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Originally published In Press as doi:10.1074/jbc.M301152200 on July 18, 2003

J. Biol. Chem., Vol. 278, Issue 41, 40041-40049, October 10, 2003
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Cellular Heparan Sulfate Participates in the Metabolism of Prions*

Olga Ben-Zaken {ddagger} §, Salit Tzaban § ¶, Yuval Tal ¶, Lior Horonchik ¶, Jeffrey D. Esko ||, Israel Vlodavsky ** and Albert Taraboulos ¶ {ddagger}{ddagger}

From the {ddagger}Department of Oncology, Hadassah-University Hospital, Jerusalem 91120, Israel, Department of Molecular Biology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel, ||Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California 92093, and **Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel

Received for publication, February 3, 2003 , and in revised form, July 15, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
During prion diseases, the host protein PrPC is refolded into an abnormal conformer "prion" PrPSc. Histological and pharmacological data have suggested that glycosaminoglycans may be involved in the development of prion diseases. Here we present the first direct evidence that cellular glycosaminoglycans play a role in the biogenesis of PrPSc in prion-infected ScN2a cells. When ScN2a cells were incubated with estradiol {beta}-D-xyloside to inhibit the glycosylation of proteoglycans, PrPSc was vastly reduced. Treating ScN2a-M cells with heparinase III, but not with heparinase I or chondroitinase ABC, caused a profound reduction of PrPSc. In contrast, neither the amount of PrPC nor its subcellular distribution were affected as assayed by immunofluorescence microscopy and flotation procedures. In vitro treatment of ScN2a membranes with heparinase III at either neutral or acidic pH did not reduce the level of protease-resistant PrPSc. The inhibitor of sulfation, sodium chlorate, vastly reduces PrPSc in ScN2a cells (Gabizon, R., Meiner, Z., Halimi, M., and Ben-Sasson, S. A. (1993) J. Cell. Physiol. 157, 319–325). Both soluble heparan sulfate and chondroitin sulfate partially restored the level of PrPSc in chlorate-treated cells. We conclude that heparinase III-sensitive, presumably undersulfated, cellular heparan sulfate plays a significant role in the biogenesis of PrPSc in ScN2a cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The transmissible spongiform encephalopathies, which comprise infectious, familial, and sporadic neurodegenerative diseases such as Creutzfeldt-Jakob disease of humans (1), scrapie of sheep, and bovine spongiform encephalopathy (2), are caused by prions (3). These proteinaceous agents are thought to propagate by refolding a normal cell surface glycoprotein of the host, the cellular prion protein PrPC,1 into an abnormal {beta}-sheet rich (4, 5) conformation (reviewed in Ref. 6). Whether this process occurs through a seeding mechanism, such as that involved in the fibrillogenesis of amyloidogenic peptides (7), remains to be established. The resulting pathological conformer, PrPSc, is in turn the only known component of the infectious prion.

Little is known about the molecular and cellular mechanisms that lead to the transformation of PrPC into PrPSc. The PrP isoforms possess disparate biochemical properties. In contrast to PrPC, most PrPSc possesses a protease-resistant core (PrP27–30) and is insoluble in detergents (8, 9). Kinetic analyses of prion-infected cells in culture (10) have shown that protease-resistant PrPSc is formed slowly within hours of the synthesis of PrPC, probably in a post-endoplasmic reticulum compartment of the endomembrane system (5, 11, 12). Transgenetic studies of species barriers (13) have indicated that accessory molecules of the host are probably involved in this process. Although these putative prion cofactors have not yet been identified, several candidates have been put forward including the laminin receptors (14–17), stress-induced protein 1 (18), and glycosaminoglycans (GAGs).

Diverse indirect lines of evidences have linked proteoglycans (PGs) and their GAG chains and especially heparan sulfate (HS) to the metabolism of the PrP isoforms (reviewed in Ref. 19). First, HS accumulates in cerebral prion amyloid plaques (20) and it also associates with the more diffuse PrPSc deposits that appear in early stages of prion diseases (21). Second, a variety of sulfated glycans, such as low molecular weight heparin (22), suramin (23), pentosan polysulfate, and dextran sulfate (23–25), and dextran-based HS analogs reduce the formation of PrPSc in infected cells and in some cases prolong the incubation time of experimental prion diseases. Because PrP binds heparin (22, 26, 27), it has been suggested that sulfated glycans exert their anti-prion effects by competing with the binding of PrPC to putative cellular pro-prion GAGs (22). Paradoxically, however, the cell-free conversion of PrPC to a protease-resistant form, PrP-res (which is biochemically analogous to PrPSc), is stimulated rather than inhibited by soluble HS analogs (28). Third, long term incubation of cells with chlorate, which affects the sulfation of GAGs as well as of proteins and glycolipids, reduces PrPSc (22). Fourth, a heparinase-sensitive brain fraction promotes the reconstitution of infectivity of dimethyl sulfoxide-solubilized PrPSc (29). Finally, several sulfated glycans increase the endocytosis of PrP, suggesting that PGs may also modulate the subcellular trafficking of PrP (30, 31).

During the biogenesis of PGs, the synthesis of GAG chains is initiated by the linking of a D-xylose to serine residues within the protein core. The chains are then elongated and modified to yield the highly complex GAGs. Several {beta}-D-xylosides (D-xylopyranose attached to an aglycone) (32, 33) can inhibit the initiation reaction by serving as soluble primers for GAG biosynthesis. These xylosides thus interfere with the proper glycosylation of PGs, but they also promote the synthesis of soluble xylosides-primed GAG analogs that are secreted into the medium. The aglycone structure of the xyloside can determine the type of soluble GAG that is produced (as well as the extent to which PG glycosylation is inhibited). Thus, the lipophilic xyloside estradiol {beta}-D-xyloside (EDX) primes both HS and chondroitin sulfate (CS), and the type of GAG that is primed depends on the concentration of the xyloside (34).

In this paper, we studied the involvement of proteoglycans in the metabolism of PrP in mouse neuroblastoma cells chronically infected with prions (ScN2a-M). At first, we used EDX (34) to interfere with the biosynthesis of PGs. In cells treated for 4 days with EDX, there was a strong reduction of PrPSc, whereas in uninfected cells PrPC was unchanged. This result strengthened the contention that cellular GAGs are involved in the metabolism of PrPSc. To start characterizing these GAGs, we used GAG-degrading enzymes of bacterial origin. ScN2a-M cells treated for 4 days with heparinase III showed a dramatic decrease in PrPSc, whereas chondroitinase ABC and heparinase I had no effect. None of these enzymes affected the amount, the subcellular distribution, or the association of the precursor PrPC to membrane rafts as demonstrated by immunofluorescent microscopy and by flotation assays. Incubation of purified ScN2a-M membranes with heparinase III did not reduce the protease-resistance of PrPSc, suggesting that heparinase III does not directly perturb the properties of this protein. The decrease in PrPSc caused by treatment of cells with chlorate (22) was partially restored by both the addition of soluble HS and CS. Taken together, these results strongly indicate that one or more heparinase III-sensitive cellular HS regulate the metabolism of PrPSc in ScN2a cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture reagents were from Biological Industries (Beit Haemek, Israel). Opti-MEM was from Invitrogen. Tissue culture dishes were from Miniplast (Ein Shemer, Israel). Multiwell plates and plastic chamber slides for immunofluorescence were from Nunc (Roskilde, Denmark). G418 was from Calbiochem. (35S)Na2SO4 (specific activity 540–590 mCi/mmol) and (125I)-labeled bFGF (specific activity 1.2 x 105 cpm/ng) were from Amersham Biosciences. Estradiol-{beta}-D-xyloside was prepared as described previously (33). Porcine mucosa HS was kindly provided by Dr. K. M. Shwann (Kabi-Pharmacia, Stockholm, Sweden). Bovine trachea CS (catalog number C8529) was from Sigma. Sepharose CL-6B was from Amersham Biosciences. Recombinant human bFGF was kindly provided by Takeda Chemical Industries (Osaka, Japan). Other chemicals were from Sigma.

