WO2006026977A2 - Polypeptides et procede de liaison specifique de prions - Google Patents

Polypeptides et procede de liaison specifique de prions Download PDF

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Publication number
WO2006026977A2
WO2006026977A2 PCT/DE2005/001574 DE2005001574W WO2006026977A2 WO 2006026977 A2 WO2006026977 A2 WO 2006026977A2 DE 2005001574 W DE2005001574 W DE 2005001574W WO 2006026977 A2 WO2006026977 A2 WO 2006026977A2
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Prior art keywords
prp
sequence
polypeptide
prion protein
gly
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German (de)
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WO2006026977A3 (fr
Inventor
Carsten Korth
Sirik Rutger Leliveld
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Heinrich Heine Universitaet Duesseldof
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Heinrich Heine Universitaet Duesseldof
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag

Definitions

  • the invention relates to polypeptides having at least one sequence motif which bind specifically to the pathological isoform of a prion protein.
  • the invention also relates to nucleic acids encoding these polypeptides, as well as vectors and cells containing these molecules.
  • the invention further relates to an antigen-binding peptide which binds specifically to the polypeptides.
  • the invention further relates to a kit and a pharmaceutical composition, and uses thereof, and a method for specific binding of the pathological isoform of a prion protein (PrP sc ) to a biomolecule, wherein the binding by incubation of the biomolecule with a on the presence of the pathological prion protein to examining sample is performed.
  • Transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats, CWD in deer and BSE in cattle are neurodegenerative diseases of the central nervous system, which are characterized by long incubation periods and can be transmitted across species.
  • CJD Creutzfeldt-Jakob disease
  • the causative agent of these diseases is an infectious molecule that consists of the misfolded isoform of a normal prion protein (PrP) and increasingly converts the physiological prion protein (PrP c ) into the pathogenic, misfolded isoform (PrP s0 ) during the disease process .
  • the pathological or pathogenic prion protein (PrP sc ) agrees with the normal host protein (PrP 0 ) in terms of molecular weight and amino acid sequence.
  • PrP 0 predominantly has ⁇ -helical secondary structures, is soluble and digestible by means of protease.
  • PrP sc mainly has ⁇ -sheet structures, is insoluble and can only be degraded by proteases to a limited extent.
  • protease-sensitive PrP S0 has also been described (Safar et al., 1998).
  • the pathogenic isoform PrP sc is able to convert the physiological PrP 0 into the pathological isoform PrP SG by a previously unknown mechanism, ie to change the folding or secondary structure of the prion protein.
  • the N-terminal domain of the prion protein contains four highly conserved copper-binding octamers (octarepeats), such as the amino acid sequence Pro-His-Gly-Gly-Gly-Trp-Gly-Gln from position 60 to 91 of the amino acid sequence of the prion protein of the Syrian hamster ( Burns et al., 2003).
  • This octarepeate region binds copper cooperatively (Brown et al., 1997, Garnett and Viles, 2003, Hornshaw et al., 1995, Stöckel et al., 1998) and alters its conformation by binding copper (Viles et al. 1999).
  • slaughterhouses post-mortem tests are carried out for the presence of pathogens in slaughtered animals so that infected animals can not enter the food chain.
  • BSE bovine
  • vCJD human
  • the transmission of the disease can also be via infected blood
  • WO 01/00235 A1 discloses isolated cell surface proteins which bind prion proteins. It is proposed to use these proteins for the diagnosis and therapy of prion-induced diseases.
  • EP 0 822 828 B1 likewise discloses an isolated protein which binds to prion proteins.
  • the 17 amino acid residue amino acid sequence Tyr-His-Val-Ala-Thr-Lys-Ala-Pro-His-His-Gly-Pro-Cys-Arg-Ser-Ser-Ala contained in the protein may be isolated as an isolated Peptide used for binding prion proteins.
  • EP 0 616 613 B1 discloses fragments of prion proteins with at least one antigenic domain of a prion protein.
  • the fragments are described in the form of synthetic polypeptides, disclosing numerous antigenic amino acid sequences within these polypeptides. With the aid of the synthetic polypeptides described, it is possible to detect prion proteins or antibodies to prion proteins.
  • DE 199 17 838 A1 discloses an oligopeptide of the amino acid sequence Met-Ile-Phe-Ile-Val-Ala-Glu-Arg-Arg-Gln-Gly-Val-Leu-Thr-Leu-Gly-Ala-Val, which was used for diagnosis and therapeutic treatment of transmissible spongiform encephalopathies and related neurodegenerative diseases.
  • Synthetic polypeptides for the diagnosis and therapy of prion diseases are also known from DE 197 41 607 A1, which have immunogenic properties or binding properties which correspond to those of PrP sc , but which are not infectious.
  • the polypeptides contain at least one sequence, which are arranged in the native PrP sc on its surface and form a binding site there.
  • various amino acid sequences are disclosed which may be included in the synthetic polypeptides.
  • One of these amino acid sequences is, for example, the sequence Gly-TrP-Gly-GIn-PrO-HiS-Gly-Gly-Gly-TrP-Gly-GIn-PrO-HiS-Gly, which corresponds to a sequence from the octarepeate region of a prion protein. It is proposed to specifically bind PrP sc possibly contained in sample material by means of the described polypeptides and then to detect it by suitable tests. It is further proposed to use the polypeptides for therapeutic purposes.
  • the polypeptides described in the cited prior art do not have sufficient specificity for the pathological PrP sc , so that a reliable distinction between the pathogenic isoform and the normal host protein (PrP c ) is not possible with sufficient reliability.
  • the binding to the PrP sc is in each case relatively weak and not comparable to the stability of a bond, for example by antibodies.
  • the object of the invention is therefore to provide polypeptides and methods which reliably enable the specific binding of the pathological isoform of a prion protein (PrP sc ) and thus a clear distinction between this and the normally folded isoform of the prion protein (PrP 0 ).
  • polypeptides of the type mentioned in which the at least one sequence motif of at least 9 consecutive octamers of a copper-binding, repetitive octamer of a prion protein (PrP) at least homologous and / or overlapping amino acid sequence, and in addition a contains soluble ingredient.
  • PrP prion protein
  • the polypeptides according to the invention bind PrP Sc with a very high association constant, while PrP 0 is only weakly bound under these conditions, so that PrP Sc can be specifically detected after differential washing .
  • a particular advantage of the polypeptides according to the invention lies in the fact that, in contrast to the previously known detection methods for PrP Sc , the treatment of the samples with protease can be dispensed with. As a result, the corresponding detection methods are much more sensitive and also simplified.
  • sequence motif with increasing number of octamers is compensated by the fusion with a soluble component, so that overall there is a soluble polypeptide which is suitable for use in standardized test methods.
  • the polypeptides of the invention allow a reliable distinction between the pathogenic isoform (PrP Sc ) and the normal host protein (PrP c ) and thus can be used in particular for the diagnosis of spongiform encephalopathies, for example for the detection of PrP Sc in blood samples.
  • PrP Sc pathogenic isoform
  • PrP c normal host protein
  • rapid tests can be carried out on a large number of animals (herd investigations). The investigation may be based on the presence of an infection with PrP Sc at an early stage (ante mortem), ie not only on brain homogenates, but also, for example, on samples of body fluids.
  • PrP Sc Due to the strength of the binding of PrP Sc can be purified using the polypeptides of the invention samples, such as blood preserves, further completely from the pathogenic prion proteins. It would also be conceivable to administer the polypeptides according to the invention, which for this purpose would have to be coupled with a suitable substance, to a person to be examined and to use them in an imaging method for in vivo diagnostics, such as, for example, NMR spectroscopy. In this case, besides the L-forms of the polypeptides, the D-forms (enantiomers) could also be used.
  • the octamers of the polypeptides according to the invention which form the sequence motif essential for binding to PrP Sc as a repetitive sequence, are homologs of the copper-binding, repetitive octamers of natural prion proteins (PrP) and / or overlap with their amino acid sequence.
  • the octameres of the prion protein (PrP) have the basic sequence Pro-His-Gly-Gly-Gly-Trp-Gly-Gln, wherein within the repetitive sequence, ie the 4 Octarepeats, the starting point of the repeating sequence can vary.
  • Octamers according to the invention can therefore all be selected from the sequence Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His- GIy-GIy-GIy be excitable octamers.
  • Homologous octamers within the meaning of the invention are all octamers which differ from the corresponding sequence of a natural prion protein by the exchange of one or more amino acids, the structure and / or functionality of the sequence motif being retained.
  • the amino acid after the second glycine residue may be either glycine or serine.
  • At least one octamer of the polypeptides of the invention is preferably selected from the group of amino acid sequences according to SEQ ID Nos. 9 to 24. Within the sequence motif, either identical oligomers can be connected to one another repetitively or different oligomers can be combined with one another.
  • the structure of the polypeptides of the invention is designed to be specific to one of the amino acid sequences Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp, Gly-Gln-Pro-His-Gly-Gly-Gly-Trp or Trp-Gly-Gln-Pro-His-Gly-Gly-Gly, or a repeating sequence of one of these sequences, preferably 4 times.
  • These sequence regions within the octarepeat region of the PrP Sc have been found to be preferred binding sites for the polypeptides of the invention. Consequently, the interaction of the sequence motif of the polypeptides according to the invention with these sequences is of particular importance for the specific binding of the PrP Sc .
  • the sequence motif in a preferred embodiment of the invention should consist of 10 to 32, preferably 14 or 16, octamers.
  • Such a high number of octarepeats is particularly advantageous for the specific distinction between PrP c and PrP Sc , whereby the specificity of the polypeptides according to the invention can be improved with increasing number of octamers.
  • the soluble constituent is glutathione-S-transferase (GST).
  • Preferred according to the invention are polypeptides which have one of the amino acid sequences according to Seq ID Nm. 1 to 8 have at least 90% homology.
  • detectable constituent preferably a fluorescent, radioactive or enzymatically detectable constituent
  • this can be detected in a simple manner by means of standardized methods and thus be used in any form of detection method.
  • the invention further includes a nucleic acid molecule having a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is selected from the group consisting of
  • nucleotide sequence which codes for the polypeptide according to one of claims 1 to 9, b) a nucleotide sequence which, in addition to a sequence which codes for a soluble polypeptide, at least 9 consecutive sections according to SEQ ID NO: 25 Insert contains, and c) a nucleotide sequence, which is different from the nucleotide sequences according to a) or b) by the exchange of at least one codon against a synonymous
  • the invention is not limited to a specific nucleic acid molecule that encodes a polypeptide according to the invention, but includes all nucleic acid molecules which code for a functional polypeptide.
  • the invention further includes nucleic acid molecules derived from distinguish alternative splicing of a common pre-mRNA molecule from the sequence recognized as "wild type".
  • Such splicing mechanisms include the alternative use of exons, ie, nucleic acid sequences that encode an amino acid sequence, the alternative arrangement of exons, and the "non-removal" of introns, ie, intervening sequences that do not normally code for an amino acid sequence, from the mRNA - molecule.
  • the nucleic acid molecules of the invention may be operatively linked to a regulatory sequence to facilitate expression of the nucleic acid molecule.
  • a nucleic acid molecule is said to be "capable of expressing a nucleic acid sequence” if it comprises sequence elements that contain information regarding the regulation of transcription and / or translation, and these elements are “operably linked” to the nucleic acid sequence encoding the polypeptide.
  • An operative linkage is a linkage in which the regulatory sequence elements and the protein coding sequence are linked such that gene expression is possible. The exact nature of the regulatory regions required for gene expression can vary between different species.
  • these regions comprise a promoter which consists of prokaryotes from the promoter per se, ie DNA elements which control the initiation of transcription, and of DNA elements which regulate the start of translation after their transcription into mRNA.
  • promoters normally include 5 'non-coding sequences involved in the initiation of transcription and translation, such as the -35 / -10 elements and the Shine-Dalgarno element in prokaryotes, or the TATA box, CAAT- Sequences and 5'-capping elements in eukaryotes.
  • These regions may also contain enhancer or repressor elements as well as translated signal sequences to direct the native polypeptide chain into a specific compartment of the host cell.
  • the 3 'noncoding regions may also contain regulatory elements involved in the termination of transcription, polyadenylation, and the like. If these termination sequences in a particular host cell are not or only insufficiently functional, they can be replaced by signals that are present in the particular host cell Cell are sufficiently functional.
  • a nucleic acid molecule according to the invention may accordingly comprise a regulatory sequence, in particular a promoter sequence.
  • the nucleic acid molecule according to the invention comprises a promoter sequence and a transcription termination sequence.
  • Suitable prokaryotic promoters are, for example, the / acUV5 promoter or the T7 promoter. Examples of suitable eukaryotic promoters are the SV40 promoter or the CMV promoter.
  • nucleic acid molecules according to the invention may furthermore be contained in a vector or another cloning vehicle, such as, for example, phages,
  • nucleic acid molecule is contained in a vector, in particular in an expression vector.
