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Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1
P Nash1, A Whitty, J Handwerker
1Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Abstract:
SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, and factor Xa. N-terminal sequencing confirmed Arg319-Asn320 as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr315-Leu316 seen with elastase and with cathepsin G. Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and C1s showed second-order rate constants to vary over 3 logs, from kinh = 3 x 10(5) M-1 s-1 with thrombin to approximately 600 M-1 s-1 with C1s, while steady-state inhibition constants ranged from KI = 10 pM with thrombin to approximately 100 nM with C1s. Stoichiometries of inhibition varied between SI = 1.4 +/- 0.1 for uPA to SI = 13 +/- 3 for thrombin. Analysis of the variations in inhibition kinetics shows that when serpins act at low concentrations, comparable with the target protease or with KI (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by kinh and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.
Insights
Myxoma virus serpin-1 (SERP-1) inhibits key proteases like thrombin and plasmin, crucial for virulence. Its inhibitory specificity depends on reaction kinetics and complex stability at low concentrations.
Area of Science:
- Virology
- Biochemistry
- Immunology
Background:
- Myxoma virus serpin-1 (SERP-1) is a secreted protein essential for virulence.
- SERP-1 exhibits anti-inflammatory properties.
- Serpins are known inhibitors of serine proteases.
Purpose of the Study:
- To characterize the inhibitory activity of recombinant SERP-1.
- To identify the reactive site of SERP-1.
- To determine the kinetic parameters of SERP-1 inhibition against various proteases.
Main Methods:
- Purification of recombinant SERP-1.
- Formation of SDS-stable complexes with target proteases.
- N-terminal sequencing to identify the reactive site.
- Kinetic analysis (second-order rate constants, inhibition constants, stoichiometries).
Main Results:
- SERP-1 forms stable complexes with urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, and factor Xa.
- Arg319-Asn320 was identified as the reactive site, mutation of which abolished inhibitory activity.
- Kinetic analysis revealed varying inhibition efficiencies ( kinh and KI) across different proteases.
- Inhibitory specificity is influenced by reaction partitioning and complex lifetime at low concentrations.
Conclusions:
- SERP-1 effectively inhibits multiple serine proteases involved in coagulation and fibrinolysis.
- The reactive site and kinetic properties dictate SERP-1's inhibitory specificity.
- Understanding SERP-1 inhibition mechanisms provides insights into viral virulence and host-pathogen interactions.