Enzymes—Heparinase I (heparinase, EC 4.2.2.7 [EC] ) and heparinase III (heparitinase I, EC 4.2.2.8 [EC] ) were kindly provided by Dr. Robert Heft (IBEX Technologies, Montreal, Canada). Chondroitinase ABC (EC 4.2.2.4 [EC] ) from Seikagaku Corp. (Tokyo, Japan) and from Sigma (catalog number C2905) yielded identical results (Fig. 2A).



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  		FIG. 2.
Heparinase III but not other GAG-degrading enzymes reduces PrPSc in ScN2a-M cells (Western blots). Panel A, N2a-M and ScN2a-M cells growing in 6-well plates were incubated for 4 days with chondroitinase ABC (panel 1) or with heparinase I or III (panels 2 and 3), and the level of PrP was analyzed by Western blots developed with mAb 3F4. Samples were normalized for their protein content. In some cases (+PK), the lysates were digested with proteinase K (20 µg/ml, 37 °C, 30 min) prior to electrophoresis to eliminate PrPC. Panel 1, ScN2a-M cells were either left untreated or were treated with increasing concentrations of chondroitinase ABC (0.01–0.5 units/ml). PrPSc level was unaltered. Panels 2 and 3, ScN2a-M cells (panel 2) and N2a-M cells (panel 3) were incubated with 0.1 unit/ml heparinase I, heparinase III, or a mixture of both at a final concentration of 0.1 unit/ml (i.e. 0.05 units/ml of each enzyme) as indicated. PrPSc (panel 2, +PK) but not PrPC (panel 3) was reduced specifically by heparinase III. Protease-resistant PrPSc content after heparinase I or heparinase III treatment was analyzed by densitometry and plotted. Each data point represents the mean ± S.D. of six independent experiments. B, the conditioned medium of N2a cells treated for 4 days with either 0.1 unit/ml heparinase I, heparinase III, or chondroitinase ABC or left untreated was analyzed by an ECM-degrading assay. The cell medium still contained a glycosaminoglycans-degrading activity. C, upper graph, 35S-labeled N2a-M cells were left untreated or incubated with either one of the heparinases (0.1 unit/ml, 3 h), and the cell supernatants were fractionated by gel filtration to detect released radiolabeled HS fragments. Both enzymes liberated radiolabeled HS. Lower graph, N2a-M cells were treated with either one of the heparinases or both and then incubated with 125I-labeled FGF to determine its binding to the cell surface. Both enzymes markedly reduced bFGF binding. D, upper gel, a dose-response curve of heparinase III treatment. ScN2a-M cells were treated for 4 days with increasing concentrations of heparinase III as indicated. Lower gel, time-dependent effect of heparinase III. ScN2a-M cells were treated with 0.1 unit/ml heparinase III for up to 4 days as indicated.

 

Cells—Mouse N2a-M and ScN2a-M neuroblastoma cells (35) stably express the MHM2-PrP chimera that reacts with the mAb 3F4 (36). In some cases, N2a-M cells that have been cured from prions by treatment with pentosan polysulfate were also added as control (37). These cells were maintained free of pentosan for at least 6 months before the experiment. Cells were grown at 37 °C in DMEM-16 (1 g of glucose/liter) supplemented with 10% fetal calf serum (FCS). Treatments with EDX and with GAG-degrading enzymes were performed on cells grown in 6-well dishes in DMEM-16/Opti-MEM (1:1) supplemented with 5% FCS. Cultures of bovine corneal endothelial cells were established from steer eyes as described previously (38). Stock cultures were maintained in DMEM-16 supplemented with 10% newborn calf serum and 5% FCS.

Antibodies—MAb 3F4 (36) binds to residues Met108 and Met111 (39) in chimeric MHM2-PrP but does not recognize the wild-type mouse PrP endogenous to N2a cells (40). This antibody was used at a dilution of 1:5000 for Western blot or at the dilution of 1:1000 for immunofluorescence experiments. Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA).

PrP Isoforms and PrP Analysis—The PrP isoforms were characterized and separated as described previously (9). PrPSc was defined as the PrP fraction resistant to proteolysis catalyzed by proteinase K (20 µg/ml, 37 °C, 30 min). The protease activity had been stopped by adding phenylmethylsulfonyl fluoride to 2 mM. SDS-PAGE and Western immunoblotting of the PrP isoforms were all carried out as described previously (41, 42). Cells were lysed in ice-cold "standard" lysis buffer (0.5% Triton X-100 (TX-100), 0.23% sodium deoxycholate, 150 mM NaCl, 10 mM Tris-Cl, pH 7.5, 10 mM EDTA), and the lysates were immediately centrifuged for 40 s at 14,000 rpm in a microcentrifuge. All of the biochemical analyses were performed on this postnuclear fraction. Protein concentrations in cell lysates were measured using the Bradford assay (Bio-Rad), and in each experiment the samples were normalized for their protein content prior to loading on SDS-PAGE gels. Densitometric analysis was performed using the TINA software (Raytest Isotopenmellgerilte GmbH, Straubenhardt, Germany).

Digestion of Membranes with Heparinase III—In vitro digestion was performed on total cell membranes prepared as follows: ~3 x 107 cells were resuspended in HEPES buffer (25 mM HEPES, 150 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, pH 7.4) and sonicated in a bath sonicator (3 x 15 s). The suspension was then spun in a microcentrifuge to remove debris (10,000 rpm, 3 min), and membranes were pelleted out of the supernatant by ultracentrifugation through a sucrose cushion (300 µlof 15% sucrose) in a Beckman Optima TL ultracentrifuge at 55,000 rpm (gav = 200,000 x g) for 1 h at 4 °C in a TLS-55 rotor. Membranes were incubated with 0.1 units/ml heparinase III for 2 h at 37 °C in either HEPES buffer containing 0.1% TX-100 or MES buffer (25 mM MES, 150 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, pH 5.7) containing 0.1% TX-100. Heparinase activity was stopped by the addition of proteinase K (20 µg/ml for 30 min at 37 °C).