  • the expression vector may further comprise at least one selection marker conferring a selectable phenotype on a transformed cell, include.
  • suitable vectors e.g. pBluescript, pUC18, pET or pcDNA3, is described in detail and commercially available.
  • DNA molecules encoding a polypeptide according to the invention can be transformed into a cell suitable for expression of these DNA molecules.
  • the transformation can be carried out using established standard methods.
  • the invention therefore also relates to a cell which contains a nucleic acid molecule according to the invention or a vector containing it.
  • the transformed host cells are cultured under conditions suitable for expression of the nucleotide sequences encoding a polypeptide according to the invention.
  • the host cells used may be of prokaryotic origin, such as Escherichia coli (E. coli) or Bacillus subtilis, or of eukaryotic origin, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High ⁇ insect cells, immortalized mammalian cell lines (eg, HeLa cells or CHO cells) or primary mammalian cells.
  • polypeptides according to the invention can be prepared, for example, by means of a process comprising the following steps:
  • polypeptides according to the invention can also be produced synthetically.
  • Step (a) can be carried out with a nucleic acid molecule which codes only for the polypeptide. Alternatively, however, it can also be carried out with a nucleic acid molecule in which the polypeptide is operatively linked to a regulatory sequence.
  • the nucleic acid molecule according to the invention may also be linked to a fusion partner, such as an affinity epitope, which allows for easy purification and / or detection of the recombinant protein. Expression of the thus-cloned nucleic acid molecules may be in a recombinant cell or cell extract containing all the factors required for transcription and translation.
  • the invention also encompasses an antigen-binding peptide, in particular antibodies or fragments of antibodies, which specifically binds to the polypeptide according to the invention. Preference is given to monoclonal antibodies and corresponding F a t > - or scFv fragments, with which the polypeptides of the invention can be specifically detected.
  • the invention further encompasses a kit which comprises the polypeptide according to the invention, a nucleic acid molecule according to the invention, a vector containing it, at least one cell as described above, and / or the antigen-binding Contains peptide.
  • a kit is preferably used to detect the pathological isoform of a prion protein (PrP S0 ), for example in the form of an "ante mortem" rapid test for herd examination.
  • the invention likewise encompasses a pharmaceutical composition which comprises the polypeptide according to the invention, the nucleic acid molecule according to the invention, a vector containing it and / or the antigen-binding peptide, and preferably conventional excipients and / or excipients.
  • the pharmaceutical composition is preferably used for the therapeutic or preventive treatment of spongiform encephalopathies.
  • the object is further achieved according to the invention by a method of the type mentioned, in which at least the sequence motif of the polypeptide according to the invention is used as the biomolecule and in which the incubation takes place in the presence of a chelating substance, wherein the fraction of the sample with the bound pathological prion protein (PrP Sc ) is then separated from the fraction of bound normal prion protein sample (PrP 0 ).
  • the biomolecules bind specifically and very stably to the disease-specific isoform PrP Sc
  • the pathological form of the prion protein can be detected specifically and reliably.
  • the binding of the biomolecules to the PrP Sc is inducible by the addition of the chelating substance, ie by the trapping of metal ions.
  • the inventive method is thus selectively and selectively controllable.
  • the sequence motif of the polypeptides according to the invention binds PrP Sc with very high association constant, while PrP 0 is only weakly bound under these conditions, so that PrP Sc for example after differential washing, ie the treatment with a defined washing solution , can be specifically detected.
  • the washing should be carried out under harsh conditions, ie conditions under which bound PrP c is completely washed out, while the binding to the PrP Sc is retained.
  • the method according to the invention makes possible a reliable differentiation between the pathogenic isoform (PrP Sc ) and the normal host protein (PrP c ) and thus serves in particular for the diagnosis of spongiform encephalopathies, for example the detection of PrP Sc in blood samples.
  • a complexing agent for divalent cations preferably ethylenediaminetetraacetic acid (EDTA)
  • EDTA ethylenediaminetetraacetic acid
  • the method according to the invention is intended to carry out the separation of the fractions by washing with a solution containing 0.1 to 2.0% sodium dodecyl sulfate (SDS), preferably 0.5% SDS, and / or 1 1 to 10 mol / l contains urea.
  • SDS sodium dodecyl sulfate
  • the bound by the biomolecule prion protein is detected by the subsequent specific binding of a detectable molecule or that bound by the prion protein biomolecule by the subsequent specific binding of a detectable molecule, in particular of the antigen-binding peptide according to the invention , is demonstrated.
