Epitope mapping of the anti-urokinase monoclonal antibody 5B4 by isolated domains of urokinase.
The amino terminal fragment (ATF) of urokinase-type plasminogen activator (uPA) is a degradation product comprising the entire growth factor-like and kringle domains. It has been previously shown that ATF is able to bind to the u-PA receptor through the growth factor-like domain and that the anti u-PA monoclonal antibody 5B4 (Mab 5B4) binds to ATF preventing u-PA receptor binding. To localize more precisely the epitope recognized by Mab 5B4, ATF was subfragmented by controlled enzymatic proteolysis with V8 protease. Three subfragments of 4,000 Mr (F-4k), 11,000 Mr (F-11k) and 12,000 Mr (F-12k) were purified from the reaction mixture and characterized. SDS-PAGE under reducing and non-reducing conditions, N-terminal aminoacid sequence analysis and C-terminal aminoacid analysis of each fragment indicate that F-4k and F-11k correspond to intact growth factor-like domain and kringle domain (residues 4-43 and 44-135 respectively) while F-12k corresponds to the kringle domain cleaved in the first loop at the glu52-gly53 bond. By Western blot and competitive binding experiments we show that Mab 5B4 recognizes an epitope located on the kringle domain of u-PA and that the binding is strongly reduced when the kringle contains an additional cleavage in its first loop. Since the receptor binding site of u-PA has been previously shown to be located on the growth factor-like domain, Mab 5B4 inhibits the binding of uPA to its cellular receptor likely by steric hindrance. Besides the proven utility in epitope localization of anti u-PA monoclonal antibodies, these u-PA fragments may represent powerful tools for studies of structure-function relationship of u-PA.
Corti
A
A
Merrell Dow Research Institute, Lepetit Research Center, Gerenzano (Varese), Italy.
Sarubbi
E
E
Soffientini
A
A
Nolli
M L
ML
Zanni
A
A
Galimberti
M
M
Parenti
F
F
Cassani
G
G
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis; D000939:Epitopes / Q000032:analysis*; D005343:Fibrinolytic Agents / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010960:Plasminogen Activators / Q000276:immunology* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005343:Fibrinolytic Agents; C524898:PLAUR protein, human; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator