Identification and Characterization of Human Endometase (Matrix Metalloproteinase-26) from Endometrial Tumor

Abstract

We report the discovery, cloning, and characterization of a novel human matrix metalloproteinase 26 (MMP-26) (matrixin) gene, endometase, an endometrial tumor-derived metalloproteinase. Among more than three million expressed sequence tags sequenced, the endometase gene was only obtained from human endometrial tumor cDNA library. Endometase mRNA was expressed specifically in human uterus, not in other tissues/cells tested, e.g. testis, heart, brain, lungs, liver, thymus, and melanoma G361. Endometase protein has a signal peptide, a propeptide domain, and a catalytic domain with a unique "cysteine switch" propeptide sequence, PHCGVPDGSD, and a zinc-binding motif, VAT-HEIGHSLGLQH. Endometase is 43, 41, 41, and 39% identical to human metalloelastase, stromelysin, collagenase-3, and matrilysin, respectively. The zymogen was expressed and isolated from Escherichia coli as inclusion bodies with a molecular mass of 28 kDa. The identity and homogeneity of the recombinant protein was confirmed by protein N-terminal sequencing, silver stain, and immunoblot analyses. The pro-enzyme was partially activated during the folding process. Endometase selectively cleaved type I gelatin and ␣ 1 -proteinase inhibitor; however, it did not digest collagens, laminin, elastin, ␤-casein, plasminogen, soybean trypsin inhibitor, or Bowman-Birk inhibitor. It hydrolyzed peptide substrates of matrixins and tumor necrosis factor-␣ converting enzyme. Endometase may selectively cleave extracellular matrix proteins, inactivate serpins, and process cytokines. Matrix metalloproteinases (MMPs)1 (also known as matrixins) are a family of highly homologous zinc metalloenzymes that digest extracellular matrix proteins and process progrowth factors and cytokines; their activities are inhibited by tissue inhibitors of metalloproteinases (1). Matrixins may play many important physiological and pathological functions, including reproduction, angiogenesis, development, morphogenesis, tissue remodeling, arthritis, cardiovascular diseases, neurological diseases, and cancer progression and metastasis (1-4). Knowledge about the structure-function relationship of the matrixins has been growing rapidly during past decade. New members of the matrixin family have been discovered continuously (1). So far, at least 22 MMPs have been reported; the most recent two are MT5-MMP/MMP-24 (5, 6) and MT6-MMP/ MMP-25 (7, 8). All the matrixins have a propeptide domain containing the highly conserved cysteine switch sequence PRCG(V/N)PD and a catalytic domain with a zinc-binding motif, HEXXHXXGXXH (1).We report a novel member of the matrixin family, endometase (MMP-26), which was cloned from human endometrial tumor cDNA library. Its mRNA was detected only in human uterus tissue among all the human tissues tested. Endometase has a pro-domain with a unique cysteine switch sequence, PHCGVPDGSD, and a catalytic domain with the common zincbinding motif. This new MMP has a different substrate profile from other closely similar MMPs. Endometase selectively cleaves rat tail tendon type I...

Coordinated peak expression of MMP-26 and TIMP-4 in preinvasive human prostate tumor

“…MMP-26 is a multifaceted enzyme with many direct ECM targets as well as non-ECM protein targets including insulin-like growth factor-binding protein-1 and α-1 protease inhibitor [7][8][9][10]. Previous results from our group revealed that MMP-26 promoted the invasion of human prostate cancer cells through both type IV collagen and fibronectin, not only through the direct proteolytic cleavage of these proteins but also through the activation of pro-MMP-9, a highly efficient enzyme with multiple targets in the ECM [12].…”

“…Endometase/matrilysin-2 (MMP-26) is a member of the MMP family recently cloned by our group and others [7][8][9][10]. MMP-26 is one of the two smallest members of this family, exhibiting minimal domain structure consisting of a catalytic domain and a prodomain, which maintains the enzyme in a latent form prior to its activation.…”

“…MMP-26 is one of the two smallest members of this family, exhibiting minimal domain structure consisting of a catalytic domain and a prodomain, which maintains the enzyme in a latent form prior to its activation. Once active, MMP-26 has been shown to cleave multiple components of the ECM, including fibronectin, type IV collagen, vitronectin, gelatins, and fibrinogen, as well as non ECM proteins such as insulin-like growth factor-binding protein-1 and α-1 protease inhibitor [7][8][9][10][11]. MMP-26 is also able to activate progelatinase B (pro-MMP-9), an enzyme that plays a critical role in ECM remodeling [12].…”

“…MMP-26 mRNA is primarily expressed in epithelial cancers, such as lung, breast, endometrial, and prostate carcinomas [7][8][9][10]. Our previous studies have shown that MMP-26 expression in human prostate carcinoma is significantly higher than that in prostatitis, benign prostate hyperplasia (BPH), and normal prostate tissue [12].…”

Activation of Pro-gelatinase B by Endometase/Matrilysin-2 Promotes Invasion of Human Prostate Cancer Cells

“…More than 23 human MMPs, and numerous homologues from other species, have been reported (5), and matrix metalloproteinase-26 (MMP-26)/endometase/matrilysin-2 is a novel member of this enzyme family that was recently cloned and characterized by our group (6) and others (7)(8)(9). MMP-26 mRNA is primarily expressed in epithelial cancers, such as lung, breast, endometrial, and prostate carcinomas, in their corresponding cell lines (6 -9), and in a very limited number of normal adult tissues, such as the uterus (6,8), placenta (7,8), and kidney (9).…”