Article pubs.acs.org/biochemistry
Covalent Surface Modification of Prions: A Mass Spectrometry-Based Means of Detecting Distinctive Structural Features of Prion Strains Christopher J. Silva,* Melissa L. Erickson-Beltran, and Irina C. Dynin Western Regional Research Center, United States Department of Agriculture, Albany, California 94710, United States S Supporting Information *
ABSTRACT: Prions (PrPSc) are molecular pathogens that are able to convert the isosequential normal cellular prion protein (PrPC) into a prion. The only demonstrated difference between PrPC and PrPSc is conformational: they are isoforms. A given host can be infected by more than one kind or strain of prion. Five strains of hamster-adapted scrapie [Sc237 (=263K), drowsy, 139H, 22AH, and 22CH] and recombinant PrP were reacted with five different concentrations (0, 1, 5, 10, and 20 mM) of reagent (N-hydroxysuccinimide ester of acetic acid) that acetylates lysines. The extent of lysine acetylation was quantitated by mass spectrometry. The lysines in rPrP react similarly. The lysines in the strains react differently from one another in a given strain and react differently when strains are compared. Lysines in the C-terminal region of prions have different straindependent reactivity. The results are consistent with a recently proposed model for the structure of a prion. This model proposes that prions are composed of a four-rung β-solenoid structure comprised of four β-sheets that are joined by loops and turns of amino acids. Variation in the amino acid composition of the loops and β-sheet structures is thought to result in different strains of prions. that a prion is composed of a four-rung β-solenoid structure.20 Because the protein remains intact, the four β-helices must be joined by unstructured loops of amino acids.21 The structural differences in strains should be observable in that the flexibility of the loops allows for differences in the composition of the βhelical regions.21 This would result in changes in the surface exposure of the side chains of the amino acids. Those that were part of the loops and turns would be expected to be more surface-exposed. Those amino acids in the β-helical region could be either surface-exposed or directed to the inner face of the β-solenoid. An alternative structure proposes that prions are composed of parallel in-register intermolecular β-sheets (PIRIBS).22 In such a structure, the amino acid side chains would all be surface-exposed. Hamsters are susceptible to a number of prion strains, including the Sc237,23,24 139H,25 drowsy,26 22CH, and 22AH27,28 strains. Hamsters possess only one PrP polymorphism, so these strains must differ in terms of their conformation. The Sc237 and 263K strains of hamster-adapted scrapie are thought to be the same strain.23 Each of these five strains has a distinctive phenotype and incubation period. The PrP 27−30 cores for the 139H and Sc237 strains are identical.15 PrP 27−30 from the drowsy strain is approximately 2 kDa smaller than that of the other strains. The Dy strain is a cloned strain derived from the passaging of transmissible mink
A
prion (PrPSc) is a molecular pathogen. It is a multimer comprised of a single protein constrained in a distinct conformation.1,2 Prions are able to induce a normal cellular prion protein (PrPC) to adopt the prion conformation and thereby propagate the infectious conformation. A detailed analysis of the structures of PrPSc and PrPC showed that the only demonstrable differences between the two forms were noncovalent.3−12 A given host can be infected by more than one strain of prion, each having a characteristic incubation period, disease phenotype, and pathology.13 Strains can be differentiated by their distinct physiological characteristics and biochemical properties. Strains have been characterized by their distinct incubation periods and affinities for different tissue types. They have been distinguished by their unfolding properties in guanidine hydrochloride as measured by the conformation-dependent immunoassay or by susceptibility to proteinase K digestion.14,15 These results indicate that conformational differences define prion strains. Recent evidence has resulted in a substantial revision of the structure of prions. Reinterpretation of spectral evidence has led researchers to conclude that prions are composed almost entirely of a β-sheet structure, with little or no α-helical character.16−18 This spectral evidence is supported by additional experimental evidence using deuterium exchange and mass spectrometry.18 Previous work using X-ray fiber diffraction studies revealed that the prion structure contained β-strands that were consistent with an amyloid structure, nearly perpendicular to the fibril axis and composed of four β-strand repeats.19 These lines of evidence coupled with the physical constraints observed by electron microscopy strongly suggest This article not subject to U.S. Copyright. Published 2016 by the American Chemical Society
Received: September 30, 2015 Revised: December 24, 2015 Published: January 19, 2016 894
DOI: 10.1021/acs.biochem.5b01068 Biochemistry 2016, 55, 894−902
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encephalopathy into Syrian golden hamsters.26 The Sc237 and 139H strains were cloned from a sample obtained from passage of SSBP/1 into goats to yield a drowsy goat strain.28 The 22AH and 22CH strains were cloned from a SSBP/1 sheep sample.28 The amino acids may reside in different chemical environments, which would mean that they may react differently with the same reagent under the same reaction conditions. Mass spectrometry-based approaches have been used to identify and to quantitate covalent oxidation of methionines in prions.5,29−32 Such differences are observed when small molecules are reacted with the prion isoform and analyzed by antibodies and mass spectrometry.33,34 A variety of reagents have been used to covalently modify proteins for the purpose of structural studies.35−40 Other small molecule reagents have been used to covalently modify the lysines present in prions, and these differences have been detected using mass spectrometry and other techniques.33,34,41 Prion infectivity is unaffected by reaction with N-hydroxysuccinimide (NHS)-based reagents.42 Prions reacted with β-propiolactone or diethyl pyrocarbonate show reductions in infectivity.43,44 NHS-based chemistry coupled with mass spectrometry-based analysis can be used to quantitate the extent of the reaction of lysines in the wellestablished hamster-adapted scrapie strains described above without destroying prion infectivity. We report the results of experiments using mass spectrometry to analyze the products of the covalent surface modification of prions (CSMP) from these hamster-adapted scrapie strains.
(pH 8.5)] and transferred to an ultracentrifuge tube (4.2 mL, 16 mm × 38 mm). The contents of the tube were underlaid with 1 mL of 20% (w/v) sucrose and sealed. The sample in the sealed tube was centrifuged for 75 min at 150000g (46000 rpm and 20 °C) with a floating Noryl spacer in a Beckman 70.1 Ti rotor to obtain an insoluble pellet. The supernatant was carefully removed and discarded. The pellet was retained for future experiments. Preparation of Samples for ic Inoculation. A 10% brain homogenate was prepared in PBS from brains of hamsters infected with either the Sc237, 139H, drowsy, 22CH, or 22AH strain of hamster-adapted scrapie. Some samples were treated with proteinase K (50 μg/mL, 37 °C, 1 h). Fifty microliters of the sample was injected (ic) into an animal. The animals were observed for clinical signs and were humanely euthanized when they were no longer able to feed or drink. The appearance of clinical signs and the date of euthanization were recorded. Reaction of Purified Prions with Ac-NHS. Six pellets (vide supra) from each of the five hamster-adapted scrapie strains (Sc237, 139H, Dy, 22AH, and 22CH) were individually resuspended in 500 μL of buffer [2% (w/v) β-octyl glucopyranoside (BOG) and 50 mM phosphate buffer (pH 8.0)] by brief sonication using closed tubes in a cup horn (four 45 s bursts, Misonix 3000 sonicator). Ninety microliters of the sonicated pellet solution was aliquoted into five microfuge tubes. This was performed for each of the six pellets to yield six sets of five tubes for each of