Fusion of the C-terminal triskaidecapeptide of hirudin variant 3 to alpha1-proteinase inhibitor M358R increases the serpin-mediated rate of thrombin inhibition.
BACKGROUND
Alpha-1 proteinase inhibitor (API) is a plasma serpin superfamily member that inhibits neutrophil elastase; variant API M358R inhibits thrombin and activated protein C (APC). Fusing residues 1-75 of another serpin, heparin cofactor II (HCII), to API M358R (in HAPI M358R) was previously shown to accelerate thrombin inhibition over API M358R by conferring thrombin exosite 1 binding properties. We hypothesized that replacing HCII 1-75 region with the 13 C-terminal residues (triskaidecapeptide) of hirudin variant 3 (HV354-66) would further enhance the inhibitory potency of API M358R fusion proteins. We therefore expressed HV3API M358R (HV354-66 fused to API M358R) and HV3API RCL5 (HV354-66 fused to API F352A/L353V/E354V/A355I/I356A/I460L/M358R) API M358R) as N-terminally hexahistidine-tagged polypeptides in E. coli.


RESULTS
HV3API M358R inhibited thrombin 3.3-fold more rapidly than API M358R; for HV3API RCL5 the rate enhancement was 1.9-fold versus API RCL5; neither protein inhibited thrombin as rapidly as HAPI M358R. While the thrombin/Activated Protein C rate constant ratio was 77-fold higher for HV3API RCL5 than for HV3API M358R, most of the increased specificity derived from the API F352A/L353V/E354V/A355I/I356A/I460L API RCL 5 mutations, since API RCL5 remained 3-fold more specific than HV3API RCL5. An HV3 54-66 peptide doubled the Thrombin Clotting Time (TCT) and halved the binding of thrombin to immobilized HCII 1-75 at lower concentrations than free HCII 1-75. HV3API RCL5 bound active site-inhibited FPR-chloromethyl ketone-thrombin more effectively than HAPI RCL5. Transferring the position of the fused HV3 triskaidecapeptide to the C-terminus of API M358R decreased the rate of thrombin inhibition relative to that mediated by HV3API M358R by 11-to 14-fold.


CONCLUSIONS
Fusing the C-terminal triskaidecapeptide of HV3 to API M358R-containing serpins significantly increased their effectiveness as thrombin inhibitors, but the enhancement was less than that seen in HCII 1-75-API M358R fusion proteins. HCII 1-75 was a superior fusion partner, in spite of the greater affinity of the HV3 triskaidecapeptide, manifested both in isolated and API-fused form, for thrombin exosite 1. Our results suggest that HCII 1-75 binds thrombin exosite 1 and orients the attached serpin scaffold for more efficient interaction with the active site of thrombin than the HV3 triskaidecapeptide.
Leigh Ann
LA
Roddick
Varsha
V
Bhakta
Pathology and Molecular Medicine, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4 K1, Canada. sheffiel@mcmaster.ca.
William P
WP
Sheffield
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D006629:Hirudins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011486:Protein C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D015843:Serpins / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism*
D006629:Hirudins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010455:Peptides; D011486:Protein C; D011993:Recombinant Fusion Proteins; D015843:Serpins; D000515:alpha 1-Antitrypsin; D006639:Histidine; D013917:Thrombin