ECM-degrading Assay—GAG-degrading activity was assayed by the release of radiolabeled products from 35S-labeled ECM plates produced by cultured corneal endothelial cells and prepared as described previously (38). 35S-Labeled ECM was incubated for 6 h with the same conditioned media as used in the relevant experiment. The radiolabeled material released from these ECM plates was then fractionated by gel filtration on Sepharose CL-6B columns as described previously (43).

Flotation of PrP—Detergent-insoluble complexes were analyzed on flotation gradients (44) as described previously (41). Confluent ScN2a-M cells growing in two 90-mm plates (~3 x 107 cells) were incubated with 450 µl of TNE (150 mM NaCl, 25 mM Tris-HCl, 5 mM EDTA, pH 7.5) supplemented with 1% TX-100 for 30 min. Lysates were then adjusted to 35% Nycodenz with ice-cold 70% Nycodenz prepared in TNE and loaded at the bottom of TLS-55 Beckman ultracentrifuge tubes. A 8–35% Nycodenz linear step gradient in TNE was overlaid above the lysate (200 µl each of 25, 22.5, 20, 18, 15, 12, and 8% Nycodenz). Tubes were spun at 55,000 rpm (gav = 200,000 x g) for 4 h at 4 °C in a TLS-55 rotor. Thirteen fractions of 180 µl each were collected from the top of the tube.

Immunofluorescence of PrPc—PrPC clustering and visualization on the surface of cells was performed as described previously (45). PrPC molecules were labeled with the mAb 3F4 on the cell surface of living and then clustered by adding a polyclonal second antibody (goat anti-mouse IgG) conjugated to fluorescein isothiocyanate. The cells were then fixed with 10% formalin prior to examination with a Zeiss Axiovert microscope equipped with epifluorescence.

Binding of 125I-labeled bFGF—Confluent N2a-M cultures growing in 24-well plates were treated with heparinases (0.1 unit/ml, 37 °C, 4 h). They were then rinsed twice with ice-cold binding medium (RPMI 1640 medium containing 0.1% bovine serum albumin) and incubated for 2 h on ice in binding medium containing 5 ng/ml 125I-labeled bFGF. The cells were further rinsed three times with cold binding medium and lysed with 300 µl of 0.1 N NaOH (1 h, room temperature). Both the cell lysates and supernatants (the combined three rinses) were counted in a {gamma}-counter to determine the "bound" and "unbound" values, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Lipophilic Xyloside EDX Reduces PrPSc in Prion-infected Cells—The general inhibitor of sulfation, chlorate, reduces PrPSc in scrapie-infected cells. To see whether this is attributable to its effect on sulfated GAGs, we used the membrane-permeant xyloside EDX, which specifically inhibits PG glycosylation (34). Prion-infected murine neuroblastoma ScN2a-M cells (Fig. 1A) and uninfected N2a-M cells (Fig. 1B) were treated with EDX (10–50 µM) for 4 days in DMEM/Opti-MEM medium supplemented with 5% FCS (PrPSc is very stable in cells, so that several days are required to eliminate preexisting PrPSc). There were no detectable cytopathic or cytostatic effects. To specifically detect the protease-resistant PrPSc, half of each ScN2a-M lysate was digested with proteinase K (20 µg/ml, 30 min, 37 °C) prior to electrophoresis (Fig. 1A, +PK). The three protease-resistant PrP bands are the glycoforms that are characteristic of mouse PrPSc. The other half of the ScN2a-M lysates were analyzed without proteolysis (Fig. 1A, –PK) for "total PrP." The Western blots were developed with the mAb 3F4. EDX caused a profound, dose-dependent decrease in protease-resistant PrP27–30. However, the amount of the cellular isoform, PrPC, was unaltered in uninfected N2a-M cells treated with EDX (Fig. 1B). Interestingly, EDX also reduced the high molecular mass PrP species (33–35 kDa) in the ScN2a samples not treated with proteinase K (Fig. 1A, –PK). In ScN2a cells, the high molecular mass PrP bands include both PrPC and full-length PrPSc molecules. Two high molecular mass PrPSc populations have been described in ScN2a cells. First, there is a minority of protease-resistant PrPSc molecules that have not yet been trimmed by cellular proteases. Upon digestion with PK, these species are converted to PrP27–30 (12). Second, 31–35-kDa PrP also comprises protease-sensitive PrPSc species, which are fully digested by the stringent proteinase K treatment performed here (8, 46).



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  		FIG. 1.
EDX reduces PrPSc but not PrPC in neuroblastoma cells (Western blots). ScN2a-M (panel A) and N2a-M (panel B) cells growing in 6-well plates were incubated either with EDX (25 or 50 µM) or left untreated for 4 days in DMEM/Opti-MEM supplemented with 5% FCS. Cells were then lysed, and the postnuclear fraction (PNS) (normalized for protein content) were analyzed by Western blots using the mAb 3F4. In panel A, 50% of the sample was digested with proteinase K (+PK, 20 µg/ml) at 37 °C for 30 min prior to electrophoresis to eliminate PrPC. Protease-resistant PrPSc content after EDX treatment was analyzed by densitometry and plotted. Each data point represents the mean ± S.D. of four independent experiments.

 

The decrease in PrPSc in EDX-treated cells was not due to the aglycone moiety (estradiol), since {beta}-estradiol at sub-toxic concentrations did not reduce PrPSc in ScN2a-M cells (not shown). These results support the contention that cellular proteoglycans are involved, directly or indirectly, in the metabolism of PrPSc (22, 28).

Heparinase III, but Not Heparinase I nor Chondroitinase ABC, Reduces PrPSc in Cells—At the concentration used above, EDX affects both HS and CS chains of proteoglycans (34). To further determine which type of GAG is involved in the biogenesis of prions, we used bacterial heparinases I and III as well as chondroitinase ABC to digest specific cellular GAGs. None of the enzymatic treatments described below caused detectable cytopathic or cytostatic effects.

We first used chondroitinase ABC to digest CS and dermatan sulfate in ScN2a-M cells (Fig. 2A). Cells were incubated for 4 days with 0–0.5 units/ml chondroitinase ABC. They were then lysed, and their total protein concentration was normalized before analyzing total PrP (Fig. 2A, panel 1, –PK) or protease-resistant PrPSc (Fig. 2A, panel 1, +PK) by Western blotting. This treatment had no effect on either total PrP or on protease-resistant PrPSc, suggesting that neither CS nor dermatan sulfate plays critical roles in the biogenesis of PrPSc in ScN2a-M cells. To confirm that these enzymes were active throughout the experiment, we measured their residual activity in the cell medium at the end of the experiment by ECM-degrading assay (see "Experimental Procedures") (Fig. 2B, graph). The chondroitinase was still active after 4-day incubation as confirmed by the production of low molecular weight sulfate-labeled GAG degradation fragments (fractions 25–35).

To see whether HS chains might be involved in the metabolism of PrPSc, cells were treated with heparinase I or heparinase III. Neither enzyme altered the level of PrPC in uninfected N2a-M cells (Fig. 2A, panels 2 and 3). In contrast, heparinase III almost completely eliminated protease-resistant PrPSc from ScN2a-M cells (Fig. 2B, panel 2, +PK), whereas no change was observed with heparinase I. That both enzymes were still active at the end of the incubation was again confirmed by using an ECM-degrading assay (Fig. 2B) as explained above for the chondroitinase.

