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

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

^* present address: Paion GmbH, Forschungszentrum Berlin, Berlin, Germany

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