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• W1999545332 abstract "Heparanase is a β-d-endoglucuronidase that cleaves heparan sulfate, an important structural component of the extracellular matrix (ECM) and vascular basement membrane (BM). The cleavage of heparan sulfate by heparanase-expressing cells, such as activated leukocytes, metastatic tumor cells, and proliferating endothelial cells, facilitates degradation of the ECM/BM to promote cell invasion associated with inflammation, tumor metastasis, and angiogenesis. In addition to its enzymatic function, heparanase has also recently been shown to act as a cell adhesion and/or signaling molecule upon interaction with cell surfaces. Despite the obvious importance of the mechanisms for the binding of heparanase to cell surfaces, the receptor(s) for heparanase remain poorly defined. In this study, we identify the 300-kDa cation-independent mannose 6-phosphate receptor (CIMPR) as a cell surface receptor for heparanase. Purified platelet heparanase was shown to bind the human CIMPR expressed on the surface of a transfected mouse L cell line. Optimal binding was determined to be at a slightly acidic pH (6.5-7.0) with heparanase remaining on the cell surface for up to 10 min at 37 °C. In contrast, mouse L cells or Chinese hamster ovary cells expressing the cation-dependent mannose 6-phosphate receptor (CDMPR) showed no binding of heparanase. Interestingly, the binding of heparanase to CIMPR was independent of Man-6-P moieties. Significantly, primary human T cells upon activation were shown to dramatically up-regulate levels of cell surface-expressed CIMPR, which showed a concomitant increase in their capacity to bind heparanase. Furthermore, the tethering of heparanase to the surface of cells via CIMPR was found to increase their capacity to degrade an ECM or a reconstituted BM. These data suggest an important role for CIMPR in the cell surface presentation of enzymatically active heparanase for the efficient passage of T cells into an inflammatory site and have implications for the use of this mechanism by other cell types to enhance cell invasion. Heparanase is a β-d-endoglucuronidase that cleaves heparan sulfate, an important structural component of the extracellular matrix (ECM) and vascular basement membrane (BM). The cleavage of heparan sulfate by heparanase-expressing cells, such as activated leukocytes, metastatic tumor cells, and proliferating endothelial cells, facilitates degradation of the ECM/BM to promote cell invasion associated with inflammation, tumor metastasis, and angiogenesis. In addition to its enzymatic function, heparanase has also recently been shown to act as a cell adhesion and/or signaling molecule upon interaction with cell surfaces. Despite the obvious importance of the mechanisms for the binding of heparanase to cell surfaces, the receptor(s) for heparanase remain poorly defined. In this study, we identify the 300-kDa cation-independent mannose 6-phosphate receptor (CIMPR) as a cell surface receptor for heparanase. Purified platelet heparanase was shown to bind the human CIMPR expressed on the surface of a transfected mouse L cell line. Optimal binding was determined to be at a slightly acidic pH (6.5-7.0) with heparanase remaining on the cell surface for up to 10 min at 37 °C. In contrast, mouse L cells or Chinese hamster ovary cells expressing the cation-dependent mannose 6-phosphate receptor (CDMPR) showed no binding of heparanase. Interestingly, the binding of heparanase to CIMPR was independent of Man-6-P moieties. Significantly, primary human T cells upon activation were shown to dramatically up-regulate levels of cell surface-expressed CIMPR, which showed a concomitant increase in their capacity to bind heparanase. Furthermore, the tethering of heparanase to the surface of cells via CIMPR was found to increase their capacity to degrade an ECM or a reconstituted BM. These data suggest an important role for CIMPR in the cell surface presentation of enzymatically active heparanase for the efficient passage of T cells into an inflammatory site and have implications for the use of this mechanism by other cell types to enhance cell invasion. The extracellular matrix (ECM) 3The abbreviations used are: ECMextracellular matrixBMbasement membraneHSPGheparan sulfate proteoglycanHSheparan sulfateTGFβtransforming growth factor βuPAurokinase plasminogen activatorCIMPRcation-independent mannose 6-phosphate receptorCDMPRcation-dependent mannose 6-phosphate receptorMan-6-Pmannose 6-phosphateMPRmannose 6-phosphate receptorIGF-IIinsulin-like growth factor IIPPMEpolyphosphomannan esterStrep-PEstreptavidin-phycoerythrinMS9-IImouse L cells stably transfected with human CIMPRMSmouse L cells stably transfected with expression vectorDMEMDulbecco's modified Eagle's mediumpgSA-745Chinese hamster ovary cells unable to synthesize glycosaminoglycanspgCDMPRpgSA-745 cells stably transfected with human CDMPRPBSphosphate-buffered salineBSAbovine serum albuminFITCfluorescein isothiocyanateFCSfetal calf serumCIPcalf intestinal alkaline phosphatasePEphycoerythrin.3The abbreviations used are: ECMextracellular matrixBMbasement membraneHSPGheparan sulfate proteoglycanHSheparan sulfateTGFβtransforming growth factor βuPAurokinase plasminogen activatorCIMPRcation-independent mannose 6-phosphate receptorCDMPRcation-dependent mannose 6-phosphate receptorMan-6-Pmannose 6-phosphateMPRmannose 6-phosphate receptorIGF-IIinsulin-like growth factor IIPPMEpolyphosphomannan esterStrep-PEstreptavidin-phycoerythrinMS9-IImouse L cells stably transfected with human CIMPRMSmouse L cells stably transfected with expression vectorDMEMDulbecco's modified Eagle's mediumpgSA-745Chinese hamster ovary cells unable to synthesize glycosaminoglycanspgCDMPRpgSA-745 cells stably transfected with human CDMPRPBSphosphate-buffered salineBSAbovine serum albuminFITCfluorescein isothiocyanateFCSfetal calf serumCIPcalf intestinal alkaline phosphatasePEphycoerythrin. and its specialized form known as basement membranes (BM) represent a major physical barrier to migrating cells (1Yurchenco P.D. Schittny J.C. FASEB J. 1990; 4: 1577-1590Crossref PubMed Scopus (788) Google Scholar, 2Erickson A.C. Couchman J.R. J. Histochem. Cytochem. 2000; 48: 1291-1306Crossref PubMed Scopus (242) Google Scholar). An important ubiquitous structural component of the ECM/BM is heparan sulfate proteoglycan (HSPG). HSPGs are a diverse family of complex macromolecules that consist of a protein core to which are attached linear side chains of the glycosaminoglycan heparan sulfate (HS). They contribute to the assembly and stability of the ECM/BM by interacting with multiple matrix proteins, including collagen, laminin, nidogen, and fibronectin (3Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1676) Google Scholar, 4Iozzo R.V. San Antonio J.D. J. Clin. Investig. 2001; 108: 349-355Crossref PubMed Scopus (402) Google Scholar). Heparanase is a β-d-endoglucoronidase that cleaves HS and has been proposed to have an important role in facilitating the disassembly of the ECM/BM by acting in concert with the various matrix proteases. Heparanase is synthesized as a 65-kDa inactive proenzyme that is processed by removal of a 6-kDa linker fragment into an active form consisting of a heterodimer between the remaining 50- and 8-kDa polypeptide subunits (5Fairbanks M.B. Mildner A.M. Leone J.W. Cavey G.S. Mathews W.R. Drong R.F. Slightom J.L. Bienkowski M.J. Smith C.W. Bannow C.A. Heinrikson R.L. J. Biol. Chem. 1999; 274: 29587-29590Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The proteolytic cleavage of heparanase is mediated by L-cathepsin and possibly other protease species (6Abboud-Jarrous G. Rangini-Guetta Z. Aingorn H. Atzmon R. Elgavish S. Peretz T. Vlodavsky I. J. Biol. Chem. 2005; 280: 13568-13575Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), with the site of processing suggested as being localized to lysosomes (7Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar, 8Cohen E. Atzmon R. Vlodavsky I. Ilan N. FEBS Lett. 2005; 579: 2334-2338Crossref PubMed Scopus (37) Google Scholar). Heparanase shows optimal catalytic activity in slightly acidic environments (pH 6.0-7.0); however, it retains its HS binding capacity at neutral pH (9Gilat D. Hershkoviz R. Goldkorn I. Cahalon L. Korner G. Vlodavsky I. Lider O. J. Exp. Med. 1995; 181: 1929-1934Crossref PubMed Scopus (112) Google Scholar) and has been proposed to also have a noncatalytic function as an adhesion or signaling molecule (7Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar, 9Gilat D. Hershkoviz R. Goldkorn I. Cahalon L. Korner G. Vlodavsky I. Lider O. J. Exp. Med. 1995; 181: 1929-1934Crossref PubMed Scopus (112) Google Scholar, 10Goldshmidt O. Zcharia E. Cohen M. Aingorn H. Cohen I. Nadav L. Katz B.Z. Geiger B. Vlodavsky I. FASEB J. 2003; 17: 1015-1025Crossref PubMed Scopus (168) Google Scholar, 11Sotnikov I. Hershkoviz R. Grabovsky V. Ilan N. Cahalon L. Vlodavsky I. Alon R. Lider O. J. Immunol. 2004; 172: 5185-5193Crossref PubMed Scopus (74) Google Scholar, 12Gingis-Velitski S. Zetser A. Kaplan V. Ben-Zaken O. Cohen E. Levy-Adam F. Bashenko Y. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 44084-44092Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). For many years it has been postulated that heparanase is utilized by cells such as activated leukocytes, metastatic tumor cells, and proliferating vascular endothelial cells to promote cell invasion associated with inflammation, tumor metastasis, and angiogenesis (14Vlodavsky I. Friedmann Y. J. Clin. Investig. 2001; 108: 341-347Crossref PubMed Scopus (546) Google Scholar, 15Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar, 16Ilan N. Elkin M. Vlodavsky I. Int. J. Biochem. Cell Biol. 2006; 38: 2018-2039Crossref PubMed Scopus (462) Google Scholar). In addition heparanase has been implicated in the liberation of HS-bound growth factors, e.g. basic fibroblast growth factor and vascular endothelial growth factor, from ECM depots to initiate growth factor-dependent responses such as angiogenesis and wound healing (17Ishai-Michaeli R. Eldor A. Vlodavsky I. Cell Regul. 1990; 1: 833-842Crossref PubMed Scopus (171) Google Scholar, 18Elkin M. Ilan N. Ishai-Michaeli R. Friedmann Y. Papo O. Pecker I. Vlodavsky I. FASEB J. 2001; 15: 1661-1663Crossref PubMed Scopus (285) Google Scholar). The recent cloning of heparanase (19Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (725) Google Scholar, 20Hulett M.D. Freeman C. Hamdorf B.J. Baker R.T. Harris M.J. Parish C.R. Nat. Med. 1999; 5: 803-809Crossref PubMed Scopus (486) Google Scholar, 21Toyoshima M. Nakajima M. J. Biol. Chem. 1999; 274: 24153-24160Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 22Kussie P.H. Hulmes J.D. Ludwig D.L. Patel S. Navarro E.C. Seddon A.P. Giorgio N.A. Bohlen P. Biochem. Biophys. Res. Commun. 1999; 261: 183-187Crossref PubMed Scopus (182) Google Scholar, 23Dempsey L.A. Plummer T.B. Coombes S.L. Platt J.L. Glycobiology. 2000; 10: 467-475Crossref PubMed Scopus (85) Google Scholar) has enabled experimental confirmation that the enzyme plays a key role in these processes. Heparanase overexpression strategies and mRNA knockdown approaches have demonstrated in experimental animal models a direct role for heparanase in tumor metastasis and angiogenesis (19Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (725) Google Scholar, 24Uno F. Fujiwara T. Takata Y. Ohtani S. Katsuda K. Takaoka M. Ohkawa T. Naomoto Y. Nakajima M. Tanaka N. Cancer Res. 2001; 61: 7855-7860PubMed Google Scholar, 25Edovitsky E. Elkin M. Zcharia E. Peretz T. Vlodavsky I. J. Natl. Cancer Inst. 2004; 96: 1219-1230Crossref PubMed Scopus (227) Google Scholar), as well as inflammation (26Edovitsky E. Lerner I. Zcharia E. Peretz T. Vlodavsky I. Elkin M. Blood. 2006; 107: 3606-3616Crossref Scopus (113) Google Scholar). The clinical relevance of heparanase in tumor growth and metastasis is further supported by the comprehensive documentation of heparanase up-regulation in many human tumors, with heparanase expression correlating with increased metastasis, tumor vascularization, and reduced post-operative survival of patients (27Gohji K. Hirano H. Okamoto M. Kitazawa S. Toyoshima M. Dong J. Katsuoka Y. Nakajima M. Int. J. Cancer. 2001; 95: 295-301Crossref PubMed Scopus (110) Google Scholar, 28Koliopanos A. Friess H. Kleeff J. Shi X. Liao Q. Pecker I. Vlodavsky I. Zimmermann A. Buchler M.W. Cancer Res. 2001; 61: 4655-4659PubMed Google Scholar, 29Rohloff J. Zinke J. Schoppmeyer K. Tannapfel A. Witzigmann H. Mossner J. Wittekind C. Caca K. Br. J. Cancer. 2002; 86: 1270-1275Crossref PubMed Scopus (107) Google Scholar, 30Maxhimer J.B. Quiros R.M. Stewart R. Dowlatshahi K. Gattuso P. Fan M. Prinz R.A. Xu X. Surgery. 2002; 132: 326-333Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 31Kim A.W. Xu X. Hollinger E.F. Gattuso P. Godellas C.V. Prinz R.A. J. Gastrointest. Surg. 2002; 6: 167-172Crossref PubMed Scopus (59) Google Scholar, 32Takaoka M. Naomoto Y. Ohkawa T. Uetsuka H. Shirakawa Y. Uno F. Fujiwara T. Gunduz M. Nagatsuka H. Nakajima M. Tanaka N. Haisa M. Lab. Investig. 2003; 83: 613-622Crossref PubMed Scopus (148) Google Scholar). Furthermore, heparanase expression is up-regulated in inflammatory disease and diabetic nephropathy (33Vlodavsky I. Eldor A. Haimovitz-Friedman A. Matzner Y. Ishai-Michaeli R. Lider O. Naparstek Y. Cohen I.R. Fuks Z. Invasion Metastasis. 1992; 12: 112-127PubMed Google Scholar, 34Katz A. Van-Dijk D.J. Aingorn H. Erman A. Davies M. Darmon D. Hurvitz H. Vlodavsky I. Isr. Med. Assoc. J. 2002; 4: 996-1002PubMed Google Scholar, 35Levidiotis V. Freeman C. Tikellis C. Cooper M.E. Power D.A. J. Am. Soc. Nephrol. 2004; 15: 68-78Crossref PubMed Scopus (81) Google Scholar, 36Maxhimer J.B. Somenek M. Rao G. Pesce C.E. Baldwin Jr., D. Gattuso P. Schwartz M.M. Lewis E.J. Prinz R.A. Xu X. Diabetes. 2005; 54: 2172-2178Crossref PubMed Scopus (102) Google Scholar, 37de Mestre A.M. Khachigian L.M. Santiago F.S. Staykova M.A. Hulett M.D. J. Biol. Chem. 2003; 278: 50377-50385Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The importance of heparanase in these disease settings, together with the identification of only a single heparanase, makes the enzyme a highly promising target for the development of anti-cancer and anti-inflammatory drugs. extracellular matrix basement membrane heparan sulfate proteoglycan heparan sulfate transforming growth factor β urokinase plasminogen activator cation-independent mannose 6-phosphate receptor cation-dependent mannose 6-phosphate receptor mannose 6-phosphate mannose 6-phosphate receptor insulin-like growth factor II polyphosphomannan ester streptavidin-phycoerythrin mouse L cells stably transfected with human CIMPR mouse L cells stably transfected with expression vector Dulbecco's modified Eagle's medium Chinese hamster ovary cells unable to synthesize glycosaminoglycans pgSA-745 cells stably transfected with human CDMPR phosphate-buffered saline bovine serum albumin fluorescein isothiocyanate fetal calf serum calf intestinal alkaline phosphatase phycoerythrin. extracellular matrix basement membrane heparan sulfate proteoglycan heparan sulfate transforming growth factor β urokinase plasminogen activator cation-independent mannose 6-phosphate receptor cation-dependent mannose 6-phosphate receptor mannose 6-phosphate mannose 6-phosphate receptor insulin-like growth factor II polyphosphomannan ester streptavidin-phycoerythrin mouse L cells stably transfected with human CIMPR mouse L cells stably transfected with expression vector Dulbecco's modified Eagle's medium Chinese hamster ovary cells unable to synthesize glycosaminoglycans pgSA-745 cells stably transfected with human CDMPR phosphate-buffered saline bovine serum albumin fluorescein isothiocyanate fetal calf serum calf intestinal alkaline phosphatase phycoerythrin. It is evident from recent studies that heparanase can bind efficiently to the surface of cells, a process that has been postulated as important in a number of key aspects of heparanase function (15Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar, 16Ilan N. Elkin M. Vlodavsky I. Int. J. Biochem. Cell Biol. 2006; 38: 2018-2039Crossref PubMed Scopus (462) Google Scholar). Cell surface display of heparanase has been implicated in cell adhesion, whereby heparanase can bind to T cells and aid adhesion to ECM components under shear flow conditions (9Gilat D. Hershkoviz R. Goldkorn I. Cahalon L. Korner G. Vlodavsky I. Lider O. J. Exp. Med. 1995; 181: 1929-1934Crossref PubMed Scopus (112) Google Scholar, 10Goldshmidt O. Zcharia E. Cohen M. Aingorn H. Cohen I. Nadav L. Katz B.Z. Geiger B. Vlodavsky I. FASEB J. 2003; 17: 1015-1025Crossref PubMed Scopus (168) Google Scholar, 11Sotnikov I. Hershkoviz R. Grabovsky V. Ilan N. Cahalon L. Vlodavsky I. Alon R. Lider O. J. Immunol. 2004; 172: 5185-5193Crossref PubMed Scopus (74) Google Scholar). The interaction of heparanase with the surface of endothelial cells has also been shown to trigger signaling cascades, including the enhancement of protein kinase B/Akt signaling and stimulation of phosphatidylinositol 3-kinase- and p38-dependent cell migration and invasion (38Gingis-Velitski S. Zetser A. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 23536-23541Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), as well as activation of the Src pathway that induces vascular endothelial growth factor expression to promote an angiogenic response (39Zetser A. Bashenko Y. Edovitsky E. Levy-Adam F. Vlodavsky I. Ilan N. Cancer Res. 2006; 66: 1455-1463Crossref PubMed Scopus (216) Google Scholar). In addition, the ability of cells to bind and internalize exogenous heparanase via cell surface receptors has been suggested as important for the cellular uptake, processing, and storage of the enzyme (7Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar, 12Gingis-Velitski S. Zetser A. Kaplan V. Ben-Zaken O. Cohen E. Levy-Adam F. Bashenko Y. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 44084-44092Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 40Nadav L. Eldor A. Yacoby-Zeevi O. Zamir E. Pecker I. Ilan N. Geiger B. Vlodavsky I. Katz B.Z. J. Cell Sci. 2002; 115: 2179-2187Crossref PubMed Google Scholar). Furthermore, the tethering of degradative enzymes to the surface of migrating cells promotes the efficient local and directed degradation of ECM/BM barriers (15Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar, 41Chen W.T. Curr. Opin. Cell Biol. 1992; 4: 802-809Crossref PubMed Scopus (153) Google Scholar), and this is likely to also be the case with heparanase (42Sasaki N. Higashi N. Taka T. Nakajima M. Irimura T. J. Immunol. 2004; 172: 3830-3835Crossref PubMed Scopus (72) Google Scholar, 43Benhamron S. Nechushtan H. Verbovetski I. Krispin A. Abboud-Jarrous G. Zcharia E. Edovitsky E. Nahari E. Peretz T. Vlodavsky I. Mevorach D. J. Immunol. 2006; 176: 6417-6424Crossref PubMed Scopus (45) Google Scholar). The identification of heparanase receptor(s) expressed on the surface of cells is of fundamental importance in understanding the above processes; however, in most instances, these receptors remain undefined. A number of lines of evidence indicate that HS is important in the uptake of heparanase (12Gingis-Velitski S. Zetser A. Kaplan V. Ben-Zaken O. Cohen E. Levy-Adam F. Bashenko Y. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 44084-44092Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 44Levy-Adam F. Abboud-Jarrous G. Guerrini M. Beccoti D. Vlodavsky I. Ilan N. J. Biol. Chem. 2005; 280: 20457-20466Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 45Vreys V. Delande N. Zhang Z. Coomans C. Roebroek A. Durr J. David G. J. Biol. Chem. 2005; 280: 33141-33148Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). More recently, the low density lipoprotein receptor-related protein and an unidentified mannose 6-phosphate receptor (MPR) were also suggested to mediate heparanase uptake; however, with the latter receptor it was only shown to be the case when in cooperation with HSPGs and receptor-associated protein-sensitive receptors (45Vreys V. Delande N. Zhang Z. Coomans C. Roebroek A. Durr J. David G. J. Biol. Chem. 2005; 280: 33141-33148Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Here, we identify the 300-kDa CIMPR (CD222) as a novel receptor for heparanase. Interestingly, the binding of heparanase to CIMPR was independent of Man-6-P. The tethering of heparanase to cell surfaces via CIMPR was found to significantly enhance the ability of cells to degrade the ECM/BM. Human primary T cells upon stimulation were shown to up-regulate the cell surface expression of CIMPR, and concomitantly the binding of heparanase, suggesting that this is a mechanism utilized by T cells and possibly other cells types to mediate efficient ECM degradation. Proteins and Reagents–Polyphosphomannan ester (PPME) and 5-polyphosphomannan ester were the gifts of G. Bartell (John Curtin School of Medical Research, Canberra, Australia). Streptavidin-phycoerythrin (Strep-PE) and streptavidin-TRICOLOR were purchased from Caltag Laboratories (Burlingame, CA). Heparanase was purified from human platelets as described previously (46Freeman C. Parish C.R. Biochem. J. 1998; 330: 1341-1350Crossref PubMed Scopus (173) Google Scholar) and consisted of the majority (∼95%) in an active processed form. Purified heparanase was labeled with biotin using biotin-NHS (Sigma) according to the manufacturer's instructions, and it retained an activity comparable with that of unlabeled heparanase. Methotrexate, heparin (from porcine intestinal mucosa; Mr ∼ 15,000), the monosodium salts of Man-6-P and glucose-6 phosphate, mannose, and bacterial heparinase were purchased from Sigma. The anti-CIMPR antibody JT-CIMPR was a generous gift of J. Trapani (Peter MacCallum Cancer Centre, Melbourne, Australia), and the anti-CIMPR antibody MEM-328 was from Abcam (Cambridge UK). The anti-heparanase antibody (Hpa1) was obtained from Insight Biopharmaceuticals (Rehovot, Israel). The iduronidase and anti-iduronidase antibody (47Clements P.R. Brooks D.A. Saccone G.T. Hopwood J.J. Eur. J. Biochem. 1985; 152: 21-28Crossref PubMed Scopus (35) Google Scholar) were the kind gifts of D. Brooks (Women's and Children's Hospital, Adelaide, Australia). The anti-HS antibody (F58-10E4) was from Seikagaku Corp. (Tokyo, Japan). Cells and Cell Culture–Mouse L cells stably overexpressing human CIMPR (MS9-II) or transfected with DNA vector alone (MS), have been described previously (48Kyle J.W. Nolan C.M. Oshima A. Sly W.S. J. Biol. Chem. 1988; 263: 16230-16235Abstract Full Text PDF PubMed Google Scholar) (obtained by permission of W. Sly (Washington University, St. Louis) from J. Trapani (Peter MacCallum Cancer Centre, Melbourne, Australia)) and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) (CSL, Parkville, Victoria, Australia) and 3.2 μm methotrexate (Sigma). The MS9-II cells express similar levels of cell surface CIMPR when compared with physiological cell settings such as activated primary human T cells and endothelial cells. 4R. J. Wood and M. D. Hulett, unpublished observations. Xylosyltransferase-deficient pgSA-745 Chinese hamster ovary cells (express no cell surface heparan sulfate) (49Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (485) Google Scholar) stably transfected with a mammalian expression vector for human CDMPR (pKC4-CDM6R) or vector alone (pKC4) were maintained in DMEM supplemented with 10% FCS and 0.4 mg/ml geneticin (Invitrogen). Transient transfection of MS cells with pCR3.1-CDMPR or pCR3.1 was carried out using GeneJuice transfection reagent as described by the manufacturer (Novagen). HR9 cells (50Chung A.E. Estes L.E. Shinozuka H. Braginski J. Lorz C. Chung C.A. Cancer Res. 1977; 37: 2072-2081PubMed Google Scholar) (provided by A. Tester, University of Melbourne, Australia) were cultured on 1% gelatin-coated flasks and maintained in DMEM supplemented with 10% FCS and daily addition of 50 μg/ml ascorbic acid. Purified human primary T cells (see below) were maintained in RPMI 1640 medium supplemented with 10% FCS. All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Cell Surface Binding Assays–Cells were analyzed for the binding of heparanase by immunofluorescence flow cytometry. Typically 2 μg/ml of biotinylated heparanase was added to 2 × 105 cells in 1 ml of PBS, pH 6.0 to 7.2, containing 0.1% fraction V bovine serum albumin (BSA) on ice for 60 min, and cells were washed three times with ice-cold PBS and 0.1% BSA, pH 6.0 to 7.2. Biotinylated anti-rabbit Ig (Amersham Biosciences) was used as a control for background binding. Cell bound heparanase-biotin or anti-rabbit Ig-biotin was detected using Strep-PE (Caltag Laboratories) by flow cytometry using a FACScan (BD Biosciences) with Weasel software (Walter and Eliza Analysis Software Eclectic and Lucid version 2.2.2). Assays to assess the binding of heparanase to CDMPR were performed as above in the presence of 10 mm CaCl2, pH 6.5. Binding assays were performed on transiently transfected MS cells 48 h post-transfection. As no antibody was available to human CDMPR, expression of the transfected gene was confirmed by reverse transcriptase-PCR using the primers 5′-ctactccagtttcccacgac-3′ and 5′-ctgcttgagaaatctggctg-3′ (data not shown), and cell surface expression of the receptor was demonstrated by binding of the Man-6-P containing molecule PPME (Fig. 1). Heparanase binding inhibition assays were carried out as above with the exception that the cells were first incubated in the presence or absence of various concentrations of different inhibitors (see figures legends) for 60 min on ice or pretreated with active bacterial heparinase (to remove cell surface HS) for 60 min at 37 °C. Iduronidase binding experiments were carried out in the same manner with 1 μg of iduronidase instead of biotinylated heparanase and detected using a mouse anti-iduronidase