 13   ) , wherein Glu in the  1   - position is pGlu and the C - terminus is amidated.     
     
     
       13. The conotoxin according to  claim 12  comprising the formula:
   Gly - Xaa - Ser - Phe - Cys - Lys - Ala - Asp - Glu - Lys - Xaa - Cys - Glu - Tyr - His - Ala - Asp - Cys - Cys - Asn - Cys - Cys - Leu - Ser - Gly - Ile - Cys - Ala - Xaa - Ser - Thr - Asn - Trp - Ile - Leu - Pro - Gly - Cys - Ser - Thr - Ser - Ser - Phe - Phe - Lys - Ile  ( SEQ ID NO: 7   )  wherein Xaa is  4 Hyp.     
     
     
       14. The isolated conotoxin according to  claim 12  comprising the formula:
   Gly - Cys - Cys - Ser - His - Pro - Ala - Cys - Ser - Gly - Lys - Tyr - Gln - Xaa - Tyr - Cys - Arg - Xaa - Ser  ( SEQ ID NO: 8   )  wherein Xaa is Gla and the C - terminus is amidated.     
     
     
       15. The isolated conotoxin according to  claim 12  comprising the formula:
   His - Xaa - Xaa - Cys - Cys - Leu - Tyr - Gly - Lys - Cys - Arg - Arg - Tyr - Xaa - Gly - Cys - Ser - Ser - Ala - Ser - Cys - Cys - Gln  ( SEQ ID NO: 9   )  wherein Xaa is  4 Hyp.     
     
     
       16. The isolated conotoxin according to  claim 12  comprising the formula:
   Cys - Lys - Thr - Tyr - Ser - Lys - Tyr - Cys - Xaa - Ala - Asp - Ser - Xaa - Cys - Cys - Thr - Xaa - Gln - Cys - Val - Arg - Ser - Tyr - Cys - Thr - Leu - Phe  ( SEQ ID NO: 10   )  wherein Xaa is Gla and the C - terminus is amidated.     
     
     
       17. The isolated conotoxin according to  claim 12  comprising the formula:
   Ser - Thr - Ser - Cys - Met - Glu - Ala - Gly - Ser - Tyr - Cys - Gly - Ser - Thr - Thr - Arg - Ile - Cys - Cys - Gly - Tyr - Cys - Ala - Tyr - Phe - Gly - Lys - Lys - Cys - Ile - Asp - Tyr - Pro - Ser - Asn  ( SEQ ID NO: 11   ).   
     
     
       18. The isolated conotoxin according to  claim 12  comprising the formula:
   Gly - Glu - Xaa - Xaa - Val - Ala - Lys - Met - Ala - Ala - Xaa - Leu - Ala - Arg - Xaa - Asn - Ile - Ala - Lys - Gly - Cys - Lys - Val - Asn - Cys - Tyr - Pro  ( SEQ ID NO: 12   )  wherein Xaa is Gla.     
     
     
       19. The isolated conotoxin according to  claim 12  comprising the formula:
   Glu - Ser - Glu - Glu - Gly - Gly - Ser - Asn - Ala - Thr - Lys - Lys - Pro - Tyr - Ile - Leu  ( SEQ ID NO: 13   ) , wherein Glu in the  1   - position is pGlu and the C - terminus is amidated.     
     
     
       20. The isolated conotoxin according to  claim 19  wherein the Thr residue is glycosylated. 
     
     
       21. An isolated conotoxin comprising the amino acid sequence Glu- Ser - Glu - Glu - Gly - Gly - Ser - Asn - Ala - Thr - Lys - Lys - Pro - Tyr - Ile - Leu  ( SEQ ID NO: 13   ) , wherein Glu in the  1   - position is pGlu.   
     
     
       22. An isolated conotoxin comprising the amino acid sequence Glu- Ser - Glu - Glu - Gly - Gly - Ser - Asn - Ala - Thr - Lys - Lys - Pro - Tyr - Ile - Leu  ( SEQ ID NO: 13   ) , wherein Glu in the  1   - position is pGlu and the Thr residue is glycosylated.