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Activation of progelatinase A and progelatinase A/TIMP-2 complex by membrane type 2-matrix metalloproteinase
H Kolkenbrock1, A Hecker-Kia, D Orgel
1Deutsches Rheumaforschungszentrum Berlin, Germany.
Abstract:
C-terminal truncated membrane-type 2 matrix metalloproteinase (MT2-MMP1-269), comprising prodomain and catalytic domain, was expressed as a soluble protein in Escherichia coli. Unlike the corresponding form of MT1-MMP, which can be isolated as a 31 kDa protein, MT2-MMP1-269 proved to be comparatively instable, and already the freshly isolated preparation displayed several proteins in SDS-PAGE representing MT2-MMP1-269 (33 kDa) and four N-truncated forms with N-termini methionine32 (30 kDa), isoleucine37 (30 kDa), leucine84 (24 kDa), and leucine93 (22 kDa), the catalytic domain. After thawing of frozen preparations the 33 and the 30 kDa proforms were no longer detectable in SDS-PAGE, and only the 24 and 22 kDa forms remained. The catalytic domain of MT2-MMP activated progelatinase A as well as the progelatinase A/TMP-2 complex by cleaving the 72 kDa progelatinase A to yield 67 kDa gelatinase A, which is then transformed into 62 kDa gelatinase A. The 62 kDa form is about twice as active as the 67 kDa form towards the synthetic substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. No significant difference in activity was found between free and complexed gelatinase A forms. the activation of the progelatinase A/TIMP-2 complex proceeds in two steps: At first MT2-MMP is inhibited by the progelatinase A/TIMP-2/MT2-MMP, complex, whereby a ternary complex, progelatinase A/TIMP-2/ MT-2MMP is generated. This ternary complex is then activated by excess MT2-MMP. Our results suggest a mechanism for spatially regulated extracellular gelatinase A activity mediated by activation with membrane-type MMPs; Free gelatinase A is released into the extracellular space, while gelatinase A/TIMP-2 bound to MT-MMP remains anchored on the cell surface.
Insights
The catalytic domain of membrane-type 2 matrix metalloproteinase (MT2-MMP) activates progelatinase A, influencing its extracellular activity. MT2-MMP facilitates the release of active gelatinase A into the extracellular space.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a well-studied enzyme.
- The stability and activity of truncated forms of matrix metalloproteinases (MMPs) require further investigation.
Purpose of the Study:
- To characterize the C-terminal truncated MT2-MMP1-269, including its stability and enzymatic activity.
- To investigate the activation of progelatinase A by MT2-MMP and its implications for extracellular matrix remodeling.
Main Methods:
- Expression of soluble C-terminal truncated MT2-MMP (MT2-MMP1-269) in Escherichia coli.
- Analysis of protein stability using SDS-PAGE.
- Assay of catalytic activity on progelatinase A and its complex with TIMP-2.
Main Results:
- MT2-MMP1-269 was expressed as a soluble protein but showed instability, yielding several N-truncated forms.
- The catalytic domain of MT2-MMP activated progelatinase A, generating active gelatinase A forms (67 kDa and 62 kDa).
- MT2-MMP mediated a two-step activation of the progelatinase A/TIMP-2 complex, with the 62 kDa gelatinase A being twice as active as the 67 kDa form.
Conclusions:
- MT2-MMP plays a role in the spatial regulation of extracellular gelatinase A activity.
- Free gelatinase A is released extracellularly, while TIMP-2 bound to MT-MMP remains cell-surface anchored, suggesting distinct roles in matrix degradation.