Because the two heparinase yielded such contrasting results, we decided to compare the amount of cellular HS that they released from N2a cells. N2a-M cells were radiolabeled for 2 days with Na2 35 SO4 (40 µCi/ml). After thorough rinses, the cells were treated for 3 h with either one of the two heparinases (0.1 unit/ml) in DMEM/Opti-MEM containing 5% FCS (in a CO2 incubator). The incubation medium was then cleared from cells and debris by centrifugation (5 min, 450 x g) and subjected to gel filtration on Sepharose CL-6B columns (Fig. 2C, upper panel). Both heparinases released radiolabeled, low molecular weight degradation fragments from the cells, indicating that they both digested similar amount of cellular HS chains. This conclusion was confirmed independently by a bFGF-binding assay. HS is a low affinity receptor for this growth factor (47–49). After digestion with heparinases (60 min, 37 °C, serum-free RPMI 1640 medium), N2a-M cells were exposed to radioiodinated bFGF (on ice, 1 h) and thoroughly rinsed and the amount of bound 125I was counted (Fig. 2C, lower panel). Both heparinases I and III reduced bFGF binding by approximately the same extent. Taken together, these results show that both heparinase I and heparinase III released sulfated fragments from the cell surface. Their different cleavage specificities may explain their contrasting action on PrPSc (see Fig. 6 and "Discussion").



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  		FIG. 2.
Heparinase III but not other GAG-degrading enzymes reduces PrPSc in ScN2a-M cells (Western blots). Panel A, N2a-M and ScN2a-M cells growing in 6-well plates were incubated for 4 days with chondroitinase ABC (panel 1) or with heparinase I or III (panels 2 and 3), and the level of PrP was analyzed by Western blots developed with mAb 3F4. Samples were normalized for their protein content. In some cases (+PK), the lysates were digested with proteinase K (20 µg/ml, 37 °C, 30 min) prior to electrophoresis to eliminate PrPC. Panel 1, ScN2a-M cells were either left untreated or were treated with increasing concentrations of chondroitinase ABC (0.01–0.5 units/ml). PrPSc level was unaltered. Panels 2 and 3, ScN2a-M cells (panel 2) and N2a-M cells (panel 3) were incubated with 0.1 unit/ml heparinase I, heparinase III, or a mixture of both at a final concentration of 0.1 unit/ml (i.e. 0.05 units/ml of each enzyme) as indicated. PrPSc (panel 2, +PK) but not PrPC (panel 3) was reduced specifically by heparinase III. Protease-resistant PrPSc content after heparinase I or heparinase III treatment was analyzed by densitometry and plotted. Each data point represents the mean ± S.D. of six independent experiments. B, the conditioned medium of N2a cells treated for 4 days with either 0.1 unit/ml heparinase I, heparinase III, or chondroitinase ABC or left untreated was analyzed by an ECM-degrading assay. The cell medium still contained a glycosaminoglycans-degrading activity. C, upper graph, 35S-labeled N2a-M cells were left untreated or incubated with either one of the heparinases (0.1 unit/ml, 3 h), and the cell supernatants were fractionated by gel filtration to detect released radiolabeled HS fragments. Both enzymes liberated radiolabeled HS. Lower graph, N2a-M cells were treated with either one of the heparinases or both and then incubated with 125I-labeled FGF to determine its binding to the cell surface. Both enzymes markedly reduced bFGF binding. D, upper gel, a dose-response curve of heparinase III treatment. ScN2a-M cells were treated for 4 days with increasing concentrations of heparinase III as indicated. Lower gel, time-dependent effect of heparinase III. ScN2a-M cells were treated with 0.1 unit/ml heparinase III for up to 4 days as indicated.

 


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  		FIG. 6.
A model for the opposing influence of heparinases I and III on PrPSc. HS chains are composed of a linker tetrasaccharide (dotted bars) followed by a linear chain composed of iduronic/glucuronic acid and glucosamine disaccharide repeats. Extensive enzymatic processing results in the formation of clustered structures consisting of hypersulfated regions (S) separated by non-sulfated regions (lines) with partially sulfated segments (diagonal lines) often flanking the S regions (74). Because of their disparate specificities, digestion with heparinase I (arrow) and heparinase III (arrows) produces completely different GAG fragments attached to the core protein of cell-surface HSPGs. A, by cleaving preferentially hypersulfated regions, heparinase I generates relatively long GAG fragments attached to the core protein. B, in the case of undersulfated chains, heparinase I may even leave the whole GAG chain attached to the core protein. In contrast, by cleaving non-sulfated or undersulfated regions, heparinase III leaves very short GAG regions and in some cases only the tetrasaccharide linker remains attached (74).

 

To examine the dose response of the anti-prion effect of heparinase III, we repeated the experiment described above with increasing concentrations of heparinase III ranging between 0 and 0.5 units/ml (Fig. 2D, upper panel). The minimal heparinase III concentration that reduced PrPSc was 0.01 unit/ml. In all of the subsequent experiments, we used 0.1 unit/ml as the standard heparinase III concentration. We next determined the time course of heparinase III-induced PrPSc reduction in ScN2a-M cells (Fig. 2D, lower panel). For this purpose, the enzyme (at 0.1 unit/ml) was added to separate dishes at days 1, 2, 3, 4, or 4.5 and the cells were harvested on day 5. The anti-prion effect of heparinase III could be observed between 2 and 3 days of inhibition. Taken together, the results observed with the GAG-degrading enzymes strongly suggest that HS, but not CS or dermatan sulfate, plays a role in the biogenesis of PrPSc in ScN2a-M cells.

Heparinase III Does Not Change the Cellular Location of PrPC—One way by which heparinase III could reduce the level of PrPSc in cells is by decreasing the pool of the substrate PrPC that is available for conversion. Although the overall level of PrPC was not altered by the enzymatic treatment (Fig. 2A and see below), the pool of PrPC available for conversion could be decreased by changing its subcellular localization. Indeed, in some cases, soluble GAG analogs and polyanions increase PrP internalization (30) and even completely remove PrP from the cell surface (31).

To investigate whether heparinases change the distribution of PrPC in cells, we applied immunofluorescence microscopy (Fig. 3A). N2a-M cells grown on plastic slides were either left untreated or were treated with heparinase I or heparinase III (0.1 unit/ml) for either 1.5 or 12 h as indicated or with suramin (200 µg/ml, 12 h) (23, 31). The cell surface PrPC was then examined by 3F4 mAb immunofluorescence on non-permeabilized cells. There was no major difference in the level of PrP on the cell surface after treatment with each of the heparinases (Fig. 3A, panels a and c–f). In contrast, suramin completely abolished the cell-surface PrP labeling (Fig. 3A, panel b), in agreement with previous results (31).



