Amyloid-β (Aβ) 42, one of the causes of Alzheimer's disease (AD), is produced by the cleavage of amyloid precursor protein (APP) by β- or γ-secretases. Since Aβ42 oligomers exhibit strong neurotoxicity, Aβ42 is predicted to be a potentially efficient target for drug therapies. Recently, we screened peptides that activate MMP7 using our peptide library and found that the synthetic peptide JAL-TA9 (YKGSGFRMI), which is derived from the BoxA region of Tob1 protein, showed proteolytic activity. It is generally accepted that an enzyme should be a large molecular protein consisting of more than thousands of amino acids. Thus, this is the first finding that a small synthetic peptide has protease activity, and we termed Catalytide as the general name of peptides with protease activity [1]. In this study, we demonstrate the cleavage activity of JAL-TA9 not only against the authentic soluble form of Aβ42 but also against the solid type of Aβ42 in the central region [2]. In addition, we demonstrated the cleavage activity using brain slices of AD patients. JAL-TA9 decreased the amount of accumulated Aβ42 in the brain of Alzheimer's patients. Taken together, JAL-TA9 is an attractive seed for the development of peptide drugs with a new strategy for Alzheimer's disease.
Human matrix metalloproteinase-7 (MMP-7=matrilysin) was overproduced in Escherichia coli; as a recombinant zymogen (31 kDa), the C-terminus of which bears artificial hexa-histidines. Most of the enzyme was isolated from the insoluble fraction of the cell lysate and purified by a single step using Ni-NTA resin after solubilization of the precipitates with 8 M urea solution. The resin-bound recombinant protein was refolded into a form that is activatable by p-amino-phenylmercuric acetate in an autocatalytic manner. The activated enzyme cleaved a synthetic peptide substrate at the reported site for MMP-7. Digestion of carboxymethylated transferrin (a natural substrate of MMP-7) by the recombinant proteinase generated fragments with the same peptide map as in the case of native purified MMP-7. The autocatalytic activation and enzyme reaction were entirely dependent on the presence of calcium and zinc ions. The enzyme activity to cleave carboxymethylated transferrin was inhibited by tissue inhibitors of metalloproteinases−1 and −2, MMP-specific inhibitors. The activity of the recombinant MMP-7 was also inhibited by a synthetic peptide derived from a part of the cysteine switch that maintains the zymogen in an inactive state. Thus, we report here a simple means of preparing a large quantity of recombinant proMMP-7 that can be used to study the activation mechanism and to screen synthetic inhibitors.
MT6-MMP/MMP-25 is the latest member of the membrane-type matrix metalloproteinase (MT-MMP) subgroup in the MMP family and is expressed in neutrophils and some brain tumors. The proteolytic activity of MT6-MMP has been studied using recombinant catalytic fragments and shown to degrade several components of the extracellular matrix. However, the activity is possibly modulated further by the C-terminal hemopexin-like domain, because some MMPs are known to interact with other proteins through this domain. To explore the possible function of this domain, we purified a recombinant MT6-MMP with the hemopexin-like domain as a soluble form using a Madin-Darby canine kidney cell line as a producer. Mature and soluble MT6-MMP processed at the furin motif was purified as a 45-kDa protein together with a 46-kDa protein having a single cleavage in the hemopexin-like domain. Interestingly, 73- and 70-kDa proteins were co-purified with the soluble MT6-MMP by forming stable complexes. They were identified as clusterin, a major component of serum, by N-terminal amino acid sequencing. MT1-MMP that also has a hemopexin-like domain did not form a complex with clusterin. MT6-MMP forming a complex with clusterin was detected in human neutrophils as well. The enzyme activity of the soluble MT6-MMP was inactive in the clusterin complex. Purified clusterin was inhibitory against the activity of soluble MT6-MMP. On the other hand, it had no effect on the activities of MMP-2 and soluble MT1-MMP. Because clusterin is an abundant protein in the body fluid in tissues, it may act as a negative regulator of MT6-MMP in vivo. MT6-MMP/MMP-25 is the latest member of the membrane-type matrix metalloproteinase (MT-MMP) subgroup in the MMP family and is expressed in neutrophils and some brain tumors. The proteolytic activity of MT6-MMP has been studied using recombinant catalytic fragments and shown to degrade several components of the extracellular matrix. However, the activity is possibly modulated further by the C-terminal hemopexin-like domain, because some MMPs are known to interact with other proteins through this domain. To explore the possible function of this domain, we purified a recombinant MT6-MMP with the hemopexin-like domain as a soluble form using a Madin-Darby canine kidney cell line as a producer. Mature and soluble MT6-MMP processed at the furin motif was purified as a 45-kDa protein together with a 46-kDa protein having a single cleavage in the hemopexin-like domain. Interestingly, 73- and 70-kDa proteins were co-purified with the soluble MT6-MMP by forming stable complexes. They were identified as clusterin, a major component of serum, by N-terminal amino acid sequencing. MT1-MMP that also has a hemopexin-like domain did not form a complex with clusterin. MT6-MMP forming a complex with clusterin was detected in human neutrophils as well. The enzyme activity of the soluble MT6-MMP was inactive in the clusterin complex. Purified clusterin was inhibitory against the activity of soluble MT6-MMP. On the other hand, it had no effect on the activities of MMP-2 and soluble MT1-MMP. Because clusterin is an abundant protein in the body fluid in tissues, it may act as a negative regulator of MT6-MMP in vivo. Cells are continuously communicating with the extracellular environment through the cell surface where molecules mediating signals exist. Proteolysis is an important part of the regulation of these transmembrane signals by activation, inactivation, or functional conversion of the molecules (1Lopez-Otin C. Overall C.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 509-519Crossref PubMed Scopus (618) Google Scholar, 2Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5072) Google Scholar, 3Seiki M. Curr. Opin. Cell Biol. 2002; 14: 624-632Crossref PubMed Scopus (190) Google Scholar). However, our knowledge about the proteases involved in such processes remains limited compared with that of the intracellular signaling events and responsive molecules. Recently, a number of cell-associated proteases have been identified by cDNA cloning techniques and from the whole genome sequencing projects, and this has gradually opened the way to addressing the proteolytic events in the pericellular space.One such example is MT6-MMP, the latest member of the MT-MMP 1The abbreviations used are: MT-MMP, membrane-type matrix metalloproteinase; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; Mca, (7-methoxycoumarin-4-yl)acetyl; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PVDF, polyvinylidene difluoride; FN, fraction; PMN, polymorphonuclear leukocytes; GPI, glycosylphosphatidylinositol; MDCK, Madin-Darby canine kidney; mAb, monoclonal antibody.1The abbreviations used are: MT-MMP, membrane-type matrix metalloproteinase; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; Mca, (7-methoxycoumarin-4-yl)acetyl; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PVDF, polyvinylidene difluoride; FN, fraction; PMN, polymorphonuclear leukocytes; GPI, glycosylphosphatidylinositol; MDCK, Madin-Darby canine kidney; mAb, monoclonal antibody. subgroup in the MMP family (matrixins) (4Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (168) Google Scholar, 5Velasco G. Cal S. Merlos-Suarez A. Ferrando A.A. Alvarez S. Nakano A. Arribas J. Lopez-Otin C. Cancer Res. 2000; 60: 877-882PubMed Google Scholar). Among the six members, MT6-MMP tethers to the cells through a covalent link to the glycosylphosphatidylinositol (GPI) in the plasma membrane (6Kojima S. Itoh Y. Matsumoto S. Masuho Y. Seiki M. FEBS Lett. 2000; 480: 142-146Crossref PubMed Scopus (116) Google Scholar, 7Kang T. Yi J. Guo A. Wang X. Overall C.M. Jiang W. Elde R. Borregaard N. Pei D. J. Biol. Chem. 2001; 276: 21960-21968Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) like MT4-MMP (8Itoh Y. Kajita M. Kinoh H. Mori H. Okada A. Seiki M. J. Biol. Chem. 1999; 274: 34260-34266Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), whereas other MT-MMPs are integrated into the plasma membrane through a C-terminal transmembrane sequence (MT1, MT2, MT3, and MT5-MMP) (3Seiki M. Curr. Opin. Cell Biol. 2002; 14: 624-632Crossref PubMed Scopus (190) Google Scholar, 9Seiki M. APMIS. 