Abstract
(Deutsch)
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Pathophysiologische Untersuchungen zur Interaktion zwischen PrPSc und Plasminogen bzw. verwandten Proteinen: Diese Forschungsarbeiten finden im Rahmen einer auf 3 Jahre geplanten Dissertation statt. Sie beschäftigte sich mit dem Nachweis, dass Plasminogen nicht nur - wie bei Fischer et al. 2000 gezeigt - PrPSc aus Hirnhomogenate von Mäusen, sondern auch PrPSc aus Hirnhomogenaten von Schafen, Kühen, und Menschen interagiert, nicht jedoch mit PrPC derselben Hirnhomogenate. Zudem soll untersucht werden, ob Plasminogen-Beads als Abreicherungs-Tool für PrPSc in Lösung verwendet werden können. Weiter soll untersucht werden, ob Plasminogen die 3 verschiedenen Glycoformen von PrPSc mit derselben Affinität bindet. Es wird untersucht, ob der von der Forschungsgruppe entwickelte "Plasminogen Affinity Assay" (PAA) die Glykosilierungsmuster der untersuchten PrPSc-Proben mit gleicher Zuverlässigkeit wiedergibt, wie der dazu klassischerweise verwendete Westernblot nach Proteinase-K Verdau. Ein detaillierter Arbeitsbericht zu diesen Resultaten steht zur Verfügung - entsprechende wissenschaftliche Publikationen sind geplant.
Schlussbeurteilung zum Vertrag 02.001233 (01.12.2002 - 30.11.2003) von Adriano Aguzzi, Pathophysiologische Untersuchungen zur Interaktion zwischen PrP(sc) und Plasminogen bzw. verwandten Proteinen
"Executive Summary' of Manuela Maissen PhD thesis"
Human plasminogen was the first reagent found which was reliably able to discriminate between the pathological folded form of the prion protein (PrPSc) and the cellular protein (PrPC) (Fischer, Roeckl et al. 2000). Due to the astonishing efficiency with which solid phase-bound plasminogen precipitate PrPSc, we reasoned that it could be of interest to exploit this property for diagnostic tests. Current transmissible spongiform encephalopathy (TSE) test assays require pre-digestion of the sample with proteinase K which not only decreases the sensitivity of the assays but also increases their complexity and impairs their amenability to automation. As hplg precipitates specifically PrPSc, PK treatment is unnecessary. Furthermore, due to the precipitation the sensitivity of the assay is increased as PrPSc is enriched. This phenomena might form the basis for a diagnostic assay; however, further characterization of the biochemical properties of the binding are essential.
One main question was whether hplg is able to precipitate PrPSc from other instances of transmissible spongiform encephalopathies as it would be of high importance to develop a highly sensitive screening test which would be a standard assay for all different types of TSE. Therefore, we performed plasminogen-mediated precipitations followed by Western blotting on brain homogenates of patients with sporadic CJD, as well as of sheep with scrapie and of cows of various breeds with BSE and could show that plasminogen interacts with the disease-associated form but not cellular form of several species (murine, bovine, ovine and human) (Maissen, Roeckl et al. 2001).
To get some insight into the interaction of plasminogen and PrPSc, we tested several recombinant plasminogen fragments consisting of different kringle domains for their ability to bind to PrPSc but not PrPC. Judging form the results we got, it seems that kringle I of plasminogen is sufficient for the interaction with PrPSc but not PrPC as the plasminogen fragment consisting of kringle I and V bound PrPSc but the fragment consisting of kringle V only did not. These results might be helpful at some point for the design of artificial plasminogen analogue with optimised PrPSc capturing properties.
Several human PrPSc types, which are observed on Western blot after limited PK digestion, resulting in PrP fragment of differing molecular mass and band intensities, have been identified and associated with different phenotypes of CJD (Parchi, Giese et al. 1999). Therefore preliminary classification of sporadic CJD bases on clinical and pathological criteria as well as molecular subtypes of PrPSc. In case, the interaction between the pathologically folded form of the prion protein and plasminogen is used as a diagnostic tool one day, we considered it important to investigate whether each glycoform of the infectious prion protein has got the same affinity for plasminogen. Therefore, we assessed whether performing the Plasminogen Affinity Assay (PAA) on sporadic CJD or vCJD brain homogenates would lead to the same PrPSc glycoform pattern as after Proteinase K treatment. Quantification of the relative intensities of the high molecular mass and low molecular mass glycoforms seen on Western blot after PAA or proteinase K treatment showed little variations. From these results we conclude that using plasminogen for diagnostic applications would not lead to any loss of information about the TSE strain.
Due to the relative low sensitivity of current TSE tests, it cannot be excluded that contaminated material is processed into dietary and pharmaceutical products. Besides the food and pharmaceutical industry, suppliers of blood products are interested in methods for prion removal. Because beads coated with plasminogen pull out PrPSc and infectivity from murine brain homogenate, it might be possible to remove prions by exposing the potentially contaminated material to a solid phase covered with plasminogen or plasminogen derivates with augmented PrPSc affinity. To investigate this issue, we incubated infectious murine brain homogenate (RML5) several times with plasminogen or BSA coupled beads and subsequently inoculated the supernatants into tga20 mice. We expected the mice inoculated with supernatants of the BSA-beads-assay would succumb to scrapie as the BSA-beads should bind no infectivity. On the other hand, the mice inoculated with dilutions of the supernatants of the plasminogen-beads-assay should not die in case plasminogen would pull all infectivity out of the infectious brain homogenate. We found that we were able to reduce the infectivity by incubating the infectious brain homogenates five times with plasminogen beads by about 4 logs. The 1:5 diluted supernatants of the assay with plasminogen beads contained some border line infectivity which might have been removed by an additional incubation with plasminogen beads meaning that plasminogen seems to be able to bind all forms of PrPSc.
In summary, one might conclude from the results we got so far that plasminogen has some potential to be exploited as a PrPSc-binding protein for diagnostic purposes and as a prion-binding agent to reduce the infectious load of contaminated material.
In patients with sporadic Creutzfeldt-Jakob disease (CJD), PrPSc has been identified only in the central nervous system and olfactory-nerve tissue. However, it has been reported that in patients with variant CJD (vCJD), PrPSc accumulates in tonsils and other lymphoreticular organs (Hill, Zeidler et al. 1997; Wadsworth, Joiner et al. 2001). Refinements in the sensitivity of the PrPSc detection methods (Safar, Wille et al. 1998) prompted us to reinvestigate the distribution of PrPSc in humans with sporadic CJD. We used a highly sensitive detection method (NaPTA precipitation) - involving the concentration of PrPSc by differential precipitation with sodium phosphotungstic acid (NaPTA), which increased the sensitivity of Western blot analysis by up to three orders of magnitude - to search for PrPSc in extraneural organs of 36 patients with sporadic Creutzfeldt-Jakob disease who died between 1996 and 2002. Of 28 spleen samples analysed, 10 contained PrPSc, as did 8 of 32 skeletal muscle samples. PrPSc was found in both spleen and muscles of 3 people. Additionally, PrPSc accumulation did not seem to be specific to particular muscle groups (Glatzel, Abela et al. 2003). Concluding from these results, it seems that extraneural deposits of PrPSc are much more frequent in patients with sporadic CJD than previously thought, what might have some biosafety implications. Furthermore, one could suggest that muscle biopsies could be used as specific, and relatively non-invasive, diagnostic tests for sCJD. However, although a positive result would be definitive, a negative result would be inconclusive, because less than a third of the samples tested positive for PrPSc in muscles.
Fischer, M. B., C. Roeckl, et al. (2000). "Binding of disease-associated prion protein to plasminogen." Nature 408(6811): 479-83.
Glatzel, M., E. Abela, et al. (2003). "Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease." N Engl J Med 349(19): 1812-20.
Hill, A. F., M. Zeidler, et al. (1997). "Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy." Lancet 349: 99.
Maissen, M., C. Roeckl, et al. (2001). "Plasminogen binds to disease-associated prion protein of multiple species." Lancet 357(9273): 2026-8.
Parchi, P., A. Giese, et al. (1999). "Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects." Ann Neurol 46(2): 224-33.
Safar, J., H. Wille, et al. (1998). "Eight prion strains have PrP(Sc) molecules with different conformations " Nat Med 4(10): 1157-65.
Wadsworth, J. D. F., S. Joiner, et al. (2001). "Tissue distribution of protease resistant prion protein in variant CJD using a highly sensitive immuno-blotting assay." Lancet 358: 171-180.
The research work for the PhD-thesis contributed significantly to the following publications:
Maissen M, Roeckl C, Glatzel M, Goldmann W, Aguzzi A.(2001)."Plasminogen binds to disease-associated prion protein of multiple species." Lancet. 357(9273): 2026-8.
Glatzel M, Abela E, Maissen M, Aguzzi A. (2003). "Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease" N Engl J Med. 349(19): 1812-20.
Praus M, Kettelgerdes G, Baier M, Holzhutter HG, Jungblut PR, Maissen M, Epple G, Schleuning WD, Kottgen E, Aguzzi A, Gessner R. (2003). "Stimulation of plasminogen activation by recombinant cellular prion protein is conserved in the NH2-terminal fragment PrP23-110" Thromb Haemost. 89(5): 812-9
Meier P, Genoud N, Prinz M, Maissen M, Rulicke T, Zurbriggen A, Raeber AJ, Aguzzi A.(2003). "Soluble dimeric prion protein binds PrP(Sc) in vivo and antagonizes prion disease." Cell. 113(1): 49-60.
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