Both lysine-clusters of the NH2-terminal prion-protein fragment PrP23-110 are essential for t-PA mediated plasminogen activation.
We have recently shown that the NH(2)-terminal fragment (PrP23-110) of the human cellular prion protein (PrP(c) ) stimulates t-PA mediated plasminogen activation. PrP23-110 contains an N-terminal lysine cluster (LC1; K(23),K(24), K(27)) and a C-terminal one (LC2; K(101),K(104),K(106),K(110)). To study their biological function we have substituted all lysine residues of each cluster by alanine and generated the recombinant PrP proteins PrP23-110sLC1 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 PrP23-110 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.
Institut für Laboratoriumsmedizin und Pathobiochemie, Charité Berlin, Medizinische Fakultät der Humboldt Universität zu Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany.
Guido
G
Epple
Kristina
K
Langfeld
Michael
M
Baier
Hermann-Georg
HG
Holzhütter
Wolf-Dieter
WD
Schleuning
Eckart
E
Köttgen
Reinhard
R
Gessner
Michael
M
Praus
D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D009154:Mutation; D010960:Plasminogen Activators / Q000378:metabolism*; D011328:Prions / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000737:chemistry*
D006025:Glycosaminoglycans; D011328:Prions; D011994:Recombinant Proteins; D006493:Heparin; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D008239:Lysine