Function-stabilizing mechanism of plasminogen activator inhibitor type 1 induced upon binding to alpha1-acid glycoprotein.
Recently, we found that alpha(1)-acid glycoprotein (AGP), one of the major acute-phase proteins, forms a function-stabilizing complex with plasminogen activator inhibitor 1 (PAI-1). In this study we describe the mechanism by which AGP, as well as its recombinant fragment AGP(118)(-)(201), interacts exclusively with the active form of PAI-1 and stabilizes its conformation. The binding domain of PAI-1 for AGP was initially mapped by antibodies reacting with the well-defined PAI-1 epitopes and then verified in binding assays utilizing a library of PAI-1 mutants. The latter consisted of PAI-1 molecules with individual, tandem, or grouped mutations in the epitope region of MA-55F4C12, MA-33B8, MA-33H1F7, MA-44E4, and MA-8H9D4. Solid-phase binding experiments showed that only MA-8H9D4 did not bind to the PAI-1/AGP complex, indicating that its epitope is hidden upon binding of PAI-1 to AGP. Consistently, only PAI-1 mutants with substitutions in the region of R300-D305, constituting the MA-8H9D4 epitope, showed a lack of binding or severe deficit in both the capacity and affinity of binding to AGP. These results support a location of the binding site close to the epitope within the segment connecting the regions hI with S5A. In conclusion, our present data suggest that AGP binding stabilizes the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.
Smolarczyk
Katarzyna
K
Center of Medical Biology, Polish Academy of Sciences, Lodz, Poland.
Gils
Ann
A
Boncela
Joanna
J
Declerck
Paul J
PJ
Cierniewski
Czeslaw S
CS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D009961:Orosomucoid / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D000939:Epitopes; D009961:Orosomucoid; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins