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• W2017454736 abstract "Membrane-type 1 matrix metalloproteinase 1 (MT1-MMP) is a potent modulator of the pericellular microenvironment and regulates cellular functions in physiological and pathological settings in mammals. MT1-MMP mediates its biological effects through cleavage of specific substrate proteins. However, our knowledge of MT1-MMP substrates remains limited. To identify new substrates of MT1-MMP, we purified proteins associating with MT1-MMP in human epidermoid carcinoma A431 cells and analyzed them by mass spectrometry. We identified 163 proteins, including membrane proteins, cytoplasmic proteins, and functionally unknown proteins. Sixty-four membrane proteins were identified, and they included known MT1-MMP substrates. Of these, eighteen membrane proteins were selected, and we confirmed their association with MT1-MMP using an immunoprecipitation assay. Co-expression of each protein together with MT1-MMP revealed that nine proteins were cleaved by MT1-MMP. Lutheran blood group glycoprotein (Lu) is one of the proteins cleaved by MT1-MMP, and we confirmed the cleavage of the endogenous Lu protein by endogenous MT1-MMP in A431 cells. Mutation of the cleavage site of Lu abrogated processing by MT1-MMP. Lu protein expressed in A431 cells bound to laminin-511, and knockdown of MT1-MMP in these cells increased both their binding to laminin-511 and the amount of Lu protein on the cell surface. Thus, the identified membrane proteins associated with MT1-MMP are an enriched source of physiological MT1-MMP substrates. Membrane-type 1 matrix metalloproteinase 1 (MT1-MMP) is a potent modulator of the pericellular microenvironment and regulates cellular functions in physiological and pathological settings in mammals. MT1-MMP mediates its biological effects through cleavage of specific substrate proteins. However, our knowledge of MT1-MMP substrates remains limited. To identify new substrates of MT1-MMP, we purified proteins associating with MT1-MMP in human epidermoid carcinoma A431 cells and analyzed them by mass spectrometry. We identified 163 proteins, including membrane proteins, cytoplasmic proteins, and functionally unknown proteins. Sixty-four membrane proteins were identified, and they included known MT1-MMP substrates. Of these, eighteen membrane proteins were selected, and we confirmed their association with MT1-MMP using an immunoprecipitation assay. Co-expression of each protein together with MT1-MMP revealed that nine proteins were cleaved by MT1-MMP. Lutheran blood group glycoprotein (Lu) is one of the proteins cleaved by MT1-MMP, and we confirmed the cleavage of the endogenous Lu protein by endogenous MT1-MMP in A431 cells. Mutation of the cleavage site of Lu abrogated processing by MT1-MMP. Lu protein expressed in A431 cells bound to laminin-511, and knockdown of MT1-MMP in these cells increased both their binding to laminin-511 and the amount of Lu protein on the cell surface. Thus, the identified membrane proteins associated with MT1-MMP are an enriched source of physiological MT1-MMP substrates. Cells in tissues are surrounded by an extracellular cellular matrix that interacts with cells to regulate their activity (1Lukashev M.E. Werb Z. Trends Cell Biol. 1998; 8: 437-441Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 2Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar). Matrix metalloproteinases (MMPs) 3The abbreviations used are: MMPmatrix metalloproteinaseDoxdoxycyclineFACSfluorescence-activated cell sorterLC/MS/MSliquid chromatography tandem mass spectrometryLuLutheran blood group glycoproteinMMImatrix metalloproteinase inhibitorMT-MMPmembrane type-matrix metalloproteinaseshRNAshort hairpin RNAsiRNAsmall interference RNAE/Acatalytically inactive mutant containing an amino acid substitution from Glu240 to AlaEMMPRINextracellular matrix metalloproteinase inducer. 3The abbreviations used are: MMPmatrix metalloproteinaseDoxdoxycyclineFACSfluorescence-activated cell sorterLC/MS/MSliquid chromatography tandem mass spectrometryLuLutheran blood group glycoproteinMMImatrix metalloproteinase inhibitorMT-MMPmembrane type-matrix metalloproteinaseshRNAshort hairpin RNAsiRNAsmall interference RNAE/Acatalytically inactive mutant containing an amino acid substitution from Glu240 to AlaEMMPRINextracellular matrix metalloproteinase inducer. are endopeptidases responsible for extracellular matrix degradation and thereby regulate turnover of the extracellular matrix. However, recent studies have demonstrated that substrates of MMPs are expanded to a variety of pericellular proteins. matrix metalloproteinase doxycycline fluorescence-activated cell sorter liquid chromatography tandem mass spectrometry Lutheran blood group glycoprotein matrix metalloproteinase inhibitor membrane type-matrix metalloproteinase short hairpin RNA small interference RNA catalytically inactive mutant containing an amino acid substitution from Glu240 to Ala extracellular matrix metalloproteinase inducer. matrix metalloproteinase doxycycline fluorescence-activated cell sorter liquid chromatography tandem mass spectrometry Lutheran blood group glycoprotein matrix metalloproteinase inhibitor membrane type-matrix metalloproteinase short hairpin RNA small interference RNA catalytically inactive mutant containing an amino acid substitution from Glu240 to Ala extracellular matrix metalloproteinase inducer. MT1-MMP/MMP14 is an integral membrane proteinase that cleaves multiple proteins in the pericellular milieu and thereby regulates various cell functions. Substrates of MT1-MMP identified to date include extracellular matrix proteins (type I collagen, fibronectin, vitronectin, laminin-1 and -5, and others), cell adhesion molecules (CD44, syndecan-1, and αv integrin), cytokines (SDF-1 and transforming growth factor-β and others), and latent forms of pro-MMPs (pro-MMP-2 and pro-MMP13) (3Itoh Y. Seiki M. J. Cell Physiol. 2006; 206: 1-8Crossref PubMed Scopus (415) Google Scholar, 4Overall C.M. Mol. Biotechnol. 2002; 22: 51-86Crossref PubMed Google Scholar, 5Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5113) Google Scholar). Processing of these proteins by MT1-MMP alters their activities and thereby regulates a variety of cellular functions, such as motility, invasion, growth, differentiation, and apoptosis. Consistent with these functions, forced expression of MT1-MMP in tumor cells enhances behavior consistent with increased malignancy, such as rapid tumor growth, invasion, and metastasis (6Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (361) Google Scholar). However, MT1-MMP is normally expressed in various types of cell and mice deficient in MT1-MMP expression (MT1−/−) display pleiotropic defects (7Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar, 8Zhou Z. Apte S.S. Soininen R. Cao R. Baaklini G.Y. Rauser R.W. Wang J. Cao Y. Tryggvason K. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 4052-4057Crossref PubMed Scopus (685) Google Scholar, 9Chun T.H. Hotary K.B. Sabeh F. Saltiel A.R. Allen E.D. Weiss S.J. Cell. 2006; 125: 577-591Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 10Ohtake Y. Tojo H. Seiki M. J. Cell Sci. 2006; 119: 3822-3832Crossref PubMed Scopus (102) Google Scholar). However, we as yet have only limited knowledge of the physiological substrates of MT1-MMP that could explain such pleiotropic effects. Proteases interact with their substrates at least transiently, but in some cases such interaction is more stable. For instance, type I collagen binds MT1-MMP via a hemopexin-like domain and is cleaved (11Tam E.M. Wu Y.I. Butler G.S. Stack M.S. Overall C.M. J. Biol. Chem. 2002; 277: 39005-39014Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 12Tam E.M. Morrison C.J. Wu Y.I. Stack M.S. Overall C.M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6917-6922Crossref PubMed Scopus (249) Google Scholar). Cleavage of collagen by MT1-MMP regulates cell growth and invasion in a collagen-rich environment (13Hotary K.B. Allen E.D. Brooks P.C. Datta N.S. Long M.W. Weiss S.J. Cell. 2003; 114: 33-45Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). CD44, a hyaluronic acid receptor, also binds to the hemopexin of MT1-MMP and is cleaved (14Kajita M. Itoh Y. Chiba T. Mori H. Okada A. Kinoh H. Seiki M. J. Cell Biol. 2001; 153: 893-904Crossref PubMed Scopus (614) Google Scholar). Expression of CD44 and MT1-MMP in tumor cells promotes cell migration, accompanied by the shedding of CD44 by MT1-MMP (14Kajita M. Itoh Y. Chiba T. Mori H. Okada A. Kinoh H. Seiki M. J. Cell Biol. 2001; 153: 893-904Crossref PubMed Scopus (614) Google Scholar, 15Nakamura H. Suenaga N. Taniwaki K. Matsuki H. Yonezawa K. Fujii M. Okada Y. Seiki M. Cancer Res. 2004; 64: 876-882Crossref PubMed Scopus (116) Google Scholar). pro-MMP-2, which is cleaved by MT1-MMP for activation, forms a tri-molecular complex with MT1-MMP and TIMP-2 (3Itoh Y. Seiki M. J. Cell Physiol. 2006; 206: 1-8Crossref PubMed Scopus (415) Google Scholar, 16Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3877) Google Scholar). Therefore, screening of proteins that associate with MT1-MMP may provide a systematic method to identify potential substrates of MT1-MMP in cells. In addition, these proteins may also be regulatory proteins of MT1-MMP. To identify proteins associating with MT1-MMP in different types of tumor cells, we first studied conditions for cell lysis using malignant melanoma A375 cells and following purification method of the proteins as reported recently (17Tomari T. Koshikawa N. Uematsu T. Shinkawa T. Hoshino D. Egawa N. Isobe T. Seiki M. Cancer Sci. 2009; 100: 1284-1290Crossref PubMed Scopus (27) Google Scholar). Proteins purified in this manner were analyzed by high-throughput proteomic analysis (18Gavin A.C. Bösche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Höfert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (3993) Google Scholar, 19Ho Y. Gruhler A. Heilbut A. Bader G.D. Moore L. Adams S.L. Millar A. Taylor P. Bennett K. Boutilier K. Yang L. Wolting C. Donaldson I. Schandorff S. Shewnarane J. Vo M. Taggart J. Goudreault M. Muskat B. Alfarano C. Dewar D. Lin Z. Michalickova K. Willems A.R. Sassi H. Nielsen P.A. Rasmussen K.J. Andersen J.R. Johansen L.E. Hansen L.H. Jespersen H. Podtelejnikov A. Nielsen E. Crawford J. Poulsen V. Sorensen B.D. Matthiesen J. Hendrickson R.C. Gleeson F. Pawson T. Moran M.F. Durocher D. Mann M. Hogue C.W. Figeys D. Tyers M. Nature. 2002; 415: 180-183Crossref PubMed Scopus (3072) Google Scholar, 20Kaji H. Saito H. Yamauchi Y. Shinkawa T. Taoka M. Hirabayashi J. Kasai K. Takahashi N. Isobe T. Nat. Biotechnol. 2003; 21: 667-672Crossref PubMed Scopus (570) Google Scholar, 21Natsume T. Yamauchi Y. Nakayama H. Shinkawa T. Yanagida M. Takahashi N. Isobe T. Anal. Chem. 2002; 74: 4725-4733Crossref PubMed Scopus (181) Google Scholar). Interestingly, approximately one-half of the membrane proteins identified in our previous study could be cleaved by MT1-MMP at least in vitro. Here, we applied this approach to human carcinoma cells (A431) that originate from epidermoid cells and further validated the systemic whole cell analysis method. To evaluate whether the MT1-MMP-associated membrane proteins so identified include physiological targets of MT1-MMP activity, we select one of them, Lutheran blood group glycoprotein (Lu), and evaluate its processing in A431 cells. The human epidermoid carcinoma cell line A431 was obtained from the American Type Culture Collection (Manassas, VA), and the African green monkey kidney cell line COS-7 was obtained from Health Science Research Resources Bank (Osaka, Japan). MT1-MMP constructs were prepared as previously described (14Kajita M. Itoh Y. Chiba T. Mori H. Okada A. Kinoh H. Seiki M. J. Cell Biol. 2001; 153: 893-904Crossref PubMed Scopus (614) Google Scholar). Catalytically inactive MT1-MMP mutant (MT1 E/A) has a substitution of Ala for Glu240 in the active site as reported previously (22Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (340) Google Scholar). A cDNA sequence corresponding to the open reading frame of human Lu was amplified using PCR. Expression constructs for C-terminally FLAG-tagged Lu were generated as previously described using the Gateway system (Invitrogen) (23Egawa N. Koshikawa N. Tomari T. Nabeshima K. Isobe T. Seiki M. J. Biol. Chem. 2006; 281: 37576-37585Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For inducible expression of wild-type or catalytically inactive MT1-MMP mutant (MT1 E/A) in A431 cells we used the RevTet-OffTM Gene Expression Systems (Clontech) according to the manufacturer's instructions. MMI270 (a synthetic hydroxamic MMP inhibitor, a kind gift of Novartis Pharma AG, Basel, Switzerland) (24MacPherson L.J. Bayburt E.K. Capparelli M.P. Carroll B.J. Goldstein R. Justice M.R. Zhu L. Hu S. Melton R.A. Fryer L. Goldberg R.L. Doughty J.R. Spirito S. Blancuzzi V. Wilson D. O'Byrne E.M. Ganu V. Parker D.T. J. Med. Chem. 1997; 40: 2525-2532Crossref PubMed Scopus (326) Google Scholar) was added when the medium was changed 24 h after transfection. A431 cells that stably expressed FLAG-tagged MT1-MMP were gently lysed by a lysis buffer containing 1% (w/v) Brij-98 as described previously (17Tomari T. Koshikawa N. Uematsu T. Shinkawa T. Hoshino D. Egawa N. Isobe T. Seiki M. Cancer Sci. 2009; 100: 1284-1290Crossref PubMed Scopus (27) Google Scholar). Lysate containing the FLAG-tagged MT1-MMP was subjected to affinity purification using a column packed with agarose beads conjugated to the anti-FLAG M2 antibody. Proteins bound to the column were eluted with successive fractions of buffer containing increasing concentrations of NaCl followed by elution buffer containing FLAG peptide (17Tomari T. Koshikawa N. Uematsu T. Shinkawa T. Hoshino D. Egawa N. Isobe T. Seiki M. Cancer Sci. 2009; 100: 1284-1290Crossref PubMed Scopus (27) Google Scholar). To label the cell surface proteins with biotin, cells were incubated with sulfo-LC-NHS-biotin (Pierce) at 0.1 mg/ml for 30 min at 4 °C. The reaction was terminated by washing three times with phosphate-buffered saline (+). The eluted fractions were digested with endoproteinase Lys-C, and subjected to Nano-Flow liquid chromatography (nano-LC), which was linked online to tandem mass spectrometry (MS/MS). The details of the system were previously described (17Tomari T. Koshikawa N. Uematsu T. Shinkawa T. Hoshino D. Egawa N. Isobe T. Seiki M. Cancer Sci. 2009; 100: 1284-1290Crossref PubMed Scopus (27) Google Scholar). All MS/MS spectra were correlated with a search engine, Mascot program (Matrix Science), against the non-redundant protein sequence data base at the National Center for Biotechnology Information. We followed the criteria used for match acceptance as described previously (21Natsume T. Yamauchi Y. Nakayama H. Shinkawa T. Yanagida M. Takahashi N. Isobe T. Anal. Chem. 2002; 74: 4725-4733Crossref PubMed Scopus (181) Google Scholar). When the match scores exceeded 10 above the threshold, identifications were accepted without further consideration. When scores were lower than 10 or identifications were based on single matched MS/MS spectra, we manually inspected the raw data for confirmation prior to acceptance. Peptides assigned by less than three y series ions and those with a +4 charge state were all eliminated regardless of scores. Immunoprecipitation of cell lysates prepared with RIPA buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% (v/v) Nonidet P-40, 0.5% (v/w) sodium deoxycholate, 0.1% (v/w) SDS) and using agarose beads conjugated to an anti-FLAG M2 antibody was performed as previously described (23Egawa N. Koshikawa N. Tomari T. Nabeshima K. Isobe T. Seiki M. J. Biol. Chem. 2006; 281: 37576-37585Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Proteins in the conditioned medium were precipitated with 10% (v/w) trichloroacetic acid, and prepared for SDS-PAGE. Total cell lysates (trichloroacetic acid), immunoprecipitated products, proteins in the conditioned medium, and purified laminin-511 were separated by SDS-PAGE under reducing or non-reducing conditions. Western blot analysis was performed as previously described (23Egawa N. Koshikawa N. Tomari T. Nabeshima K. Isobe T. Seiki M. J. Biol. Chem. 2006; 281: 37576-37585Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Primary antibodies included an anti-human MT1-MMP mouse monoclonal antibody (222-1D8, Daiichi-fine-Chemical, Takaoka, Japan), an anti-FLAG M2 mouse monoclonal antibody, an anti-FLAG rabbit polyclonal antibody (Sigma), an anti-human Lu goat polyclonal antibody (AF148, R & D Systems), an anti-c-Myc mouse monoclonal antibody (Roche Applied Science), an anti-FAS Ligand mouse monoclonal antibody (BD Biosciences), an anti-transferrin receptor mouse monoclonal antibody (Invitrogen), and an anti-CD44 rabbit polyclonal antibody (prepared as previously described (14Kajita M. Itoh Y. Chiba T. Mori H. Okada A. Kinoh H. Seiki M. J. Cell Biol. 2001; 153: 893-904Crossref PubMed Scopus (614) Google Scholar)). The secondary antibodies were horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). The ECL Plus Western blot detection system (Amersham Biosciences) was used for development of the blots. We established HEK293 cells expressing laminin α5 (C-FLAG-tagged), β1 and γ1 (25Kikkawa Y. Sasaki T. Nguyen M.T. Nomizu M. Mitaka T. Miner J.H. J. Biol. Chem. 2007; 282: 14853-14860Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Purification of recombinant laminin-511 was performed as previously described (26El Nemer W. Gane P. Colin Y. Bony V. Rahuel C. Galactéros F. Cartron J.P. Le Van Kim C. J. Biol. Chem. 1998; 273: 16686-16693Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Cells were incubated on ice with the primary antibody followed by the secondary antibody for 30 min each. In the laminin-511 binding assay, cells were incubated with 20 μg/ml purified laminin-511 on ice for 30 min prior to application of the primary antibody. Samples were analyzed with FACS Aria (BD Biosciences). The primary antibodies used were an anti-human Lu mouse monoclonal antibody (BRIC221) (AbD), an anti-FLAG M2 mouse monoclonal antibody (Sigma). The secondary antibodies used were an Alexa 488-conjugated goat anti-mouse IgG, an Alexa 488-conjugated goat anti-rabbit IgG, and an Alexa 488-conjugated goat anti-rat IgG (Invitrogen). The entry and destination vectors were generated according to the manufacturer's instruction using a BLOCK-it U6 Entry Vector Kit and the Gateway system (Invitrogen). Lu knockdown cells were established using the Virapower lentiviral expression system (Invitrogen), and the cells were maintained in the presence of 10 μg/ml blasticidin (Invitrogen). Small interfering RNA (siRNA) targeting MT1-MMP mRNA was designed and prepared by B-Bridge (Sunnyvale, CA), and transfection was carried out using Lipofectamine RNAi MAX Reagent (Invitrogen). The conditioned medium or cells were harvested 72 h after transfection. To isolate proteins associating with MT1-MMP, we first prepared tetracycline-inducible expression constructs for MT1-MMP FLAG-tagged at either the N (MT1-NF) or C terminus (MT1-CF) (Fig. 1A). To prevent the possible degradation of protein components in the complex by the proteolytic activity of MT1-MMP, we used a catalytically inactive mutant (E/A) containing an amino acid substitution from Glu240 to Ala (E/A) in the catalytic site (22Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (340) Google Scholar). The viral vectors encoding the tagged MT1-MMPs were transduced into A431 cells as described under “Experimental Procedures,” and stable clones were isolated. Expression of MT1-MMP was induced by the depletion of doxycycline (Dox) in the culture medium, and the identities of the MT1-MMP proteins were confirmed by Western blot analysis using anti-FLAG antibody. We detected both pro and active forms of MT1-MMP (Fig. 1B, pro and active, respectively). An excessive amount of MT1-MMP to the endogenously expressed protein was induced as presented in supplemental Fig. S1. Protein fragments corresponding to those predicted by autodegradation of MT1-MMP were detected even in cells expressing the E/A mutant (Fig. 1B, fragment), presumably because A431 cells express endogenous MT1-MMP (refer to Fig. 5B).FIGURE 5Processing of Lu by endogenous MT1-MMP in A431 cells. A, domain structure of Lu protein. Lu comprises five Ig-like domains within its extracellular region, a transmembrane domain, and a cytoplasmic tail. C-terminally