title: The transferable tail: fusion of the N-terminal acidic extension of heparin cofactor II to alpha1-proteinase inhibitor M358R specifically increases the rate of thrombin inhibition.
abstract: The conversion of the reactive center bond of the serpin alpha1-proteinase inhibitor (alpha1-PI, also known as alpha1-antitrypsin) from Met-Ser to Arg-Ser decreases the rate at which it inhibits neutrophil elastase and endows it with the ability to inhibit thrombin and activated protein C (APC). Another serpin, heparin cofactor II (HCII), contains a unique N-terminal extension that binds thrombin exosite 1. We fused residues 1-75 of HCII to the N-terminus of alpha1-PI M358R, forming an HCII-alpha1-PI chimera (HAPI M358R). It inhibited alpha-thrombin 21-fold faster than alpha1-PI M358R, with second-order rate constants of 2.3 x 10(8) M(-1) min(-1) versus 1.1 x 10(7) M(-1) min(-1), respectively. When gammaT-thrombin, which lacks an intact exosite 1, was substituted for alpha-thrombin, the kinetic advantage of HAPI M358R over alpha1-PI M358R was reduced to 9-fold, whereas APC and trypsin, proteases lacking exosite 1-like regions, were inhibited only 1.3- and 2-fold more rapidly by HAPI M358R than by alpha1-PI M358R, respectively. Maximal enhancement of alpha1-PI M358R activity required the acidic residues found between HCII residues 55 and 75, because no enhancement was observed either by fusion of residues 1-54 alone or by fusion of a mutated HCII acidic extension in which all Glu and Asp residues between positions 55 and 75 were neutralized by mutation. Fusing residues 55-75 to alpha1-PI M358R yielded a relative rate enhancement of only 6-fold, suggesting a need for the full tail region to achieve maximal enhancement. Our results suggest that transfer of the N-terminal acidic extension of HCII to alpha1-PI M358R enhanced its inhibition of thrombin by conferring the ability to bind exosite 1 on HAPI M358R. This enhancement may aid in efforts to tailor this inhibitor for therapeutic use.
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, HSC 4N66, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
authors.forename: Jason S
authors.initials: JS
authors.lastname: Sutherland
authors.forename: Varsha
authors.initials: V
authors.lastname: Bhakta
authors.forename: Marc L
authors.initials: ML
authors.lastname: Filion
authors.forename: William P
authors.initials: WP
authors.lastname: Sheffield
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry*; D005337:Fibrin / Q000378:metabolism; D015844:Heparin Cofactor II / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D008958:Models, Molecular; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012995:Solubility; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013917:Thrombin / Q000037:antagonists & inhibitors*; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin; D015844:Heparin Cofactor II; D005337:Fibrin; D001120:Arginine; D008715:Methionine; D013917:Thrombin