NR ARCX
AU Epple,G.; Geßner,R.; Praus,M.
TI Interaction of the Cellular Prion Protein with the Fibrinolytic System
QU TSE-Forum, 4. Kongress - Nationale TSE-Forschungsplattform, Düsseldorf 28.10.-29.10.2004, Vortrag V-14
PT Konferenz-Vortrag
AB
The pathological conformer of the human prion protein binds tightly to human plasminogen. The binding is, however, not specific for the pathological conformer. Interaction of the cellular form, PrPc, with t-PA, a fibrin-cofactor dependent plasminogen activator, and plasminogen stimulates t-PA mediated plasmin generation. We have shown that this stimulating activity is conserved in the NH2-terminal plasmatic cleavage product PrP23-110. Cleavage at lysine residue 110 appears to be part of the physiological turnover of PrPc, as it has been shown to occur naturally in post mortem specimens of healthy individuals.
PrP23-110 contains clusters of lysine residues (K23,24,27 und K101,104,106,110) at both ends. Both clusters are involved in the binding of PrPc to t-PA and plasminogen and must be present in order to stimulate plasminogen activation. To further study the molecular mechanism we compared the stimulatory effect of PrPc on plasmin generation by different plasminogen activators, that have a distinct composition of functional domains and are unique with respect to their cofactor-requirements. Using plasmon resonance technology to study the protein / protein interaction directly, we can show that PrPc binds to plasminogen activators that carry kringle domains. Binding of PrP to kringle domains is however not sufficient to stimulate plasmin generation as only t-PA that carries a lysine binding site in the kringle two domain is stimulated by PrP.
Using plasmon resonance technology we could show, that recombinant PrP23-110 binds to itself and forms aggregates in solution. The involvement of the N-terminus in PrP self-association has also been shown by others and could serve as a model for the initiation of prion aggregates. We have identified the glycosamino-glycan, heparin, as a necessary cofactor for efficient stimulation of plasmin generation by PrP23-110. Heparin also alters the binding properties of PrP23-110 to itself, suggesting that it alters the prion aggregates in a way that the lysine clusters become accessible for the interaction with t-PA and plasminogen. Using this functional assay we have tested various substances, that have been described in cell culture assays to posses the ability to cure PrPsc infection. From the substances tested in our activity assay, only pentosan polysulfate had a reproducibly stronger stimulatory effect compared to heparin. Pentosan polysulfate is also the only substance with a reproducible anti prion effect in vivo. Thus this plasmin generation assay might prove to be a useful tool to screen large substance libraries for anti-prion agents.
To study the interaction of PrPc with the fibrinolytic system in a more complex model we have stably transfected 293 cells with human PrPc. Using this model we can show that PrPc expressing cells display a higher binding affinity for t-PA coated fluorenscent-beads than mock transfected cells. The binding could be specifically inhibited by a monoclonal antibody directed towards PrPc. We also studied the effect of PrPc expression on the stimulation of plasmin generation in cell culture. Plasmin generation at a physiological t-PA concentration of 70 pM was higher in PrP expressing compared to mock transfected 293 cells.
In conclusion our data support our general hypothesis, that PrPc is involved in the regulation of cellular proteolysis in the central nervous system.
Praus et al. Stimulation of Plasminogen Activation is Conserved in the NH2-terminal fragment PrP23-110. Thromb Haemostasis 2003;89:812
Epple et al. Both Lysine Clusters of the NH2-terminal Prion Protein Fragment PrP23-110 are Essential for t-PA mediated Plasminogen Activation. Thromb Haemostasis 2004;91:465
Epple et al. Prion Protein Stimulates t-PA mediated Plasmin Generation via a Lysine Binding Site in Kringle 2. J Thromb Haemostasis 2004;2:962-8
AD Guido Epple, Reinhard Geßner, Michael Praus, Institut für Laboratoriumsmedizin und Pathobiochemie, Charité Universitätsmedizin Berlin, Campus Virchow Klinikum, Augustenburger Platz 1, 13353 Berlin
SP englisch
PO Deutschland
OR Tagungsband