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Definitions

• botulinum neurotoxin A protein receptor The botulinum neurotoxin A protein receptor and its applications
• the present invention relates to a polypeptide which binds the botulinum neurotoxin A (BoNT / A) formed by Clostridium botulinum.
• This polypeptide is a polypeptide consisting of an amino acid sequence which is at least 70% identical to the amino acid sequence of the synaptic vesicle glycoprotein 2C (SV2C) from Homo sapiens so that the polypeptide binds the Hc fragment of botulinum neurotoxin A, with the proviso in that the polypeptide is not the synaptic vesicle glycoprotein 2C from Homo sapiens.
• the present invention further relates to the peptide portions of the SV2C protein which interact with BoNT / A.
• the invention relates to the use of the polypeptide and the peptide portions thereof as an antagonist for reducing the neurotoxicity of BoNT / A, as an aid for the identification of substances which decrease the binding of BoNT / A to nerve cells and for the detection of BoNT / A in different matrices.
• Exocytosis is the fusion of the membranes of intracellular vesicles with the plasma membrane. In this process, the vesicular content is simultaneously released into the synaptic cleft. The fusion of the two membranes is regulated by calcium, which reacts with the protein synaptotagmin. Together with other cofactors, synaptotagmin controls the status of three so-called fusion proteins, the SNAP-25, synaptobrevin 2 and syntoxin IA. While syntaxin IA and synaptobrevin 2 are integrated into the plasma or vesicle membrane, SNAP-25 is only weakly bound to the plasma membrane.
• the above-mentioned fusion proteins are the light chain (LC) target molecules of the clostridial neurotoxins formed by the bacteria C. botulinum, C. butyricum, C. baratii and C. tetani.
• LC light chain
• the anaerobic, gram-positive bacterium C. botulinum produces seven different serotypes of clostridial neurotoxins. These are referred to as the botulinum neurotoxins (BoNT / A to BoNT / G). Of these, BoNT / A and BoNT / B in particular cause a neuroparalytic disorder in humans and animals called botulism.
• the spores of C. botulinum are found in the soil, but may develop into improperly sterilized and sealed home-grown canned foods, which are responsible for many of the botulism cases.
• BoNT / A is the most active of all known biological substances. Only about 5-6 pg of purified BoNT / A represents an MLD (Minimum Jet Dose). A Unit (U) of BoNT / A is defined as the MLD, which after intraperitoneal injection kills half of female Swiss Webster mice weighing 18-20 g each. Seven immunologically different BoNTs were characterized. They bear the names BoNT / A, B, Cl, D, E, F and G and can be distinguished by neutralization with serotype-specific antibodies. The different serotypes of BoNT differ in affected species with regard to the severity and duration of the caused paralysis. So z. B. in the rat with respect to the paralysis BoNT / A 500 times more effective than BoNT / B.
• BoNT / B has been shown to be non-toxic in primates at a dose of 480 U / kg body weight. The same amount of BoNT / A is equivalent to 12 times the lethal dose of this substance in primates. On the other hand, in mice the paralysis time after injection of BoNT / A is 10 times longer than after injection of BoNT / E. BoNTs are used to treat neuromuscular disorders characterized by hyperactivity in skeletal muscle caused by pathologically overactive peripheral nerves. BoNT / A is approved by the US Food and Drug Administration for the treatment of blepharospasm, strabismus, hyperhidrosis, wrinkles and hemifacial spasm.
• BoNT / A Compared with BoNT / A, the remaining BoNT serotypes obviously have lower potency and shorter duration of action. Clinical effects of peripheral intramuscularly administered BoNT / A usually occur within one week. The duration of symptom suppression by a single intramuscular injection of BoNT / A is usually about three to six months.
• BoNT / A, Cl and E cleave SNAP-25, while BoNT / B, D, F, G and tetanus neurotoxin (TeNT) attack the vesicle-associated membrane protein (VAMP) 2 - also called synaptobrevin 2.
• VAMP vesicle-associated membrane protein
• BoNT / Cl also splits syntaxin IA.
• H N fragment The amino-terminal half (H N fragment) integrates at low pH in membranes and translocates the LC into the cytosol of the nerve cell.
• the carboxyl-terminal half (Hc Fragment) binds to complex polysialogangliosides, which only occur in nerve cell membranes, and to previously only partially identified protein receptors. This explains the high neuroselectivity of clostridial neurotoxins.
• BoNT / A does not show any kind of interaction with any of the 13 members of the synaptotagmin protein family, either in PC12 cells or in in vitro protein binding studies.
• the present invention is therefore based on the object to provide means and methods for influencing the neurotoxicity of BoNT / A.
• the object is achieved by providing a polypeptide consisting of an amino acid sequence which is at least 70% identical to the amino acid sequence of the synaptic vesicle glycoprotein 2C from Homo sapiens and that the polypeptide binds the Hc fragment of botulinum neurotoxin A, with the proviso that the polypeptide is not the synaptic vesicle glycoprotein 2C from Homo sapiens.
• the present invention further relates to the Use of the polypeptide and its luminal domain as an antagonist for the reduction of the neurotoxicity of BoNT / A, as an aid for the identification of substances that reduce the binding of BoNT / A to nerve cells and for the detection of BoNT / A in different matrices.
• the present invention now introduces synaptic vesicle protein 2C (SV2C) as a receptor for BoNT / A.
• SV2C synaptic vesicle protein 2C
• TeNT were expressed both recombinantly in E. coli and with
• HDA HDA
• the luminal domain of SV2C represents the fragment for interaction with BoNT / A.
• the isolated 125mer peptide of the luminal domain can interact without ganglioside with the Hc fragment of BoNT / A.
• the present invention involves a 125mer peptide comprising the luminal domain of SV2C, or a peptide consisting of an amino acid sequence of at least 80% identity to the luminal domain of SV2C or which is posttranslationally modified. These agents can be used for specific binding to the Hc fragment of BoNT / A.
• BoNT / A This occupies the receptor binding site of BoNT / A and inhibits its physiological interaction with the SV2C present in the plasma membrane.
• these agents can be used in competitive binding studies to search for other molecules that also insert into the receptor binding site in the Hc fragment of BoNT / A and thus act as antagonists.
• labeling the agents for example with fluorophores or specific recognition sequences, or immobilization on solid phases, it is possible to detect this specifically specifically after binding of these agents to BoNT / A. This gives the possibility to detect BoNT / A specifically in different environments and matrices.
• SV2C is a neuronal cell and neuroendocrine cell glycoprotein (for review, Janz, R. and Suedhof TC, Neuroscience 94 (1999), 1279-1290). It consists of 727 amino acids with a molecular weight of 86 kDa and is incorporated with 12 transmembrane domains into the membrane of synaptic vesicles. The approximately 160 amino acids long amino terminus and the 1 1 amino acids short carboxyl terminus are as cytosol as a 90 amino acids long section between the transmembrane domains 6 and 7.
• Intravesikulär found only between the transmembrane domains 7 and 8 a 125 amino acids long area (amino acids 454 -579), the intravesicular or luminal domain (LD), which contains three putative N-glycosylation sites and two putative disulfide bridges.
• LD luminal domain
• SV2C as an ionic or sugar transporter in synaptic vesicles has not been confirmed, but an interaction of the three SV2 isoforms with synaptotagmine, which is dependent on the phosphorylation of the amino terminus, indicates that the amount of free synapse over SV2 totagmin is influenced to bind Ca 2+ and subsequent initiation of exocytosis.
• the membrane of the synaptic vesicles fused with the presynaptic plasma membrane, whereby the synaptic vesicle proteins are briefly in the presynaptic membrane.
• the intravesical domains of the synaptic vesicle proteins are exposed extracellularly.
• the binding of the Hc fragment of BoNT / A to the numerous occurring complex polysialogangliosides on the nerve cell surface is not sufficient for the uptake of the neurotoxin alone.
• accumulation of the BoNT / A molecules on the nerve cell surface may diffuse laterally in the membrane and increase the likelihood of productive interaction with the rarely exposed protein receptor.
• the 125 amino acid luminal domain is exposed extracellularly after vesicle fusion and is thus available for BoNT / A as a protein receptor. Since the neurotoxin is located very close above the membrane due to the ganglioside binding, the approximately 30 first and last amino acids of the luminal domain are preferably available as receptor, analogous to the BoNT / B / G synaptotagmin interaction.
• the translocation domain is inserted into the endosomal membrane and translocates the partially unfolded LC into the cytosol, where in the final step it cleaves its specific substrate.
• BoNT serotypes The cycle of complex formation and dissociation of the fusion proteins is interrupted, thus inhibiting the release of acetylcholine. As a result, striped muscles are paralyzed, and sweat glands cease their secretion. The duration of action of the individual BoNT serotypes varies and depends on the presence of intact LC in the cytosol.
• the preferential blocking of cholinergic transmission can be explained by the HC entering the neuron at the periphery.
• BoNT / A botulinum neurotoxin A
• E. coli which u. a. which is identical in amino acid sequence to the native botulinum neurotoxin A, behaves pharmacologically identically as the native BoNT / A and is called recombinant botulinum neurotoxin wild type.
• the recombinantly produced Hc fragment of BoNT / A has the same amino acid sequence as the corresponding native Hc fragment and the same binding properties as the native BoNT / A.
• the nerve cells mentioned are cholinergic motor neurons.
• the BoNT / A specifically binds to the plasma membrane associated molecules, transmembrane proteins, synaptic vesicle proteins, a protein of the SV2 family, preferably SV2C, most preferably the luminal domain of SV2C.
• the binding is preferably determined in vitro. Particularly preferably, the determination is carried out by using GST-PuIl-down experiments, which are carried out in detail in the examples.
• the sequence of SV2C is available to anyone from databases.
• the sequence ID for SV2C from Homo sapiens is u. a. GenBank NP_055794, for SV2C from Rattus norvegicus u. a. GenBank NP_1 13781, for SV2C from Mus musculus u. a. GenBank XP_ 127490.
• polypeptide in this context means amino acid polymers of at least two monomer units, the monomers may be natural or non-naturally occurring amino acids, Preferably, a polypeptide has at least 10 amino acid monomers, the individual amino acids may be modified Be originated (eg, post-translationally) or synthetically introduced as z. As glycosylation, di- and oligomerization, and modifications of the Cys residues.
• antibody includes classical antibodies, single-chain antibodies and antibody fragments, preferred fragments being F (ab) 2 and F (ab).
• composition also includes fusion proteins
• the polypeptide contained in the composition may be in the form of a conjugate, with conjugates of dyes, iron particles, epitopes such as Flag or HA tag, crosslinkers, affinity peptides or radioactive isotopes being preferred ,
• the amino acid sequence of the polypeptide is at least 80% identical to the amino acid sequence of the synaptic - lO -
• amino acid sequence which is at least 90% identical to the amino acid sequence of the synaptic vesicle glycoprotein 2C from Homo sapiens.
• amino acid sequence which is at least 95% identical to the amino acid sequence of the synaptic vesicle glycoprotein 2C from Homo sapiens.
• the amino acid sequence of the polypeptide differs from the amino acid sequence of the synaptic vesicle glycoprotein 2C from H. sapiens by the addition, substitution, deletion, insertion and / or inversion of at least one amino acid, preferably not more than 5 amino acids, in particular not more than 1 amino acid.
• the addition, substitution, deletion, insertion or inversion can be carried out in a manner known per se.
• the amino acid sequence of the polypeptide is at least 70% identical to the amino acid sequence of the luminal domain (amino acids 454-579) of the synaptic vesicle glycoprotein 2C from Homo sapiens.
• the amino acid sequence which is at least 80% identical to the amino acid sequence of the luminal domain (amino acids 454-579) of the synaptic vesicle glycoprotein 2C from Homo sapiens is preferred.
• the amino acid sequence which is at least 95% identical to the amino acid sequence of the luminal domain (amino acids 454-579) of the synaptic vesicle glycoprotein 2C from Homo sapiens.
• the polypeptide is the isolated polypeptide of the luminal domain (amino acids 454-579) of the synaptic vesicle glycoprotein 2C from Homo sapiens, wherein the isolated polypeptide binds the Hc fragment of botulinum neurotoxin A. - I l -
• the present invention further provides nucleic acids encoding the polypeptide of the invention.
• vectors which contain the nucleic acid according to the invention and for replication and optionally expression under the control of a suitable promoter in a suitable host cell.
• a process for producing the polypeptide according to the invention comprising recombinantly expressing a nucleic acid encoding the polypeptide in a suitable host and optionally isolating the polypeptide produced in a manner known per se.