  • the detectable molecule is preferably a, in particular monoclonal, antibody or a fragment of an antibody that binds specifically to the prion protein or the biomolecule.
  • the detectable molecule can either bind specifically to the biomolecule or the prion protein.
  • the detection of the pathological prion protein (PrP sc ) preferably takes place by means of ELISA or dipstick technology.
  • the pathological prion protein can also be concentrated or purified, for example, by binding to the, preferably immobilized, biomolecule.
  • the biomolecule is in an advantageous embodiment of the
  • sequence motif of the polypeptide according to the invention can be used in immobilized form for the detection of the pathological isoform of a prion protein (PrP SG ) or for the purification of a sample infected with the pathological isoform of a prion protein (PrP sc ). Since the solubility of the sequence motif does not have any particular influence on the functionality of the sequence motif Peptide has, can be dispensed in appropriate applications on the fusion with a soluble peptide. Immobilization may be accomplished, for example, by coupling to a suitable column material, a membrane, Sepharose beads or the like. Peptides immobilized in this way can be used to detect prion diseases or to purify prion protein-infected material.
  • the most sensitive tests for prions include bioassays. These are so far only carried out in suitable animals, since cell culture assays for unknown reasons are so far unsuitable. Since the polypeptides or sequence motifs according to the invention stabilize the PrP Sc conformation, they can also be used, for example, as cofactors, in order to improve the sensitivity of bioassays and also to enable the performance of assays in cell cultures. For this purpose, polypeptides or sequence motifs according to the invention having nine or more octamers (preferably> 10OR) are added to the inoculum and then inoculated into suitable animals or cell cultures.
  • polypeptides or sequence motifs according to the invention have a shorter incubation time and can thus be diluted more highly (infective titre).
  • a particularly advantageous possibility of using the polypeptides according to the invention and / or the sequence motif of a polypeptide according to the invention is therefore an increase in the sensitivity of bioassays.
  • the polypeptide according to the invention and / or the sequence motif of the polypeptide according to the invention can also be used to detect a subpopulation of the pathological isoform of a prion protein (PrP sc ) or to purify a sample infected with a subpopulation of the pathological isoform of a prion protein (PrP sc ) be used.
  • PrP sc prion protein
  • at least some of the polypeptides according to the invention exclusively bind a small proportion of the total amount of proteinase K-stable PrP Sc in a sample.
  • the 16-octamer polypeptide of the present invention specifically binds a subpopulation of PrP Sc that is clearly correlated with infectivity. Consequently, that can In an advantageous embodiment of the invention, the polypeptide or sequence motif according to the invention can also be used for the specific detection of particular subpopulations of the PrP Sc .
  • GST glutathione-S-transferase
  • the extended octarepeat domain (expOR), i. the binding-specific sequence motif of a polypeptide according to the invention, which consists of 14 consecutive octamers, was composed and cloned from the following oligomers:
  • the "wild-type" -PrP-octarepeate (wtOR) fragment was amplified by PCR from the vector pET-11a (SyHaPrP (23-231)) and isolated from an agarose gel, and after extension from the agarose gel, extended
  • Clones were detected by automated fluorescence sequencing.
  • GST-OR fusion proteins and uncoupled GST were expressed in Escherichia coli BL21 ( ⁇ DE3).
  • Fresh transformed cells were grown in Luria-Bertoni medium until mid-log phase and induced with 1 mM isopropyl- ⁇ -D-thiogalactopyranoside. After 3 hours the cells were harvested, (30 min, 30 0 C) in 50 mM Tris pH 8, 2 mM EDTA, 1% NP-40, 0.1 mg / ml lysozyme lysed and followed by 30 minutes with DNase I, 5 mM MgCl 2 at 3O 0 C incubated.
  • the clarified lysate (30 min at 10,000 g) was loaded with a Glutathione Sepharose column (Amersham) washed with 50 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 0.2% Sarkosyl, 1 mM EDTA, and eluted with 50 mM Tris pH 8.5, 10 mM reduced glutathione (GSH), 2 mM dithiothreitol (DTT) at room temperature. All GST proteins were then treated with 50 mM lodoaetamide for 30 min at room temperature to block free cysteine residues in the GST segment.
  • the GST wtOR and GST expOR eluates were brought to 300 mM NaCl, clarified by filtration and loaded onto a Zn 2+ nitriloacetic acid agarose column (approximately 1 and 5 mg protein per ml beads for GST-expOR resp GST-wtOR).
