Both lysine-clusters of the NH2-terminal prion-protein fragment PrP23-110 are essential for t-PA mediated plasminogen activation

Guido Epple; Kristina Langfeld; Michael Baier; Hermann-Georg Holzhütter; Wolf-Dieter Schleuning; Eckart Köttgen; Reinhard Geßner; Michael Praus

doi:10.1160/TH03-06-0382

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 91(03): 465-472
DOI: 10.1160/TH03-06-0382

Theme Issue Article

Schattauer GmbH

Both lysine-clusters of the NH₂-terminal prion-protein fragment PrP23-110 are essential for t-PA mediated plasminogen activation

Authors

Guido Epple

¹Institut für Laboratoriumsmedizin und Pathobiochemie, Charité Berlin, Medizinische Fakultät der Humboldt-Universität zu Berlin, Berlin, Germany
Kristina Langfeld

¹Institut für Laboratoriumsmedizin und Pathobiochemie, Charité Berlin, Medizinische Fakultät der Humboldt-Universität zu Berlin, Berlin, Germany
Michael Baier

²Robert-Koch-Institut, Berlin, Germany
Hermann-Georg Holzhütter

³Institut für Biochemie, Charité, Medizinische Fakultät, Humboldt-Universität zu Berlin, Berlin, Germany
Wolf-Dieter Schleuning^*

⁴Schering Research Laboratories, Berlin, Germany
Eckart Köttgen

¹Institut für Laboratoriumsmedizin und Pathobiochemie, Charité Berlin, Medizinische Fakultät der Humboldt-Universität zu Berlin, Berlin, Germany
Reinhard Geßner

¹Institut für Laboratoriumsmedizin und Pathobiochemie, Charité Berlin, Medizinische Fakultät der Humboldt-Universität zu Berlin, Berlin, Germany
Michael Praus

¹Institut für Laboratoriumsmedizin und Pathobiochemie, Charité Berlin, Medizinische Fakultät der Humboldt-Universität zu Berlin, Berlin, Germany

Abstract

Summary

We have recently shown that the NH₂-terminal fragment (PrP23-110) of the human cellular prion protein (PrPc) stimulates t-PA mediated plasminogen activation. PrP23-110 contains an N-terminal lysine cluster (LC1; K₂₃, K₂₄, K₂₇) and a C-terminal one (LC2; K₁₀₁, K₁₀₄, K₁₀₆, K₁₁₀). To study their biological function we have substituted all lysine residues of each cluster by alanine and generated the recombinant PrP proteins PrP23110sLC1 and PrP23-110sLC2. The ability of the mutant proteins to stimulate plasminogen activation was assayed. We found that both lysine clusters are essential for t-PA mediated plasminogen activation. We further studied the binding of soluble PrP23110 to immobilized t-PA or plasminogen using surface plasmon resonance. The recorded binding curves could not be modeled by classical 1:1 binding kinetics suggesting oligomerisation of PrP23-110. Further plasmon resonance studies show that indeed PrP23-110 binds to itself and that glycosaminoglycans modify this interaction. Binding of t-PA or plasminogen to PrP23-110 was no longer influenced by glycosaminoglycans when PrP23-110 was immobilized on the chip surface. Thus a possible role of heparin as a cofactor in the stimulation of plasminogen activation by t-PA could be the generation of a PrP23-110 form with both lysine clusters accessible for binding of t-PA and plasminogen.

Keywords

Plasminogen activators - proteolysis/pericellular - heparins/glycosaminoglycans - protein function/activity

