Heparan Sulfate Is a Cellular Receptor for Purified Infectious Prions
2005; Elsevier BV; Volume: 280; Issue: 17 Linguagem: Inglês
10.1074/jbc.m500122200
ISSN1083-351X
AutoresLior Horonchik, Salit Tzaban, Olga Ben-Zaken, Yifat Yedidia, Alex Rouvinski, Dulce Papy-García, Denis Barritault, Israël Vlodavsky, Albert Taraboulos,
Tópico(s)RNA regulation and disease
ResumoPrions replicate in the host cell by the self-propagating refolding of the normal cell surface protein, PrPC, into a β-sheet-rich conformer, PrPSc. Exposure of cells to prion-infected material and subsequent endocytosis can sometimes result in the establishment of an infected culture. However, the relevant cell surface receptors have remained unknown. We have previously shown that cellular heparan sulfates (HS) are involved in the ongoing formation of scrapie prion protein (PrPSc) in chronically infected cells. Here we studied the initial steps in the internalization of prions and in the infection of cells. Purified prion “rods” are arguably the purest prion preparation available. The only proteinaceous component of rods is PrPSc. Mouse neuroblastoma N2a, hypothalamus GT1–1, and Chinese hamster ovary cells efficiently bound both hamster and mouse prion rods (at 4 °C) and internalized them (at 37 °C). Treating cells with bacterial heparinase III or chlorate (a general inhibitor of sulfation) strongly reduced both binding and uptake of rods, whereas chondroitinase ABC was inactive. These results suggested that the cell surface receptor of prion rods involves sulfated HS chains. Sulfated glycans inhibited both binding and uptake of rods, probably by competing with the binding of rods to cellular HS. Treatments that prevented endocytosis of rods also prevented the de novo infection of GT1–1 cells when applied during their initial exposure to prions. These results indicate that HS are an essential part of the cellular receptor used both for prion uptake and for cell infection. Cellular HS thus play a dual role in prion propagation, both as a cofactor for PrPSc synthesis and as a receptor for productive prion uptake. Prions replicate in the host cell by the self-propagating refolding of the normal cell surface protein, PrPC, into a β-sheet-rich conformer, PrPSc. Exposure of cells to prion-infected material and subsequent endocytosis can sometimes result in the establishment of an infected culture. However, the relevant cell surface receptors have remained unknown. We have previously shown that cellular heparan sulfates (HS) are involved in the ongoing formation of scrapie prion protein (PrPSc) in chronically infected cells. Here we studied the initial steps in the internalization of prions and in the infection of cells. Purified prion “rods” are arguably the purest prion preparation available. The only proteinaceous component of rods is PrPSc. Mouse neuroblastoma N2a, hypothalamus GT1–1, and Chinese hamster ovary cells efficiently bound both hamster and mouse prion rods (at 4 °C) and internalized them (at 37 °C). Treating cells with bacterial heparinase III or chlorate (a general inhibitor of sulfation) strongly reduced both binding and uptake of rods, whereas chondroitinase ABC was inactive. These results suggested that the cell surface receptor of prion rods involves sulfated HS chains. Sulfated glycans inhibited both binding and uptake of rods, probably by competing with the binding of rods to cellular HS. Treatments that prevented endocytosis of rods also prevented the de novo infection of GT1–1 cells when applied during their initial exposure to prions. These results indicate that HS are an essential part of the cellular receptor used both for prion uptake and for cell infection. Cellular HS thus play a dual role in prion propagation, both as a cofactor for PrPSc synthesis and as a receptor for productive prion uptake. The transmissible spongiform encephalopathies that comprise infectious, familial, and sporadic neurodegenerations such as Creutzfeldt-Jakob disease of humans (1Gibbs Jr., C.J. Gajdusek D.C. Science. 