monoclonal antibody. Similarly, cell surface expression of CIMPR was determined using a mouse monoclonal antibody (JT-CIMPR) to the human CIMPR protein. The expression of cell surface HS was determined by immunofluorescence flow cytometry using the mouse monoclonal antibody to HS (F58-10E4). In each case the binding of the primary antibody was detected with a rabbit anti-mouse antibody conjugated to PE (Dako, Denmark). Cell surface binding of PPME was detected using fluorescein isothiocyanate (FITC)-labeled PPME by immunofluorescence flow cytometry. Direct Binding Assay–Washed protein A-Sepharose (30-μl packed volume) (Amersham Biosciences) was incubated with 50 μl of culture supernatant of a mouse monoclonal antibody to human CIMPR (JT-CIMPR), 10 μg/ml of the mouse monoclonal antibody to CIMPR MEM-238 (Abcam, Cambridge UK), or 50 μl of a culture supernatant of an IgG1 isotype control antibody to mouse anti-trinitrobenzene sulfonate, at 4 °C for 120 min on a rotating wheel in PBS, 3% BSA. MS9-II cells (5 × 106) were incubated with 2 μg/ml HSPE at 4 °C. The cells were washed in two cycles of ice-cold PBS, 0.1% BSA and then resuspended in 100 μl of 150 mm NaCl, 20 mm Tris-Cl, pH 8.2, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 0.1% aprotinin, 1% Nonidet P-40 (Sigma), and Complete Protease Inhibitors (Roche Diagnostics) and lysed by three cycles of rapid freeze/thaw incubation. The cell debris was pelleted by centrifugation at 6500 × g for 10 min at 4 °C. The protein A-Sepharose was pelleted by centrifugation at 2000 × g for 5 min at 4 °C followed by two cycles of incubation in PBS, 5% BSA, 0.005% Tween for 1 h each cycle. The protein A-Sepharose was then incubated for 2 h with 100 μl of the cleared cell lysate. The beads were than washed with two cycles of PBS, 3% BSA, 0.005% Tween, and binding of heparanase to the CIMPR was assessed by Western blotting using a rabbit polyclonal antibody to heparanase (Hpa1) followed by an anti-rabbit Ig conjugated to horseradish peroxidase (Chemicon, Melbourne, Australia). Alkaline Phosphatase Assay–Biotinylated heparanase (2 μg/ml), fluorescein isothiocyanate PPME-FITC (5 μg/ml), or iduronidase (1 μg/ml) were pretreated with 1 unit of calf intestinal alkaline phosphatase (CIP) for 60 min at 37 °C in both normal saline and PO4 Buffer (64.5 mm Na2HPO4, 21 mm KH2PO4). MS and MS9-II cells (1 × 105/well) were incubated with the pretreated heparanase or PPME-FITC in PBS and 0.1% BSA, pH 7.2, for 60 min on ice. The cells were washed three times with ice-cold PBS and 0.1% BSA, pH 7.2. The cell surface binding of heparanase-biotin was detected with Strep-PE by incubation for 30 min on ice. Changes in binding because of CIP treatment were detected by immunofluorescence flow cytometry using a FACScan (BD Biosciences) with Weasel software (Walter and Eliza Analysis Software Eclectic" @default.
• W1999545332 created "2016-06-24" @default.
• W1999545332 creator A5081955081 @default.
• W1999545332 creator A5089817671 @default.
• W1999545332 date "2008-02-01" @default.
• W1999545332 modified "2023-10-18" @default.
• W1999545332 title "Cell Surface-expressed Cation-independent Mannose 6-Phosphate Receptor (CD222) Binds Enzymatically Active Heparanase Independently of Mannose 6-Phosphate to Promote Extracellular Matrix Degradation" @default.
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• W1999545332 doi "https://doi.org/10.1074/jbc.m708723200" @default.
• W1999545332 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18073203" @default.
• W1999545332 hasPublicationYear "2008" @default.
• W1999545332 type Work @default.
• W1999545332 sameAs 1999545332 @default.

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