Full Text

References

References
1 Ellis V, Daniels M, Misra R. et al.. Plasminogen activation is stimulated by prion protein and regulated in a copper-dependent manner. Biochemistry 2002; 41 (22) 6891-6.
2 Praus M, Kettelgerdes G, Baier M. et al.. Stimulation of plasminogen activation by recombinant cellular prion protein is conserved in the NH(2)-terminal fragment PrP23-110. Thromb Haemost 2003; 89 (05) 812-9.
3 Chen SG, Teplow DB, Parchi P. et al.. Truncated Forms of the Human Prion Protein in Normal Brain and in Prion Diseases. J Biol Chem 1995; 270 (32) 19173-80.
4 Vincent B, Paitel E, Saftig P. et al.. The Disintegrins ADAM10 and TACE Contribute to the Constitutive and Phorbol Ester-regulated Normal Cleavage of the Cellular Prion Protein. J Biol Chem 2001; 276 (41) 37743-6.
5 Madani R, Hulo S, Toni N. et al.. Enhanced hippocampal long-term potentiation and learning by increased neuronal expression of tissue-type plasminogen activator in transgenic mice. Embo J 1999; 18 (11) 3007-12.
6 Hayden SM, Seeds NW. Modulated expression of plasminogen activator system components in cultured cells from dissociated mouse dorsal root ganglia. J Neurosci 1996; 16 (07) 2307-17.
7 Chen ZL, Strickland S. Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell 1997; 91 (07) 917-25.
8 Seeds NW, Williams BL, Bickford PC. Tissue plasminogen activator induction in Purkinje neurons after cerebellar motor learning. Science 1995; 270 5244 1992-4.
9 Muller CM, Griesinger CB. Tissue plasminogen activator mediates reverse occlusion plasticity in visual cortex. Nat Neurosci 1998; 01 (01) 47-53.
10 Wiman B, Collen D. Molecular mechanism of physiological fibrinolysis. Nature 1978; 272 5653 549-50.
11 Bringmann P, Gruber D, Liese A. et al.. Structural features mediating fibrin selectivity of vampire bat plasminogen activators. J Biol Chem 1995; 270 (43) 25596-603.
12 van Zonneveld AJ, Veerman H, Pannekoek H. On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin. Inhibition of kringle-2 binding to fibrin by epsilon-amino caproic acid. J Biol Chem 1986; 261 (30) 14214-8.
13 Bok RA, Mangel WF. Quantitative characterization of the binding of plasminogen to intact fibrin clots, lysine-sepharose, and fibrin cleaved by plasmin. Biochemistry 1985; 24 (13) 3279-86.
14 Ryou C, Prusiner SB, Legname G. Cooperative binding of dominant-negative prion protein to kringle domains. J Mol Biol 2003; 329 (02) 323-33.
15 Warner RG, Hundt C, Weiss S. et al.. Identification of the heparan sulfate binding sites in the cellular prion protein. J Biol Chem 2002; 277 (21) 18421-30.
16 Kovalchuk O, Tzaban S, Tal Y. et al.. Cellular heparan sulfate participates in the metabolism of prions. J Biol Chem 2003; 18: 18.
17 Ben-Zaken O, Tzaban S, Tal Y. et al.. Cellular heparan sulfate participates in the metabolism of prions. J Biol Chem 2003; 278 (41) 40041-9.
18 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227 (259) 680-5.
19 Schagger H, von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 1991; 199 (02) 223-31.
20 Brimacombe DB, Bennett AD, Wusteman FS. et al.. Characterization and polyanion-binding properties of purified recombinant prion protein. Biochem J 1999; 342 Pt (03) 605-13.
21 Andrade-Gordon P, Strickland S. Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator. J Biol Chem 1989; 264 (26) 15177-81.
22 Medved L, Nieuwenhuizen W. Molecular mechanisms of initiation of fibrinolysis by fibrin. Thromb Haemost 2003; 89 (03) 409-19.
23 Fischer MB, Roeckl C, Parizek P. et al.. Binding of disease-associated prion protein to plasminogen. Nature 2000; 408 6811 479-83.
24 Allen RA. An enhancing effect of poly-lysine on the activation of plasminogen. Thromb Haemost 1982; 47 (01) 41-5.
25 Zahn R. The Octapeptide Repeats in Mammalian Prion Protein Constitute a pHdependent Folding and Aggregation Site. J Mol Biol 2003; 334 (03) 477-88.
26 Caughey B, Raymond GJ. Sulfated polyanion inhibition of scrapie-associated PrP accumulation in cultured cells. J Virol 1993; 67 (02) 643-50.
27 Mayor S. Small improvement seen in teenager with vCJD. BMJ 2003; 327 7418 765.