  • the metal affinity column was washed with 20 mM Tris pH 8, 5 mM imidazole, 300 mM NaCl and eluted with 20 mM Tris pH 8, 200 mM imidazole, 300 mM imidazole.
  • GST-wtOR and GST-expOR were dialysed twice against ultrapure water (1: 100, SnakeSkin, Pierce).
  • GST, GST-WTOR and GST-Expor were in 50 mM NaHCO 3, pH 8.3, 1% NP40 (0.1-0.5 mg / ml protein, 4 mg protein per ml beads) for 2 hours at room temperature covalently attached to NHS-activated Sepharose ( Amersham) coupled.
  • the "beads” were blocked in 50 mM hydroxylamine, pH 7.5 (30 min, room temperature), and washed successively with 50 mM Tris, pH 8.8, 50 mM Na acetate, pH 4.6, 50 mM HEPES, pH 7.5, 1% sarcosyl, 2 mM EDTA and then 50 mM HEPES, pH 7.5, 300 mM NaCl, 0.6% NP-40, 0.3% Sarkosyl.
  • Brain homogenates from healthy or scrapie-infected hamsters were incubated in 50 mM HEPES / Na acetate, pH 7.5 / 4.6, 300 mM NaCl, 0.6% NP40, 0.3% sarcosyl (buffer B / 7.5 or B / 4.5) to 1% ( w / v), added with protease inhibitors (Roche), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0-200 ⁇ M CuSO 4 VZnSO 4 or 5 mM EDTA (final concentrations) and clarified at 10,000 g.
  • PMSF protease inhibitors
  • FIG. 1 shows a Western blot analysis of the binding of PrP c by GST wtOR fusion peptides ("wild-type") and polypeptides according to the invention (GST-expOR fusion peptides) as a function of the metal ion concentration at physiological (A and B) and low (D) pH and the dissolution of the bond (C).
  • FIG. 2 shows a Western blot analysis of the binding of PrP Sc by GST wtOR fusion peptides ("wild-type") and polypeptides according to the invention (GST-expOR fusion peptides) as a function of the presence of metal ions at physiological (A) and low ( B) pH.
  • FIG. 3 shows a graphic summary of FIG
  • Binding properties of the GST-OR fusion peptides for "wild type” (A) and polypeptides (B) according to the invention Binding properties of the GST-OR fusion peptides for "wild type” (A) and polypeptides (B) according to the invention.
  • FIG. 4 shows a Western blot analysis for determining the critical length of the sequence motif which is necessary for the stable binding of the polypeptides according to the invention to PrP Sc in the presence of EDTA.
  • FIG. 5 shows a Western blot for detecting the differential washout of PrP 0 under conditions in which PrP Sc of terminally ill hamsters remains bound to immobilized polypeptides according to the invention.
  • Figure 6 shows a Western blot for detecting differential washout of PrP c under conditions where PrP Sc of healthy hamsters remains bound to immobilized polypeptides of the invention 42 days after infection.
  • FIG. 7 shows a western blot of a peptide library for mapping potential binding sites for the polypeptide of the invention in FIG. 7
  • FIG. 9 shows a Western blot analysis for the detection of the specific
  • PrP Sc with GST-16OR bound to Sepharose beads, PK proteinase K).
  • FIG. 1 Binding of native PrP 0 by immobilized polypeptides as a function of metal ion concentration and pH.
  • C binding of PrP c by normal (PrP c ) mouse PrP (GST-MoPrP (52-98) coupled to GSH-Sepharose and polypeptide of the invention with 14 octamers (GST-MoPrP-14OR (52-98)) in 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 0.5% DOC, 0-200 .mu.M ZnSO4 and peeling of the bound PrP with 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.3% Sarkosyl, 10 mM EDTA.
  • Lanes 1-3 Brain extract and Sepharose-coupled GST, GST-MoPrP (52-98) and GST-MoPrP-14OR (52-98) were preincubated with copper at pH 7.5 and then combined in acetate buffer at pH 4.6.
  • FIG. 2 Binding of PrP sc by immobilized polypeptides as a function of the pH and the addition of protease.
  • MoPrP (52-98), wt) and a polypeptide according to the invention with 14 octamers (GST-MoPrP-14OR (52-98), exp) in 50 mM HEPES 1 pH 7.5, 300 mM NaCl, 0.6% NP40, 0.3% Sarkosyl 5 mM EDTA or 200 ⁇ M CuSO 4 .
