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Fluorogenic MMP activity assay for plasma including MMPs complexed to alpha 2-macroglobulin
B Beekman1, J W Drijfhout, H K Ronday
1Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands.
Abstract:
Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor alpha 2-macroglobulin (alpha 2M). Reconstituted MMP-13/alpha 2M complex was unable to degrade collagen (MW 300,000) in contrast to the low-molecular-weight fluorogenic substrate (MW < 1500). Limited access of high-MW substrates to the active site of MMPs captured by alpha 2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW approximately 28,000) was unable to inhibit MMP/alpha 2M enzyme activity, whereas the low-MW inhibitor BB94 (MW approximately 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin-ation, sensitive MMP-activity assays in plasma were achieved. Spiking of active MMP-13 and MMP-13/alpha 2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by alpha 2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.
Insights
Matrix metalloproteinase (MMP) activity in plasma is influenced by alpha 2-macroglobulin (alpha 2M). This study developed sensitive assays to detect MMP activity in plasma, finding elevated levels in rheumatoid arthritis patients.
Area of Science:
- Biochemistry
- Enzymology
- Proteomics
Background:
- Matrix metalloproteinases (MMPs) play crucial roles in tissue degradation, implicated in diseases like arthritis and cancer.
- Active MMPs in circulation are rapidly inhibited by alpha 2-macroglobulin (alpha 2M), a major plasma proteinase inhibitor.
Purpose of the Study:
- To develop and validate sensitive assays for measuring MMP enzyme activity directly in human plasma.
- To investigate the interaction between MMPs and alpha 2M in plasma and its effect on enzyme activity.
- To assess MMP activity levels in plasma from healthy individuals and rheumatoid arthritis patients.
Main Methods:
- Utilized fluorogenic substrates with quencher/fluorophore pairs (Dabcyl/Fluorescein) for sensitive detection of MMP activity.
- Investigated the impact of alpha 2M binding on MMP-13 activity using high- and low-molecular-weight substrates and inhibitors.
- Performed spiking experiments with active MMP-13 and MMP-13/alpha 2M complexes, and inhibitor studies using TIMP-1 and BB94.
Main Results:
- MMP-13 complexed with alpha 2M showed significantly reduced degradation of high-molecular-weight collagen compared to low-molecular-weight substrates.
- High-molecular-weight inhibitor TIMP-1 did not inhibit MMP/alpha 2M activity, while low-molecular-weight inhibitor BB94 was effective.
- Sensitive MMP activity assays in plasma were successfully developed, confirming efficient capture of active MMPs by alpha 2M.
- MMP activity was detectable in control plasma and significantly elevated in plasma from rheumatoid arthritis patients.
Conclusions:
- Alpha 2-macroglobulin efficiently captures and modulates the activity of matrix metalloproteinases in human plasma.
- The molecular weight of substrates and inhibitors dictates their interaction with MMPs bound to alpha 2M.
- Developed plasma-based MMP activity assays provide a valuable tool for studying MMP dysregulation in diseases such as rheumatoid arthritis.