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

We report the discovery, cloning, and characterization of a novel human matrix metalloproteinase 26 (MMP-26) (matrixin) gene, endometase, an endometrial tumor-derivedmetalloproteinase. 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, VATHEIGHSLGLQH. 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 α1-proteinase inhibitor; however, it did not digest collagens, laminin, elastin, β-casein, plasminogen, soybean trypsin inhibitor, or Bowman-Birk inhibitor. It hydrolyzed peptide substrates of matrixins and tumor necrosis factor-α 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)(2)(3)(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 (RTTI) gelatin and human plasma ␣ 1 -proteinase inhibitor (␣ 1 -PI). Our data show that endometase is an authentic new member of the MMP family with unique biochemical characteristics and substrate specificities. Endometase may be tissue-specific and may have unique biological and pathological functions.

EXPERIMENTAL PROCEDURES
Materials-Human ␣ 1 -antitrypsin (␣ 1 -PI) and most chemicals and reagents were purchased from Sigma. Human matrilysin, stromelysin, and recombinant catalytic domain of collagenase-3 (cd-collagenase-3) were kindly provided by Drs. Harold E. van Wart of Roche Bioscience, L. Jack Windsor of Indiana University, and Harald Tschesche of the University of Bielefeld, respectively. Recombinant rabbit catalytic domain of metalloelastase (cd-metalloelastase) was kindly provided by Dr. C. Bruun Schiødt, OsteoPro A/S. Molecular Cloning of Endometase Full-length cDNA Sequence-A data base of more than three million expressed sequence tags (ESTs) was obtained from over 750 different cDNA libraries. This data base has been generated through the combined efforts of Human Genome Science Inc. and the Institute for Genomic Research using high throughput automated DNA sequence analysis of randomly selected human cDNA clones (9). Sequence homology comparisons of each EST were performed against the GenBank TM data base using the Basic Local Alignment Search Tool (BLAST) and BLASTN (BLAST for nucleic acid) algorithms (10). A specific homology and motif search using the known amino acid sequence of matrilysin against this human EST data base revealed several ESTs that had translated sequences with Ͼ38% identity to that of matrilysin. One clone, HETBW05, from human endometrial tumor cDNA library encoding an intact N-terminal signal peptide and the * This work was supported by NCI, National Institutes of Health Grant CA78646, a grant from Elsa U. Pardee Foundation, and a grant from the Gustavus and Louise Pfeiffer Research Foundation (to Q.-X. A. S.) and by a Postdoctoral Research Training Grant from the National Science Foundation (to H. I. P.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The full-length protein was selected for further investigation. This EST was sequenced on both strands to the 3Ј end, and its homology to matrilysin was confirmed.
Northern Blot Analysis-Five Northern blot membranes containing 2 g of poly(A) ϩ RNAs per lane from human multiple tissues, human immune system, and human cancer cell lines were purchased from CLONTECH. Two g of 2.0-kb human ␤-actin poly(A) ϩ RNAs per lane was a positive control for all the Northern blot membranes. The membrane was probed with 32 P-labeled HETBW05 cDNA probe and the human ␤-actin cDNA positive control probe. Northern blotting was performed with the ExpressHyb TM hybridization solution (PT1190 -1) from CLONTECH following the manufacturer's manual.
Expression of Pro-endometase in Escherichia coli Cells and Refolding of the Denatured Protein-The full-length open reading frame of HETBW05 was amplified using standard polymerase chain reaction techniques with primers corresponding to the 5Ј and 3Ј sequences of the open reading frame (5Ј restriction site, BglII; 5Ј primer, GAGCGCa-gatctGCCATCATGCAGCTCGTCATCTT; 3Ј restriction site, XbaI; 3Ј primer, CGGTGCtctagaTTAAGGTATGTCAGATGAACAT). The amplified fragment was purified and digested with BglII and XbaI followed by a second purification. A tag-free construct for expression of proendometase in pET-20b(ϩ) (Novagen) was generated by restriction enzyme digestion using the NdeI and XhoI restriction sites. Doubly cleaved vector and inserts with cohesive ends were purified by agarose gel electrophoresis and ligated by T4 DNA ligase. All the clones were verified by DNA sequencing. Plasmids were propagated in TOP10FЈ competent cells (Invitrogen) and then transformed into BL21 (DE3)competent E. coli cells (Novagen) for expression according to the manufacturer's instructions. The majority of endometase was expressed in the form of inclusion bodies. The inclusion bodies were isolated and purified using the B-PER TM bacterial protein extraction reagent according to the manufacturer's instructions. The insoluble protein was dissolved in 8 M urea at about 5 mg/ml. The protein was diluted to 100 g/ml in 8 M urea and 10 mM dithiothreitol for 1 h, dialyzed in 4 M urea, 1 mM dithiothreitol, 50 mM Tricine, pH 7.5, for 2 h, and then folded by dialysis in 50 mM HEPES, 0.2 M NaCl, 10 mM CaCl 2 , 20 M ZnSO 4 , 0.05% Brij-35, pH 7.5, for 16 h. Several other folding conditions were also tested.
