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Ernst Habermann - One of the best experts on this subject based on the ideXlab platform.
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Limited Proteolysis of single-chain tetanus toxin by tissue enzymes, in cultured brain tissue and during retrograde axonal to the spinal cord
Naunyn-Schmiedeberg's Archives of Pharmacology, 1991Co-Authors: Ernst Habermann, U. Weller, M. HudelAbstract:Single-chain toxin was investigated in vitro and in vivo for Limited Proteolysis into the fully active two-chain toxin. Plasmin from serum, elastase and gelatinase from leucocytes, as well as clostripain from C. histolyticum cleaved single-chain toxin and increased by that way its ability to inhibit [^3H]noradrenaline release in vitro. Cultured mouse brain generated fragments from ^125I-single-chain toxin which were cell-associated. Some of them comigrated in electrophoresis with light and heavy chain after mercaptolysis. When injected i. v. into rats, ^125I-single-chain-toxin disappeared from the blood with a half-life of about 11 h without signs of nicking. However, after its injection into the triceps surae muscle both single- and two-chain toxin were found in the ipsilateral ventral horn of the spinal cord. Thus single-chain toxin is subjected to Limited Proteolysis by enzymes involved in tissue damage, by cultured brain tissue, and during or after its retrograde axonal transport to the spinal cord. Limited Proteolysis is necessary for the release of the light chain known to mediate the action of toxin on several systems.
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Limited Proteolysis of tetanus toxin Relation to activity and identification of cleavage sites
European Journal of Biochemistry, 1991Co-Authors: Kerstin G. Krieglstein, Agnes Henschen, Ulrich Weller, Ernst HabermannAbstract:Tetanus toxin is synthesized by Clos/ridium tetarii as a 151-kDa peptide chain. The primary gene product is processed post-translationally by removal of the initiating methionine residue, formation of disulfide bridges and Limited Proteolysis by bacterial or exogenous proteinases. The mature toxins consist of a 52-kDa light chain and a 98-kDa heavy chain, linked together by a disulfide bond. Proteolytic nicking is accompanied by increased pharmacological potency. To identify the structural alterations involved, single-chain toxin has been subjected to Limited Proteolysis with various enzymes. The new N-termini have been determined by Edman degradation and the C-termini by isolation of short C-terminal peptide fragments and subsequent analysis of the sequence and composition. All two-chain toxins result from proteolytic nicking within the 17-residue segment of residues 445 461. Thus, the protease(s) of the culture broth cleave on the C-terminal side of Glu449 and partially AIa456. giving rise to two heavy chain N-termini. Trypsin and clostripain first attack the C-terminal of Arg454 and later Arg448, whereas endoproteinase Arg-C cleaves the former bond only. Chymotrypsin and endoproteinase Glu-C each split a single peptide bond, i.e. that located after Tyr452 and (3111449, respectively. Papain gives rise to a large number of cleavages within the 17-residue segment, the new C-terminus being Thr445 or Asn446 and the new N-terminus being Asp460 or Leu461. Further papain digestion leads to an additional cleavage within the heavy chain between Ser863 and Lys864. The original N-terminal Pro1 and C-terminal Asp1314, predicted from the nucleotide sequence, are conserved in all proteolytic digests. The pharmacological activity of the various twochain toxins was 5 - 11 times that of the single-chain toxin, as estimated from the inhibition of [3H]noradrenaline release from rat-brain homogenate. The present data on the processing and activation by Limited Proteolysis prove the existence of several active tetanus isotoxins. These data, together with our previous data on the localization of disulfide bridges and sulfhydryl groups (Krieglstein, K., Henschen, A,, Weller, U. & Habermann, E. (1990) Eur. J. Biochem. 188, 39 -45), provide the detailed protein chemical characterization of the tetanus isotoxins.
Jesus R Requena - One of the best experts on this subject based on the ideXlab platform.
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scrapie prion protein structural constraints obtained by Limited Proteolysis and mass spectrometry
Journal of Molecular Biology, 2008Co-Authors: Gustavo Sajnani, Miguel A Pastrana, Irina Dynin, Bruce Onisko, Jesus R RequenaAbstract:Abstract Elucidation of the structure of scrapie prion protein (PrP Sc ), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrP Sc . Limited Proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in β-strands. We treated PrP Sc isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrP Sc was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and Limited Proteolysis after partial unfolding of PrP Sc confirmed that only the mentioned cleavage sites at the N-terminal side of the PrP Sc are susceptible to PK. Our results indicate that besides the “classic” amino-terminal PK cleavage points, PrP Sc contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short β-sheet stretches connected by loops and turns.
M. Hudel - One of the best experts on this subject based on the ideXlab platform.
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Limited Proteolysis of single-chain tetanus toxin by tissue enzymes, in cultured brain tissue and during retrograde axonal to the spinal cord
Naunyn-Schmiedeberg's Archives of Pharmacology, 1991Co-Authors: Ernst Habermann, U. Weller, M. HudelAbstract:Single-chain toxin was investigated in vitro and in vivo for Limited Proteolysis into the fully active two-chain toxin. Plasmin from serum, elastase and gelatinase from leucocytes, as well as clostripain from C. histolyticum cleaved single-chain toxin and increased by that way its ability to inhibit [^3H]noradrenaline release in vitro. Cultured mouse brain generated fragments from ^125I-single-chain toxin which were cell-associated. Some of them comigrated in electrophoresis with light and heavy chain after mercaptolysis. When injected i. v. into rats, ^125I-single-chain-toxin disappeared from the blood with a half-life of about 11 h without signs of nicking. However, after its injection into the triceps surae muscle both single- and two-chain toxin were found in the ipsilateral ventral horn of the spinal cord. Thus single-chain toxin is subjected to Limited Proteolysis by enzymes involved in tissue damage, by cultured brain tissue, and during or after its retrograde axonal transport to the spinal cord. Limited Proteolysis is necessary for the release of the light chain known to mediate the action of toxin on several systems.
David Gil - One of the best experts on this subject based on the ideXlab platform.
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recombinant prpsc shares structural features with brain derived prpsc insights from Limited Proteolysis
PLOS Pathogens, 2018Co-Authors: Alejandro M Sevillano, Natalia Fernandezborges, Neelam Younas, Fei Wang, Saioa R Elezgarai, Susana B Bravo, Ester Vazquezfernandez, Isaac Rosa, Hasier Erana, David GilAbstract:Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used Limited Proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited Proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.
Gustavo Sajnani - One of the best experts on this subject based on the ideXlab platform.
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scrapie prion protein structural constraints obtained by Limited Proteolysis and mass spectrometry
Journal of Molecular Biology, 2008Co-Authors: Gustavo Sajnani, Miguel A Pastrana, Irina Dynin, Bruce Onisko, Jesus R RequenaAbstract:Abstract Elucidation of the structure of scrapie prion protein (PrP Sc ), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrP Sc . Limited Proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in β-strands. We treated PrP Sc isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrP Sc was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and Limited Proteolysis after partial unfolding of PrP Sc confirmed that only the mentioned cleavage sites at the N-terminal side of the PrP Sc are susceptible to PK. Our results indicate that besides the “classic” amino-terminal PK cleavage points, PrP Sc contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short β-sheet stretches connected by loops and turns.