1999; 107: 137-143Crossref PubMed Scopus (273) Google Scholar). MT6-MMP is expressed almost specifically in neutrophils (polymorphonuclear leukocytes, PMN) (4Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (168) Google Scholar, 7Kang T. Yi J. Guo A. Wang X. Overall C.M. Jiang W. Elde R. Borregaard N. Pei D. J. Biol. Chem. 2001; 276: 21960-21968Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), although some brain tumors also express it (5Velasco G. Cal S. Merlos-Suarez A. Ferrando A.A. Alvarez S. Nakano A. Arribas J. Lopez-Otin C. Cancer Res. 2000; 60: 877-882PubMed Google Scholar). Neutrophils express other MMPs such as MMP-8 (neutrophil collagenase) and MMP-9 (gelatinase B) as well (10Owen C.A. Campbell E.J. J. Leukocyte Biol. 1999; 65: 137-150Crossref PubMed Scopus (350) Google Scholar), but these are also expressed in other tissues and cell types (2Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5072) Google Scholar). Given this specificity in its expression, MT6-MMP seems to play a pivotal role in the neutrophil function. It is also interesting that stimulation of neutrophils with phorbol myristate acetate or interleukin-8 caused MT6-MMP to be released as a soluble enzyme from the cell surface or secretory vesicles (7Kang T. Yi J. Guo A. Wang X. Overall C.M. Jiang W. Elde R. Borregaard N. Pei D. J. Biol. Chem. 2001; 276: 21960-21968Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Because MMPs are responsible for the degradation of most of the components of the extracellular matrix, the MMPs produced by neutrophils are presumably important for invasion and migration of the cells to inflammatory sites and/or destruction of the host tissue. On the other hand, the substrates of MMPs are not restricted to the matrix components (11McCawley L.J. Matrisian L.M. Curr. Opin. Cell Biol. 2001; 13: 534-540Crossref PubMed Scopus (1088) Google Scholar). Recent studies in the field have revealed their targets also include non-matrix type molecules, such as cell adhesion molecules, cytokines, growth factors, and receptors (2Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5072) Google Scholar).The proteolytic activity of MT6-MMP has been studied using recombinant catalytic fragments and was found to degrade type IV collagen, gelatin, fibrin, fibronectin, chondroitin sulfide proteoglycan, and dermatin sulfide proteoglycan but not types I–III collagens (7Kang T. Yi J. Guo A. Wang X. Overall C.M. Jiang W. Elde R. Borregaard N. Pei D. J. Biol. Chem. 2001; 276: 21960-21968Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 12English W.R. Velasco G. Stracke J.O. Knauper V. Murphy G. FEBS Lett. 2001; 491: 137-142Crossref PubMed Scopus (71) Google Scholar). Although the substrate specificity of MMP is basically attributable to the structure of the catalytic cleft, the hemopexin-like domain is also known to modulate the activity by binding the substrates, interacting with other proteins that may function as an adaptor for possible substrates, or affecting the enzyme structure (13Murphy G. Allan J.A. Willenbrock F. Cockett M.I. O'Connell J.P. Docherty A.J. J. Biol. Chem. 1992; 267: 9612-9618Abstract Full Text PDF PubMed Google Scholar, 14Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 15McQuibban G.A. Gong J.H. Tam E.M. McCulloch C.A. Clark-Lewis I. Overall C.M. Science. 2000; 289: 1202-1206Crossref PubMed Scopus (638) Google Scholar, 16Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (337) Google Scholar, 17Rozanov D.V. Ghebrehiwet B. Postnova T.I. Eichinger A. Deryugina E.I. Strongin A.Y. J. Biol. Chem. 2002; 277: 9318-9325Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Mori H. Tomari T. Koshikawa N. Kajita M. Itoh Y. Sato H. Tojo H. Yana I. Seiki M. EMBO J. 2002; 21: 3949-3959Crossref PubMed Scopus (277) Google Scholar). Thus, it is important to examine MT6-MMP with a hemopexin-like domain.In this study, we established a Madin-Darby canine kidney (MDCK) cell line that stably expresses a soluble form of MT6-MMP with a hemopexin-like domain to study the properties of the intact MT6-MMP focusing on the function of the C-terminal domain. Mature MT6-MMP processed at the furin motif was purified as a 45-kDa protein and a 46-kDa protein having a single cleavage in the hemopexin-like domain. During the purification, 73- and 70-kDa proteins were found to associate with MT6-MMP by forming stable complexes. These proteins were identified as clusterin, a major component in serum, by N-terminal amino acid sequencing. This study examined the specificity and roles of the complex formation.EXPERIMENTAL PROCEDURESMaterials—Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, FLAG peptide, monoclonal mouse anti-FLAG M2 antibody, and anti-FLAG M2-agarose were purchased from Sigma. Monoclonal mouse anti-human clusterin antibody was from Research Diagnostics, Inc. (Flanders, NJ). G418 (geneticin) and hygromycin B were from Invitrogen. Protease inhibitor mixture and FuGENE™ 6 were from Roche Applied Science. A quenched fluorescent peptide substrate, Mca-PLGL-Dpa-AR-NH2, was obtained from Peptide Institute, Inc. (Osaka, Japan). Soluble form of MT1-MMP was kindly provided by Dr. Y. Okada at Keio University (Tokyo, Japan). MMP-2 was purified as described previously (16Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (337) Google Scholar). TIMP-1 and -2 were a kind gift from Dr. K. Iwata (Dai-ichi Pure Chemical, Takaoka, Japan).Cell Culture and Transfection—Madin-Darby canine kidney (MDCK) cells and human breast carcinoma MCF-7 cells were purchased from the American Type Culture Collection (Manassas, VA). MDCK cells were maintained in DMEM (Sigma) supplemented with 10% fetal bovine serum and 0.1 mg/ml of kanamycin (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2. MCF-7 cells were maintained in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum and 0.1 mg/ml kanamycin. For transfection, cells were seeded in 6-well plates at 1 × 105 cells/well, and transfection was performed after 16 h. Expression plasmids for proteins were transfected using FuGENE™ 6 (Roche Applied Science) according to the manufacturer's instructions.Construction of Expression Plasmids—cDNA of MT6-MMP was used as a template to generate DNA fragments for sMT6-F (Met1–Gly514) by PCR employing two primers: a forward primer (5′-AGT GGA TCC CCA CCA TGC GGC TGC GGC TCC G-3′) and a reverse FLAG insertion primer (5′-ACT CTC GAG TCA TCA CTT GTC ATC GTC GTC CTT GTA GTC ACC AGA GCT CGG GGC GG-3′). The fragment was sub-cloned into the pcDNA3.1(+) expression vector (Invitrogen).FLAG epitope (DYKDDDDK)-tagged MT1-MMP (MT1F) and MT6-MMP (MT6F) were constructed as described previously (6Kojima S. Itoh Y. Matsumoto S. Masuho Y. Seiki M. FEBS Lett. 2000; 480: 142-146Crossref PubMed Scopus (116) Google Scholar) and sub-cloned into the pCEP4 expression vector (Invitrogen). The MT6F plasmid was used as a template to generate DNA fragments for MT6FdCAT and MT6FdPEX. MT6FdCAT and MT6FdPEX were catalytic domain (Tyr108–Gly280)-deleted and hemopexin-like domain (Cys317–Cys518)-deleted mutants of MT6F, respectively. All the mutant constructs were generated by PCR using the overlap extension method as described previously. All PCR products were confirmed by DNA sequencing.Establishment of Stable Cell Lines of sMT6-F—The expression vector for sMT6-F was transfected into MDCK cells using FuGENE™ 6 (Roche Applied Science). At 2 days after transfection, the cells were selected in DMEM supplemented with 10% fetal bovine serum, 0.1 mg/ml kanamycin (Invitrogen), and 800 μg/ml G418. After culture for 10 days, each single clone was selected by limiting dilution. Positive clones were then used for protein purification.Purification of sMT6-F—The stable sMT6-F transfectant was cultured in a cell factory (Nunc, Rochester, NY) until confluent. The cells were washed three times with PBS and replenished with serum-free DMEM. After incubation for 72 h, the conditioned medium (CM) was collected, clarified by centrifugation, and then concentrated using ammonium sulfate to a final saturation of 80%. The precipitated protein was collected by centrifugation and then dissolved in and dialyzed against Tris-buffered saline (TBS: 50 mm Tris-HCl (pH 7.5), 150 mm NaCl) containing 0.05% Brij 35. This fraction was then applied to an anti-FLAG M2-agarose column, and the column was washed with the same buffer. Specific elution was carried out using FLAG peptide (100 μg/ml).Western Blot Analysis—Samples were subjected to SDS-PAGE under reducing or non-reducing conditions (16Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (337) Google Scholar), and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, MA). After blocking with 10% fat-free dry milk in PBS for 1 h, the membrane was probed with primary antibody specific to each antigen. The membrane was further probed with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Biosciences) and detected using ECL Plus (Amersham Biosciences).Gelatin Zymography—Gelatin zymography was performed as described previously (19Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (214) Google Scholar). The samples were mixed with SDS sample buffer without a reducing agent and separated on 7.5% acrylamide gels containing gelatin (0.8 mg/ml). The gelatin-containing gel was renatured by washing with 2.5% Triton X-100-containing buffer for 1 h and incubated for 12 h at 37 °C. The gelatin remaining in the gel was stained with Coomassie Brilliant Blue R-250, and gelatinolytic activity was detected as clear bands against a blue background.N-terminal Amino Acid Sequencing of Purified sMT6-F and Its Binding Proteins—The sample was subjected to SDS-PAGE under reducing conditions and transferred to PVDF membrane (Millipore). After the staining of the membrane with Coomassie Brilliant Blue R-250, the stained bands were excised, and the Beckman Coulter LF3000 amino acid sequencer was used.Gel Permeation Chromatography—The sMT6-F-clusterin complex was subjected to gel permeation chromatography on HiLoad 16/60 Superdex 200 pg (Amersham Biosciences) equilibrated with TBS buffer containing 0.05% Brij 35 at a flow rate of 0.5 ml/min, and a 1-ml fraction was collected. The calibration was performed using thyroglobulin (669 kDa), ferritin (440 kDa), adolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa) as protein standards.Dissociation of sMT6-F from sMT6-F-Clusterin Complex—The sMT6-F-clusterin complex was separated by gel permeation chromatography. The complex-containing fractions were pooled and then dialyzed against 50 mm Tris-HCl (pH 7.5) buffer. After 12 h dialysis, the pooled fraction was denatured using 6 m urea. The denatured complex was applied to a Q-anion exchange column (Bio-Rad). The dissociated clusterin was eluted with the same buffer containing 0.1 m NaCl, whereas clusterin-free sMT6-F was eluted with buffer containing 0.2 m NaCl. Subsequently, the separated sMT6-F and clusterin were individually dialyzed against 50 mm Tris-HCl (pH 7.5) containing 150 mm NaCl, 5 mm CaCl2, 50 μm ZnCl2, and 0.01% Brij 35 at 4 °C for 2 and 12 h, respectively. The final preparation was frozen at –80 °C before use.Assay of Enzyme Activities—Purified enzyme was assayed using a fluorescence-quenched peptide substrate (Mca-PLGL-Dpa-AR-NH2) (20Knight C.G. Willenbrock F. Murphy G. FEBS Lett. 1992; 296: 263-266Crossref PubMed Scopus (671) Google Scholar). Purified enzyme (0.7 nm) was incubated with substrate (2 μm)in50 mm Tris-HCl (pH 7.5) containing 150 mm NaCl, 5 mm CaCl2, and 0.05% Brij 35 at 37 °C for 1 h. The concentration of enzyme was determined by active site titration with TIMP-2.Reaction mixtures of enzyme and inhibitors were preincubated at 25 °C for 15 min with TIMP-1, TIMP-2, and clusterin. The apparent inhibition constant K i(app) was calculated by using Equation 1, Ki(app)=I/(Vo/Vi-1)(Eq. 1) where I is the final inhibitor concentration, Vo the rate of substrate hydrolysis without inhibitor, and Vi the rate of hydrolysis with inhibitor.Immunoprecipitation—Expression plasmids were transfected into MCF-7 cells using FuGENE™ 6. Stable transfectants were selected using hygromycin B (300 μg/ml) and lysed with RIPA buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Triton X-100, 1% deoxycholic acid, and 0.1% SDS) in the presence of a protease inhibitor mixture (Roche Applied Science). The cell lysate was clarified by centrifugation at 15,000 rpm for 15 min. The supernatant was incubated with anti-FLAG M2-agarose (Sigma) at 4 °C for 6 h. The agarose was washed three times with RIPA buffer and precipitated. The bound proteins were eluted with FLAG peptide and analyzed by Western blotting using anti-FLAG M2 antibody or anti-human clusterin antibody. Other experimental conditions were described in our previous report (18Mori H. Tomari T. Koshikawa N. Kajita M. Itoh Y. Sato H. Tojo H. Yana I. Seiki M. EMBO J. 2002; 21: 3949-3959Crossref PubMed Scopus (277) Google Scholar).Preparation of Anti-MT6-MMP Polyclonal Antibody—A polyclonal antibody against MT6-MMP was raised in rabbits immunized with a purified sMT6-F. Because the sMT6-F preparation may contain residual clusterin, the antibody was extensively absorbed using the clusterin preparation from the MDCK cells. The antibody was confirmed to be specific to MT6-MMP without showing cross-reactivity against other MT-MMPs (Fig. 10A) and not to react with clusterin using MDCK and MCF-7 cells (data not shown).Isolation of PMNs—Human PMNs with >98% purity were isolated from fresh heparinized blood of healthy volunteers by dextran sedimentation followed by separation on a density gradient (21Liu Z. Zhou X. Shapiro S.D. Shipley J.M. Twining S.S. Diaz L.A. Senior R.M. Werb Z. Cell. 2000; 102: 647-655Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). The viability was confirmed by the trypan blue exclusion assay.RESULTSPurification of MT6-MMP—The translated pro-MT6-MMP has a hydrophobic amino acid stretch at the C terminus that acts as a signal for GPI anchoring. To prepare a soluble form of MT6-MMP, the hydrophobic stretch was deleted and substituted with the FLAG tag sequence (sMT6-F) (Fig. 1A). MDCK cells that stably express sMT6-F were prepared, and the conditioned medium containing the secreted sMT6-F was collected. The sMT6-F in the culture medium was detected as a band with an expected molecular size of 47 kDa under reducing conditions as demonstrated by Western blotting using anti-FLAG M2 monoclonal antibody (anti-FLAG mAb). sMT6-F was accumulated in the medium according to the time in culture without significant degradation (Fig. 1B). A weak band of 21 kDa presumably representing a degradation product was also detected depending on the culture conditions.Fig. 1Stable expression of soluble MT6-MMP. A, schematic representation of wild-type (MT6-MMP) and soluble MT6-MMP (sMT6-F). sMT6-F was a C-terminal membrane-spanning domain deletion mutant and tagged with a FLAG epitope for detection and purification. SP, signal peptide; Pro, propeptide; Furin Motif, furin cleavage site; Cat, catalytic domain; PEX, hemopexin-like domain; GPI signal, glycosylphosphatidylinositol anchor signal; FLAG, FLAG epitope (DYKDDDDK). B, a plasmid for sMT6-F and an empty vector (mock) were stably transfected into MDCK cells. Confluent cells were washed three times with PBS and cultured further in serum-free DMEM. After 24, 48, and 72 h, each conditioned medium was collected and analyzed by Western blotting using anti-FLAG M2 antibody.View Large Image Figure ViewerDownload (PPT)After concentration of the culture medium with ammonium sulfate, sMT6-F was purified using an agarose beads column conjugated with the anti-FLAG mAb. Most of the proteins did not bind to the column as shown in Fig. 2 (lanes 1 and 2). The proteins bound to the column were eluted with FLAG peptide and analyzed by SDS-PAGE under reducing conditions. Five major bands corresponding to 47, 45, 35, 27, and 21 kDa were detected in the elution fraction (lane 3). Among them, the 47- and 21-kDa bands had the FLAG sequence from the reactivity to the antibody, but the others did not (lane 5). SDS-PAGE under non-reducing conditions