Immunological and Biochemical Characterization of Endometase-Anti-human endometase peptide polyclonal antibodies were developed in rabbits as described (11). Polyclonal antibody against endometase peptide starting from residue 188 (pAb-E188) was directed against a peptide just a few residues upstream the zinc binding site, 188 DKNE-HWSASDTGYN 201 of the prepro-enzyme. Using BLAST search method at the National Center for Biotechnology Information web site against all of the sequences in the data banks, no peptide with Ͼ45% level of identity was found. Thus, the antibody against this peptide should be very specific. Western blot analysis and SDS-PAGE experiments characterizing different refolded enodmetase samples were performed as described previously (11,12). Protein substrates were digested by en- dometase and the products were detected by SDS-PAGE gel with Coomassie Blue staining (12)(13)(14). Pro-endometase and ␣ 1 -PI digestion products were also analyzed by protein N-terminal sequencing (12)(13)(14).
Synthetic Fluorogenic Peptide Substrate Cleavage Assays-The following substrates were purchased from BACHEM Bioscience Inc.: substrate (16); and substrate 5, Mca-PLAQAVDpaRSSSR-NH 2 (17), where Nva represents norvaline. The first four are MMP substrates, and the last one is a substrate of the tumor necrosis factor-␣ converting enzyme (TACE). Substrates were prepared as 50 -500 M stock solution in 1:1 dimethyl sulfoxide (Me 2 SO) and water. Fluorescent assays were performed at excitation ϭ 328 nm and emission ϭ 393 nm using a Perkin-Elmer LS 50B luminescence spectrometer equipped with a constant-temperature water bath. The relationship between fluorescence units and nmol of product produced was determined from the fluorescence value obtained when all the substrate was hydrolyzed. Assays for obtaining kinetic parameters were performed at 25°C in 10 mM CaCl 2 , 0.2 M NaCl, and 0.05% Brij-35 in 50 mM HEPES, pH 7.5, over a substrate concentration range of 1-4 M and an enzyme concentration range of 0.06 -50 nM under steady-state conditions. Stock solutions of MMPs were diluted to 1-500 nM by adding 50 mM HEPES buffer containing 10 mM CaCl 2 , 0.2 M NaCl, and 0.05% Brij-35 or 50 mM Tricine buffer with the same constituents. A typical assay was carried out by incubating 186 l of buffer solution and 4 l of substrate solution in an assay cuvette at least for 15 min at 25°C and then adding 10 l of enzyme solution to the assay cuvette. Initial hydrolysis rates were monitored for 10 -30 min.

Molecular
Cloning of Endometase and Homology of Endometase to Other MMPs-By searching EST data bases, we found one clone, HETBW05, from a human endometrial tumor cDNA library, that encodes a MMP homologue with an intact N-terminal signal peptide. This clone was not found in any other cDNA libraries derived from other human tissues/cells. The protein expressed from this clone was selected for further investigation, and it was named endometase (for endometrial tumor-derived matrix metalloproteinase).
The cDNA nucleotide and deduced protein sequences of the full-length clone encoding human endometase are shown in Fig. 1. Those sequences were submitted to GenBank TM with accession number AF248646. The signal peptide sequence was predicted according to its homology to other MMPs and using the PSORT computer program. The activation cleavage site between the propeptide domain and the catalytic domain was predicted according to its homology to other MMPs (18). Endometase has a unique histidine residue instead of an arginine residue in the cysteine switch sequence (PHCGVPDGSD). All other MMPs have an arginine residue at that position. In addition, endometase has the MMP zinc-binding consensus sequence in the catalytic domain. The endometase protein sequence was scanned against the GenBank TM data bases, and all of the sequences with significant relatedness to the new sequence were identified. The endometase protein sequence was compared with those of five most similar human  NP_002418.1) (21), enamelysin (MMP-20, accession number NP_004762.1) (22), and matrilysin (MMP-7, accession number P09237) (23) (Fig. 2). The endometase catalytic domain is 54, 51, 49, 48, and 46% identical to those of MMP-12, -3, -13, -20, and -7, respectively. Its full length is 43, 41, 41, 39, and 39% identical to those of MMP-12, -3, -13, -20, and -7, respectively ( Table I). All of the above cDNA and protein sequence information indicates that endometase is a new member of the MMP family.
