Spatial orientation of tissue-type plasminogen activator bound at the melanoma cell surface.
Human melanoma cells produce tissue-type plasminogen activator (tPA) which is bound to the cell surface where it effectively mediates generation of plasmin. The present study is focused on analysis of involvement of the tPA domains in binding of the enzyme to the cell surface. The extent of plasminogen activation by tPA of melanoma cells was measured using an immunocapture assay. The activator anchored to solid surface via monoclonal antibodies directed to the individual domains of the activator exhibited variable enzymatic activity. The tPA was the most effective when bound by the antibodies against kringle-1 or kringle-2. Accessibility of the epitopes within cell surface-bound tPA was probed by the same set of monoclonal antibodies. FACS analysis showed that the epitopes within the finger/growth factor domain one part of the kringle-2 domain and the active site epitope were the most exposed. The kringle-1 domain epitope and the protease region epitope appeared partially exposed. Full-length melanoma-derived tPA and three recombinant domain-deletion variants of tPA were compared for their capacity to bind to the melanoma cells. The estimated IC50 value for the melanoma-derived tPA was 2.3 +/- 0.25 microM. Comparable IC50 values were found for the tPA variant lacking the finger domain (3.6 +/- 0.6 microM) as well as for the variants consisting only of the kringle-2 and protease domains (7.5 +/- 0.45 microM). In contrast the value found for a tPA variant lacking the kringle-2 domain was > 100 microM. The consistent results obtained by the three different experimental approaches provide evidence that tPA binds to melanoma cells via its kringle-2 domain but binding sites within kringle-1 domain and protease domain may support the interaction. The finger domain did not contribute to the binding.
Bizik
J
J
Haartman Institute, Department of Virology, University of Helsinki, Finland.
Trancikova
D
D
Felnerova
D
D
Verheijen
J H
JH
Vaheri
A
A
D000911:Antibodies, Monoclonal; D001665:Binding Sites; D018604:Epitope Mapping; D005434:Flow Cytometry; D006801:Humans; D008545:Melanoma / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013499:Surface Properties; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator