Heparan Sulfate and Heparin Promote Faithful Prion Replication in Vitro by Binding to Normal and Abnormal Prion Proteins in Protein Misfolding Cyclic Amplification
2016; Elsevier BV; Volume: 291; Issue: 51 Linguagem: Inglês
10.1074/jbc.m116.745851
ISSN1083-351X
AutoresMorikazu Imamura, Naoko Tabeta, Nobuko Kato, Yuichi Matsuura, Yoshifumi Iwamaru, Takashi Yokoyama, Yuichi Murayama,
Tópico(s)Viral Infections and Immunology Research
ResumoThe precise mechanism underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) remains unclear. Protein misfolding cyclic amplification (PMCA), an in vitro technique used for amplifying PrPSc, results in PrPSc replication that preserves the strain-specific characteristics of the input PrPSc; thus, PMCA mimics the process of in vivo PrPSc replication. Previous work has demonstrated that in PMCA, nucleic acids are critical for PrPSc amplification, but little information has been reported on glycosaminoglycan (GAG) participation in PrPSc replication in vitro. Here, we investigated whether GAGs play a role in the faithful replication of PrPSc by using a modified PMCA performed with baculovirus-derived recombinant PrP (Bac-PrP) as a substrate. The addition of heparan sulfate (HS) or its analog heparin (HP) restored the conversion efficiency in PMCA that was inhibited through nucleic acid depletion. Moreover, the PMCA products obtained under these conditions were infectious and preserved the properties of the input PrPSc. These data suggest that HS and HP play the same role as nucleic acids in facilitating faithful replication of prions in PMCA. Furthermore, we showed that HP binds to both Bac-PrP and Bac-PrPSc through the sulfated groups present on HP and that the N-terminal domain of Bac-PrPSc might potentially not be involved in the binding to HP. These results suggest that the interaction of GAGs such as HS and HP with PrPC and/or PrPSc through their sulfate groups is critical for the faithful replication of prions. The precise mechanism underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) remains unclear. Protein misfolding cyclic amplification (PMCA), an in vitro technique used for amplifying PrPSc, results in PrPSc replication that preserves the strain-specific characteristics of the input PrPSc; thus, PMCA mimics the process of in vivo PrPSc replication. Previous work has demonstrated that in PMCA, nucleic acids are critical for PrPSc amplification, but little information has been reported on glycosaminoglycan (GAG) participation in PrPSc replication in vitro. Here, we investigated whether GAGs play a role in the faithful replication of PrPSc by using a modified PMCA performed with baculovirus-derived recombinant PrP (Bac-PrP) as a substrate. The addition of heparan sulfate (HS) or its analog heparin (HP) restored the conversion efficiency in PMCA that was inhibited through nucleic acid depletion. Moreover, the PMCA products obtained under these conditions were infectious and preserved the properties of the input PrPSc. These data suggest that HS and HP play the same role as nucleic acids in facilitating faithful replication of prions in PMCA. Furthermore, we showed that HP binds to both Bac-PrP and Bac-PrPSc through the sulfated groups present on HP and that the N-terminal domain of Bac-PrPSc might potentially not be involved in the binding to HP. These results suggest that the interaction of GAGs such as HS and HP with PrPC and/or PrPSc through their sulfate groups is critical for the faithful replication of prions. Prions are unique proteinaceous infectious agents that are considered to be the cause of transmissible spongiform encephalopathies, including scrapie in sheep, bovine spongiform encephalopathy (BSE) 2The abbreviations used are: BSE, bovine spongiform encephalitis; mBSE, mouse-adapted BSE; PrPC, normal cellular prion protein; PrPSc, abnormal pathogenic PrP; recPrP, recombinant PrP; Bac-PrP, baculovirus-derived recPrP; PMCA, protein misfolding cyclic amplification; iPMCA, insect cell PMCA; GAG, glycosaminoglycan; HA, hyaluronic acid; HP, heparin; HS, heparan sulfate; DeN, de-N-sulfated; DeO, de-O-sulfated; NaDO, N-acetyl-de-O-sulfated; CS, chondroitin sulfate; PA, poly(A); PK, proteinase K; PHCL, PK- and heat-treated cell lysate; BH, brain homogenate; WB, Westernblotting; IMAC, immobilized metal affinity chromatography; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. in cattle, and Creutzfeldt-Jakob disease in humans (1.Collinge J. Prion diseases of humans and animals: their causes and molecular basis.Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1122) Google Scholar). Prions consist primarily of a pathogenic form (PrPSc) of the normal cellular prion protein (PrPC), and PrPSc appears to propagate itself through the autocatalytic conformational conversion of PrPC (2.Prusiner S.B. The prion diseases.Brain Pathol. 1998; 8: 499-513Crossref PubMed Scopus (317) Google Scholar). Although the mechanism of PrPC conversion into PrPSc remains unclear, host cofactors have been suggested to be necessary for efficient replication of PrPSc (3.Soto C. Prion hypothesis: the end of the controversy?.Trends Biochem. Sci. 2011; 36: 151-158Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 4.Ma J. The role of cofactors in prion propagation and infectivity.PLoS Pathog. 2012; 8: e1002589Crossref PubMed Scopus (41) Google Scholar); various biological molecules, such as nucleic acids (5.Deleault N.R. 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Presymptomatic detection of prions in blood.Science. 2006; 313: 92-94Crossref PubMed Scopus (203) Google Scholar, 20.Murayama Y. Yoshioka M. Okada H. Takata M. Yokoyama T. Mohri S. Urinary excretion and blood level of prions in scrapie-infected hamsters.J. Gen. Virol. 2007; 88: 2890-2898Crossref PubMed Scopus (81) Google Scholar21.Murayama Y. Ono F. Shimozaki N. Shibata H. l-Arginine ethylester enhances in vitro amplification of PrP in macaques with atypical L-type bovine spongiform encephalopathy and enables presymptomatic detection of PrP in the bodily fluids.Biochem. Biophys. Res. Commun. 2016; 470: 563-568Crossref PubMed Scopus (7) Google Scholar). PrPSc propagated using PMCA retains the properties of the input PrPSc, suggesting that PMCA mimics the in vivo replication of PrPSc (22.Castilla J. Saá P. Hetz C. Soto C. In vitro generation of infectious scrapie prions.Cell. 2005; 121: 195-206Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar). Therefore, PMCA has been used in studies investigating the PrPSc replication mechanism and the cofactors involved in the replication (23.Deleault N.R. Kascsak R. Geoghegan J.C. Supattapone S. Species-dependent differences in cofactor utilization for formation of the protease-resistant prion protein in vitro.Biochemistry. 2010; 49: 3928-3934Crossref PubMed Scopus (87) Google Scholar, 24.Abid K. Morales R. Soto C. Cellular factors implicated in prion replication.FEBS Lett. 2010; 584: 2409-2414Crossref PubMed Scopus (50) Google Scholar). Deleault et al. (5.Deleault N.R. Lucassen R.W. Supattapone S. RNA molecules stimulate prion protein conversion.Nature. 2003; 425: 717-720Crossref PubMed Scopus (448) Google Scholar, 25.Deleault N.R. Geoghegan J.C. Nishina K. Kascsak R. Williamson R.A. Supattapone S. Protease-resistant prion protein amplification reconstituted with partially purified substrates and synthetic polyanions.J. Biol. 