Northern Blot Analysis-Tissue expression distribution of endometase mRNA was examined. Five Northern blots were purchased from CLONTECH. Blot 1 contains poly(A) ϩ RNAs from human multiple tissues, spleen, thymus, prostate, testis, ovary, small intestine, colon (mucosal lining), and peripheral blood leukocytes. Blot 2 contains poly(A) ϩ RNAs from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. Blot 3 contains poly(A) ϩ RNAs from human immune system tissues, spleen, lymph node, thymus, appendix, peripheral blood leukocytes, bone marrow, and fetal liver. Blot 4 contains poly(A) ϩ RNAs from human cancer cell lines, promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361. Blot 5 contains poly(A) ϩ RNAs from human spleen, thymus, prostate, testis, uterus, small intestine, colon (mucosal lining), and peripheral blood leukocytes. 2 kb of human ␤-actin poly(A) ϩ RNA per lane was a positive control for all the Northern blot membranes. The membrane was probed with both 32 P-labeled HETBW05 cDNA probe and the human ␤-actin cDNA positive control probe. Except for 2-kb ␤-actin bands, no detectable band was found in blots 1-4. Endometase mRNA was expressed only by human uterus as a 1.03-kb band (Fig. 3) in blot 5; it was not expressed in any other human tissues or human cancer cells tested. Because endometrium is the mucous membrane lining of the uterus, these data demonstrate that endometase may be uterus-specific.
Biochemical and Immunological Analysis of Endometase Protein Samples-The pro-endometase inclusion bodies were isolated and purified using B-PER TM bacterial protein extraction and purification reagent. Lysozyme was used for preparation of the inclusion bodies. This method allowed us to obtain highly homogenous endometase from the inclusion bodies as demonstrated by SDS-PAGE silver stain gel and immunoblot analysis using our anti-catalytic domain peptide antibody (pAb-E188) (data not shown). The purified recombinant proendometase was partially activated containing both latent (28 kDa) and active (19 kDa) species during the folding process. The specificity of pAb-E188 was verified because it did not recognize human stromelysin, collagenase-1, collagenase-3, matrilysin, MT1-MMP, gelatinase A, or gelatinase B (data not shown). Protein N-terminal sequencing further confirmed that we have isolated the pro-endometase with one extra initiation codon coded methionine residue, MVPVPPAADH.
Hydrolysis of Protein Substrates by Endometase-Heat-denatured RTTI collagen (gelatin) was cleaved slowly by endometase compared with that by matrilysin (Fig. 4). 275 ng of endometase and matrilysin was incubated with 4 mg/ml gelatin for 0, 1, 2, and 3 days at 37°C in 100 l of assay buffer containing 50 mM HEPES, 0.2 M NaCl, 10 mM CaCl 2 , and 0.05% Brij-35, pH 7.5. Endometase cleaved RTTI gelatin and generated three-quarter, one-quarter, and other fragments of ␣ 1 (I) and ␣ 2 (I) subunits. However, endometase did not cleave RTTI collagen, mouse type IV collagen, laminin, bovine elastin, ␤-casein, or human milk lactoferrin, demonstrating that the endometase cleavage of type I gelatin is specific. Human plasma ␣ 1 -PI (63 kDa) was cleaved by endometase and generated a visible band at about 58 kDa (Fig. 5). Both the 63-and the 58-kDa protein bands have the same sequence, EDPQG-DAAQK, as determined by the N-terminal sequencing technique, suggesting that ␣ 1 -PI was cleaved near the C terminus by removing a 5-kDa fragment. This sequence represents the N-terminal sequence of the mature ␣ 1 -PI protein (24). However, endometase did not cleave other two serine proteinase inhibitors (serpins) tested, soybean trypsin inhibitor and trypsin-chymotryspin inhibitor (Bowman-Birk inhibitor). Moreover, endometase did not digest any of the other human plasma proteins tested (albumin, ␣ 1 -acid glycoprotein, apo-transferrin,   (Table II). It hydrolyzed the common MMP substrate, Mca-PLGLDpaAR-NH 2 (15), with a k cat /K m value of 1850 M Ϫ1 s Ϫ1 , which is similar to those of stromelysin and the cd-collagenase-3 but is much lower than that of matrilysin and cd-metalloelastase. Endometase also digested the other three MMP substrates. Interestingly, it has the