Related Experiment Videos
Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules
1Department of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920, Japan.
Abstract:
Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed on cancer cell membranes and activates the zymogen of MMP-2 (gelatinase A). We have recently isolated MT1-MMP complexed with tissue inhibitor of metalloproteinases 2 (TIMP-2) and demonstrated that MT1-MMP exhibits gelatinolytic activity by gelatin zymography (Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710). In the present study, we have further purified to homogeneity a deletion mutant of MT1-MMP lacking the transmembrane domain (DeltaMT1) and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231 cells) and examined their substrate specificities. Both proteinases are active, without any treatment for activation, and digest type I (guinea pig), II (bovine), and III (human) collagens into characteristic 3/4 and 1/4 fragments. The cleavage sites of type I collagen are the Gly775-Ile776 bond for alpha1(I) chains and the Gly775-Leu776 and Gly781-Ile782 bonds for alpha2(I) chains. DeltaMT1 hydrolyzes type I collagen 6.5- or 4-fold more preferentially than type II or III collagen, whereas MMP-1 (tissue collagenase) digests type III collagen more efficiently than the other two collagens. Quantitative analyses of the activity of DeltaMT1 and MMP-1 indicate that DeltaMT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of DeltaMT1 is 8-fold higher than that of MMP-1. DeltaMT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as alpha1-proteinase inhibitor and alpha2-macroglobulin. The activity of DeltaMT1 on type I collagen is synergistically increased with co-incubation with MMP-2. These results indicate that MT1-MMP is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases, and suggest that MT1-MMP plays a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of proMMP-2.
Insights
Membrane type 1 matrix metalloproteinase (MT1-MMP) degrades various extracellular matrix components, including collagens, and activates proMMP-2. This enzyme plays a dual role in matrix digestion and cancer progression.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Membrane type 1 matrix metalloproteinase (MT1-MMP) is a key enzyme involved in cancer cell invasion and metastasis.
- MT1-MMP activates proMMP-2 and degrades extracellular matrix components.
- Previous studies demonstrated MT1-MMP's gelatinolytic activity.
Purpose of the Study:
- To purify and characterize a deletion mutant of MT1-MMP (DeltaMT1) and native MT1-MMP.
- To investigate the substrate specificities of purified MT1-MMP and DeltaMT1.
- To elucidate the role of MT1-MMP in extracellular matrix degradation and proMMP-2 activation.
Main Methods:
- Purification of DeltaMT1 and native MT1-MMP from human breast carcinoma cells (MDA-MB-231).
- Gelatin zymography to assess gelatinolytic activity.
- Collagenase assays using type I, II, and III collagens to determine substrate specificity and cleavage sites.
- Analysis of digestion of other extracellular matrix proteins and inhibitors.
Main Results:
- Both DeltaMT1 and native MT1-MMP are active proteinases that digest type I, II, and III collagens.
- DeltaMT1 shows preferential hydrolysis of type I collagen over types II and III.
- DeltaMT1 exhibits significantly higher gelatinolytic activity compared to MMP-1.
- MT1-MMP activity on type I collagen is enhanced by co-incubation with MMP-2.
- DeltaMT1 degrades cartilage proteoglycan, fibronectin, vitronectin, laminin-1, alpha1-proteinase inhibitor, and alpha2-macroglobulin.
Conclusions:
- MT1-MMP functions as an extracellular matrix-degrading enzyme with substrate specificity similar to interstitial collagenases.
- MT1-MMP plays a critical role in pathophysiological extracellular matrix digestion through direct substrate cleavage and proMMP-2 activation.
- These findings highlight MT1-MMP's dual function in matrix remodeling and cancer progression.