Plasminogen binding properties of macrophage inflammatory protein (MIP)-2alpha
1Institut de Recerca Oncològica, Hospital Duran i Reynals, Barcelona, Spain.
Abstract:
The chemokine macrophage inflammatory protein (MIP)-2alpha was identified as a plasminogen binding protein by phage display analysis. MIP-2alpha and a truncated form lacking 5 lysine residues in the COOH-terminal region (mut-MIP-2alpha) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2alpha but not mut-MIP-2alpha bound specifically to plasminogen, with K(A) of 3.7 X 10(5) M(-1) for the interaction of plasminogen with surface-bound MIP-2alpha. Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2alpha. Activation of plasminogen bound to surface-associated MIP-2alpha by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency k(cat)K(M) of 0.1 microM(-1)s(-1), as compared to 0.04 microM(-1)s(-1). In contrast, binding of plasminogen to MIP-2alpha in solution was very weak, as evidenced by the absence of competition of MIP-2alpha with lysine-Sepharose or with human THP-1 cells for binding of plasminogen. In agreement with this finding, addition of excess MIP-2alpha did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissue-type plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by alpha2-antiplasmin. Thus, association of MIP-2alpha with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.
Insights
Macrophage inflammatory protein (MIP)-2alpha binds plasminogen, enhancing its activation when MIP-2alpha is surface-associated. This interaction may promote local plasmin generation, impacting biological processes.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Signaling
Background:
- Chemokines, such as macrophage inflammatory protein (MIP)-2alpha, play roles in inflammation and immune responses.
- Plasminogen is a key protein in the fibrinolytic system, involved in blood clot breakdown.
- Understanding protein-protein interactions is crucial for elucidating biological pathways.
Purpose of the Study:
- To identify plasminogen-binding proteins using phage display.
- To characterize the interaction between MIP-2alpha and plasminogen.
- To investigate the functional consequences of this interaction on plasmin generation.
Main Methods:
- Phage display analysis to identify MIP-2alpha as a plasminogen-binding protein.
- Bacterial expression and purification of MIP-2alpha and a mutant form (mut-MIP-2alpha).
- Binding assays, competition studies, and enzymatic activity measurements (plasminogen activation).
Main Results:
- MIP-2alpha specifically binds plasminogen with a moderate affinity (K(A) = 3.7 x 10(5) M(-1)) when surface-bound.
- The interaction involves the COOH-terminal lysine-rich domain of MIP-2alpha and the kringle regions of plasminogen.
- Surface-associated MIP-2alpha enhances plasminogen activation by tcu-PA approximately 2.5-fold compared to solution-phase interactions.
- MIP-2alpha binding to plasminogen in solution is weak and does not affect plasminogen/plasmin functions in solution.
Conclusions:
- Surface association of MIP-2alpha exposes its plasminogen-binding site.
- This localized interaction can lead to enhanced plasmin generation at specific sites.
- MIP-2alpha may play a role in modulating fibrinolysis and other plasmin-dependent processes through surface-mediated plasminogen binding.
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