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The authors developed MAMBI, a multiplexed bead-based immunoassay for measuring metalloproteinase activities, demonstrating its sensitivity and utility in characterizing active native MMPs in uterine tissue and menstrual effluent relevant to endometriosis.
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This study introduces the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI), a novel method for simultaneously measuring the activity of multiple zinc-dependent endopeptidases. The researchers validated the assay using recombinant proteins and demonstrated its sensitivity and selectivity across various samples, including conditioned cell culture medium, menstrual effluent, and uterine tissue. Key findings indicate that MAMBI achieves detection sensitivities comparable to leading single-plex strategies while enabling high-throughput inhibitor screening in complex patient-derived samples. Relevance to endometriosis: MMPs are explicitly listed as playing significant roles in the pathogenesis of endometriosis, and the paper includes analysis of menstrual effluent, which is a relevant biofluid for studying this condition.
Abstract
Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 μL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.
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Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI)
Name
Lauffenburger-MAMBIFinalCCdalLGG copy.pdf
Description
Accepted version
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5.65 MB
Format
Adobe PDF
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Author(s) • • • • • • •
Chopko Ahrens, Caroline
Chiswick, Evan
Ravindra, Kodihalli C.
Miller, Miles Aaron
Ramseier, Julie Y.
Isaacson, Keith B.
Lauffenburger, Douglas A
Griffith, Linda G
Date Issued
September 2019
Journal
Biochemistry
Publisher
American Chemical Society (ACS)
Citation
Ahrens, Caroline C. et al. "Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI)." Biochemistry 58, 38 (September 2019): 3938–3942 © 2019 American Chemical Society
Version
Author's final manuscript
Abstract
Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 μL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
https://doi.org/10.1021/acs.biochem.9b00584
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