• the coding nucleic acid may be RNA, DNA or mixtures thereof.
• the nucleic acid may also be modified with respect to its nuclease resistance, such as e.g. B. by incorporation of phosphorothioate bonds.
• the nucleic acid can be prepared from an initial nucleic acid, wherein the starting nucleic acid z. B. is accessible by cloning from genomic or cDNA libraries. Furthermore, the nucleic acid can be prepared directly by solid phase synthesis. Suitable methods are known to the person skilled in the art. If it is assumed that an initial nucleic acid, z. B. by localized mutagenesis a targeted change can be performed, which leads to at least one addition, insertion, deletion and / or substitution at the amino acid level.
• the nucleic acid is then operably linked to a suitable promoter.
• suitable promoters for expression in known expression systems are known to the person skilled in the art. The choice of the promoter depends on the expression system used for the expression. In general, constitutive promoters are preferred, but inducible promoters are also useful.
• the construct thus produced comprises at least part of a vector, in particular regulatory elements, the vector being selected, for example, from ⁇ derivatives, adenoviruses, baculoviruses, vaccinia viruses, SV40 viruses and retroviruses.
• the vector is preferably capable of expressing the nucleic acid in a given host cell.
• the invention provides Wirtszel len containing the vector and which are suitable for the expression of the vector.
• the host cells are for example selected from prokaryotic cells such as E. coli or B. subtilis, eukaryotic cells such as S. cerevisiae and P. pastoris or higher eukaryotic cells such as insect cells len or mammalian cells.
• the peptide or polypeptide can also be obtained directly by synthesis or fragment condensation. Corresponding methods are known to the person skilled in the art.
• the peptide or polypeptide is subsequently purified.
• the skilled worker known methods are used, such. As chromatography or electrophoresis.
• composition comprising at least one polypeptide of the invention.
• the composition may be present as a mixture or as a conjugate.
• the polypeptide may be conjugated to dye molecules or carriers.
• an antibody or a fragment thereof which is at least 70% identical to the amino acid sequence of the luminal domain (amino acids 454-579) of the synaptic vesicle glycoprotein 2C from Homo sapiens binds.
• the antibody may be monoclonal or polyclonal.
• Monoclonal antibodies can be prepared according to methods known to those skilled in the art by immunizing animals such as mice and subsequently isolating and screening hybridoma.
• the antibody is capable of blocking the binding of synaptic vesicle glycoprotein 2C to botulinum neurotoxin. This can be demonstrated in known competitive assays such as radioimmunoassays or ELISAs.
• a pharmaceutical composition comprising at least one polypeptide of the invention and / or at least one antibody of the invention.
• the pharmaceutical composition may optionally contain a pharmaceutically acceptable carrier, diluent and / or additive.
• the pharmaceutical composition is suitable for oral, intravenous, subcutaneous, intramuscular and topical administration.
• the pharmaceutical composition is indicated for the treatment of botulism, overdose in therapeutic treatment or cosmetic application of BoNT / A, intoxication or for prophylaxis.
• a method of reducing the neurotoxicity of BoNT / A in mammals comprising administering to the mammal an agent, the agent comprising binding BoNT / A to at least 70% identical amino acid sequence to the luminal domain (amino acids 454-579) of the synaptic vesicle glycoprotein 2C from Homo sapiens reduced by at least 10%, preferably reduced by at least 50%, in particular by at least 80%.
• the agent can be determined by the screening method described below.
• the mammal is given a polypeptide having at least 70% identical amino acid sequence to the luminal domain (amino acids 454-579) of the synaptic vesicle glycoprotein 2C (SV2C) from Homo sapiens and which reduces the binding of BoNT / A to SV2C.
• SV2C synaptic vesicle glycoprotein 2C
• the mammal is given an antibody which reduces the binding of BoNT / A to an at least 70% identical amino acid sequence to the luminal domain (amino acids 454-579) of the synaptic vesicle glycoprotein 2C from Homo sapiens.
• the mammal is given an agent which reduces the expression of the luminal domain (amino acids 454-579) of the synaptic vesicle glycoprotein 2C from Homo sapiens.
• the agent may be an antisense molecule. Methods of making antisense molecules are known to those skilled in the art.
• the method can be used to reduce the neurotoxicity of BoNT / A in botulism, after overdose in therapeutic treatment or cosmetic application of BoNT / A, intoxication or for prophylaxis.
