Botulinum neurotoxin a receptor and the use thereof
AssigneeWisconsin Alumni Research Foundation
InventorsEdwin R. Chapman, Min Dong
Life of this patent
Estimating the term…
Abstract
The present invention is based on the identification of synaptic vessel glycoprotein SV2 as the BoNT/A receptor and the further identification of various BoNT/A-binding fragments of SV2. The disclosure here provides new tools for diagnosing and treating botulism.
Claims43
1-41. (canceled)
An isolated polypeptide comprising an amino acid sequence selected from (i) amino acids 529-562 of rat SV2C (SEQ ID NO:6), (ii) amino acids 529-562 of human SV2C (SEQ ID NO:18), (iii) amino acids 486 to 519 of rat SV2B (SEQ ID NO:4), (iv) amino acids 486 to 519 of mouse SV2B (SEQ ID NO:10), (v) amino acids 486 to 519 of human SV2B (SEQ ID NO:16), (vi) amino acids 543 to 576 of rat SV2A (SEQ ID NO:2), (vii) amino acids 454-546 of rat SV2C (SEQ ID NO:6), (viii) amino acids 454-546 of human SV2C (SEQ ID NO:18), (ix) amino acids 411 to 503 of rat SV2B (SEQ ID NO:4), (x) amino acids 411 to 503 of human SV2B (SEQ ID NO:16), (xi) amino acids 468 to 560 of rat SV2A (SEQ ID NO:2), (xii) amino acids 468 to 560 of human SV2A (SEQ ID NO:14), and (xiii) an amino acid sequence that is at least 95% identical to (iii), (iv), (v), (ix), or (x) and is capable of binding to botulinum neurotoxin A (BoNT/A), with the proviso that a polypeptide comprising a full length SV2 protein, a polypeptide consisting of an SV2 luminal domain, and a polypeptide comprising an SV2 luminal domain wherein the domain is flanked at one or both ends by a non-native flanking amino acid sequence are excluded.
An isolated polypeptide comprising an amino acid sequence selected from (i) amino acids 529-566 of rat SV2C (SEQ ID NO:6), (ii) amino acids 529-566 of human SV2C (SEQ ID NO:18), (iii) amino acids 486 to 523 of rat SV2B (SEQ ID NO:4), (iv) amino acids 486 to 523 of mouse SV2B (SEQ ID NO:10), (v) amino acids 486 to 523 of human SV2B (SEQ ID NO:16), (vi) amino acids 543 to 576 of rat SV2A (SEQ ID NO:2), (vii) amino acids 454 to 580 of rat SV2C (SEQ ID NO:6), (viii) amino acids 454 to 580 of human SV2C (SEQ ID NO:18), (ix) amino acids 411 to 536 of rat SV2B (SEQ ID NO:4), (x) amino acids 411 to 536 of mouse SV2B (SEQ ID NO:10), (xi) amino acids 411 to 536 of human SV2B (SEQ ID NO:16), (xii) amino acids 468 to 595 of rat SV2A (SEQ ID NO:2), (xiii) amino acids 468 to 595 of human SV2A (SEQ ID NO:14), and an amino acid sequence that is at least 95% identical to (iii), (iv), (v), (ix), (x), or (xi) and is capable of binding to botulinum neurotoxin A (BoNT/A).
An isolated polypeptide consisting of an amino acid sequence selected from (i) amino acids 529-562 of rat SV2C (SEQ ID NO:6), (ii) amino acids 529-562 of mouse SV2C (SEQ ID NO:12), (iii) amino acids 529-562 of human SV2C (SEQ ID NO:18), (iv) amino acids 486 to 519 of rat SV2B (SEQ ID NO:4), (v) amino acids 486 to 519 of mouse SV2B (SEQ ID NO:10), (vi) amino acids 486 to 519 of human SV2B (SEQ ID NO:16), (vii) amino acids 543 to 576 of rat SV2A (SEQ ID NO:2), (viii) amino acids 543 to 576 of human SV2A (SEQ ID NO:14), (ix) amino acids 529-566 of rat SV2C (SEQ ID NO:6), (x) amino acids 529-566 of mouse SV2C (SEQ ID NO:12), (xi) amino acids 529-566 of human SV2C (SEQ ID NO:18), (xii) amino acids 486 to 523 of rat SV2B (SEQ ID NO:4), (xiii) amino acids 486 to 523 of mouse SV2B (SEQ ID NO:10), (xiv) amino acids 486 to 523 of human SV2B (SEQ ID NO:16), (xv) amino acids 543 to 580 of rat SV2A (SEQ ID NO:2), (xvi) amino acids 543 to 580 of human SV2A (SEQ ID NO:14), (xvii) amino acids 454-546 of rat SV2C (SEQ ID NO:6), (xviii) amino acids 454-546 of mouse SV2C (SEQ ID NO:12), (xix) amino acids 454-546 of human SV2C (SEQ ID NO:18), (xx) amino acids 411 to 503 of rat SV2B (SEQ ID NO:4), (xxi) amino acids 411 to 503 of human SV2B (SEQ ID NO:16), (xxii) amino acids 468 to 560 of rat SV2A (SEQ ID NO:2), (xxiii) amino acids 468 to 560 of human SV2A (SEQ ID NO:14), and (xxiv) an amino acid sequence that is at least 95% identical to (iv), (v), (vi), (xii), (xiii), (xiv), (xx), or (xxi) and is capable of binding to botulinum neurotoxin A (BoNT/A).