revealed four major bands of 73-, 70-, 46-, and 45-kDa proteins (lane 4). Both the 46- and 45-kDa proteins were detected by anti-FLAG mAb (lane 6) and showed gelatinolytic activity, whereas the 73- and 70-kDa proteins did not (lane 7, and also refer to Fig. 5 for better resolution). As the 73- and 70-kDa proteins were eluted from the column by FLAG peptide and were not detected in the preparations from the non-transfected MDCK cells (data not shown), they are presumably associating with sMT6-F.Fig. 2Purification of soluble MT6-MMP by affinity chromatography. Purified proteins on an anti-FLAG monoclonal affinity column were subjected to SDS-PAGE (left panel, lanes 3 and 4) and Western blotting (center panel, lanes 5 and 6) using anti-FLAG M2 antibody under reducing or non-reducing conditions. Purified fraction (lane 4) was also analyzed by gelatin zymography (lane 7). Lane 1, conditioned medium; lane 2, flow-through.View Large Image Figure ViewerDownload (PPT)Fig. 5Separation of sMT6-F-clusterin complex by gel permeation chromatography. A, separation of clusterin-bound and -free sMT6-F by gel permeation chromatography. Purified proteins (clusterin-bound and -free sMT6-F) were subjected to chromatography on a gel permeation column of Superdex 200 at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected. Protein standards (MW Markers indicated by arrowheads) were used to calibrate the column: thyroglobulin (669 kDa), ferritin (440 kDa), adolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa). B, starting material (starting) and purified samples (FN. 47, 50, 53, 57, 60, 64, 70, 75, 78, 80, 83, and 87) were analyzed by SDS-PAGE under non-reducing conditions. Each protein was detected by silver staining. Bands corresponding to clusterin and sMT6-F are indicated by arrows. Asterisks indicate the fractions used for the following assay. C, comparison of proteolytic activity between clusterin-bound sMT6-F (FN 57 and 60) and -free sMT6-F (FN 80 and 83). Each fraction was assayed using fluorescence-quenched peptide substrate. D, the same fractions were analyzed by gelatin zymography.View Large Image Figure ViewerDownload (PPT)Identification of Canine Clusterin as the Protein Binding to sMT6-F—To analyze the polypeptide components of the bands detected under non-reducing conditions (Fig. 3A), they were extracted from the gel and examined further under reducing conditions (Fig. 3B). The 73-kDa protein in Fig. 3A was composed of two polypeptide chains of 45 and 35 kDa possibly linked with disulfide bonds (Fig. 3B, 2nd lane), and the 70-kDa protein also contained two polypeptides of 45 and 27 kDa (Fig. 3B, 3rd lane). The 46-kDa protein in Fig. 3A was separated into 27- and 21-kDa polypeptides (Fig. 3B, 4th lane). The smaller polypeptide has the FLAG tag from the reactivity to the antibody (Fig. 2, lane 5) and is thought to contain the C-terminal fragment of sMT6-F. The 27-kDa polypeptide presumably corresponds to the fragment containing the catalytic domain of sMT6-F, because the 46-kDa protein, from which this fragment is derived, retained the gelatinolytic activity (Fig. 2, lane 7, and also refer to Fig. 5 for better resolution). The 45-kDa protein in Fig. 3A was composed of a single polypeptide of sMT6-F (Fig. 3B, 5th lane). A shift of molecular mass from 45 kDa under non-reducing conditions to 47 kDa under reducing conditions was observed, and this reflects the disruption of the intramolecular disulfide bond in the hemopexin-like domain.Fig. 3Identification of purified proteins that shifted their molecular weight on SDS-PAGE under non-reducing conditions. A, purified sMT6-F and associated proteins were separated by SDS-PAGE under non-reducing conditions and stained with Coomassie Brilliant Blue R-250. B, the 73-, 70-, 46-, and 45-kDa proteins (1st 4 bands) were excised from the gel and subjected to SDS-PAGE under reducing conditions. Each protein was detected by silver staining. Total, purified sMT6-F and associated proteins.View Large Image Figure ViewerDownload (PPT)Then each polypeptide was extracted from the gel and subjected to N-terminal amino acid sequencing (Fig. 4). The N terminus of the 47-kDa