FLAG-tagged Lu (Lu-CF) is also presented. B, shedding of Lu fragments in A431 cells. Proteins in the conditioned medium of A431 cells treated with the indicated siRNA were collected and analyzed by Western blot analysis (bottom panel) using a polyclonal antibody against the extracellular portion of Lu (Lu (pAb)). NT, non-treated; cont., control siRNA; siMT1, siRNA for knockdown of MT1-MMP. Total cell lysates were examined by Western blot analysis using anti-MT1-MMP antibody (top panel), Lu (pAb) (second panel), and anti-actin antibody (third panel). Knockdown of MT1-MMP in the cells diminished the amount of the shed Lu fragment with smaller apparent molecular weight (arrowhead). C, processing of Lu-CF by MT1-MMP in COS-7 cells. Lu-CF and MT1-MMP (MT1) were expressed in the cells in the indicated combinations, and cell lysates were analyzed by immunoprecipitation followed by Western blot analysis using anti-FLAG antibody. Total cell lysate (TCL) were examined for MT1-MMP expression using anti-MT1-MMP antibody. D, amino acid sequence of the cleavage site and design of a non-cleavable Lu mutant (mLu-CF). Two amino acids flanking the original Lu cleavage site were substituted by alanine. E, processing of mLu-CF was tested in COS-7 cells and analyzed by immunoprecipitation/Western blotting using an anti-FLAG antibody. The fragment processed by MT1-MMP is indicated by an arrowhead.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We examined different detergents for preparation of the cell lysate and determined to use Brij-98, because this reagent does not disrupt the reported association between MT1-MMP and CD44 (14Kajita M. Itoh Y. Chiba T. Mori H. Okada A. Kinoh H. Seiki M. J. Cell Biol. 2001; 153: 893-904Crossref PubMed Scopus (614) Google Scholar, 27Suenaga N. Mori H. Itoh Y. Seiki M. Oncogene. 2005; 24: 859-868Crossref PubMed Scopus (83) Google Scholar). Cleared lysate was applied to an affinity column packed with agarose beads conjugated to an anti-FLAG M2 antibody (refer to “Experimental Procedures” for a detailed purification procedure). The column was washed with increasing concentrations of NaCl, and finally the proteins bound to the beads were eluted with a FLAG peptide. Aliquots of each step sample were examined by Coomassie Brilliant Blue staining after SDS-PAGE (Fig. 2A). Most of the proteins that were nonspecifically retained in the column were eluted with washing buffer containing 0.5 m NaCl. The major polypeptides detected by Coomassie Brilliant Blue staining in the final elution fraction corresponded to MT1-MMP, because they could be detected by Western blot analysis using an anti-FLAG antibody (Fig. 2B). Unexpectedly, we observed autodegraded MT1-MMP fragments in the final FLAG eluate fraction even when we used cells expressing MT1-NF (Fig. 2A). We speculate that this is due to the ability of MT1-MMP to form homophilic dimers through the hemopexin-like domain (HPX), as we reported previously (22Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (340) Google Scholar). Multiple proteins in the final elution became visible with silver staining (Fig. 2C, left). The proteins that eluted with MT1-MMP showed a distinct pattern from that of the starting material. Almost no proteins were detected in the elution fraction from lysates prepared from non-induced cells (Dox(+)). We further examined whether a specific set of membrane proteins eluted with MT1-MMP by labeling them with biotin prior to cell lysis and affinity purification. The labeled proteins were detected by Western blot analysis using Avidin-horseradish peroxidase (Fig. 2C, right). Only a distinct subset of cell surface proteins eluted with MT1-MMP. We next subjected the membrane proteins that eluted with MT1-MMP to Western blot analysis using antibodies specific for CD44 and the transferrin receptor, which have both been reported to interact with MT1-MMP (Fig. 2D) (14Kajita M. Itoh Y. Chiba T. Mori H. Okada A. Kinoh H. Seiki M. J. Cell Biol. 2001; 153: 893-904Crossref PubMed Scopus (614) Google Scholar, 15Nakamura H. Suenaga N. Taniwaki K. Matsuki H. Yonezawa