1969; 165: 1023-1025Crossref PubMed Scopus (108) Google Scholar), scrapie of sheep, and bovine spongiform encephalopathy (2Wilesmith J.W. Wells G.A. Cranwell M.P. Ryan J.B. Vet. Rec. 1988; 123: 638-644Crossref PubMed Google Scholar) are caused by prions (3Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4124) Google Scholar). These proteinaceous agents are thought to propagate by refolding a normal cell surface glycoprotein of the host, the cellular prion protein (PrPC) 1The abbreviations used are: PrPC, cellular prion protein; PrPSc, scrapie prion protein; CHO, Chinese hamster ovary; GAG, glycosaminoglycan; HS, heparan sulfate; HM, heparan mimetic; PBS, phosphate-buffered saline; mAb, monoclonal antibody; WB, Western blot; DS, dextran sulfate. , into an abnormal β-sheet-rich (4Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Abstract Full Text PDF PubMed Google Scholar, 5Pan K.M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2084) Google Scholar) conformation (reviewed in Ref. 6Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholar). The resulting pathological conformer, PrPSc, is in turn the only known component of the infectious prion. The formation of PrPSc is thought to involve a direct contact between “seed” PrPSc and “substrate” PrPC (7Prusiner S.B. Scott M. Foster D. Pan K.M. Groth D. Mirenda C. Torchia M. Yang S.L. Serban D. Carlson G.A. Hoppe P.C. Westaway D. DeArmond S.J. Cell. 1990; 63: 673-686Abstract Full Text PDF PubMed Scopus (731) Google Scholar, 8Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (792) Google Scholar) and probably involves cellular cofactors (9Telling G.C. Scott M. Mastrianni J. Gabizon R. Torchia M. Cohen F.E. DeArmond S.J. Prusiner S.B. Cell. 1995; 83: 79-90Abstract Full Text PDF PubMed Scopus (768) Google Scholar) including the laminin receptors (10Hundt C. Peyrin J.M. Haik S. Gauczynski S. Leucht C. Rieger R. Riley M.L. Deslys J.P. Dormont D. Lasmezas C.I. Weiss S. EMBO J. 2001; 20: 5876-5886Crossref PubMed Scopus (255) Google Scholar, 11Gauczynski S. Peyrin J.M. Haik S. Leucht C. Hundt C. Rieger R. Krasemann S. Deslys J.P. Dormont D. Lasmezas C.I. Weiss S. EMBO J. 2001; 20: 5863-5875Crossref PubMed Scopus (365) Google Scholar, 12Rieger R. Edenhofer F. Lasmezas C.I. Weiss S. Nat. Med. 1997; 3: 1383-1388Crossref PubMed Scopus (379) Google Scholar) and cellular heparan sulfate proteoglycans (13Gabizon R. Meiner Z. Halimi M. Ben-Sasson S.A. J. Cell. Physiol. 1993; 157: 319-325Crossref PubMed Scopus (121) Google Scholar, 14Caughey B. Raymond G.J. J. Virol. 1993; 67: 643-650Crossref PubMed Google Scholar, 15Ben-Zaken O. Tzaban S. Tal Y. Horonchik L. Esko J.D. Vlodavsky I. Taraboulos A. J. Biol. Chem. 2003; 278: 40041-40049Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Although several cell lines are susceptible to prion infection (16Clarke M.C. Haig D.A. Nature. 1970; 225: 100-101Crossref PubMed Scopus (80) Google Scholar) (reviewed in Ref. 17Solassol J. Crozet C. Lehmann S. Br. Med. Bull. 2003; 66: 87-97Crossref PubMed Scopus (70) Google Scholar), the molecular mechanisms involved remain largely obscure. Infection is usually started by exposing cells to prion-infected material, such as brain homogenate. Many cell types (including cell lines (18Taraboulos A. Serban D. Prusiner S.B. J. Cell Biol. 1990; 110: 2117-2132Crossref PubMed Scopus (232) Google Scholar) and primary dendritic cells (19Luhr K.M. Wallin R.P. Ljunggren H.G. Low P. Taraboulos A. Kristensson K. J. Virol. 2002; 76: 12259-12264Crossref PubMed Scopus (46) Google Scholar)) can internalize prion-infected material, but the cellular receptors for prions have not been identified. One factor that is likely to complicate the study of how prions enter cells is their notorious association with heterogeneous cellular membranes and aggregates (which contain, in addition, other cellular components). Thus, prions in crude tissue homogenates are likely to “hitch-hike” their way into the cell using aggregates and microsomes as vehicles via a variety of cell surface receptors. However, prions can be extracted from membranes to yield purer preparations. Prion “rods” are arguably the purest form of prions known (20Prusiner S.B. McKinley M.P. Bowman K.A. Bolton D.C. Bendheim P.E. Groth D.F. Glenner G.G. Cell. 1983; 35: 349-358Abstract Full Text PDF PubMed Scopus (833) Google Scholar, 21McKinley M.P. Braunfeld M.B. Bellinger C.G. Prusiner S.B. J. Infect. Dis. 1986; 154: 110-120Crossref PubMed Scopus (47) Google Scholar). These infectious, unbranched amyloidic structures are prepared from prion-infected tissues by the combined action of detergents and proteases (22McKinley M.P. Meyer R.K. Kenaga L. Rahbar F. Cotter R. Serban A. Prusiner S.B. J. Virol. 1991; 65: 1340-1351Crossref PubMed Google Scholar) (often supplemented by nucleases), and their only proteinaceous component is PrP27–30 (23Bolton D.C. McKinley M.P. Prusiner S.B. Biochemistry. 1984; 23: 5898-5906Crossref PubMed Scopus (102) Google Scholar), the protease-resistant core of PrPSc. The size of rods is very heterogeneous, and they may contain up to several thousands PrP molecules (20Prusiner S.B. McKinley M.P. Bowman K.A. Bolton D.C. Bendheim P.E. Groth D.F. Glenner G.G. Cell. 1983; 35: 349-358Abstract Full Text PDF PubMed Scopus (833) Google Scholar). Although purified prion rods are efficiently taken up by a variety of cells and often lead to productive infections (18Taraboulos A. Serban D. Prusiner S.B. J. Cell Biol. 1990; 110: 2117-2132Crossref PubMed Scopus (232) Google Scholar), even in this highly simplified situation the relevant cellular receptors have not yet been identified. Here we set out to characterize receptors for rods in two infectible mouse cell lines, the neuroblastoma N2a (24Butler D.A. Scott M.R. Bockman J.M. Borchelt D.R. Taraboulos A. Hsiao K.K. Kingsbury D.T. Prusiner S.B. J. Virol. 1988; 62: 1558-1564Crossref PubMed Google Scholar) and the hypothalamic cell line GT1–1 (25Mellon P.L. Windle J.J. Goldsmith P.C. Padula C.A. Roberts J.L. Weiner R.I. Neuron. 1990; 5: 1-10Abstract Full Text PDF PubMed Scopus (900) Google Scholar, 26Weiner R.I. Wetsel W. Goldsmith P. Martinez de la Escalera G. Windle J. Padula C. Choi A. Negro-Vilar A. Mellon P. Front Neuroendocrinol. 1992; 13: 95-119PubMed Google Scholar, 27Schätzl H.M. Laszlo L. Holtzman D.M. Tatzelt J. DeArmond S.J. Weiner R.I. Mobley W.C. Prusiner S.B. J. Virol. 1997; 71: 8821-8831Crossref PubMed Google Scholar), and Chinese hamster ovary (CHO) cells, which seem to be refractive to prion infection. Glycosaminoglycans (GAGs) such as heparan sulfate (HS) are long, unbranched side chains of proteoglycans that are found in several cellular compartments, including the cell surface and endosomes (reviewed in Ref. 28Turnbull J. Powell A. Guimond S. Trends Cell Biol. 2001; 11: 75-82Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). Three arguments put forward GAGs as candidates for cellular receptors for prion rods. First, PrP has several heparin-binding sites both in the N-terminal unstructured region and in the PrP27–30 core (13Gabizon R. Meiner Z. Halimi M. Ben-Sasson S.A. J. Cell. Physiol. 1993; 157: 319-325Crossref PubMed Scopus (121) Google Scholar, 29Warner R.G. Hundt C. Weiss S. Turnbull J.E. J. Biol. Chem. 2002; 277: 18421-18430Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 30Caughey B. Brown K. Raymond G.J. Katzenstein G.E. Thresher W. J. Virol. 1994; 68: 2135-2141Crossref PubMed Google Scholar). Second, cellular HS are required for PrPSc formation in persistently infected mouse neuroblastoma ScN2a cells (15Ben-Zaken O. Tzaban S. Tal Y. Horonchik L. Esko J.D. Vlodavsky I. Taraboulos A. J. Biol. Chem. 2003; 278: 40041-40049Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Because these HS prion cofactors probably perform their task by binding the endogenous PrP isoforms, it is plausible that they can also bind PrP27–30 molecules found in exogenous rods. Another indication that GAGs may serve as prion receptors is the finding that certain soluble dextran-based heparan mimetics (HMs) (31Ledoux D. Papy-Garcia D. Escartin Q. Sagot M.A. Cao Y. Barritault D. Courtois J. Hornebeck W. Caruelle J.P. J. Biol. Chem. 2000; 275: 29383-29390Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) reduce the internalization of prion rods in both N2a and CHO cells (32Schonberger O. Horonchik L. Gabizon R. Papy-Garcia D. Barritault D. Taraboulos A. Biochem. Biophys. Res. Commun. 2003; 312: 473-479Crossref PubMed Scopus (74) Google Scholar). Conceivably, this inhibition could result from the competition of soluble HM molecules with putative HS receptors of rods. To evaluate the role of cellular HS in the binding and endocytosis of prion rods, we used GAG-degrading enzymes, the sulfation inhibitor chlorate, and soluble glycans, including several HM species. Our results indicate that heparinase III-sensitive HS on the cell surface are involved in both the binding and the uptake of rods in N2a, CHO, and GT1–1 cells. Treatments that prevented the binding and internalization of rods also prevented the de novo infection of GT1–1 cells. Cellular HS are thus an essential component of cellular receptors for the uptake of prions and the infection of cells. Materials—Cell culture reagents were purchased from Biological Industries (Beit Haemek, Israel). OptiMem was from Invitrogen. Dextran sulfate 500 (150821) was from ICN (Costa Mesa, CA). Heparinase I (heparinase EC 4.2.2.7) and heparinase III (heparitinase I, EC 4.2.2.8) were from IBEX Technologies (Montreal, Canada). Chondroitinase ABC (EC 4.2.2.4) was from Seikagaku Corp. (Tokyo, Japan). The dextran-based HMs (32Schonberger O. Horonchik L. Gabizon R. Papy-Garcia D. Barritault D. Taraboulos A. Biochem. Biophys. Res. Commun. 2003; 312: 473-479Crossref PubMed Scopus (74) Google Scholar) were obtained from OTR3 Sarl (Creteil, France). Micrococcal nuclease (N-5386) was from Sigma. Recombinant mouse PrP23–231 and recombinant R1 and D13 PrP antibodies were from InPro (South San Francisco, CA). Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Porcine mucosa HS was kindly provided by Dr. K. M. Shwann (Kabi-Pharmacia, Stockholm, Sweden). Preparation of Prion Rods—Prion rods were purified from the brains of Syrian hamsters and of C57/bl mice infected with experimental Sc237 and RML scrapie, respectively, using a procedure modified from Prusiner et al. (33Prusiner S.B. Groth D.F. Bolton D.C. Kent S.B. Hood L.E. Cell. 1984; 38: 127-134Abstract Full Text PDF PubMed Scopus (376) Google Scholar) and Diringer et al. (34Diringer H. Hilmert H. Simon D. Werner E. Ehlers B. Eur. J. Biochem. 