A complex of a ligand and a polypeptide, wherein the polypeptide comprises a member to which the ligand binds, the member being selected from (i) amino acids 529-562 of rat SV2C (SEQ ID NO:6), (ii) amino acids 486 to 519 of rat SV2B (SEQ ID NO:4), (iii) amino acids 543 to 576 of rat SV2A (SEQ ID NO:2), (iv) a fragment of a homolog of the rat SV2C, SV2B, or SV2A wherein the fragment corresponds to amino acids 529-562 of rat SV2C, amino acids 486 to 519 of rat SV2B, or amino acids 543 to 576 of rat SV2A, respectively, (v) amino acids 454-546 of rat SV2C (SEQ ID NO:6), (vi) amino acids 411 to 503 of rat SV2B (SEQ ID NO:4), (vii) amino acids 468 to 560 of rat SV2A (SEQ ID NO:2), (viii) a fragment of a homolog of the rat SV2C, SV2B, or SV2A wherein the fragment corresponds to amino acids 454-546 of rat SV2C, amino acids 411 to 503 of rat SV2B, or amino acids 468 to 560 of rat SV2A, respectively, (ix) an amino acid sequence that is at least 70% identical to any of the amino acid sequences in (i) to (viii) and is capable of binding to BoNT/A, and (x) an amino acid sequence from (i) to (viii) with conservative substitutions and is capable of binding to BoNT/A, with the proviso that where the polypeptide is a full length SV2 protein, the ligand is not a botulinum toxin.
A method for reducing BoNT/A toxicity in a human or non-human animal comprising the step of:
- administering to the human or non-human animal an agent that reduces binding between BoNT/A and an SV2 protein in the human or non-human animal wherein the agent is selected from a polypeptide comprising the polypeptide of claim 5 and an antibody of claim 6.
A method for identifying an agent that can block binding between BoNT/A and an SV2 protein, the method comprising the steps of:
- measuring binding between BoNT/A and a polypeptide that comprises the polypeptide of claim 5 in the presence of a test agent;
- 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/A and the SV2 protein.
A method for identifying an agent that can bind to a BoNT/A-binding sequence of an SV2 protein, the method comprising the steps of:
- exposing a polypeptide that comprises the polypeptide of claim 5 to a test agent;
- and
- determining whether the agent binds to the polypeptide.
A method for detecting BoNT/A or Clostridium botulinum comprising the steps of:
- exposing a sample suspected of containing BoNT/A to a polypeptide that comprises the polypeptide of claim 5;
- optionally, exposing the sample to a ganglioside;
- and
- detecting binding of the polypeptide to BoNT/A.
A method for identifying polypeptide that can bind to BoNT/A comprising the steps of:
- providing a polypeptide that comprises a BoNT/A-binding sequence of an SV2 protein;
- modifying the polypeptide at the BoNT/A-binding sequence;
- and
- determining whether the modified polypeptide can bind to BoNT/A.
An isolated polypeptide comprising an amino acid sequence NTYFKNCTFIDT(X1)F(X2)NTDFEPYKFIDSEF(X3)NCSF (SEQ ID NO:
- 23), where X1=L or V, X2=D or E, and X3=Q or K;
- wherein the polypeptide is capable of binding to botulinum neurotoxin A (BoNT/A).
Dependencies are inferred from the claim text.