Botulinum Neurotoxin E Receptors and Uses Thereof

Abstract

An isolated polypeptide comprising an amino acid sequence selected from amino acids 506-582 of SV2A, wherein position 573 is N and is glycosylated, or amino acids 449-525 of SV2B, wherein position 516 is N and is glycosylated. The present invention also provides an antibody that binds specifically to the polypeptide, an isolated nucleic acid comprising a polynucleotide that encodes the polypeptide; a method for reducing BoNT/E toxicity in an animal; a method for identifying an agent that blocks or inhibits binding between BoNT/E and an SV2A or SV2B protein; a method for monitoring synaptic vesicle endo- or exocytosis, a method for specifically delivering a chemical entity to a cell which has a specific receptor to a BoNT toxin. Also provided are a chimeric toxin for targeting a proteolytic domain of a toxin to a cell, the chimeric toxin comprising a catalytic or proteolytic domain of the BoNT toxin, and a ligand or a fragment thereof for a non-BoNT receptor on the cell; a method for targeting a proteolytic domain of a BoNT toxin to a cell, an isolated non-neuronal cell comprising a BoNT toxin receptor; and a method for screening for an inhibitor of a BoNT toxin.

We claim: 
     
         1 . An isolated polypeptide comprising an ammo acid sequence selected from (i) amino acids 506-582 of SV2A (SEQ ID NO: 1), wherein position 573 is Nand is glycosylated; (ii) amino acids 449-525 of SV2B (SEQ ID NO: 2), wherein position 516 is N and is glycosylated; and (iii) an amino acid sequence that is at least 70% identical to either of the amino acid sequences in (i) to (ii) and is capable of binding to  botulinum  neurotoxin E (BoNT/E), provided that full-length SV2A and SV2B proteins are excluded. 
     
     
         2 . The isolated polypeptide of  claim 1 , wherein the amino acid sequence in (iii) is at least 80% identical to either of the amino acid sequences in (i) to (ii). 
     
     
         3 . The isolated polypeptide of  claim 1 , wherein the amino acid sequence in (iii) is at least 90% identical to either of the amino acid sequences in (i) to (ii). 
     
     
         4 . The isolated polypeptide of  claim 1 , wherein the amino acid sequence in (iii) is at least 95% identical to either of the amino acid sequences in (i) to (ii). 
     
     
         5 . The isolated polypeptide of  claim 1 , wherein the amino acid sequence in (iii) is at least 97% identical to either of the amino acid sequences in (i) to (ii). 
     
     
         6 . The isolated polypeptide of  claim 1 , wherein the amino acid sequence in (iii) is at least 98% identical to either of the amino acid sequences in (i) to (ii). 
     
     
         7 . The isolated polypeptide of  claim 1 , wherein the polypeptide is soluble. 
     
     
         8 . The isolated polypeptide of  claim 1 , wherein the polypeptide further comprises a ganglioside binding site. 
     
     
         9 . An antibody that binds specifically to the polypeptide of  claim 1 . 
     
     
         10 . An isolated nucleic acid comprising a polynucleotide or its complement wherein the polynucleotide encodes the polypeptide of  claim 1 . 
     
     
         11 . A vector comprising the nucleic acid of  claim 10  operably linked to a normative promoter. 
     
     
         12 . A host cell comprising the vector of  claim 11 . 
     
     
         13 . A method for identifying an agent that blocks or inhibits binding between BoNT/E and an SV2A or SV2B protein, the method comprising:
 measuring binding between BoNT/E and a polypeptide in the presence of a test agent wherein the polypeptide is selected from a polypeptide of  claim 1 , a polypeptide that comprises a full length SV2A or Sv2B protein, a polypeptide consisting of an SV2A L4 domain, and a polypeptide that comprises an SV2B L4 domain wherein the SV2B L4 domain is flanked at one or both ends by a non-native flanking amino acid sequence; 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/E and the SV2A or SV2B protein.   
     
     
         14 . The method of  claim 13 , wherein all steps are performed in vitro. 
     
     
         15 . The method of  claim 13 , wherein the polypeptide is provided on a cell surface and the cell is exposed to the test agent. 
     
     
         16 . The method of  claim 15 , wherein the binding between BoNT/E and the polypeptide is measured indirectly by monitoring the entry of BoNT/E into the cell. 
     
     
         17 . A method for monitoring synaptic vesicle endo- or exocytosis, comprising administering to synaptic cells a BoNT/A, B, E or G toxin, or a fragment thereof that contains a receptor binding domain as a marker, allowing the marker to bind to a specific receptor for the BoNT, and detecting the marker. 
     
     
         18 . The method according to  claim 17 , wherein the BoNT/A, B, E or G toxin, or a fragment thereof is fluorescently labeled. 
     
     
         19 . A method according to  claim 17 , wherein the marker is detected with a suitably labeled antibody against the BoNT. 
     
     
         20 . The method according to  claim 19 , wherein the antibody is fluorescently labeled.