1983; 134: 555-560Crossref PubMed Scopus (59) Google Scholar). All steps were performed at 4 °C. One hamster brain or three mouse brains (a total of ∼1 g) were homogenized in 20 ml of 10% sucrose in PBS (buffer A). A 3220 × g, 10-min pellet was rehomogenized in 10 ml of buffer A and then repelleted, and the supernatants were united and cleared by a 3220 × g, 30-min spin. The supernatant was made 1 mm with each EDTA and dithiothreitol, and Triton X-100 and Na-deoxycholate were added to 4 and 2% final concentrations, respectively. The supernatant was then stirred for 30 min, and the following reagents were added dropwise while stirring: Tris acetate (pH 8.3) to 30 mm, KCl to 100 mm, glycerol to 20%, and polyethylene glycol 10000 to 8% w/v. After an additional 30 min of stirring, the homogenate was pelleted at 4500 × g for 30 min. The pellet was resuspended in 20 mm Tris acetate, pH 8.3, 0.02% TX-100, 1 mm dithiothreitol, and 2 mm CaCl2 and then digested with micrococcal nuclease (12.5 units/ml) for 16 h at 4 °C. The nuclease reaction was stopped by the addition of 2 mm EDTA and 0.2% Sarkosyl. The homogenate was subjected to proteolysis with proteinase K (100 μg/ml, 8 h, 4 °C), and the reaction was stopped by incubating for 30 min with 100 mm phenylmethylsulfonyl fluoride. Sarkosyl was added to 1%, and after 30 min of incubation on ice the homogenate was spun at 100,000 × g for 1 h. The pellet, which contains the prion rods, was resuspended in 500 μl of TNS (10 mm Tris, pH 7.5, 150 mm NaCl, 1% Sarkosyl) using a probe sonicator (Sonopuls; Dandelin Electronics, Germany) full power (4 × 1 s), and then repelleted (3×). Sarkosyl was removed by rinsing the pellet twice with 70% EtOH (100,000 × g, 30 min) and then resuspended by sonication in 600 μl of 30% sucrose in TN. A 5-μl sample of this rod preparation was analyzed by electrophoresis and silver staining (Fig. 1A, right lane) and compared with 10 ng of recombinant mouse PrP (Fig. 1A, left lane). Hamster rods contained a single 27–30-kDa band (Fig. 1A) that is characteristic of Syrian hamster Sc237 PrPSc, whereas mouse rods had three characteristic glycoforms. The PrP concentration in rod preparations was determined by comparing with dilution of recombinant mouse PrP in Western blots developed with the recombinant Fab, R1 (Fig. 1B). Rod preparation usually contained about 5 ng/ml PrP27–30. Cells—Mouse neuroblastoma ScN2a-M are ScN2a cells (24Butler D.A. Scott M.R. Bockman J.M. Borchelt D.R. Taraboulos A. Hsiao K.K. Kingsbury D.T. Prusiner S.B. J. Virol. 1988; 62: 1558-1564Crossref PubMed Google Scholar) that stably express the MHM2-PrP chimera that reacts with the mAb 3F4 (35Kascsak R.J. Rubenstein R. Merz P.A. Carp R.I. Robakis N.K. Wisniewski H.M. Diringer H. J. Virol. 1986; 59: 676-683Crossref PubMed Google Scholar). An uninfected version (N2a-M) was obtained by curing ScN2a-M cells with pentosan polysulfate (5 μg/ml, 5 days) (36Ehlers B. Diringer H. J. Gen. Virol. 1984; 65 (Pt. 8): 1325-1330Crossref PubMed Scopus (154) Google Scholar) and subsequently maintaining them without inhibitors for at least 1 month prior to use. GT1–1 are mouse hypothalamus cells (27Schätzl H.M. Laszlo L. Holtzman D.M. Tatzelt J. DeArmond S.J. Weiner R.I. Mobley W.C. Prusiner S.B. J. Virol. 1997; 71: 8821-8831Crossref PubMed Google Scholar). GT1–1-M stably express MHM2-PrP and react with 3F4. Cells were grown at 37 °C in low glucose Dulbecco's modified Eagle's medium-16 (N2a and GT1–1) or F12 (CHO-K1, (37Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (489) Google Scholar)) containing 10% fetal calf serum. In some experiments, cells were maintained in a 1:1 mixture of either of the above media and OptiMem (Invitrogen). To assess the susceptibility of GT1–1-MHM2 cells to infection, cells growing on 12-well trays were treated with the relevant inhibitors for 24 h. Thereafter, the