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Biochemical characterization of the catalytic domain of membrane-type 4 matrix metalloproteinase
H Kolkenbrock1, L Essers, N Ulbrich
1Deutsches Rheumaforschungszentrum Berlin, Germany.
Abstract:
A C-terminal truncated form of membrane-type 4 matrix metalloproteinase (MT4-MMP; MMP 17), lacking the hemopexin-like and transmembrane domain, was expressed in Escherichia coli. The catalytic domain was produced by tryptic activation of the recombinant proenzyme and proved to be catalytically active towards the fluorogenic substrate for matrix metalloproteinases (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu(3-(2,4-dinitrophenyl)-L-2,3-diaminopro-p ionyl)-Ala-Arg-NH2. In contrast to the other three MT-MMPs (MT1-, MT2-, and MT3-MMP), the catalytic domain of MT4-MMP does not activate progelatinase A, nor does it hydrolyze one of the offered extracellular matrix (ECM) proteins, such as collagen types I, II, III, IV, and V, gelatin, fibronectin, laminin or decorin. TIMP-1, a poor inhibitor of MT1-, MT2- and MT3-MMP, suppresses MT4-MMP activity effectively. The progelatinase A/TIMP-2 complex that usually reacts like TIMP-2 also inhibits MT4-MMP. TIMP-2, a strong inhibitor of other MT-MMPS, inhibits MT4-MMP at low concentrations. With increasing TIMP-2 concentration, however, activity passes through a minimum and then increases until at high TIMP-2 concentration the activity is the same as in the absence of TIMP-2. TIMP-1 or the progelatinase A/TIMP-2 complex do not prevent reactivation of MT4-MMP catalytic domain at high TIMP-2 concentrations.
Insights
The catalytic domain of membrane-type 4 matrix metalloproteinase (MT4-MMP) is active but does not degrade extracellular matrix proteins. Tissue inhibitors of metalloproteinases (TIMPs) effectively inhibit MT4-MMP activity.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Matrix metalloproteinases (MMPs) are crucial enzymes involved in extracellular matrix (ECM) remodeling.
- Membrane-type MMPs (MT-MMPs) play specific roles in cell adhesion, migration, and tissue development.
- MT4-MMP (MMP-17) function and regulation are less understood compared to other MT-MMPs.
Purpose of the Study:
- To characterize the catalytic activity and substrate specificity of the MT4-MMP catalytic domain.
- To investigate the inhibitory effects of tissue inhibitors of metalloproteinases (TIMPs) on MT4-MMP activity.
- To compare the functional properties of MT4-MMP with other MT-MMP family members.
Main Methods:
- Expression and purification of the catalytic domain of MT4-MMP in Escherichia coli.
- Tryptic activation of the recombinant proenzyme to generate the active catalytic domain.
- Assays using fluorogenic substrates to determine catalytic activity.
- Incubation with various ECM proteins (collagens, gelatin, fibronectin, laminin, decorin) to assess hydrolysis.
- Inhibition studies using TIMP-1, TIMP-2, and a progelatinase A/TIMP-2 complex.
Main Results:
- The recombinant catalytic domain of MT4-MMP was catalytically active towards a fluorogenic MMP substrate.
- Unlike other MT-MMPs, MT4-MMP catalytic domain did not activate progelatinase A or degrade tested ECM proteins.
- TIMP-1 effectively suppressed MT4-MMP activity.
- TIMP-2 exhibited complex inhibitory behavior, with activity decreasing at low concentrations and increasing at high concentrations.
- The progelatinase A/TIMP-2 complex also inhibited MT4-MMP, similar to TIMP-2 alone.
Conclusions:
- The catalytic domain of MT4-MMP possesses enzymatic activity but lacks the broad substrate specificity and progelatinase A activation capabilities of other MT-MMPs.
- MT4-MMP activity is effectively regulated by TIMP-1 and exhibits unique inhibition kinetics with TIMP-2.
- These findings highlight distinct functional characteristics of MT4-MMP within the MT-MMP family, suggesting specialized roles in biological processes.