• the mammal is Homo sapiens.
• a method of identifying an agent that reduces the binding of BoNT / A to the luminal domain (amino acids 454-579) of the synaptic vesicle homo sapiens glycoprotein 2C comprising:
• the luminal domain (amino acids 454-579) of synaptic vesicle glycoprotein 2C from Homo sapiens is incorporated into a plasma membrane of a cell.
• Suitable cells are neuroendocrine cells such as PC 12, neuroblastoma cells such as 2A, and hybridoma cells from embryonic brain tissue such as NT2.
• BoNT / A the reduced binding of BoNT / A to the synaptic vesicle glycoprotein 2C from Homo sapiens is demonstrated by a reduced neurotoxicity of the BoNT / A in the mouse hemidiaphragm test.
• Glycoproteins 2C from Homo sapiens and which reduces the binding of BoNT / A to SV2C;
• the immobilization can be carried out, for example, by BrCN coupling of the polypeptide to materials known for chromatography purposes, such as Sepharose.
• the conditions that bind the BoNT / A to the polypeptide can be determined by routine experimentation. The conditions should be chosen so that none of the binding partners denature.
• the elution can be carried out, for example, by competition or changing the pH and / or salt conditions.
• the complex or its components can be separated, for example by SDS-PAGE.
• Figure 1 The H c fragment of BoNT / A interacts with SV2C.
• A Schematic representation of synaptic vesicle glycoproteins.
• B, C GST
• Hc fragments are detected by SDS-PAGE and Coomassie blue staining or autoradiography.
• BoNT / A Hc fragment binds to the intravesicular region adjacent to the transmembrane domain 8 of SV2C.
• GST fusion proteins immobilized on GT sepharose beads are incubated with recombinant BoNT Hc fragments in the presence of gangliosides.
• Solid phase-bound Hc fragments are detected by SDS-PAGE and Coomassie blue staining or immunochemically. material and methods
• Plasmids for E. coli expression of full-length BoNT / A and B or recombinant Hc fragments of BoNT / AG and TeNT with carboxyl-terminal StrepTag for affinity purification or for in vitro transcription / translation with 3: ⁇ S- Methionine were generated by PC R methods with suitable primers, BoNT / AG and TeNT encoding cDNA and the expression vector pQe3 (Qiagen AG) or pSP72 (Promega) as the starting vector.
• cDNA sections encoding the intravesicular segments of the various synaptic vesicle protein were inserted into the vector pGEX-4T3 using appropriate oligonucleotides and a mouse embryonic cDNA library (GST-Syo-157-218, GST-Syg-I-45-98 ) or from plasmids with corresponding cDNA as a template: GST-Syg-I-127-169 (RZPD, German Resource Center for Genomic Research GmbH, www.rzpd.de; ID IRAKp961C072Q), GST-SV2A-468-618 (RZPD-ID IRAKp961024100Q), GST-Syo-22-103 (rat, TC Südhof, Dallas), GST-Syg-III-46-88, and GST-Syg-III-128-168 (mouse; TC Südhof, Dallas), GST -SV2B-413-560 (Rat, S.
• a plasmid encoding the hybrid GST-SV2-C / A contains amino acids 454-553 of SV2C and 568-594 of SV2A and was also generated with the corresponding oligonucleotides by PCR. The nucleic acid sequences of all plasmids were confirmed by DNA sequencing. The recombinant Hc fragments were produced in E. coli strain M15 [pRep4] (Qiagen) during a 10 hour induction at room temperature and purified on a StrepTactin matrix (IBA GmbH) according to the manufacturer's recommendations. The GST fusion proteins obtained from E.
• coli BL21 were isolated using glutathione immobilized on sepharose beads. Fractions containing the desired proteins were pooled and challenged with Tris-NaCl-Triton buffer (20 mM Tris-HCl, 15 mM NaCl, 0.5% Triton X-100, pH 7.2) or Kreb-Ringer buffer (1 18 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO 4, 1.2 mM KLH 2 PO 4, 25 mM NaHCO 3, 2.5 mM CaCl 2 , 1 1 mM glucose).