cells were inoculated either with purified mouse rods for 24 h or with cell supernatant of ScGT1–1 (the medium was frozen and thawed at least 3 times before addition to the cells) for 48 h at 37 °C in Dulbecco's modified Eagle's medium-OptiMem (1:1) in the presence of the inhibitors. The cells were rinsed and further grown in fresh medium (without inoculum or treatments) for either 5 or 10 days as indicated in Fig. 5. Because the mouse inocula are not recognized by the mAb 3F4, successful infection was identified by the appearance in the cells of 3F4-reactive PrPSc (as depicted in Fig. 5B). PrP Isoforms and Analysis—PrPSc was defined as the PrP fraction resistant to proteinase K (20 μg/ml, 37 °C, 30 min). Western blots (WB) were carried out as described (38Naslavsky N. Stein R. Yanai A. Friedlander G. Taraboulos A. J. Biol. Chem. 1997; 272: 6324-6331Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). The protein content of parallel samples was normalized using a Bradford kit (500–006) from Bio-Rad (Hercules, CA) prior to electrophoresis. Cell lysates (in ice-cold lysis buffer: 0.5% Triton X-100, 0.25% Na-deoxycholate, 150 mm NaCl, 10 mm Tris-Cl, pH 7.5, 10 mm EDTA) were immediately centrifuged for 40 s at 14,000 rpm in a microfuge, and biochemical analyses were performed on the post-nuclear supernatant. Rod Internalization and Binding—Assays were carried out on confluent monolayers grown in 24-well trays. Cells were treated with enzymes or inhibitors for 24 h at 37 °C prior to the assays unless otherwise indicated. Rods (1 μl/well, ∼5 ng of PrP, Fig. 1B) were then added either for 20 h at 37 °C (internalization) or for 3 h at 4 °C (binding assay) performed in PBS or in F12/OptiMem, 1:1, with identical results. The cells were rinsed with ice-cold PBS and lysed, and the protein content was normalized using the Bradford method. The samples were incubated with 2 μg/ml proteinase K (1 h, 37 °C, stopped by 2 mm phenylmethylsulfonyl fluoride), and the level of cell-associated PrPSc (from the exogenous rods) was assayed by WB developed with 3F4 mAb (for Syrian hamster rods) or D13 Fab (for mouse rods). Fluorescence Microscopy—For internalization experiments, cells growing on 8-well slides (Nunc, Roskilde, Denmark) were pretreated with the relevant inhibitors for 24 h and then exposed for 20 h to purified Syrian hamster prion rods with or without inhibitors. At the end of the incubation the cells were rinsed and further incubated in normal medium for 4 h to reduce the signal of the rods on the cell surface. The cells were fixed (8% formalin in PBS, 30 min, room temperature), denatured in situ (3 m GdnSCN, 0.1% TX-100, 50 mm Tris-HCl, pH 7.5; 5 min, room temperature) (18Taraboulos A. Serban D. Prusiner S.B. J. Cell Biol. 1990; 110: 2117-2132Crossref PubMed Scopus (232) Google Scholar) to visualize rods, immunostained with 3F4, and examined by fluorescence microscopy. Inhibition of Rod Uptake by Soluble Glycans Correlates with Their Anti-prion Activity—It is well established that certain glycans reduce the ongoing formation of PrPSc in chronically infected cells. To see whether cellular GAGs might be part of a cellular receptor for prions, we thus first studied the extent to which several glycans reduce the uptake of prion rods, as compared with their anti-prion efficacy. To this end we chose the “classical” anti-prion dextran sulfate (DS500) (36Ehlers B. Diringer H. J. Gen. Virol. 1984; 65 (Pt. 8): 1325-1330Crossref PubMed Scopus (154) Google Scholar) and three members of the HM library of substituted dextrans with vastly different anti-prion potencies (32Schonberger O. Horonchik L. Gabizon R. Papy-Garcia D. Barritault D. Taraboulos A. Biochem. Biophys. Res. Commun. 