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...

This work has explored a putative biochemical mechanism by which endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) may promote human prostate cancer cell invasion. Here, we showed that the levels of MMP-26 protein in human prostate carcinomas from multiple patients were significantly higher than those in prostatitis, benign prostate hyperplasia, and normal prostate glandular tissues. The role of MMP-26 in prostate cancer progression is unknown. MMP-26 was capable of activating pro-MMP-9 by cleavage at the Ala 93 -Met 94 site of the prepro-enzyme. This activation proceeded in a time-and dose-dependent manner, facilitating the efficient cleavage of fibronectin by MMP-9. The activated MMP-9 products generated by MMP-26 appeared more stable than those cleaved by MMP-7 under the conditions tested. During the initial phases of carcinoma cell invasion, as tumor cells begin to spread and infiltrate into the surrounding normal tissues, these cells must first degrade the basement membrane and other elements of the extracellular matrix (ECM), 1 including type IV collagen, laminin, and fibronectin (FN) (1). Multiple protease families, including the matrix metalloproteinases (MMPs), serine proteases, and cysteine proteases, are suspected of contributing to the invasive and metastatic abilities of a variety of malignant tumors (2-5), but the specific biochemical mechanisms that facilitate these invasive behaviors remain elusive.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 (7-9). 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). Recently, we have found that the levels of MMP-26 gene and protein expression are higher in a malignant choriocarcinoma cell line (JEG-3) than in normal human cytotrophoblast cells (10). Our preliminary studies indicate that expression of MMP-26 may be correlated with the malignant transformation of human prostate and breast epithelial cells. The specific expression of MMP-26 in malignant tumors and the proteolytic activity of this enzyme against 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 ␣1-protease inhibitor (6 -9), indicate that MMP-26 may possess an important function in tumor progression.Another member of the MMP family considered to be an important contributor to the processes of invasion, metastasis, and angiogenesis exhibited by tumor cells is gelatinase B (MMP-9) (11-14). Uría and López-Otín (8) have demonstrated that MMP-26 is able to cleave MMP-9, and here we examine the possibility that MMP...

Local disruption of the integrity of both the myoepithelial cell layer and the basement membrane is an indispensable prerequisite for the initiation of invasion and the conversion of human breast ductal carcinoma in situ (DCIS) to infiltrating ductal carcinoma (IDC). We previously reported that human endometase/matrilysin-2/matrix metalloproteinase (

Matrix metalloproteinases (MMPs, 1 matrixins) are believed to participate in angiogenesis, embryonic development, morphogenesis, reproduction, tissue resorption and remodeling, and tumor growth, progression, invasion, and metastasis through breakdown of the extracellular matrix, cell surface proteins, and processing growth factors, cytokines, and chemokines (1-3). Recently, human MMP-26 (endometase/matrilysin 2) was identified and its mRNA expression was detected in normal tissues of the human uterus and placenta, and in many types of malignant tumors (4 -7). Characterization of the MMP-26 promoter suggests that this proteinase may be expressed in cancer cells of epithelial origin (8). MMP-26 may play an important role in human prostate and breast cancer invasion (9 -10).MMP-26 cleaves type I gelatin, ␣ 1 -proteinase inhibitor, fibrinogen, fibronectin, vitronectin, type IV collagen, and insulin-like growth factor binding protein-1 (4, 7, 11). Studies of MMP-26 indicate that it has substrate specificity similar to other MMPs, with the exception of a preference for Ile at the P 2 and P 2 Ј positions, for small residues at the P 3 Ј and P 4 Ј positions, and Lys at the P 4 position (11). MMP-26 also hydrolyzes several synthetic fluorogenic peptide substrates designed for stromelysin-1, gelatinases, collagenases, and tumor necrosis factor-␣ converting enzyme (4, 11). According to these peptide substrate studies, MMP-26 may be capable of cleaving a broad range of substrates, although it has less catalytic efficiency than other MMPs.X-ray crystal structures of MMPs illustrate that overall topology and secondary structures are conserved (12-18). The S 1 Ј pocket, a hydrophobic pocket of variable depth, is a well defined substrate P 1 Ј-binding site in MMPs. Three types of S 1 Ј pockets can be distinguished from the available structures of . One type is a shallow pocket, as found in MMP-1 (human fibroblast collagenase; 13) and 16), where the pockets are limited by the side chains of Arg and Tyr, respectively, crossing the pockets. Many of the structurally known MMPs possess Leu at the corresponding site, and its side chain forms the top of the pocket rather than crossing the pocket. These Leu-containing MMPs may be further classified as deep and intermediate S 1 Ј pocket MMPs. A deep, tunnel-like pocket is found in MMP-3 (stromelysin-1; 12), MMP-12 (metalloelastase; 17), and MMP-14 (MT1-MMP; 21), whereas MMP-2 (gelatinase A; 22), MMP-8 (human neutrophil collagenase; 15), and MMP-9 (gelatinase B; 23) possess an intermedi-

Human endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) is a novel epithelial and cancer-specific metalloproteinase. Peptide libraries were used to profile the substrate specificity of MMP-26 from the P4 -P4 sites. The optimal cleavage motifs for MMP-26 were Lys-Pro-Ile/Leu-Ser(P1)-Leu/Met(P1)-Ile/Thr-Ser/Ala-Ser. The strongest preference was observed at the P1 and P2 sites where hydrophobic residues were favored. Proline was preferred at P3, and Serine was preferred at P1. The overall specificity was similar to that of other MMPs with the exception that more flexibility was observed at P1, P2, and P3. Accordingly Matrix metalloproteinases (MMPs)1 share a conservative metal binding sequence of HEXGHXXGXXHS and a turn containing methionine (1). Evidence suggests that MMPs may play important roles in extracellular matrix (ECM) remodeling in physiological processes (2, 3). Excessive breakdown of the ECM by MMPs is observed in pathological conditions including periodontitis, rheumatoid arthritis, and osteoarthritis. MMPs also participate in tumor cell invasion and metastasis by degrading the basement membrane and other ECM components and allowing the cancer cells to gain access to blood and lymphatic vessels (4). Analyses of a large number of peptide and protein substrates and more recent work with phage display and synthetic peptide libraries have led to the identification of consensus cleavage site motifs for a number of different MMPs (5-13). The substrate specificities of MMPs are quite similar to each other, showing strong preferences for hydrophobic residues at P1Ј. Although distinct MMPs often prefer the same type of amino acid residues at corresponding positions surrounding the cleavage site, differences in the orders of preference for specific residues at each position may more precisely determine MMP specificity for substrates.Endometase (matrilysin-2/MMP-26) is the smallest member of the MMP family, with a molecular mass of 28 kDa (14 -17). Sequence homology calculations identified metalloelastase (MMP-12) and stromelysin-1 (MMP-3) as the closest relatives. Nevertheless, the specificity constant profile of peptide substrates with MMP-26 was quite different from that with . According to protein substrate studies in vitro, MMP-26 might process matrix proteins such as fibronectin, vitronectin, fibrinogen, type IV collagen, gelatinase B (MMP-9), and gelatin (14 -17).MMP-26 has been found to be highly expressed in several cancer cell lines. A significant level of expression in normal tissues was found only in the uterus and placenta. The limited occurrence of MMP-26 in normal tissues suggests that the production of this enzyme may be strictly regulated during specific events, such as implantation, and that MMP-26 could be a target enzyme for the treatment of cancer and other pathological conditions.The biological function and substrate specificity of MMP-26 are not yet fully understood. According to the protein substrate