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  		FIG. 3.
PrPC subcellular distribution is unaffected by heparinase treatment. A, Immunofluorescence. N2a-M cells growing on plastic slides were untreated (a) or incubated with heparinase I (panels c and d) or III (panels e and f) (0.1 unit/ml) for either 1.5 (panels c and e) or 12 h (panels d and f). Cells were also treated with suramin (200 µg/ml, panel b) for 12 h. Live cells were then labeled with mAb 3F4, and their cell surface PrPC was detected by fluorescein isothiocyanate-conjugated goat-anti-mouse antibody. There was no change in the localization of the PrPC. B, cell surface patching of PrPC before (panels a and c) and after treatment of cells with 0.1 unit/ml heparinase III for 12 h (panels b and d) (N2a-M cells (panels a and b); cured N2a-M cells (panels c and d)). The patching pattern of PrPC was not altered following the enzymatic treatment. C, flotation assay. N2a-M cells were treated with heparinase III for the indicated times. TX-100 lysates were then subjected to standard Nycodenz flotation gradients that were fractionated from the top. PrP in the fractions was analyzed by Western blots developed with mAb 3F4. There was no detectable change in the flotation pattern of PrPC.

 

Although the compartments where PrPSc is formed have not been entirely identified, they seem to involve post-endoplasmic reticulum/Golgi (12) rafts (35, 41, 42, 50). One way to characterize the association of PrP with rafts is by visualizing the level of antibody-induced patching of PrP on the cell surface (51). This patching is caused by the cross-linking of raft PrPC by a divalent secondary polyclonal antibody (52). Heparinase III did not alter the patching of PrP on the surface of either N2a-M (Fig. 3B, panel b) or cured ScN2a-M (Fig. 3B, panel d), suggesting that this enzyme does not change the association of PrPC with rafts. This conclusion was supported by the results of a diagnostic flotation assay (Fig. 3C) (44). Confluent N2a-M cells grown in 90-mm dishes were incubated with heparinase III (0.1 units/ml) for either 2 h or 3 days or left untreated. The cells were then lysed for 30 min in ice-cold TX-100 and subjected to a standard flotation in Nycodenz. As shown in Fig. 3C, heparinase III did not affect the flotation pattern of PrPC.

In Vitro Digestion with Heparinase III Does Not Reduce the Protease Resistance of PrPSc—The structural features that lead to the protease resistance of PrPSc are not entirely known. Whereas PrPSc is clearly conformationally different from PrPC, it is possible that accessory molecules also participate in the formation and/or the maintenance of protease-resistant PrPSc. Because PrP possesses several heparin-binding sites (27), we wondered whether HS might stabilize the protease-resistant conformation of PrPSc. Such a role has been recently proposed on the basis of infectivity reconstitution experiments (29). To investigate this possibility, we incubated a total cell membranes preparation of ScN2a-M cells with heparinase III. Because the PrP isoforms enter acidic compartments in the course of their trafficking (12, 53), we performed the enzymatic hydrolysis at both acidic (pH 5.7) and neutral (pH 7.4) conditions (we confirmed that heparinase III is inactive under pH 5.5 as indicated by the manufacturer) (data not shown). Membranes were prepared from ScN2a-M cells by sonication in a bath sonicator followed by incubation with or without heparinase III (0.1 unit/ml, 37 °C, 2 h) at the indicated pH in the presence of 0.1% TX-100. The membranes were then incubated with or without proteinase K (20 µg/ml, 37 °C, 30 min) prior to Western analysis (Fig. 4). That there was no change in the level of protease-resistant PrPSc in heparinase III-treated membranes argues against the structural involvement of heparinase-sensitive HS in stabilizing PrPSc. This result suggests that the decrease in PrPSc in heparinase III-treated cells is not the result of direct action of this enzyme on preformed PrPSc. Rather, the effect appears to occur during the biosynthesis of PrPSc.



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  		FIG. 4.
In vitro digestion of membrane-bound PrPSc by heparinase III. Membranes prepared from ScN2a-M cells were resuspended in 0.1% TX-100 at the pH indicated. Heparinase III (0.1 unit/ml) was added to the lanes indicated (+), and the samples were incubated at 37 °C for 2 h. 50% of the sample was digested with proteinase K (+PK, 20 µg/ml) at 37 °C for 30 min. The heparinase treatments did not alter the PrPSc content of the membranes.

 

Soluble HS and CS Partially Reverse the Chlorate-induced Decrease in PrPSc—Sodium chlorate, an inhibitor of ATP-sulfurylase and hence of the production of phosphoadenosine phosphosulfate (the active sulfate donor for sulfotransferases) (54), reduces the sulfation of proteins and carbohydrates in intact cells without inhibiting cell growth or protein synthesis (54, 55). Treatment with chlorate decreases the amount of protease-resistant PrPSc in ScN2a cells (22). Assuming that the anti-PrPSc effect of chlorate is caused by the loss of cellular sulfated GAGs, we asked whether soluble HS or CS preparations would restore PrPSc in chlorate-treated cells. We thus incubated cells for 4 days with 30 mM chlorate alone or chlorate supplemented with 50 µg/ml of either porcine mucosa HS or bovine trachea CS (Fig. 5). Interestingly, both GAGs partially reversed the chlorate-induced reduction of PrPSc (compare lanes 3 and 4 in both panels of Fig. 5). This indicates that the role of cellular HS in assisting prions can be also played, at least in part, by soluble GAGs. This result is in line with the demonstration of Wong et al. (28) that soluble HS-like molecules (and to a lesser extent soluble CS) can promote the formation of PrP-res in a cell-free reaction. That CS could also restore PrPSc in these cells is surprising in view of the inability of chondroitinase ABC to reduce PrPSc. This apparent paradox is discussed below. It is interesting that high molecular weight CS increased PrPSc even with cells not treated with chlorate (Fig. 5B). This result confirms previous observations by Gabizon et al., (22) and is also in line with early data from Caughey and Raymond (25) showing that high molecular weight CS is much less inhibitory (by 3 orders of magnitude), if at all, than low molecular weight sulfated glycans.



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  		FIG. 5.
Soluble HS and CS partially rescue the decrease in PrPSc caused by sodium chlorate. ScN2a-M cells were left untreated or treated for 4 days with sodium chlorate (30 mM), supplemented in some cases with porcine mucosa HS (50 µg/ml) (A) or bovine trachea CS (50 µg/ml) (B). Proteinase K-digested (20 µg/ml at 37 °C for 30 min) cell lysates normalized for protein content were analyzed by Western blot (3F4). Soluble HS and soluble CS partially restored the decrease in PrPSc caused by sodium chlorate. Protease-resistant PrPSc content after porcine mucosa HS (A) or bovine trachea CS (B) chlorate treatment or a combination of both treatments was analyzed by densitometry and plotted. Each data point represents the mean ± S.D. of three independent experiments.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Both the cell-permeant xyloside EDX and heparinase III strongly reduced the amount of protease-resistant PrPSc in scrapie-infected cells. In contrast, neither the amount nor the subcellular distribution of PrPC was affected. Heparinase I and chondroitinase ABC had no effect on either of the PrP isoforms. These results suggest that one (or more) heparinase III-sensitive and heparinase I-insensitive cellular HSPGs is involved in the formation of PrPSc. This conclusion is supported by the finding that soluble exogenous GAGs partially compensate for the loss of sulfation in chlorate-treated cells. Possible pro-PrPSc mechanisms of HS are discussed below.

