Limits...
A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers.

Noble GP, Wang DW, Walsh DJ, Barone JR, Miller MB, Nishina KA, Li S, Supattapone S - PLoS Pathog. (2015)

Bottom Line: Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163.Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers.Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Medicine, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.

ABSTRACT
Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

No MeSH data available.


Related in: MedlinePlus

Immunoprecipitation with conformation-specific mAb 15B3 distinguishes between cofactor and protein-only PrPSc.Converted sPMCA products were purified by ultracentrifugation with nOG washes to remove unconverted α-PrP and excess lipid, and immunoprecipation was performed using 15B3-coated or uncoated rat anti-mouse IgM-conjugated magnetic beads, as indicated. The location of the discontinuous, 15B3 conformational epitope is shown in Fig 2A. Arrows indicate an Mr of ~23 kDa, the expected mobility of full-length recombinant PrP. By densitometry, the efficiency of 15B3 immunoprecipitation in this experiment is 79%, 71% and 74% for 301C-seeded, ME7-seeded, and OSU-seeded cofactor PrPSc, respectively, and 15% for OSU-seeded protein-only PrPSc.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4488359&req=5

ppat.1005017.g003: Immunoprecipitation with conformation-specific mAb 15B3 distinguishes between cofactor and protein-only PrPSc.Converted sPMCA products were purified by ultracentrifugation with nOG washes to remove unconverted α-PrP and excess lipid, and immunoprecipation was performed using 15B3-coated or uncoated rat anti-mouse IgM-conjugated magnetic beads, as indicated. The location of the discontinuous, 15B3 conformational epitope is shown in Fig 2A. Arrows indicate an Mr of ~23 kDa, the expected mobility of full-length recombinant PrP. By densitometry, the efficiency of 15B3 immunoprecipitation in this experiment is 79%, 71% and 74% for 301C-seeded, ME7-seeded, and OSU-seeded cofactor PrPSc, respectively, and 15% for OSU-seeded protein-only PrPSc.

Mentions: Having identified the α1-β2 domain by DXMS as a region of conformational divergence in our cofactor and protein-only PrPSc samples, we sought to confirm this finding using additional biochemical and biophysical approaches. The α1-β2 region contains a portion of the epitope for 15B3, a well-characterized PrPSc-specific conformational antibody [18]. The regions of PrP primary structure that comprise the discontinuous 15B3 epitope are shown schematically in Fig 2A. In a single immunoprecipitation experiment, 15B3 efficiently pulled down all three of our cofactor PrPSc samples (Fig 3, top three panels), but only weakly bound protein-only PrPSc (Fig 3, bottom panel), indicating a disruption of the 15B3 conformational epitope, consistent with our DXMS results.


A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers.

Noble GP, Wang DW, Walsh DJ, Barone JR, Miller MB, Nishina KA, Li S, Supattapone S - PLoS Pathog. (2015)

Immunoprecipitation with conformation-specific mAb 15B3 distinguishes between cofactor and protein-only PrPSc.Converted sPMCA products were purified by ultracentrifugation with nOG washes to remove unconverted α-PrP and excess lipid, and immunoprecipation was performed using 15B3-coated or uncoated rat anti-mouse IgM-conjugated magnetic beads, as indicated. The location of the discontinuous, 15B3 conformational epitope is shown in Fig 2A. Arrows indicate an Mr of ~23 kDa, the expected mobility of full-length recombinant PrP. By densitometry, the efficiency of 15B3 immunoprecipitation in this experiment is 79%, 71% and 74% for 301C-seeded, ME7-seeded, and OSU-seeded cofactor PrPSc, respectively, and 15% for OSU-seeded protein-only PrPSc.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488359&req=5

ppat.1005017.g003: Immunoprecipitation with conformation-specific mAb 15B3 distinguishes between cofactor and protein-only PrPSc.Converted sPMCA products were purified by ultracentrifugation with nOG washes to remove unconverted α-PrP and excess lipid, and immunoprecipation was performed using 15B3-coated or uncoated rat anti-mouse IgM-conjugated magnetic beads, as indicated. The location of the discontinuous, 15B3 conformational epitope is shown in Fig 2A. Arrows indicate an Mr of ~23 kDa, the expected mobility of full-length recombinant PrP. By densitometry, the efficiency of 15B3 immunoprecipitation in this experiment is 79%, 71% and 74% for 301C-seeded, ME7-seeded, and OSU-seeded cofactor PrPSc, respectively, and 15% for OSU-seeded protein-only PrPSc.
Mentions: Having identified the α1-β2 domain by DXMS as a region of conformational divergence in our cofactor and protein-only PrPSc samples, we sought to confirm this finding using additional biochemical and biophysical approaches. The α1-β2 region contains a portion of the epitope for 15B3, a well-characterized PrPSc-specific conformational antibody [18]. The regions of PrP primary structure that comprise the discontinuous 15B3 epitope are shown schematically in Fig 2A. In a single immunoprecipitation experiment, 15B3 efficiently pulled down all three of our cofactor PrPSc samples (Fig 3, top three panels), but only weakly bound protein-only PrPSc (Fig 3, bottom panel), indicating a disruption of the 15B3 conformational epitope, consistent with our DXMS results.

Bottom Line: Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163.Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers.Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Medicine, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.

ABSTRACT
Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

No MeSH data available.


Related in: MedlinePlus