• Tris-NaCl-Triton buffer (20 mM Tris-HCl, 15 mM NaCl, 0.5% Triton X-100, pH 7.2) or Kreb-Ringer buffer (1 18 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO 4, 1.2 mM KLH 2 PO 4, 25 mM NaHCO 3, 2.5 mM Ca
• ⁇ S-labeled Hc fragments were generated in vitro from pSP72 derivatives linearized downstream of the carboxy-terminal codon of the neurotoxin using the reticulocyte lysate system (Promega, Mannheim) and Lv 0 S-methionine (840 kSq,> 37 TBq / mmol Amersham Biosciences) in a 2 ⁇ l assay
• GST fusion proteins (0.15 nmol each) immobilized on 10 ⁇ l of GT-Sepharose beads were incubated with Hc fragments (0.1 nmol) in the absence or presence of a bovine brain ganglioside mixture (18% GM l, 55% GDIa, 10% GT I b, 2% other gangliosides, Calbiochem, 20 ⁇ g each) in a total volume of 100 ⁇ l Tris-NaCl-Triton buffer for 3 h at 4 ° C. The beads were collected by centrifugation, the supernatant removed and the separated beads each washed three times with 160 ⁇ l of the same buffer. The washed pellet fractions were boiled in SDS sample buffer and assayed with the supernatant fractions by SDS-PAGE and Coomassie blue staining, autoradiography or immunoblotting.
• the mouse hemidiaphragm assay was performed as described in Habermann (Habermann E, Dreyer F, Bigalke H (1980) Tetanus toxin blocks the novel romu- cular transmission in vitro like botulinum A toxin Naunyn Schmiedebergs Arch Pharmacol 31 1: 33-40). described.
• the phrenic nerve was stimulated with a heartbeat and the contraction patterns were continuously recorded using a force gauge and the VitroDat Online Software (FMI GmbH, Seeheim-Ober Beerbach, Germany). records. After addition of the BoNT, the time was measured in which the contraction amplitude drops to 50% of the initial value (paralytic half-time).
• scBoNT / A (223 pM) and scBoNT / B (1000 pM) were treated with either GST-S V2C-454-579 or GST-S V2-C / A (final concentration 4.7 or II, 2 ⁇ M), incubated for 15 min at 20 ° C and added to the HDA.
• the z. T. extended paralytic half times converted into correspondingly lower neurotoxin doses and expressed as% toxicity.
• the luminal domains and the carboxyl-terminal transmembrane domains of the proteins synaptophysin, synaptoporin, synaptogyrin I & III, synaptotagmin II, SV2A, SV2B and SV2C were subcloned and recombinantly expressed as E. coli in the glutathione-S-transferase (GST) fusion protein isolated.
• GST glutathione-S-transferase
• the Hc fragments of the seven BoNTs and tetanus neurotoxin (TeNT) were both recombinantly expressed in E. coli and translocated in vitro with D S-methionine.
• the affinity of the Hc fragments to the luminal domains of the GST fusion proteins listed above was determined in glutathione-S-transferase (GST) pull-down experiments.
• GST glutathione-S-transferase
• the respective GST fusion protein was incubated with different Hc fragments and carried out a phase separation. Free Hc fragment remained in the separated supernatant while bound BoNT Hc fragment was detectable in the solid phase along with the GST fusion protein.
• Replacement of recombinant Hc fragments by 3 :) S-labeled Hc fragments as well as full-length BoNT / A gave the same results in the GST pull-down experiments compared to the Hc fragment of BoNT / A.
• Table 1 The intravesicular domain of SV2C blocks the activity of BoNT / A in the HDA.
• BoNT BoNT Inhibitor 3 InhibiParal Semi-toxicity versus inhibition
• BoNT / A 223 SV2-C / A 1 1.2 51 ⁇ 5 100.0 i 1, 0 0 a Inhibitors were used as GST fusion proteins b means ⁇ SD (n 3-9) c full length scBoNT / A wild-type was at least measured three times in the following final concentrations: 24.3 pM, 72.8 pM, 223 pM, and 728 pM. At this concentration-response relationship is a power function has been approximated.
• the GST fusion proteins of the SV2A and SV2B homologous to SV2C did not bind to BoNT / A, even without a carboxyl-terminal transmembrane domain.
• the generation of a hybrid consisting of GST, amino acids 454-554 of SV2C and amino acids 568-594 of SV2A also showed no further interaction with BoNT / A Hc fragment.

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