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Insect cell-derived cofactors become fully functional after proteinase K and heat treatment for high-fidelity amplification of glycosylphosphatidylinositol-anchored recombinant scrapie and BSE prion proteins.PLoS One. 2013; 8: e82538Crossref PubMed Scopus (8) Google Scholar). Thus, available data strongly suggest that nucleic acids promote PrPSc replication in PMCA, although whether nucleic acids are involved in prion replication in vivo is unknown. GAGs, like nucleic acids, are polyanions, and GAGs function in brain development and are critical determinants of brain structure and function (27.Aono S. Oohira A. Chondroitin sulfate proteoglycans in the brain.Adv. Pharmacol. 2006; 53: 323-336Crossref PubMed Scopus (5) Google Scholar28.Avram S. Shaposhnikov S. Buiu C. Mernea M. Chondroitin sulfate proteoglycans: structure-function relationship with implication in neural development and brain disorders.Biomed. Res. 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Association between vascular basement membrane components and the lesions of Alzheimer's disease.J. Neurosci. Res. 1991; 30: 673-681Crossref PubMed Scopus (104) Google Scholar), and HS is suggested to be involved in the pathogenesis of amyloidosis. Furthermore, sulfated glycans were found to alter PrPC cellular localization and stimulate PrPC endocytosis in cultured cells (33.Shyng S.L. Lehmann S. Moulder K.L. Harris D.A. Sulfated glycans stimulate endocytosis of the cellular isoform of the prion protein, PrPC, in cultured cells.J. Biol. Chem. 1995; 270: 30221-30229Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), suggesting that GAGs serve as a cellular receptor for both prion uptake and cell infection. PrPSc accumulation also influences the metabolism of GAGs and results in their accumulation and secretion in urine (34.Mayer-Sonnenfeld T. Zeigler M. Halimi M. Dayan Y. Herzog C. Lasmezas C.I. Gabizon R. The metabolism of glycosaminoglycans is impaired in prion diseases.Neurobiol. Dis. 2005; 20: 738-743Crossref PubMed Scopus (20) Google Scholar). Furthermore, sulfated GAGs containing HS stimulate the conformational change of PrPC into a PrPSc-like proteinase K (PK)-resistant form even in a cell-free conversion assay (9.Lawson V.A. Lumicisi B. Welton J. Machalek D. Gouramanis K. Klemm H.M. Stewart J.D. Masters C.L. Hoke D.E. Collins S.J. Hill A.F. Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein.PLoS One. 2010; 5: e12351Crossref PubMed Scopus (22) Google Scholar, 12.Wong C. Xiong L.W. Horiuchi M. Raymond L. Wehrly K. Chesebro B. Caughey B. Sulfated glycans and elevated temperature stimulate PrP(Sc)-dependent cell-free formation of protease-resistant prion protein.EMBO J. 2001; 20: 377-386Crossref PubMed Scopus (216) Google Scholar). Thus, GAG involvement in prion disease pathogenesis and PrPC conversion into PrPSc has been widely reported (35.Caughey B. Raymond G.J. Sulfate polyanion inhibition of scrapie-associated PrP accumulation in cultured cells.J. Virol. 1993; 67: 643-650Crossref PubMed Google Scholar, 36.Gabizon R. Meiner Z. Halimi M. Ben-Sasson S.A. Heparin-like molecules bind differentially to prion-proteins and change their intracellular metabolic fate.J. Cell. Physiol. 1993; 157: 319-325Crossref PubMed Scopus (122) Google Scholar37.Caughey B. Brown K. Raymond G.J. Katzenstein G.E. Thresher W. Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and congo red.J. Virol. 1994; 68: 2135-2141Crossref PubMed Google Scholar), but whether GAGs promote PrPSc replication in PMCA, which mimics in vivo prion replication, is unknown. We hypothesized that GAGs might play a similar role as nucleic acids in PMCA because GAGs are also polyanions and because GAGs contribute to critical molecular events in the pathogenesis of prion diseases and to both in vivo and in vitro PrPSc replication. Therefore, we investigated whether GAGs were involved in PrPSc replication by a modified PMCA performed using partially purified Bac-PrP as the PrP substrate; we denoted this as insect-cell PMCA (iPMCA). In iPMCA, PK- and heat-treated insect cell lysates were used as the conversion enhancer instead of brain homogenates (BHs), and iPMCA enabled PrPSc propagation that preserved the strain characteristics of the input PrPSc. We showed that HS and its analog heparin (HP) restored the amplification of Bac-PrPSc retaining the strain characteristics of the input PrPSc when amplification was blocked through nucleic acid depletion. Furthermore, HP bound to both Bac-PrP and Bac-PrPSc through its sulfate groups, and the degree of HP sulfation affected the conversion of Bac-PrP into Bac-PrPSc. To determine whether GAGs were involved in the conversion ofBac-PrP into Bac-PrPSc in iPMCA (26.Imamura M. Kato N. Okada H. Yoshioka M. Iwamaru Y. Shimizu Y. Mohri S. Yokoyama T. Murayama Y. Insect cell-derived cofactors become fully functional after proteinase K and heat treatment for high-fidelity amplification of glycosylphosphatidylinositol-anchored recombinant scrapie and BSE prion proteins.PLoS One. 2013; 8: e82538Crossref PubMed Scopus (8) Google Scholar), we first investigated whether treatment of PK- and heat-treated cell lysate (PHCL), which contains the cofactors necessary for Bac-PrPSc conversion, with GAG-degrading enzymes influences the activity inducing Bac-PrP conversion into Bac-PrPres. Electrophoretic separation of PHCL and Alcian blue staining of gels revealed that PHCL contains GAGs (NT; Fig. 1A). Furthermore, we found that most of the GAGs in PHCL were digested following treatment with chondroitinase ABC, which digests chondroitin sulfates (CSs), chondroitins, and hyaluronic acid (HA), but not after treatment with heparinase II, which digests HS and HP (Ch and Hp; Fig. 1A). The results of control experiments demonstrated that chondroitinase ABC and heparinase II efficiently digested purified CSB and HP, respectively, under the iPMCA reaction-buffer condition. These results suggested that PHCL contained CSs, such as CSA, CSB, and CSC, but not HS and HP. Furthermore, treatment of PHCL with both chondroitinase ABC and heparinase II did not affect Bac-PrP conversion into Bac-PrPres in iPMCA in which the reaction mixtures were seeded with ME7 strain prions and mouse-adapted BSE (mBSE) prions (Ch and Hp; Fig. 1B); however, treatment with benzonase, which digests both DNA and RNA, efficiently inhibited the conversion, as reported previously (26.Imamura M. Kato N. Okada H. Yoshioka M. Iwamaru Y. Shimizu Y. Mohri S. Yokoyama T. Murayama Y. Insect cell-derived cofactors become fully functional after proteinase K and heat treatment for high-fidelity amplification of glycosylphosphatidylinositol-anchored recombinant scrapie and BSE prion proteins.PLoS One. 2013; 8: e82538Crossref PubMed Scopus (8) Google Scholar). Next, we investigated how the addition of GAGs into the iPMCA reaction mixture affects Bac-PrP conversion into Bac-PrPres (Fig. 1C) and found that none of the seven types of GAGs added independently of the reaction mixtures increased the conversion efficiency. Therefore, we speculated that the presence of a large amount of nucleic acids in PHCL masked the effect of each GAG treatment on Bac-PrPres conversion and that the nucleic acids played a key role in the conversion in iPMCA because the amount of GAGs included in PHCL was insufficient to stimulate the conversion. To directly test whether the nucleic acids present in the iPMCA reaction mixtures masked the effects of the GAGs, we examined how GAGs influenced Bac-PrPres conversion in iPMCA under nucleic acid depletion by using three prion strains, ME7, mBSE, and Chandler. In accordance with our previous work (26.Imamura M. Kato N. Okada H. Yoshioka M. Iwamaru Y. Shimizu Y. Mohri S. Yokoyama T. Murayama Y. Insect cell-derived cofactors become fully functional after proteinase K and heat treatment for high-fidelity amplification of glycosylphosphatidylinositol-anchored recombinant scrapie and BSE prion proteins.PLoS One. 2013; 8: e82538Crossref PubMed Scopus (8) Google Scholar), benzonase treatment lowered the conversion efficiency and precluded sequential amplification (NA; Fig. 2), and the addition of synthetic poly(A) (PA) to the reaction mixtures completely restored the conversion (Fig. 2). When HP and GAGs except for HP were added at 10 and 100 μg/ml to the nucleic acid-depleted reaction mixtures, all of the GAGs enhanced the efficiencies of first round conversion relative to the basal level (NA; Fig. 2), although the effect of HA and keratan sulfate was very weak. The addition of HS or HP enabled sequential amplification of Bac-PrPres under nucleic acid-depleted conditions in the case of all three prion strains, but for the sequential amplification, HS was required at a 10-fold higher concentration than HP (50-fold in the case of ME7). This quantitative difference in the effects on conversion could be attributed to the distinct degrees of sulfation. All CSs tested were incapable of maintaining ME7-seeded sequential amplification; however, in the case of mBSE, CSA promoted at least five rounds of Bac-PrPres amplification but did not maintain the amplification until the 10th round, and, by contrast, CSB and CSC enabled sequential amplification. Last, for Chandler, all CSs facilitated Bac-PrPres amplification until the fifth round but could not maintain the amplification until the tenth round, although CSB added at 500 μg/ml enabled 10 rounds of amplification of the Chandler-seeded iPMCA products (500CSB). These results suggest that the effects of CSA, CSB, and CSC on conversion depended on the prion strains. GAG length and degree of sulfation were previously reported to affect the conversion of PrPC or recPrP into PrPSc (9.Lawson V.A. Lumicisi B. Welton J. Machalek D. Gouramanis K. Klemm H.M. Stewart J.D. Masters C.L. Hoke D.E. Collins S.J. Hill A.F. Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein.PLoS One. 2010; 5: e12351Crossref PubMed Scopus (22) Google Scholar, 10.Yokoyama T. Takeuchi A. Yamamoto M. Kitamoto T. Ironside J.W. Morita M. Heparin enhances the cell-protein misfolding cyclic amplification efficiency of variant Creutzfeldt-Jakob disease.Neurosci. Lett. 2011; 498: 119-123Crossref PubMed Scopus (42) Google Scholar, 35.Caughey B. Raymond G.J. Sulfate polyanion inhibition of scrapie-associated PrP accumulation in cultured cells.J. Virol. 1993; 67: 643-650Crossref PubMed Google Scholar36.Gabizon R. Meiner Z. Halimi M. Ben-Sasson S.A. Heparin-like molecules bind differentially to prion-proteins and change their intracellular metabolic fate.J. Cell. Physiol. 1993; 157: 319-325Crossref PubMed Scopus (122) Google Scholar, 37.Caughey B. Brown K. Raymond G.J. Katzenstein G.E. Thresher W. Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and congo red.J. Virol. 1994; 68: 2135-2141Crossref PubMed Google Scholar38.Ellett L.J. Coleman B.M. Shambrook M.C. Johanssen V.A. Collins S.J. Masters C.L. Hill A.F. Lawson V.A. Glycosaminoglycan sulfation determines the biochemical properties of prion protein aggregates.Glycobiology. 2015; 25: 745-755Crossref PubMed Scopus (11) Google Scholar). Therefore, to examine the relative effects of GAG length and sulfation degree on Bac-PrP conversion into Bac-PrPres, we added chemically modified HPs to iPMCA reaction mixtures under nucleic acid-depleted conditions (Fig. 3A). We selected HP for this experiment because structurally modified forms are obtained from only HP. Low molecular mass HPs (lane 5; 50 μg/ml. By contrast, the activity of DeN HP in the ME7 and mBSE strains peaked at 500 and 100 μg/ml, respectively, although the conversion rates measured at these concentrations were also higher than the rates of the PMCA performed in the presence of nucleic acids (filled triangles). In the case of DeO HP, the highest activities were observed at 1000 μg/ml in both ME7 and mBSE strains, but the conversion rates were ∼65% of the rate in PMCA performed in the presence of nucleic acids (filled circles); however, the conversion rates measured with DeO HP might be increased at concentrations >1000 μg/ml. Last, with NaDO HP, the conversion rates were almost equivalent to those of PMCA performed with no added HPs under nucleic acid-depleted conditions (dotted line, 24.5% for ME7 and 18.1% for mBSE), even when the input amounts were increased to 1000 μg/ml (filled squares). These results suggested that the sulfate groups on HP are critical for the conversion of Bac-PrP into Bac-PrPres. Furthermore, the diminished ability of DeN HP and DeO HP to induce Bac-PrPres conversion could be compensated for by increasing the amounts of the input. This finding suggested that the adequate amount of sulfate groups required for inducing the conversion to Bac-PrPSc does not have to be present on a single HP molecule and that the conversion can be promoted by the interaction of multiple low sulfated HP molecules with Bac-PrP and/or Bac-PrPSc through the sulfate groups from each molecule. We investigated whether GAGs bound to Bac-PrP and/or Bac-PrPres by employing a binding assay that was performed using HP-conjugated agarose beads. The iPMCA reaction mixtures before and after the reaction were incubated with HP-conjugated beads, and after centrifuging the samples, the distributions of Bac-PrP or Bac-PrPres between the pellet (HP-bound fraction) and the supernatant (unbound fraction) were determined through densitometric quantification of their signals in WB analysis. When the sample tested was the iPMCA reaction mixture before the reaction, which contained only Bac-PrP, 40% of the input Bac-PrP was located in the HP-bound fraction, but only 10% of the input Bac-PrP was in the control FLAG-bead fraction (left columns; Fig. 4), and the difference was statistically significant. Notably, the amount of Bac-PrP in the HP-bound fraction was decreased to 20% when an excess amount of free normal HP (NM; Fig. 4) was added. By contrast, when NaDO HP was added to the mixture, the binding rate of Bac-PrP with HP was 38%, and no significant difference was observed between the NaDO HP addition and no addition. These results strongly suggested that Bac-PrP specifically bound to HP. Next, we performed the HP-binding assay by using the PK-digested reaction mixture after ME7-seeded iPMCA (right columns; Fig. 4). This sample did not contain Bac-PrP because of the digestion with PK and contained only the PK-resistant core of Bac-PrPres. Approximately 60% of the PK-resistant core of Bac-PrPres was contained in the HP-bound fraction, but only ∼10% of it was in the FLAG-bead fraction. The amount of the PK-resistant core of Bac-PrPres in the HP-bound faction was reduced substantially, to ∼10%, when free HP was added, but NaDO HP addition did not markedly affect the binding of the PK-resistant core to HP-agarose beads. These results suggested that both the PK-resistant core of Bac-PrPres and Bac-PrP specifically bound to HP and that of the two, the truncated Bac-PrPres bound more efficiently to HP. Furthermore, in the case of the non-treated iPMCA mixtures after the reaction, which included both Bac-PrP and full-length Bac-PrPres, the binding rates measured for each experimental group were between those of Bac-PrP and the PK-resistant core of Bac-PrPres (middle columns; Fig. 4), which suggested that full-length Bac-PrPres also specifically bound to HP. We investigated whether the products generated from iPMCA performed using HS and HP instead of nucleic acids were infectious. We collected the products generated from 13 rounds of ME7- and mBSE-seeded iPMCA performed with HP or HS added under nucleic acid-depleted conditions and intracerebrally inoculated the
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