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Cleavage of recombinant murine interferon-gamma by plasmin and miniplasmin
S L Gonias1, W W Young, J W Fox
1Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Abstract:
Plasmin reacted readily with recombinant murine interferon-gamma (rIFN-gamma) in vitro, reducing the relative molecular mass of each monomer by approximately 1,000. The amino terminus of the rIFN-gamma remained intact and no sites of internal peptide bond hydrolysis were detected, indicating that the plasmin target region is most likely near the carboxyl terminus. Cleavage of rIFN-gamma was observed with similar concentrations of trypsin or min-plasmin. By contrast, human neutrophil elastase failed to alter the structure of rIFN-gamma. The plasma proteinase inhibitor, alpha 2-antiplasmin, protected rIFN-gamma from plasmin digestion. Purified alpha 2-macroglobulin-plasmin complex cleaved rIFN-gamma; however, the activity was greatly reduced compared with the free proteinase. The antiviral activity of the rIFN-gamma was enhanced four- to fivefold by treatment with plasmin or trypsin. By contrast, naturally occurring murine IFN-gamma was inactivated by plasmin (80%), suggesting that the effect of plasmin on IFN activity can vary depending on the preparation studied. The importance of plasmin at the site of an immune reaction is well established. This investigation identifies plasmin and miniplasmin as physiologic proteinases capable of reacting with IFN-gamma in vivo.
Insights
Plasmin and trypsin cleave recombinant murine interferon-gamma (rIFN-gamma), enhancing its antiviral activity. Natural interferon-gamma, however, is inactivated by plasmin, highlighting preparation-dependent effects.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Interferon-gamma (IFN-gamma) is a crucial cytokine in immune responses.
- The interaction of proteases with IFN-gamma and its functional consequences are not fully understood.
Purpose of the Study:
- To investigate the in vitro interaction between plasmin and recombinant murine interferon-gamma (rIFN-gamma).
- To determine the effect of plasmin and other proteases on rIFN-gamma structure and antiviral activity.
Main Methods:
- Incubation of rIFN-gamma with plasmin, trypsin, mini-plasmin, and human neutrophil elastase.
- Analysis of rIFN-gamma molecular mass and N-terminus integrity.
- Assessment of antiviral activity following protease treatment.
- Investigation of inhibition by alpha 2-antiplasmin and alpha 2-macroglobulin-plasmin complex.
Main Results:
- Plasmin and trypsin cleaved rIFN-gamma near the carboxyl terminus, reducing molecular mass.
- Alpha 2-antiplasmin inhibited plasmin cleavage of rIFN-gamma.
- Plasmin and trypsin treatment enhanced rIFN-gamma antiviral activity four- to fivefold.
- Naturally occurring murine IFN-gamma was inactivated by plasmin.
Conclusions:
- Plasmin and miniplasmin are identified as physiological proteinases that can react with IFN-gamma.
- Proteolytic cleavage can modulate IFN-gamma antiviral activity, with effects varying by preparation.
- The findings suggest a potential role for plasmin in regulating IFN-gamma function at immune sites.