Prion-promoting Cellular HS or Inhibitory HS Fragments?— Both EDX and the heparinases affect the treated cells in two different ways. First, they create a pool of potentially anti-prion, soluble GAGs (or GAG fragments) in the medium. Whether these soluble HS analogs can interfere with the formation of PrPSc remains to be determined. Second, they reduce the amount and the length of cell-associated GAGs. The relative influence of these two processes on PrPSc remains to be seen. However, the fact that chlorate, which acts on cell-associated GAGs, reduced PrPSc strongly argues in favor of the involvement of cell-associated GAGs in the metabolism of PrPSc. This conclusion is further supported by the finding that some of the chlorate-induced PrPSc loss can be restored by exogenous added soluble GAGs (Fig. 5). In addition, we have recently found that heparinase III-sensitive cell-surface HS serves as endocytosis receptors for prion rods.2 This finding indicates that cell-surface HS also play a major role in PrPSc cell binding. A supporting role of HS GAGs in the formation of PrPSc is also suggested by the results of Wong et al. (28) who showed that HS and its low molecular weight analog pentosan polysulfate promote the production of PrP-res in a cell-free system.

Possible Mechanisms for the Intervention of HS in the Metabolism of PrPSc—In scrapie-infected cells, PrPSc seems to be formed by the post-translational refolding of PrPC. Although the exact subcellular location of this process is not known, rafts (35, 41, 42, 50) of the plasma membrane or of the endocytic pathways (11, 12) have been invoked. The formation of PrPSc seems to involve a direct molecular interaction between the "substrate" PrPC and the "seed" or "template" PrPSc (56). One way that GAGs could participate in this productive interaction is by helping to bring PrPC and PrPSc within interaction range (28). A similar function of HS has been demonstrated for bFGF receptor binding and activation (47–49). In the latter case, well defined HS sequences (57–60) help approximate two (or more) high affinity receptor-bound bFGF molecules, hence promoting dimerization of the receptors and subsequent intracellular signaling. Similar to bFGF, the normal isoform PrPC binds heparin (22, 26) and its HS-binding sites have recently been identified (27) as (i) residues 23–52 and residues 53–93 (including the octarepeat sequences) in the N-terminal region and (ii) residues 110–128. In contrast to PrPC, the heparin-binding sites within the prion conformer, PrPSc, have not yet been elucidated. However, the fact that a truncated PrP that lacks the N-terminal region ({Delta}23–89) is able to form PrPSc with high efficiency (61, 62) and the finding that prion rods that also lack the N-terminal region can bind cell surface HS2 shows that the productive binding of PrPSc to HS takes place primarily within the PrP27–30 region. Whether the binding of both PrPC and PrPSc to HS indeed helps bring these molecules to a suitable physical proximity remains to be determined.

HS could also take part in PrPSc metabolism in additional ways. First, specific HS sequences could directly promote the production of PrPSc, perhaps by destabilizing the PrPSc "seed" (28), by helping to stabilize intermediate metastable conformations (PrP*) (63), or by lowering the energetic barrier proposed to hamper the refolding of PrPC. Such an effect has been noted for other amyloidogenic proteins. For instance, HS promotes the formation of amyloid fibers by A{beta} peptides (64, 65) and {alpha}-synuclein (66, 67). Second, instead of being just enzymatic facilitators, HS or fragments thereof could remain complexed with PrPSc as structural components of prions. Shaked et al. (29) have recently shown that a heparinase-sensitive brain fraction can restore the infectivity of Me2 SO-solubilized PrPSc. However, that digestion in vitro with heparinase III did not abolish the protease resistance of PrPSc (Fig. 4) argues against the structural involvement of heparinase III-sensitive HS in stabilizing PrPSc conformation in our system. Third, HS could recruit other heparin-binding proteins that could help in the formation of PrPSc (such as the proposed protein X) (13). Other possible pro-prion mechanisms include possible influence of HS on cellular trafficking including PrP endocytosis (30).

Why do large GAGs increase PrPSc, whereas small analogs thereof consistently and potently decrease PrPSc? One possibility is that small analogs bind separately to substrate PrPC and to template PrPSc so that these PrP isoforms are separated as a result, thus preventing the proteinaceous reaction leading to PrPSc. In contrast, higher molecular weight GAGs could bind both PrP isoforms, thereby recapitulating the putative action of cellular HS on the formation of PrPSc.

The finding that exogenous CS efficiently reconstituted PrPSc is surprising in view of the fact that digesting cells with chondroitinase ABC did not reduce this prion isoform. At least two mechanisms can be envisaged to explain this apparent contradiction. First, it is possible that both HS and CS can serve as pro-prion cofactors with equal efficacy, but because CS are a small minority in ScN2a-M cells, their enzymatic removal is undetectable in terms of PrPSc production. Another possibility is that the affinity of CS for PrP is smaller than that of HS. Additional experiments will be needed to determine the relative pro-prion potencies of these two GAG types.

In contrast to its action on PrPSc, heparinase III did not modify any of the properties of PrPC that were tested here. Thus, neither the overall amount of PrPC nor its attachment to membrane rafts or its subcellular distribution was disturbed. Hence, the reduction of PrPSc by this enzyme is probably not caused by changes in the precursor PrPC.

Differences between Heparinase I and III—What is the structural basis of the disparate effects of heparinase I and heparinase III on PrPSc? These two heparinases have markedly different substrate specificities. Whereas heparinase I cleaves preferentially within highly sulfated GAG regions, heparinase III cleavage takes place within regions that are either non-sulfated or undersulfated (68). These cleavage specificities suggest the following model for the contrasting actions of heparinases on PrPSc (Fig. 6). By cleaving HS chains only within hypersulfated regions (S), heparinase I leaves longer HS stubs on the protein core. These stubs are presumably sufficient for PrPSc production (Fig. 6, heparinase I, A). Undersulfated GAGs may lack heparinase I cleavage sites altogether (Fig. 6, heparinase I, B). In opposition, cleavage by heparinase III within undersulfated regions leaves shorter stubs as short as the tetrasaccharide linker (69) that are presumably inadequate for the formation of PrPSc (Fig. 6, heparinase III). Altogether, these results indicate that undersulfated regions of HS chains on proteoglycans are needed for PrPSc metabolism. Whether highly sulfated regions play any role in PrP metabolism remains to be seen.

Which HSPGs involved in PrPSc formation remains to be determined. Because both PrPC and PrPSc are largely associated with rafts (35, 41, 42, 50) and since the formation of PrPSc seems to occur within these microdomains, glypicans, which are raft-associated glycophosphatidylinositol-anchored PGs, are of course attractive candidates for such a role. Six different glypicans are now known in mammals (70). However, because GAG chains are quite long (50–150 disaccharide units) (71, 72), non-glycophosphatidylinositol-anchored cell-surface HSPGs (syndecans) found in neighboring non-raft regions of the membrane could also play such a role. Incidentally, it was recently shown that syndecan-4 can enter rafts after being engaged by ligands or by cross-linking antibody (73). Whether a specific cell-associated HSPG is involved in the genesis of prions or whether this role is played by generic HS chains present on many protein cores also remains to be established.


