Abstract
We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry. Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells. Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects. The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied. Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators. However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity. Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators. Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region.
Highlights
From the Departments of Biochemistry and Internal Medicine, University 7.52359038 and aPfizer Central Research, Groton, Connecticut 06340 of Texas Southwestern
To assess the functional significance of the reactive center and second site sequences as determinants of the inhibitory activity and protease specificity of plasminogen activator inhibitor type 1 (PAI-1), wild-type and mutant PAI- molecules have been expressed in Escherichia coli and used both in enzyme inhibition assays to measure the rate constants of inhibition of the serine proteases tPA, uPA, and thrombin, and in complementary immunochemical experiments using anti-peptide antibodies
Production and Characterization of rPAI-1 and rPAI-1 Variants- Site-directed mutants of recombinant human PAI(rPAI-1) were constructed in the reactive center and in a second site in order to assess the functional significance of these two structural regions
Summary
From the Departments of Biochemistry and Internal Medicine, University 7.52359038 and aPfizer Central Research, Groton, Connecticut 06340 of Texas Southwestern. Mutations in both the reactive center Pl and Pl’ residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI- have been engineered to assess their functional effects. Substitution of the Pl arginine residue with lysine or the Pl’ residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators. Replacement of both Pl and Pl’ by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity. Plasminogen activation is regulated through several mechanisms, including the controlled synthesis and secretion of plasminogen activators and the modulation of their enzymatic activity by specific inhibitors (Gerard and Meidell, 1989). Both plasminogen activator inhibitor 1 (PAI1) and plasminogen activator inhibitor 2 (PAI-2) are members of the serine proteinase inhibitor (serpin) superfamily
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