2003; 312: 473-479Crossref PubMed Scopus (74) Google Scholar): B103, HM2102, and HM2602 (Fig. 2). Chronically infected ScN2a-M cells were treated for 5 days with these compounds, and protease-resistant PrPSc was then analyzed by WB developed with 3F4 (panel A). As expected, the more sulfated and/or benzylaminated compounds (DS500, HM2602, and B103) reduced PrPSc more efficiently than the non-sulfated HM2102 (32Schonberger O. Horonchik L. Gabizon R. Papy-Garcia D. Barritault D. Taraboulos A. Biochem. Biophys. Res. Commun. 2003; 312: 473-479Crossref PubMed Scopus (74) Google Scholar). Next, we turned to determination of the extent to which these glycans decrease rod uptake in uninfected N2a-M cells. The cells were preincubated for 24 h with the polyanions, as in panel A. Purified Syrian hamster rods were added to the cell medium for an additional 20 h in the presence of the inhibitors. The cells were rinsed thoroughly, and their protease-resistant PrPSc was monitored in WBs with 3F4 (panel B). The ability of these compounds to reduce the internalization of rods correlated well with their anti-PrPSc potency (compare panels A and B, and see Fig. 6). (We have previously reported that HMs are not general endocytosis inhibitors (32Schonberger O. Horonchik L. Gabizon R. Papy-Garcia D. Barritault D. Taraboulos A. Biochem. Biophys. Res. Commun. 2003; 312: 473-479Crossref PubMed Scopus (74) Google Scholar).) Interestingly, higher concentrations of inhibitors were required to reduce rod uptake than to reduce endogenous PrPSc (see “Discussion”).Fig. 6Cellular heparan sulfates serve both as rod receptors and as prion cofactors: a model.A, left, rods, which comprise large aggregates of PrP27–30, can bind cell surface HS and use them for internalization. Because of their large dimension, rods are likely to bind cooperatively to a very large number of HS molecules. These HS receptors are similar to or identical with the cellular HS cofactors (right panel). However, whereas rod binding takes place on the cell surface, PrPSc synthesis (right panel) may also occur in intracellular compartments. B, digesting with heparinase or preventing sulfation with chlorate prevents both rod binding and internalization (left) and PrPSc formation (right). C, identical polyanions prevent the binding of rods and the formation of PrPSc, but a larger concentration is required to compete out the presumably more numerous binding sites of rods (left).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Anti-prion Heparinase III and Chlorate Inhibit Rod Uptake—This correlation suggested that there may be a mechanistic relationship between the two anti-prion activities of these polyanions, namely that the cellular HS that are needed for the ongoing synthesis of PrPSc (15Ben-Zaken O. Tzaban S. Tal Y. Horonchik L. Esko J.D. Vlodavsky I. Taraboulos A. J. Biol. Chem. 2003; 278: 40041-40049Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) also form parts of internalization receptors for rods. To verify that HS are involved in rod uptake, we explored other treatments that inhibit or digest cellular HS (Fig. 3). N2a-M (panel A) and CHO-K1 (panels B and C) cells were pretreated for 24 h with either heparinase I or heparinase III to digest away HS, or they were incubated with the metabolic sulfation inhibitor Na chlorate (30 mm) (39Baeuerle P.A. Huttner W.B. Biochem. Biophys. Res. Commun. 