   FOOTNOTES
 
* This work was supported by generous grants from the Israeli Center for the Study of Emerging Diseases (to I. V. and A. T.) and Grants CA46462 and GM33063 from the National Institutes of Health (to J. D. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 972-2-6757215; Fax: 972-2-6757086; E-mail: taraboul{at}cc.huji.ac.il.

1 The abbreviations used are: PrPC, the normal PrP isoform; PrPSc, the prion PrP isoform; bFGF, basic fibroblast growth factor; CS, chondroitin sulfate; EDX, estradiol {beta}-D-xyloside; GAGs, glycosaminoglycans; HS, heparan sulfate; HSPGs, heparan sulfate proteoglycans; PGs, proteoglycans; PK, proteinase K; TX-100, Triton X-100; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; MES, 4-morpholineethanesulfonic acid; ECM, extracellular matrix; PrP, the prion protein. Back

2 L. Horonchik and A. Taraboulos, manuscript in preparation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gibbs, C. J., Jr., and Gajdusek, D. C. (1969) Science 165, 1023–1025[Abstract/Free Full Text]
  2. Wilesmith, J. W., Wells, G. A., Cranwell, M. P., and Ryan, J. B. (1988) Vet. Rec. 123, 638–644[Abstract]
  3. Prusiner, S. B. (1982) Science 216, 136–144[Abstract/Free Full Text]
  4. Pan, K. M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E., et al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10962–10966[Abstract/Free Full Text]
  5. Caughey, B., and Raymond, G. J. (1991) J. Biol. Chem. 266, 18217–18223[Abstract/Free Full Text]
  6. Prusiner, S. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13363–13383[Abstract/Free Full Text]
  7. Come, J. H., Fraser, P. E., and Lansbury, P. T., Jr. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5959–5963[Abstract/Free Full Text]
  8. Tzaban, S., Friedlander, G., Schonberger, O., Horonchik, L., Yedidia, Y., Shaked, G., Gabizon, R., and Taraboulos, A. (2002) Biochemistry 41, 12868–12875[CrossRef][Medline] [Order article via Infotrieve]
  9. Meyer, R. K., McKinley, M. P., Bowman, K. A., Braunfeld, M. B., Barry, R. A., and Prusiner, S. B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2310–2314[Abstract/Free Full Text]
  10. Borchelt, D. R., Scott, M., Taraboulos, A., Stahl, N., and Prusiner, S. B. (1990) J. Cell Biol. 110, 743–752[Abstract/Free Full Text]
  11. Borchelt, D. R., Taraboulos, A., and Prusiner, S. B. (1992) J. Biol. Chem. 267, 16188–16199[Abstract/Free Full Text]
  12. Taraboulos, A., Raeber, A. J., Borchelt, D. R., Serban, D., and Prusiner, S. B. (1992) Mol. Biol. Cell. 3, 851–863[Abstract]
  13. Telling, G. C., Scott, M., Mastrianni, J., Gabizon, R., Torchia, M., Cohen, F. E., DeArmond, S. J., and Prusiner, S. B. (1995) Cell 83, 79–90[CrossRef][Medline] [Order article via Infotrieve]
  14. Rieger, R., Edenhofer, F., Lasmezas, C. I., and Weiss, S. (1997) Nat. Med. 3, 1383–1388[CrossRef][Medline] [Order article via Infotrieve]
  15. Graner, E., Mercadante, A. F., Zanata, S. M., Forlenza, O. V., Cabral, A. L., Veiga, S. S., Juliano, M. A., Roesler, R., Walz, R., Minetti, A., Izquierdo, I., Martins, V. R., and Brentani, R. R. (2000) Brain Res. Mol. Brain Res. 76, 85–92[Medline] [Order article via Infotrieve]
  16. Gauczynski, S., Peyrin, J. M., Haik, S., Leucht, C., Hundt, C., Rieger, R., Krasemann, S., Deslys, J. P., Dormont, D., Lasmezas, C. I., and Weiss, S. (2001) EMBO J. 20, 5863–5875[CrossRef][Medline] [Order article via Infotrieve]
  17. Hundt, C., Peyrin, J. M., Haik, S., Gauczynski, S., Leucht, C., Rieger, R., Riley, M. L., Deslys, J. P., Dormont, D., Lasmezas, C. I., and Weiss, S. (2001) EMBO J. 20, 5876–5886[CrossRef][Medline] [Order article via Infotrieve]
  18. Zanata, S. M., Lopes, M. H., Mercadante, A. F., Hajj, G. N., Chiarini, L. B., Nomizo, R., Freitas, A. R., Cabral, A. L., Lee, K. S., Juliano, M. A., de Oliveira, E., Jachieri, S. G., Burlingame, A., Huang, L., Linden, R., Brentani, R. R., and Martins, V. R. (2002) EMBO J. 21, 3307–3316[CrossRef][Medline] [Order article via Infotrieve]
  19. Diaz-Nido, J., Wandosell, F., and Avila, J. (2002) Peptides 23, 1323–1332[CrossRef][Medline] [Order article via Infotrieve]
  20. Snow, A. D., Kisilevsky, R., Willmer, J., Prusiner, S. B., and DeArmond, S. J. (1989) Acta Neuropathol. 77, 337–342[CrossRef][Medline] [Order article via Infotrieve]
  21. McBride, P. A., Wilson, M. I., Eikelenboom, P., Tunstall, A., and Bruce, M. E. (1998) Exp. Neurol. 149, 447–454[CrossRef][Medline] [Order article via Infotrieve]
  22. Gabizon, R., Meiner, Z., Halimi, M., and Ben-Sasson, S. A. (1993) J. Cell. Physiol. 157, 319–325[CrossRef][Medline] [Order article via Infotrieve]
  23. Ladogana, A., Casaccia, P., Ingrosso, L., Cibati, M., Salvatore, M., Xi, Y. G., Masullo, C., and Pocchiari, M. (1992) J. Gen. Virol. 73, 661–665[Abstract/Free Full Text]
  24. Ehlers, B., and Diringer, H. (1984) J. Gen. Virol. 65, 1325–1330[Abstract/Free Full Text]
  25. Caughey, B., and Raymond, G. J. (1993) J. Virol. 67, 643–650[Abstract/Free Full Text]
  26. Caughey, B., Brown, K., Raymond, G. J., Katzenstein, G. E., and Thresher, W. (1994) J. Virol. 68, 2135–2141[Abstract/Free Full Text]
  27. Warner, R. G., Hundt, C., Weiss, S., and Turnbull, J. E. (2002) J. Biol. Chem. 277, 18421–18430[Abstract/Free Full Text]
