Localization of epitopes for monoclonal antibodies to urokinase-type plasminogen activator: relationship between epitope localization and effects of antibodies on molecular interactions of the enzyme.
We localized the epitopes for several murine mAbs to human urokinase-type plasminogen activator (uPA) by Ala scanning mutagenesis and related the localization to the effects of the mAbs on the molecular interactions of uPA. Several antibodies against the serine proteinase domain (SPD) were found to have overlapping epitopes composed of variable combinations of Arg178, Arg179, His180, Arg181, Tyr209, Lys211, and Asp214 in the so-called 37-loop and 60-loop, located near the active site and taking part in the binding of uPA to plasminogen activator inhibitor-1 (PAI-1). Besides inhibiting uPA-catalysed plasminogen activation, all antibodies to SPD strongly delayed the binding of uPA to PAI-1, decreasing the second-order rate constant 15- to 6500-fold. There was no correlation between the relative effects of the 37-loop and 60-loop substitutions on the second-order rate constant and on the binding of the antibodies, indicating that the antibodies did not delay complex formation by blocking residues of specific importance for the uPA-PAI-1 reaction, but rather by steric hindrance of the access of PAI-1 to the active site. The affinity of the SPD antibodies for the uPA-PAI-1 complex was only slightly lower than that for free uPA, indicating that the 37-loop and 60-loop are exposed in the complex. The epitopes for two antibodies to the kringle included Arg108, Arg109, and Arg110. The ability of these antibodies to block the binding of uPA to polyanions correlated with a reduced uPA-polyanion affinity after substitution of the three Arg residues.
Petersen
H H
HH
Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, Aarhus University, Denmark.
Hansen
M
M
Schousboe
S L
SL
Andreasen
P A
PA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D018604:Epitope Mapping; D006801:Humans; D051379:Mice; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1; D014568:Urokinase-Type Plasminogen Activator