  • the bound material was detached by boiling the "beads" in SDS-PAGE sample buffer immediately (below, -PK) or after digestion with proteinase K (above, + PK).
  • sequence motif of the polypeptides according to the invention (14OR) binds the pathological PrP So in the presence of EDTA or in the absence of metal ions or copper at physiological pH independently of digestion with protease , It can also be seen that the binding of the 14OR polypeptide according to the invention to PrP Sc stronger than the bond between PrP So and the normal 40R peptide, as this is resistant to digestion with protease.
  • Figure 3 Summary of the binding of PrP c and PrP sc under different conditions.
  • the binding sequence motif is a kind of switch that controls the binding properties of PrP 0 and PrP So as a function of metal ion concentration and pH.
  • the polypeptide or sequence motif can assume different positions or conformations: NULL (pH -4.5), OFF (without Cu / Zn, pH 7.5)) or ON (with Cu / Zn, pH 7.5).
  • the sequence motif PrP c or PrP Sc can bind either weakly (+/-), strongly (+) or not at all (-).
  • A normal peptide (4 + 1 octamer)
  • B polypeptide according to the invention or sequence motif (14 octamer)
  • Figure 4 Determination of the critical length of the sequence motif, which is necessary for the precipitation of PrP SG in the presence of EDTA.
  • Sepharose "beads” were covalently linked to polypeptides having a different number of octamers, here GST-wtOR (HaPrP (52-98), about 5 mg / ml), GST-8OR, GST-10OR or GST-16OR, 1 ml of 1% (w / v) scrapie-infected (263K strain pre-purified at 10,000 xg, 5 min) brain homogenate of a terminally diseased Syrian hamster was then cross-linked with Sepharose octarepeal beads in 50 mM HEPES 1 pH 7.5, 300 mM NaCl, 0.5% NP40, 0.5% Sarkosyl, 5 mM EDTA and 0.2 mM CuSO 4 incubated (12h at 4 0 C).
  • Sepharose-beads were then washed in the Inkubationspufffer and in a fraction with protease (20 ⁇ g / ml, 37 ° C., 30 min) and one that was treated without protease, then the samples were analyzed by SDS-PAGE and Western blotting (gel: 12.5%, detected with mAb 3F4). It is clear here that a critical length of at least 9 octamers is necessary to sufficiently bind PrP Sc , but preferably more than 10 octamers or octarepeats, especially 16 octamers or more.
  • FIG. 5 Differential washing out of PrP 0 under conditions in which PrP Sc of terminally ill hamsters remains bound to immobilized polypeptides according to the invention.
  • Wash buffer (elution conditions): 1. 10 M urea pH3.7
  • S supernatant
  • PrP Sc remains immobilized
  • P pellet
  • FIG. 6 Differential washing out of PrP c under conditions in which PrP Sc of healthy hamsters remains bound to immobilized polypeptides according to the invention after 2/3 of the incubation time.
  • GST-160R (approximately 4 mg / ml) was incubated with 20 ⁇ l of Sepharose "Beads" overnight in 1 ml of 1% normal hamster hoim homogenate or hamster brain homogenate at 42 days (60 days) after inoculation with prions (ie hamsters are still neurologically inconspicuous, apparently “healthy") incubated (incubation buffer: 50 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM EDTA, 0.6% NP40, 0.3% sarcosyl) . Then the "beads” were washed 3 times with incubation buffer and then incubated with 60 ⁇ l of elution buffer (20 mM HEPES, pH 7.5, 1
  • FIG. 7 Mapping of the potential binding sites for the polypeptide according to the invention in the octarepeat region of PrP
  • a mapping of potential binding sites for the polypeptide according to the invention in the octarepeate region of the prion protein was carried out with the aid of a peptide bank ("gridded array of peptides", Jerini (Berlin)) in the form of a cellulose membrane on which C-terminal 13mer peptides of the prion protein of the Syrian Hamsters are immobilized, which sequentially cover the entire PrP amino acid sequence, ie a total of 121 peptides, performed. This membrane was used as an immunoblot.
  • GST alone (negative control), or a polypeptide of the invention (GST-14OR) was dissolved in a concentration of 40 ug / ml together with rabbit anti-GST serum (1: 5000) overnight at 4 0 C incubated, then washed and developed for 30 minutes with a goat anti-rabbit POD antibody and ECL / Hyperfilm (Amersham).