the hamster-adapted prion strains. Each set was reacted with a different concentration of Ac-NHS (10 μL of 0, 10, 50, 100, or 200 mM Ac-NHS in DMSO); the resulting six sets of five reaction mixtures were rotated for 15 min at room temperature (21−22 °C). Ten microliters of 1 M Tris (pH 8.0) was added to quench the reaction. The samples were rotated for 15 min at room temperature. After the reaction was completed, 330 μL of an 8 M guanidine hydrochloride solution was added, mixed, and allowed to stand for 24 h. After standing for 24 h, the samples were precipitated with 10 volumes of cold (−20 °C) methanol to precipitate the protein and remove the guanidine hydrochloride. The samples were centrifuged (15 min at 20000g and −9 °C) to isolate the precipitated protein. The supernatant was removed, and the resulting pellets were washed with cold (−20 °C) methanol and centrifuged (15 min at 20000g and −9 °C). The supernatant was removed, and the pellets were processed for mass spectrometry-based analysis (Supporting Information). Reaction of rPrP with Ac-NHS. One sample of rPrP (Supporting Information) was dissolved in 3 mL of buffer [2% BOG and 50 mM sodium phosphate (pH 8.0)] and sonicated. After being sonicated, the sample was centrifuged (20 min at 20000g) to remove any aggregated material. Thirty aliquots of 90 μL each were removed and placed into separate screw-cap Eppendorf tubes. Six sets of five tubes were reacted with a different concentration of Ac-NHS (10 μL of 0, 2, 10, 20, or 40 mM Ac-NHS in DMSO); the reaction mixtures were rotated for 15 min at room temperature (21−22 °C). Ten microliters of 1 M Tris (pH 8.0) was added to quench the reaction. The samples were rotated for 15 min at room temperature. After the reaction was complete, the samples were precipitated with 10 volumes of cold (−20 °C) methanol to precipitate the protein and remove the soluble quenched reagents. The samples were centrifuged (15 min at 20000g and −9 °C) to isolate the precipitated protein. The supernatant was removed, and the resulting pellets were washed with cold (−20 °C) methanol and centrifuged (15 min at 20000g and −9 °C). The supernatant
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EXPERIMENTAL PROCEDURES HPLC grade water was purchased from Burdick and Jackson (Muskegon, MI). HPLC grade acetonitrile was purchased from Fisher Scientific (Fairlawn, NJ). Trypsin (porcine, sequencing grade, modified) was purchased from Promega (Madison, WI). Chymotrypsin was purchased from Worthington Biochemical Corp. (Lakewood, NJ). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Animal Handling. All animal handling procedures were based on protocols reviewed and approved by the location animal care and use committee (ACUC). LVG Syrian golden hamsters were obtained from commercial sources (Charles River Laboratories, Wilmington, MA). The 139H, 22AH, and 22CH strains of hamster-adapted scrapie were a gift from R. Carp. The Sc237 and drowsy strains of hamster-adapted scrapie were obtained from InPro Biotechnology (South San Francisco, CA). All five of the strains were passaged once through LVG Syrian golden hamsters (Charles River Laboratories). Preparation of Samples. Brains were surgically removed from hamsters in the terminal stages of a prion infection following intracranial (ic) inoculation with either the Sc237, 139H, drowsy, 22CH, or 22AH strain of hamster-adapted scrapie. PrPSc was purified according to the methods of Bolton et al. with some minor modifications.45,46 Briefly, the brains were homogenized with 9 volumes of buffer [10% (w/v) Nlauroylsarcosine sodium salt and 9.5 mM sodium phosphate (pH 8.5)] using an Omni GLH general laboratory homogenizer and disposable Omni Tip plastic generator probes. This homogenate was allowed to stand for 30 min at room temperature and then centrifuged for 18 min (16000g at 20 °C) in an Eppendorf 5810R refrigerated centrifuge, to yield the clarified Sarkosyl homogenate. A 1 mL portion of this clarified homogenate was diluted to 3 mL with buffer [10% (w/v) Nlauroylsarcosine sodium salt and 9.5 mM sodium phosphate 895
