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Identification of soluble type of membrane-type matrix metalloproteinase-3 formed by alternatively spliced mRNA
S Matsumoto1, M Katoh, S Saito
1Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki, Japan. matsumot@yamanouchi.co.jp
Abstract:
Homology screening for human membrane-type MMP (MT-MMP) was carried out, and cDNA encoding a soluble type of MT3-MMP (SM3), which is considered to be an alternatively spliced variant of MT3-MMP, was obtained. SM3 had a novel sequence consisting of 50 amino acids after Lys407 instead of amino acids containing the transmembrane domain of MT3-MMP. When SM3 tagged with a FLAG epitope (SM3-flag) was expressed in COS-7 cells, SM3-flag was present in the conditioned medium in its activated form. The enzymatic activity of SM3 was studied using a recombinant enzyme expressed in E. coli (SM3-e). The fluorogenic peptide substrate hydrolyzing activity of SM3-e was inhibited by EDTA and by the tissue inhibitor of metalloproteinase-2 (TIMP-2), whereas TIMP-1 had only relatively weak inhibitory ability. SM3-e was able to activate proMMP-2, and this activity was also inhibited by TIMP-2 but not by TIMP-1. SM3-e was able to cleave type III collagen, and also digested fibronectin. In view of the homology of the primary structures, MT3-MMP was considered to have the same catalytic activity as SM3. The results of studies of SM3's activity on extracellular matrix (ECM) protein suggests that MT3-MMP plays a role in ECM turnover not only by activating proMMP-2 but also by acting directly on ECM macromolecules.
Insights
A novel soluble form of membrane-type 3 matrix metalloproteinase (MT3-MMP), named SM3, was identified. This soluble variant activates proMMP-2 and degrades extracellular matrix proteins, suggesting a role for MT3-MMP in tissue remodeling.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Membrane-type matrix metalloproteinases (MT-MMPs) play crucial roles in extracellular matrix (ECM) remodeling.
- Alternative splicing is a mechanism for generating protein diversity, potentially leading to functional variants of MT-MMPs.
Purpose of the Study:
- To identify and characterize novel variants of human MT-MMPs.
- To investigate the enzymatic activity and substrate specificity of a newly identified soluble MT3-MMP variant (SM3).
Main Methods:
- Homology screening to identify MT-MMP variants.
- Expression of SM3 in COS-7 cells and purification of recombinant SM3 (SM3-e).
- Enzymatic assays using fluorogenic substrates, ECM proteins (type III collagen, fibronectin), and inhibitors (EDTA, TIMP-1, TIMP-2).
- ProMMP-2 activation assays.
Main Results:
- A cDNA encoding a soluble MT3-MMP variant (SM3) was obtained, lacking the transmembrane domain.
- SM3 was secreted in an active form from COS-7 cells.
- SM3-e exhibited metalloproteinase activity, inhibited by EDTA and TIMP-2, but not significantly by TIMP-1.
- SM3-e activated proMMP-2 and degraded type III collagen and fibronectin.
Conclusions:
- SM3 represents an alternatively spliced variant of MT3-MMP with distinct biochemical properties.
- MT3-MMP, through its soluble form SM3, contributes to ECM turnover by activating proMMP-2 and directly degrading ECM components.