  • PrP Sc was precipitated with immobilized polypeptides of the invention (GST-OR) with different numbers of octamers from brain extracts of scrapie-infected hamsters (ScHa).
  • GST-OR immobilized polypeptides of the invention
  • ScHa brain extracts of scrapie-infected hamsters
  • brain homogenate (20% w / v) of ScHa with binding buffer containing either 50-200 mM CuSO 4 ZZnSO 4 or 5 mM EDTA was diluted to 1% and then clarified by centrifugation.
  • Sepharose beads covalently coupled to GST-OR the polypeptides being four (GST-4OR), eight (GST-8OR), ten (GST-10OR) or sixteen (GST-16OR ) Contained octamers.
  • the Sepharose beads were washed and either boiled directly or previously digested with 20 ⁇ g / ml proteinase K (PK; Merck) for 1 hour at 37 ° C in binding buffer with 5 mM EDTA (stopped with 5 mM PMSF). The samples were separated in a 12.5% SDS gel and the Western blot was developed with ⁇ -PrP mAB 3F4.
  • Figure 8 shows a clear limit of PrP Sc binding by the polypeptides of the invention between eight and ten octamers. Copper inhibits binding to PrP Sc in both GST-10OR and GST-16OR.
  • FIG. 9 Detection of the specific binding of a polypeptide according to the invention to a subpopulation of PrP Sc .
  • polypeptide GST-16OR according to the invention or the sequence motif 16OR is therefore specific for a specific type or subpopulation of the prP Sc and, consequently, in an advantageous embodiment of the invention can also be used for the detection or specific binding of at least one particular, infectious subpopulation of the PrP Sc be used.

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Abstract

L'invention concerne des polypeptides présentant au moins un motif de séquence, se liant spécifiquement à l'isoforme pathologique d'une protéine de prion, l'utilisation de ces polypeptides et un procédé de liaison spécifique de l'isoforme pathologique d'une protéine de prion (PrP<sup
PCT/DE2005/001574 2004-09-08 2005-09-08 Polypeptides et procede de liaison specifique de prions Ceased WO2006026977A2 (fr)

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EP1705182A4 (fr) * 2004-01-15 2008-03-12 Sergey Ivanovich Chernysh Peptides antitumoraux et antiviraux
US7439041B2 (en) 2003-08-13 2008-10-21 Novartis Vaccines And Diagnostics, Inc. Prion-specific peptide reagents
US7834144B2 (en) 2005-09-09 2010-11-16 Novartis Ag Prion-specific peptoid reagents
CN116953224A (zh) * 2023-05-31 2023-10-27 复旦大学附属华山医院 一种检测fgf21的发光试剂及其应用

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DE19741607A1 (de) * 1997-09-20 1999-03-25 Prionics Ag Synthetische Polypeptide zur Diagnose und Therapie von Prionerkrankungen

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LELIVELD SIRIK RUTGER ET AL: "The expanded octarepeat domain selectively binds prions and disrupts homomeric prion protein interactions." THE JOURNAL OF BIOLOGICAL CHEMISTRY. 10 FEB 2006, Bd. 281, Nr. 6, 10. Februar 2006 (2006-02-10), Seiten 3268-3275, XP002377178 ISSN: 0021-9258 *
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TODOROVA-BALVAY D ET AL: "Copper binding to prion octarepeat peptides, a combined metal chelate affinity and immunochemical approaches" JOURNAL OF CHROMATOGRAPHY B: BIOMEDICAL SCIENCES & APPLICATIONS, ELSEVIER, AMSTERDAM, NL, Bd. 818, Nr. 1, 15. April 2005 (2005-04-15), Seiten 75-82, XP004755758 ISSN: 1570-0232 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7439041B2 (en) 2003-08-13 2008-10-21 Novartis Vaccines And Diagnostics, Inc. Prion-specific peptide reagents
EP1705182A4 (fr) * 2004-01-15 2008-03-12 Sergey Ivanovich Chernysh Peptides antitumoraux et antiviraux
US8372406B2 (en) 2004-01-15 2013-02-12 Sergey Chernysh Antitumoral and antiviral peptides
US7834144B2 (en) 2005-09-09 2010-11-16 Novartis Ag Prion-specific peptoid reagents
CN116953224A (zh) * 2023-05-31 2023-10-27 复旦大学附属华山医院 一种检测fgf21的发光试剂及其应用

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