ability to hydrolyze the TACE substrate with a k cat /K m value of 185 M Ϫ1 s Ϫ1 , which is much lower than that of cdmetalloelastase (13,000 M Ϫ1 s Ϫ1 ). DISCUSSION We have cloned and identified a novel member of the MMP family, endometase/MMP-26. Its prepro-enzyme has 261 amino acid residues, making it the smallest member of the matrixin family ( Figs. 1 and 2 and Table I). Endometase has a unique cysteine switch sequence, PHCGVPDGSD, and a zinc-binding consensus sequence in the catalytic domain, HEXGHXXGXXH. It also has three potential N-glycosylation sites. One is at residue Asn 64 of the propeptide domain; the other two are at the residues Asn 133 and Asn 221 , distributed within the catalytic domain. The possibility of N-glycosylation of endometase in human and mammalian systems remains to be investigated. This unique enzyme was only cloned from a human endometrial tumor cDNA library, and its mRNA was only detected in human uterus, not in the other tissues and cancer cell lines tested, indicating a possible tissue-specific distribution and function of endometase.
Endometase has a unique protein substrate specificity. Endometase cleaved gelatin and generated three-quarter, onequarter, and other fragments of ␣ 1 (I) and ␣ 2 (I) subunits (Fig.  4), suggesting that it may have gelatin cleavage sites identical or similar to those of human interstitial collagenase (MMP-1) and frog collagenase-4 (MMP-18) (13). Although the catalytic domain of endometase was found to be 49% identical to that of human collagenase-3, it did not dissolve type I collagen, possibly due to its lack of a hemopexin-like domain. Endometase also did not cleave type IV collagen, laminin, elastin, ␤-casein, and lactoferrin, demonstrating that the endometase cleavage of type I gelatin was specific. Although the catalytic domain of endometase was found to be 54, 51, and 46% identical to those of metalloelastase, stromelysin, and matrilysin, respectively, endometase did not hydrolyze type IV collagen, elastin, laminin, plasminogen, or ␤-casein, further illustrating that endometase may have its unique substrates.
Endometase selectively hydrolyzed human plasma ␣ 1 -PI (Fig. 5). It did not cleave two other serpins, soybean trypsin inhibitor and Bowman-Birk inhibitor. Moreover, endometase did not digest any other human plasma proteins tested, e.g. albumin, ␣ 1 -acid glycoprotein, transferrin, and plasminogen, further showing that the hydrolysis of ␣ 1 -PI by endometase was specific. Our results suggested that the ␣ 1 -PI protein was cleaved by endometase near the C terminus by removing a 5-kDa fragment. Interestingly, ␣ 1 -PI protein was cleaved by MMP-1 at two sites near the C terminus, Phe 352 -Leu 353 and Pro 357 -Met 358 of the mature ␣ 1 -PI protein, which indicated that a fragment of about 4.5 kDa was removed from the C terminus (25). Stromelysin-3 also inactivated ␣ 1 -PI by cleaving Ala 350 -Met 351 within the reactive site loop in breast cancer (26). Therefore, one of the potential functions of endometase would be selective inactivation of serpin, thus promoting serine proteinase activity and enhancing extracellular matrix degradation in endometrial cancer.
The peptide substrate specificity of enodmetase was tested and compared with four most similar MMPs, cd-metalloelastase, stromelysin, cd-collagenase-3, and matrilysin (Table II). Endometase hydrolyzed four synthetic MMP peptide substrates and one TACE substrate. Endometase shares some of the same substrates as other MMPs; however, it has its own unique catalytic specificities and efficiencies. In addition, endometase hydrolyzed a TACE substrate. Interestingly, matrilysin has been reported to release tumor necrosis factor-␣ in a herniated disc resorption model (27). The possibility of processing pro-tumor necrosis factor-␣ by endometase warrants further investigation. Moreover, our preliminary studies showed that endometase was inhibited by tissue inhibitors of metalloproteinases-1 and -2, 2 further confirming that endometase is an authentic matrixin. Our data suggest that endometase may be uterus-specific and may have unique biochemical characteristics and physiological and pathological functions.