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Prion ‘virus’ particles caused by lysosomal storage disease?
Bioscience Hypotheses (2009) 2, 370e371 available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/bihy
Prion ‘virus’ particles caused by lysosomal storage disease? J.F. McEwan*, S.D. Cullis-Hill Sylvan Pharmaceuticals, 111 Bronte Rd, Bondi Junction, Sydney, NSW 2022, Australia Received 10 January 2009; received in revised form 20 January 2009; accepted 20 January 2009
KEYWORDS Prion; Virus; Mucopolysaccharidosis; Heparan; Lysosomal storage
Abstract Virus-like particles are commonly found in highly infectious scrapie brain fractions and cell lines, suggesting a viral source of prion diseases. However, new evidence indicates that these particles are likely the result of mucopolysaccharidosis; a lysosomal storage disease where similar particles are observed, along with neuronal degeneration. Heparan sulfate proteoglycans (HSPGs) have long been implicated in prion diseases including demonstrated impairment of glycosaminoglycan metabolism. In this work, unusual levels of glypican-1 and heparan sulfate in cell cultures treated with prion protein antibodies are disclosed, further evidence for a lysosomal storage disorder and suggestive of a HSPG origin for prion diseases. ª 2009 Elsevier Ltd. All rights reserved.
Introduction In highly infectious scrapie brain fractions and cell lines with high prion titres, virus-like particles are observed by ultrastructural pathology [1]. Additional data that contradict the prion hypothesis have led some researchers to propose a viral origin for prion diseases [1]. Indeed, treatment of these putative viral particles with low concentrations of SDS removed residual prion protein (PrP) but did not reduce their infectivity or size [1]. However, disruption of nucleic acideprotein complexes destroyed infectivity. Conversely to the viral theory, recent results have strengthened the implications of heparan sulfate proteoglycan (HSPG) involvement in transmissible spongiform
* Corresponding author. Tel.: þ61 2 9389 0000; fax: þ61 2 9387 5473. E-mail address: [email protected] (J.F. McEwan).
encephalopathies (TSEs). A HSPG origin for these virus-like particles is also consistent with resistance to mild SDS but susceptibility to the same conditions that disrupt nucleic acideprotein complexes. Despite a ubiquitous distribution and universal action of HSPGs in cellular processes, heparan sulfate remains relatively under-researched and largely ignored in biochemistry. Nevertheless, strong relationships between HSPGs and PrP have been uncovered. Several heparan sulfate binding sites were identified in PrP, heparan sulfate is found with amyloid deposits in TSEs, and PrP co-internalises with glypican-1 with a copper mediated mechanism [2]. Cellular heparan sulfate participates in the metabolism of prions [3], including acting as a cellular receptor for infectious prions. A change in glycosylation-related gene expression is also seen in prion diseases [4]. Even more intriguing is the observation that glycosaminoglycan (GAG) metabolism is impaired in prion diseases [5]. Secretion of large amounts of GAGs in the urine of
1756-2392/$ - see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bihy.2009.01.010
Prion ‘virus’ particles caused by lysosomal storage disease?
371
prion-infected animals was observed, a situation symptomatic of lysosomal storage disorders, prompting the authors to suggest an induction of mucopolysaccharidosis (MPS) by the prion [5]. Neurological damage in TSEs is also reminiscent of neuro-degeneration seen in MPS, adding further weight to the concept of TSE being a lysosomal storage disorder.
under ultrastructural analysis of tissue from animals with lysosomal storage disorders [6]. Tissues from animals with TSEs show some vacuolated neurons with cytoplasmic projections containing accumulations of round particles of uniform size, about 25 nm in diameter [1]. Similarly in lysosomal storage disorders, accumulation of particles of storage bodies is observed in cytosomes. Furthermore, characteristic large membrane bound vacuoles are seen in both MPS and TSEs [1,6]. Secondly, in an analogy to TSEs, human tumour cells known to overexpress PrP were treated with an antibody to PrP, and levels of glypican and heparan sulfate expression were examined. HCT116 cells were treated with anti-PrP BAR221 at 2.5 or 5 mg/mL for 24 h, then washed with PBS and proteins extracted. Mouse IgG was used as a control at 5 mg/mL. After normalisation for total protein content by BCA assay, glypican-1 levels were analysed by slot blot and western blotting. A dramatic increase in glypican-1 level that was dose-dependent was observed by both techniques (Fig. 1). Similarly, as expected, heparan sulfate levels were elevated after PrP antibody treatment (data not shown).
Hypothesis It is proposed that the ‘virus’-like particles observed in TSE infected cell lines and tissues are HSPG deposits, which result from an underlying mucopolysaccharidosis in prion disorders.
Evidence for the hypothesis Here additional evidence is presented that strengthens the argument for MPS in prion diseases. Firstly, the virus-like particles [1] seen in TSEs are similar to particles observed
Discussion Combined, this body of evidence implies a MPS involvement in TSEs. A clear relationship between PrP and glypican-1 is apparent, particularly in their combined role in cell proliferation and cell cycling at the G1/S boundary, and also during copper mediated internalisation [2]. The current work identifies a relationship between ultrastructural features in both MPS and TSEs and suggests the virus-like particles are likely to be HSPG deposits. It also demonstrates a dramatic increase in HSPG levels following PrP inhibition in cell culture. These data imply a primary function for PrP in HSPG transport/transmission and/or amplification of specific heparan sulfate signals, a proposal which also accounts for the high abundance of PrP in neurons with ongoing high plasticity. Impaired HSPG processing in TSE infected animals or cell cultures would be expected in this scenario, since misfolded PrP and aggregate formation would leave less normal PrP available for HSPG interaction.
References
Figure 1 Blots (ECL) showing increase in glypican-1 expression following treatment with PrP antibody BAR221 (Sapphire Bioscience). 1 Ab Santa Cruz sc-14648, 2 antibody anti-Goat HRP conjugate. A: WB. Lanes 1) glypican-1 recombinant fusion protein marker. 2) 2.5 mg/mL BAR221. 3) 5 mg/mL BAR221. 4) 5 mg/mL mouse IgG. 5) Untreated. B: Slot blot. Lanes 1) Untreated. 2) 5 mg/mL mouse IgG. 3) 2.5 mg/mL BAR221. 4) 5 mg/mL BAR221.
[1] Manuelidis L, Yu Z, Banquero N, Mullins B. Cells infected with scrapie and CreutzfeldteJakob disease agents produce intracellular 25-nm virus-like particles. Proc Natl Acad Sci U S A 2007;104:1965e70. [2] Cheng F, Lindqvist J, Haigh CL, Brown DR, Mani K. Copperdependent cointernalization of the prion protein and glypican1. J Neurochem 2006;98:1445e57. [3] Ben-Zaken O, Tsaban S, Tal Y, Horonchik L, Esko JD, Vlodavsky I, et al. Cellular heparan sulfate participates in the metabolism of prions. J Biol Chem 2003;278:40041e9. [4] Barret A, Forestier L, Deslys J, Julien R, Gallet P. Glycosylationrelated gene expression in prion diseases: PrPSc accumulation in scrapie infected GT1 cells depends on b-1,4-linked GalNAc-4SO4 hyposulfation. J Biol Chem 2005;280:10516e23. [5] Mayer-Sonnenfeld T, Zeigler M, Halimi M, Dayan Y, Herzog C, Lasmezas CI, et al. The metabolism of glycosaminoglycans is impaired in prion diseases. Neurobiol Dis 2005;20:738e43. [6] Jolly RD, Walkley SU. Lysosomal storage diseases of animals: an essay in comparative pathology. Vet Pathol 1997;34:527e48.
journal homepage: www.elsevier.com/locate/bihy
Prion ‘virus’ particles caused by lysosomal storage disease? J.F. McEwan*, S.D. Cullis-Hill Sylvan Pharmaceuticals, 111 Bronte Rd, Bondi Junction, Sydney, NSW 2022, Australia Received 10 January 2009; received in revised form 20 January 2009; accepted 20 January 2009
KEYWORDS Prion; Virus; Mucopolysaccharidosis; Heparan; Lysosomal storage
Abstract Virus-like particles are commonly found in highly infectious scrapie brain fractions and cell lines, suggesting a viral source of prion diseases. However, new evidence indicates that these particles are likely the result of mucopolysaccharidosis; a lysosomal storage disease where similar particles are observed, along with neuronal degeneration. Heparan sulfate proteoglycans (HSPGs) have long been implicated in prion diseases including demonstrated impairment of glycosaminoglycan metabolism. In this work, unusual levels of glypican-1 and heparan sulfate in cell cultures treated with prion protein antibodies are disclosed, further evidence for a lysosomal storage disorder and suggestive of a HSPG origin for prion diseases. ª 2009 Elsevier Ltd. All rights reserved.
Introduction In highly infectious scrapie brain fractions and cell lines with high prion titres, virus-like particles are observed by ultrastructural pathology [1]. Additional data that contradict the prion hypothesis have led some researchers to propose a viral origin for prion diseases [1]. Indeed, treatment of these putative viral particles with low concentrations of SDS removed residual prion protein (PrP) but did not reduce their infectivity or size [1]. However, disruption of nucleic acideprotein complexes destroyed infectivity. Conversely to the viral theory, recent results have strengthened the implications of heparan sulfate proteoglycan (HSPG) involvement in transmissible spongiform
* Corresponding author. Tel.: þ61 2 9389 0000; fax: þ61 2 9387 5473. E-mail address: [email protected] (J.F. McEwan).
encephalopathies (TSEs). A HSPG origin for these virus-like particles is also consistent with resistance to mild SDS but susceptibility to the same conditions that disrupt nucleic acideprotein complexes. Despite a ubiquitous distribution and universal action of HSPGs in cellular processes, heparan sulfate remains relatively under-researched and largely ignored in biochemistry. Nevertheless, strong relationships between HSPGs and PrP have been uncovered. Several heparan sulfate binding sites were identified in PrP, heparan sulfate is found with amyloid deposits in TSEs, and PrP co-internalises with glypican-1 with a copper mediated mechanism [2]. Cellular heparan sulfate participates in the metabolism of prions [3], including acting as a cellular receptor for infectious prions. A change in glycosylation-related gene expression is also seen in prion diseases [4]. Even more intriguing is the observation that glycosaminoglycan (GAG) metabolism is impaired in prion diseases [5]. Secretion of large amounts of GAGs in the urine of
1756-2392/$ - see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bihy.2009.01.010
Prion ‘virus’ particles caused by lysosomal storage disease?
371
prion-infected animals was observed, a situation symptomatic of lysosomal storage disorders, prompting the authors to suggest an induction of mucopolysaccharidosis (MPS) by the prion [5]. Neurological damage in TSEs is also reminiscent of neuro-degeneration seen in MPS, adding further weight to the concept of TSE being a lysosomal storage disorder.
under ultrastructural analysis of tissue from animals with lysosomal storage disorders [6]. Tissues from animals with TSEs show some vacuolated neurons with cytoplasmic projections containing accumulations of round particles of uniform size, about 25 nm in diameter [1]. Similarly in lysosomal storage disorders, accumulation of particles of storage bodies is observed in cytosomes. Furthermore, characteristic large membrane bound vacuoles are seen in both MPS and TSEs [1,6]. Secondly, in an analogy to TSEs, human tumour cells known to overexpress PrP were treated with an antibody to PrP, and levels of glypican and heparan sulfate expression were examined. HCT116 cells were treated with anti-PrP BAR221 at 2.5 or 5 mg/mL for 24 h, then washed with PBS and proteins extracted. Mouse IgG was used as a control at 5 mg/mL. After normalisation for total protein content by BCA assay, glypican-1 levels were analysed by slot blot and western blotting. A dramatic increase in glypican-1 level that was dose-dependent was observed by both techniques (Fig. 1). Similarly, as expected, heparan sulfate levels were elevated after PrP antibody treatment (data not shown).
Hypothesis It is proposed that the ‘virus’-like particles observed in TSE infected cell lines and tissues are HSPG deposits, which result from an underlying mucopolysaccharidosis in prion disorders.
Evidence for the hypothesis Here additional evidence is presented that strengthens the argument for MPS in prion diseases. Firstly, the virus-like particles [1] seen in TSEs are similar to particles observed
Discussion Combined, this body of evidence implies a MPS involvement in TSEs. A clear relationship between PrP and glypican-1 is apparent, particularly in their combined role in cell proliferation and cell cycling at the G1/S boundary, and also during copper mediated internalisation [2]. The current work identifies a relationship between ultrastructural features in both MPS and TSEs and suggests the virus-like particles are likely to be HSPG deposits. It also demonstrates a dramatic increase in HSPG levels following PrP inhibition in cell culture. These data imply a primary function for PrP in HSPG transport/transmission and/or amplification of specific heparan sulfate signals, a proposal which also accounts for the high abundance of PrP in neurons with ongoing high plasticity. Impaired HSPG processing in TSE infected animals or cell cultures would be expected in this scenario, since misfolded PrP and aggregate formation would leave less normal PrP available for HSPG interaction.
References
Figure 1 Blots (ECL) showing increase in glypican-1 expression following treatment with PrP antibody BAR221 (Sapphire Bioscience). 1 Ab Santa Cruz sc-14648, 2 antibody anti-Goat HRP conjugate. A: WB. Lanes 1) glypican-1 recombinant fusion protein marker. 2) 2.5 mg/mL BAR221. 3) 5 mg/mL BAR221. 4) 5 mg/mL mouse IgG. 5) Untreated. B: Slot blot. Lanes 1) Untreated. 2) 5 mg/mL mouse IgG. 3) 2.5 mg/mL BAR221. 4) 5 mg/mL BAR221.
[1] Manuelidis L, Yu Z, Banquero N, Mullins B. Cells infected with scrapie and CreutzfeldteJakob disease agents produce intracellular 25-nm virus-like particles. Proc Natl Acad Sci U S A 2007;104:1965e70. [2] Cheng F, Lindqvist J, Haigh CL, Brown DR, Mani K. Copperdependent cointernalization of the prion protein and glypican1. J Neurochem 2006;98:1445e57. [3] Ben-Zaken O, Tsaban S, Tal Y, Horonchik L, Esko JD, Vlodavsky I, et al. Cellular heparan sulfate participates in the metabolism of prions. J Biol Chem 2003;278:40041e9. [4] Barret A, Forestier L, Deslys J, Julien R, Gallet P. Glycosylationrelated gene expression in prion diseases: PrPSc accumulation in scrapie infected GT1 cells depends on b-1,4-linked GalNAc-4SO4 hyposulfation. J Biol Chem 2005;280:10516e23. [5] Mayer-Sonnenfeld T, Zeigler M, Halimi M, Dayan Y, Herzog C, Lasmezas CI, et al. The metabolism of glycosaminoglycans is impaired in prion diseases. Neurobiol Dis 2005;20:738e43. [6] Jolly RD, Walkley SU. Lysosomal storage diseases of animals: an essay in comparative pathology. Vet Pathol 1997;34:527e48.