1986; 141: 870-877Crossref PubMed Scopus (294) Google Scholar) or with soluble glycans as indicated in panel B. Rods were added to the cell medium and were allowed to enter the cells for 20 h. The cells were then harvested and proteinase K-resistant PrP was detected using either WB (Fig. 3, A and B) or immunofluorescence (Fig. 3C). Heparinase III, Na-chlorate, dextran sulfate DS500, and HM2602 almost completely abolished rod uptake, whereas heparinase I was only slightly inhibitory and the inactive HM compound HM2102 failed to reduce the uptake of rods altogether. These results correlate perfectly with the anti-prion potency of these treatments (Fig. 2 and Ref. 15Ben-Zaken O. Tzaban S. Tal Y. Horonchik L. Esko J.D. Vlodavsky I. Taraboulos A. J. Biol. Chem. 2003; 278: 40041-40049Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Similar inhibitory results were obtained when mouse rods were applied to GT1–1 cells (Fig. 5A). In contrast to heparinase III, chondroitinase ABC failed to reduce rod uptake (Fig. 3B), correlating with its inability to reduce PrPSc in chronically infected ScN2a (15Ben-Zaken O. Tzaban S. Tal Y. Horonchik L. Esko J.D. Vlodavsky I. Taraboulos A. J. Biol. Chem. 2003; 278: 40041-40049Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Whether this is caused by the paucity of chondroitin sulfate in these cells or by other mechanisms remains to be determined. Heparinase III-sensitive HS Mediate Both Binding and Internalization of Rods—These results strongly suggested that heparinase III-sensitive cellular HS are part of endocytosis receptors for purified prion rods in N2a, CHO-K1, and GT1–1 cells. Prions also bound to cell surface HS at 4 °C. N2a-M (not shown) or CHO-K1 cells (Fig. 4) were pretreated for 24 h with heparinase I, heparinase III, Na-chlorate (panel A), or dextran-based polyanions (panel B). The plates were cooled on ice (to prevent endocytosis) and further incubated with purified rods in the presence of the inhibitors (3 h on ice). At the end of the incubation, the cells were rinsed thoroughly and cell surface-bound rods were monitored by WB. All the efficient anti-PrPSc treatments also effectively reduced the binding of purified rods to the surface of these cells. Thus, only heparinase I and HM2102 left this binding intact (Fig. 4, A and B). In addition to removing HS GAGs from the cell surface, heparinase III is also likely to release HS fragments to the cell medium. By analogy with other sulfated glycans, these degradation products could also inhibit rod binding by competing with surface receptors. It was therefore necessary to verify directly that the presence of HS chains on the cell surface perform as a binding receptor for prion rods. For this purpose, we incubated CHO-K1 cells with heparinase III (0.1, 1, or 2 units/ml) for 24 h (37 °C). The cells were rinsed with ice-cold PBS to remove any soluble heparinase products and supplemented with fresh medium without heparinase, and rods were added to all cells (3 h on ice). At the end of the incubation the cells were rinsed thoroughly and the cell surface-bound rods were monitored in WB (Fig. 4C). Treatment of the cells with heparinase III inhibited rod binding to the cells (lanes 3 and 4). Taken together, the results described above indicate that (i) heparinase III-sensitive HS play a crucial role both in the binding and in the internalization of prion rods in N2a and in CHO-K1 cells, and (ii) these HS molecules are similar or identical to those that serve as cofactors for the ongoing formation of PrPSc in ScN2a cells. We next asked whether endogenous HS are required for rod binding or whether exogenous HS can mediate binding of rods to another cellular receptor. For this purpose CHO-K1 cells were treated with chlorate for 24 h to stop the sulfation of cellular HS. Exogenous HS (50 μg/ml) were then added to the cell medium along with prion rods, the cells were further incubated for 3 h on ice, and cell-b
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