K. Fujii M. Okada Y. Seiki M. Cancer Res. 2004; 64: 876-882Crossref PubMed Scopus (116) Google Scholar). We detected both CD44 and transferrin receptor in the elution fractions prepared from the induced cells. We also probed the blots for FAS ligand (FAS-L), which has not been reported to associate with MT1-MMP, and we were unable to detect its presence. These results suggest that the proteins in the final elution represent those that associate with MT1-MMP. To identify the proteins associating with MT1-MMP, the final elution fractions for MT1-NF (E/A) and MT1-CF (E/A) were digested with endoproteinase Lys-C and subjected to nano-flow liquid chromatography (nano-LC). The separated peptide fragments were automatically subjected to tandem mass spectrometry (MS/MS) connected to the nano-LC and analyzed using the Mascot program. The validity of each high scoring peptide sequence was confirmed by manual inspection of the corresponding MS/MS spectrum (refer to “Experimental Procedures”). In total, 163 proteins were identified as MT1-MMP-associating proteins (supplemental Table S1), and these were classified into membrane, cytoplasmic, secretory, and unknown proteins (Table 1A). The 64 membrane proteins included adhesion molecules, receptors, transporters, and others (Table 1B). These proteins included known MT1-MMP substrates such as CD44 (14Kajita M. Itoh Y. Chiba T. Mori H. Okada A. Kinoh H. Seiki M. J. Cell Biol. 2001; 153: 893-904Crossref PubMed Scopus (614) Google Scholar, 15Nakamura H. Suenaga N. Taniwaki K. Matsuki H. Yonezawa K. Fujii M. Okada Y. Seiki M. Cancer Res. 2004; 64: 876-882Crossref PubMed Scopus (116) Google Scholar) and EMMPRIN (23Egawa N. Koshikawa N. Tomari T. Nabeshima K. Isobe T. Seiki M. J. Biol. Chem. 2006; 281: 37576-37585Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar).TABLE 1Classifications of MT1-MMP-associating proteins by localization (A) and MT1-MMP-associating membrane proteins by those functions (B)A) Classification of MT1-MMP-associating proteins by localizationMembrane proteins64Cytoplasmic proteins87Secretory proteins5Uncharacterized proteins7Total163B) Classification of MT1-MMP-associating membrane proteins by those functionsAdhesion moleculesNM_001728, EMMPRINNM_006505, poliovirus receptorNM_005581, LuNM_002211, β1 integrinNM_000610, CD44ReceptorsNM_005228, EGFRNM_003234, transferrin receptorTransportersNM_002394, CD98OthersNM_001769, CD9NM_006670, 5T4 antigenNM_001304, carboxypeptidase D Open table in a new tab We chose 18 proteins randomly from the 64 membrane proteins and constructed vectors expressing FLAG-tagged versions of each interacting protein (Fig. 3A). We evaluated binding of these FLAG-tagged proteins to MT1-MMP by first co-expressing them individually along with Myc-tagged MT1-MMP in A431 cells. Lysates prepared from the transfected cells were then subjected to immunoprecipitation followed by Western blot analysis. We confirmed co-precipitation of MT1-MMP with each of the 18 selected test proteins (Fig. 3B) as it is expected from co-elution of the test proteins with MT1-MMP from the affinity columns. Interestingly, the ratio of pro and active forms of MT1-MMP that co-precipitated with the test proteins differed for each test protein. This may reflect the subcellular localization of the test proteins that interact with MT1-MMP. Some of these membrane proteins might be cleaved by MT1-MMP. To test this possibility, we co-expressed each of these proteins together with MT1-MMP in A431 cells. Indeed, co-expression of MT1-MMP and the test proteins generated new cleavage fragments in nine cases, and this fragmentation was inhibited by incubation of the cells with a synthetic MMP inhibitor, MMI270. Six representative cases are presented in Fig. 4, and the other twelve cases are presented in supplemental Fig. S2. Processing of JAM-1 was not affected by expression of MT1-MMP, although its processing can be inhibited by MMI270. These results indicate that approximately half of the identified membrane proteins can be cleaved by MT1-MMP" @default.
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