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30 derived from each of the strains was comparable. Analysis of the products showed no significant differences in the cleavage of the C-terminus. Hamster rPrP (Figure 4) was digested with trypsin or chymotrypsin using previously described conditions.32 Trypsin digestion yields peptides P28GGWNTGGSR37 (PGG), Y38PGQGSPGGNR48 (YPG), Y157PNQVYYR164 (YPN), Q186HTVTTTTK194 (QHT), G195ENFTETDIK204 (GEN), V209VEQMCTTQYQK220 (VVE), and E221SQAYYDGR229 (ESQ), among others. The instrument response for the YPG, GEN, VVE, and ESQ peptides has been optimized previously.5,30,46 Chymotrypsin required overnight digestion (30 °C) for optimal results and yielded peptides N100KPSKPKTNM109 (NKP) and Y157RPVDQY162 (YRP). The optimized instrument parameters for peptide YRP have been previously reported.49 The optimized instrument conditions for PGG, QHT, NKP, and YPN are described in this work (vide supra). The internal standard consists of an optimized trypsin or chymotrypsin digestion of the 15 N-labeled rPrP. This permits the simultaneous quantification of each of the tryptic or chymotryptic peptides relative to its corresponding 15N-labeled analogue. The Ac-NHS reagent also reacts with the hydroxyl groups of the various peptides. This reaction has the potential to interfere with our analysis, so we needed to determine the extent of this reaction. When rPrP (n = 3) was reacted with 20 mM Ac-NHS and then digested with trypsin, 30 ± 10% of the YPG peptide was O-acetylated. In contrast, the proportion of the tryptic YPN or chymotryptic YRP peptides that were O-acetylated was 0.6) in the slopes of the lines derived from the least-squares analysis of the intensity of the signals from the QHT, GEN, VVE, and ESQ peptides (Table 2). The same is true (Table 3) for the signal intensities after
reaction with the highest concentration of reagent (p > 0.05). For the Sc237 strain, by contrast, there is a statistically significant difference (p < 0.05) between the slopes of the leastsquares fit for the PGG, NKP, QHT, VVE, and ESQ peptides. For the Sc237, Dy, 22AH, and 22CH strains, the signal intensity for the QHT peptide is significantly (p < 0.05) lower 899
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the Sc237 strain, even though the PK resistant cores are very similar. The remaining eight lysines (K101−K220) are part of the proteinase K resistant portion of the prion (PrP 27−30). The recent revisions to the prion structure model mean that those lysines are part of a β-sheet region of the molecule. The reactivity of these lysines is observed to differ among the strains, which implies that they must be in different and straindependent chemical environments. K220 is comparably resistant to the reaction of Ac-NHS in the drowsy, 22AH, and Sc237 strains but is more susceptible in strains 139H and 22CH. The NKP peptide (K101, K104, and K106) reacts to an extent similar to that of the QHT peptide (K185 and K194) in the Sc237 strain, but to a significantly different extent (p < 0.05) in the drowsy, 139H, 22AH, and 22CH strains. K194 and K204 (GEN) react to a similar degree in the 139H, 22AH, 22CH, Sc237, and drowsy strains. K220 (VVE and ESQ) reacts to a similar extent in the Sc237, 22AH, and drowsy strains and to a greater extent in the 22CH and 139H strains. These results indicate that K101−K220, which are part of the PK resistant core, also exhibit a straindependent susceptibility to reaction with the reagent. These results are consistent with a more recent β-solenoid model for the structure of a prion,21 but not for the (PIRIBS) architecture.22 In the PIRIBS architecture, amino acid side chains alternate between different sides of an exposed β-sheet surface. If this were the case, then all of the lysines would be expected to react to a similar extent, because they would be equally exposed to the reagent. The geometric constraints of a β-solenoid structure mean that the side chains of adjacent amino acids can project inside of the solenoid or outward, which would significantly influence the reactivity of a given lysine (Figure 6). In a β-solenoid model, the β-sheet structure is not rigidly fixed; instead, there can be variations in the β-sheet