polypeptide (108 amino acids from the translation start site) exactly matched the downstream sequence of the site of processing by furin-like enzymes that generate the mature form of MT6-MMP (4Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (168) Google Scholar, 5Velasco G. Cal S. Merlos-Suarez A. Ferrando A.A. Alvarez S. Nakano A. Arribas J. Lopez-Otin C. Cancer Res. 2000; 60: 877-882PubMed Google Scholar), thus indicating that this is the intact sMT6-F. The 27-kDa polypeptide had the same sequence indicating that this is the N-terminal fragment of sMT6-F containing the catalytic domain. On the other hand, the N-terminal sequence of the 21-kDa polypeptide coincided with the sequence in the hemopexin-like domain that starts from the 348-amino acid position. Thus, the 46-kDa sMT6-F has a cleavage within the hemopexin-like domain, and the two polypeptide chains are connected to each other by a disulfide bond within the hemopexin-like domain ("nicked" sMT6-F).Fig. 4N-terminal sequence analysis of sMT6-F and its associated proteins. Purified sMT6-F and associated proteins (47, 45, 35, 27, and 21 kDa) were separated by SDS-PAGE under reducing conditions and electrotransferred onto a PVDF membrane. After the proteins were stained by Coomassie Brilliant Blue R-250, each protein band was excised and subjected to N-terminal amino acid sequencing analysis. Other experimental conditions were described under "Experimental Procedures."View Large Image Figure ViewerDownload (PPT)The N-terminal amino acid sequence of the 45-kDa polypeptide derived from the 73- and 70-kDa proteins exactly coincided with the β subunit of the canine clusterin, whereas the sequences of the 35- and 27-kDa polypeptides corresponded to the α subunit of clusterin. Clusterin is known to be composed of α and β subunits connected by five disulfide bonds (22Jones S.E. Jomary C. Int. J. Biochem. Cell Biol. 2002; 34: 427-431Crossref PubMed Scopus (449) Google Scholar, 23Wilson M.R. Easterbrook-Smith S.B. Trends Biochem. Sci. 2000; 25: 95-98Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). The difference in the molecular size of the two forms of the α subunit sharing the same N terminus
In order to investigate whether a vasa-like protein is present in germ line cells of Xenopus, antibodies were produced which react specifically with synthetic oligopeptides of sequences from near the N- or C-termini or with one including the DEAD box of the Drosophila vasa protein. Only the antibody against the oligopeptide including the DEAD box reacted strongly with germ plasm (GP) or with cytoplasm of germ line cells of Xenopus embryos by immunofluorescence microscopy. By immunoelectron microscopy, the antibody was demonstrated to react with the GP-specific structure, germinal granules, in cleaving embryos, and with their derivatives in the germ line cells of embryos at stages extending from gastrula to feeding tadpole. It also reacted with mitochondria not only in the GP and the germ line cells but also in somatic cells, and with myofibrils in muscle cells. By Western blotting, the antibody was shown to react with several bands of Mr 42-69 ± 103 in protein samples from Xenopus embryos. In samples from Drosophila ovaries, it reacted with a Mr 71 ± 103 band which was probably the vasa protein. This indicates the possibility that Xenopus embryos contain several DEAD family proteins. One of these is present on germinal granules, resembling the vasa protein on polar granules of Drosophila.
Background We evaluated the safety and efficacy of darbepoetin alfa (DA), an attractive alternative to recombinant human erythropoietin (rHuEPO) in managing renal anemia, in Japanese children with chronic kidney disease (CKD) on peritoneal dialysis (PD) and hemodialysis (HD), and not on dialysis (ND). Methods A total of 31 pediatric CKD patients (13 PD, 2 HD, and 16 ND) were enrolled. DA was administered biweekly intravenously (IV) or subcutaneously (SC) for PD or ND patients, and weekly IV for HD patients for 24 weeks. The target Hb was defined as 11.0 to B13.0 g/dl. In patients receiving rHuEPO, the initial DA dose was calculated at 1 lg DA for 200 IU rHuEPO. The initial DA
