Identification of soluble type of membrane-type matrix metalloproteinase-3 formed by alternatively spliced mRNA.
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.
Matsumoto
S
S
Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki, Japan. matsumot@yamanouchi.co.jp
Katoh
M
M
Saito
S
S
Watanabe
T
T
Masuho
Y
Y
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005109:Extracellular Matrix / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D053513:Matrix Metalloproteinase 16; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012995:Solubility; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000494:pharmacology; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000494:pharmacology
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; C507102:MMP16 protein, human; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D003094:Collagen; D017364:Collagenases; D018093:Gelatinases; D053513:Matrix Metalloproteinase 16; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C069860:progelatinase; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9