  28. Wong, C., Xiong, L. W., Horiuchi, M., Raymond, L., Wehrly, K., Chesebro, B., and Caughey, B. (2001) EMBO J. 20, 377–386[CrossRef][Medline] [Order article via Infotrieve]
  29. Shaked, G. M., Meiner, Z., Avraham, I., Taraboulos, A., and Gabizon, R. (2001) J. Biol. Chem. 276, 14324–14328[Abstract/Free Full Text]
  30. Shyng, S. L., Lehmann, S., Moulder, K. L., and Harris, D. A. (1995) J. Biol. Chem. 270, 30221–30229[Abstract/Free Full Text]
  31. Gilch, S., Winklhofer, K. F., Groschup, M. H., Nunziante, M., Lucassen, R., Spielhaupter, C., Muranyi, W., Riesner, D., Tatzelt, J., and Schatzl, H. M. (2001) EMBO J. 20, 3957–3966[CrossRef][Medline] [Order article via Infotrieve]
  32. Schwartz, N. B., Galligani, L., Ho, P. L., and Dorfman, A. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4047–4051[Abstract/Free Full Text]
  33. Fritz, T. A., Lugemwa, F. N., Sarkar, A. K., and Esko, J. D. (1994) J. Biol. Chem. 269, 300–307[Abstract/Free Full Text]
  34. Lugemwa, F. N., and Esko, J. D. (1991) J. Biol. Chem. 266, 6674–6677[Abstract/Free Full Text]
  35. Naslavsky, N., Stein, R., Yanai, A., Friedlander, G., and Taraboulos, A. (1997) J. Biol. Chem. 272, 6324–6331[Abstract/Free Full Text]
  36. Kascsak, R. J., Rubenstein, R., Merz, P. A., Tonna-DeMasi, M., Fersko, R., Carp, R. I., Wisniewski, H. M., and Diringer, H. (1987) J. Virol. 61, 3688–3693[Abstract/Free Full Text]
  37. Birkett, C. R., Hennion, R. M., Bembridge, D. A., Clarke, M. C., Chree, A., Bruce, M. E., and Bostock, C. J. (2001) EMBO J. 20, 3351–3358[CrossRef][Medline] [Order article via Infotrieve]
  38. Vlodavsky, I. (1999) in Current Protocols in Cell Biology (Bonifacino, J. S., Dasso, M., Hartford, J. B., Lippincott-Schwartz, J., and Yamada, K. M., eds) Vol. 1, pp. 10.14.11–10.14.14, John Wiley & Sons, Inc., New York
  39. Rogers, M., Serban, D., Gyuris, T., Scott, M., Torchia, T., and Prusiner, S. B. (1991) J. Immunol. 147, 3568–3574[Abstract]
  40. Scott, M. R., Kohler, R., Foster, D., and Prusiner, S. B. (1992) Protein Sci. 1, 986–997[Abstract]
  41. Naslavsky, N., Shmeeda, H., Friedlander, G., Yanai, A., Futerman, A. H., Barenholz, Y., and Taraboulos, A. (1999) J. Biol. Chem. 274, 20763–20771[Abstract/Free Full Text]
  42. Taraboulos, A., Scott, M., Semenov, A., Avrahami, D., Laszlo, L., Prusiner, S. B., and Avraham, D. (1995) J. Cell Biol. 129, 121–132[Abstract/Free Full Text]
  43. Vlodavsky, I., Fuks, Z., Bar-Ner, M., Ariav, Y., and Schirrmacher, V. (1983) Cancer Res. 43, 2704–2711[Abstract/Free Full Text]
  44. Brown, D. A., and Rose, J. K. (1992) Cell 68, 533–544[CrossRef][Medline] [Order article via Infotrieve]
  45. Yanai, A., Meiner, Z., Gahali, I., Gabizon, R., and Taraboulos, A. (1999) FEBS Lett. 460, 11–16[CrossRef][Medline] [Order article via Infotrieve]
  46. Safar, J., Wille, H., Itri, V., Groth, D., Serban, H., Torchia, M., Cohen, F. E., and Prusiner, S. B. (1998) Nat. Med. 4, 1157–1165[CrossRef][Medline] [Order article via Infotrieve]
  47. Rapraeger, A. C., Krufka, A., and Olwin, B. B. (1991) Science 252, 1705–1708[Abstract/Free Full Text]
  48. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991) Cell 64, 841–848[CrossRef][Medline] [Order article via Infotrieve]
  49. Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E., and Leder, P. (1992) Mol. Cell. Biol. 12, 240–247[Abstract/Free Full Text]
  50. Vey, M., Pilkuhn, S., Wille, H., Nixon, R., DeArmond, S. J., Smart, E. J., Anderson, R. G., Taraboulos, A., and Prusiner, S. B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14945–14949[Abstract/Free Full Text]
  51. Harder, T., Scheiffele, P., Verkade, P., and Simons, K. (1998) J. Cell Biol. 141, 929–942[Abstract/Free Full Text]
  52. Mayor, S., Rothberg, K. G., and Maxfield, F. R. (1994) Science 264, 1948–1951[Abstract/Free Full Text]
  53. McKinley, M. P., Taraboulos, A., Kenaga, L., Serban, D., Stieber, A., DeArmond, S. J., Prusiner, S. B., and Gonatas, N. (1991) Lab. Invest. 65, 622–630[Medline] [Order article via Infotrieve]
  54. Baeuerle, P. A., and Huttner, W. B. (1986) Biochem. Biophys. Res. Commun. 141, 870–877[CrossRef][Medline] [Order article via Infotrieve]
  55. Keller, K. M., Brauer, P. R., and Keller, J. M. (1989) Biochemistry 28, 8100–8107[CrossRef][Medline] [Order article via Infotrieve]
  56. Prusiner, 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., and DeArmond, S. J. (1990) Cell 63, 673–686[CrossRef][Medline] [Order article via Infotrieve]
  57. Turnbull, J. E., Fernig, D. G., Ke, Y., Wilkinson, M. C., and Gallagher, J. T. (1992) J. Biol. Chem. 267, 10337–10341[Abstract/Free Full Text]
  58. Maccarana, M., Casu, B., and Lindahl, U. (1993) J. Biol. Chem. 268, 23898–23905[Abstract/Free Full Text]
  59. Ishai-Michaeli, R., Svahn, C. M., Weber, M., Chajek-Shaul, T., Korner, G., Ekre, H. P., and Vlodavsky, I. (1992) Biochemistry 31, 2080–2088[CrossRef][Medline] [Order article via Infotrieve]
  60. Aviezer, D., Levy, E., Safran, M., Svahn, C., Buddecke, E., Schmidt, A., David, G., Vlodavsky, I., and Yayon, A. (1994) J. Biol. Chem. 269, 114–121[Abstract/Free Full Text]
  61. Rogers, M., Yehiely, F., Scott, M., and Prusiner, S. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3182–3186[Abstract/Free Full Text]
  62. Flechsig, E., Shmerling, D., Hegyi, I., Raeber, A. J., Fischer, M., Cozzio, A., von Mering, C., Aguzzi, A., and Weissmann, C. (2000)