register that comprises the β-solenoid. Changes in the β-sheet register would necessitate variations in loops and turns necessary to join the β-sheet secondary structure.21 Thus, shifts in the β-sheet register could result in significant changes in the surface exposure of those shifted lysines. Consequences of such flexibility are the existence of strains and the fact that the same lysine will have a strain-dependent susceptibility to reaction with the reagent. Changes in the register of the PIRIBS architecture could also account for strains, but the lysines would react similarly because they are still similarly exposed to the reagent. The differences in chemical reactivity of amino acids other than lysine in the protein could provide further insight into the structure of prions and prion strains.34 Different reagents could be used to covalently modify these amino acids in PrPSc. For example, tetranitromethane has been successfully used to specifically nitrate tyrosines.33 Reagents could be used to covalently modify glutamic or aspartic acids. The resulting covalent modifications can be quantitated and used to determine the relative reactivity of those other amino acids. In principle, this could provide a more refined understanding of the prion structure and a better understanding of prion strains. The proteins associated with other misfolding protein diseases, such as Alzheimer’s disease (AD), amylotrophic lateral sclerosis (ALS), and Parkinson’s disease (PD), have prion-like features and contain a number of lysines.50,51 In an analogous way, those lysines could be acetylated and the resulting acetylation quantitated. Those results could then be used to determine the differences in the extent of acetylation of amyloid β, tau, superoxide dismutase, or α-synuclein.29 In
than that of the GEN, VVE, and ESQ peptides when the samples are reacted with the highest concentration of the reagent. For the Sc237 and 139H strains, the VVE and ESQ peptides have greater intensity (p < 0.05), but the GEN peptide does not (p > 0.05) at the highest reagent concentration. For all of the strains, at least one peptide has a signal intensity that is significantly (p < 0.01) different from that of the PGG peptide at the highest reagent concentration. These results indicate that the chemical environments of the lysines in five prion conformations are different from those in the rPrP conformation. These observations are consistent with other data that show these α-helices of PrPC are converted to β-sheet when the PrPC isoform (Figure 6) is converted to the PrPSc isoform.16−18 Furthermore, this is true for strains other than Sc237.
Figure 6. Cartoon showing the structural transformation of a PrPC monomer into the PrPSc conformation. The structure of PrPC is derived from the crystal structure of the Syrian hamster PrPC and the POM1 FAB.52 The image was derived from the 4YXL structure (PDB entry 4YXL) and displayed using JSMOL. The unstructured Nterminus is added (red dotted line) to complete the molecule. The monomeric PrPSc structure is qualitative and derived from physical constraints19,20 and PK digestion data.21 The image is based on a βhelix (PDB entry 1M8N) and is displayed using JSMOL. The proposed extra loops and turns are indicated in their approximate locations as dotted lines. The N-terminus is added as a green dotted line to complete the molecule.
The reactivity of the N-terminal lysines is strain-dependent. There is a significant (p < 0.05) difference in the slopes of lines relating the log10 of the relative intensity of the PGG peptide versus the reagent concentration for the 139H, drowsy, 22AH, and 22CH strains when compared to that of the Sc237 strain. This indicates that the N-terminal lysines of the 139H, drowsy, 22AH, and 22CH strains are prevented from reacting with the reagent. This presumably occurs as a result of the N-terminal lysines binding to a portion of the prion. It is unlikely that the N-terminal lysines (K23, K24, and K27) are binding to another molecule, because all of the samples originate from a hamster brain and, therefore, must contain similar amounts of non-prion molecules. In addition, removal of the N-terminal lysines from the Sc237, drowsy, 139H, and 22AH prion strains of hamster by PK results in a consistent elongation of the survival time, but not an elimination of infectivity. This indicates that the Nterminal lysines are not essential for the propagation of prion strains.47,48 After Sc237, 22AH, and 139H are digested with PK, they all possess an identical core, based on Western blot analysis. This indicates that structural features common to the other, longer incubation prion strains (139H, drowsy, 22AH, and 22CH) prevent the reactivity of those lysines. The extent of this prevention of lysine reactivity is considerably reduced in 900
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(15) Peretz, D., Scott, M. R., Groth, D., Williamson, R. A., Burton, D. R., Cohen, F. E., and Prusiner, S. B. (2001) Strain-specified relative conformational stability of the scrapie prion protein. Protein Sci. 10, 854−863. (16) Baron, G. S., Hughson, A. G., Raymond, G. J., Offerdahl, D. K., Barton, K. A., Raymond, L. D., Dorward, D. W., and Caughey, B. (2011) Effect of glycans and the glycophosphatidylinositol anchor on strain dependent conformations of scrapie prion protein: improved purifications and infrared spectra. Biochemistry 50, 4479−4490. (17) Cobb, N. J., Sonnichsen, F. D., McHaourab, H., and Surewicz, W. K. (2007) Molecular architecture of human prion protein amyloid: a parallel, in-register beta-structure. Proc. Natl. Acad. Sci. U. S. A. 104, 18946−18951. (18) Smirnovas, V., Baron, G. S., Offerdahl, D. K., Raymond, G. J., Caughey, B., and Surewicz, W. K. (2011) Structural organization of brain-derived mammalian prions examined by hydrogen-deuterium exchange. Nat. Struct. Mol. Biol. 18, 504−506. (19) Wille, H., Bian, W., McDonald, M., Kendall, A., Colby, D. W., Bloch, L., Ollesch, J., Borovinskiy, A. L., Cohen, F. E., Prusiner, S. B., and Stubbs, G. (2009) Natural and synthetic prion structure from Xray fiber diffraction. Proc. Natl. Acad. Sci. U. S. A. 106, 16990−16995. (20) Requena, J. R., and Wille, H. (2014) The structure of the infectious prion protein: experimental data and molecular models. Prion 8, 60−66. (21) Silva, C. J., Vazquez-Fernandez, E., Onisko, B., and Requena, J. R. (2015) Proteinase K and the structure of PrP(Sc): The good, the bad and the ugly. Virus Res. 207, 120−126. (22) Groveman, B. R., Dolan, M. A., Taubner, L. M., Kraus, A., Wickner, R. B., and Caughey, B. (2014) Parallel in-register intermolecular beta-sheet architectures for prion-seeded prion protein (PrP) amyloids. J. Biol. Chem. 289, 24129−24142. (23) Kimberlin, R. H., and Walker, C. (1977) Characteristics of a short incubation model of scrapie in the golden hamster. J. Gen. Virol. 34, 295−304. (24) Marsh, R. F., and Kimberlin, R. H. (1975) Comparison of scrapie and transmissible mink encephalopathy in hamsters. II. Clinical signs, pathology, and pathogenesis. J. Infect. Dis. 131, 104−110. (25) Kimberlin, R. H., Cole, S., and Walker, C. A. (1987) Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters. J. Gen. Virol. 68, 1875−1881. (26) Bessen, R. A., and Marsh, R. F. (1992) Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters. J. Gen. Virol. 73, 329−334. (27) Carp, R. I., Kim, Y. S., and Callahan, S. M. (1990) Pancreatic lesions and hypoglycemia-hyperinsulinemia in scrapie-injected hamsters. J. Infect. Dis. 161, 462−466. (28) Kimberlin, R. H., Walker, C. A., and Fraser, H. (1989) The genomic identity of different strains of mouse scrapie is expressed in hamsters and preserved on reisolation in mice. J. Gen. Virol. 70, 2017− 2025. (29) Silva, C. J. (2014) Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure. Prion 8, 42−50. (30) Silva, C. J., Dynin, I., Erickson, M. L., Requena, J. R., Balachandran, A., Hui, C., Onisko, B. C., and Carter, J. M. (2013) Oxidation of Methionine 216 in Sheep and Elk Prion Protein Is Highly Dependent upon the Amino Acid at Position 218 but Is Not Important for Prion Propagation. Biochemistry 52, 2139−2147. (31) Requena, J. R., Dimitrova, M. N., Legname, G., Teijeira, S., Prusiner, S. B., and Levine, R. L. (2004) Oxidation of methionine residues in the prion protein by hydrogen peroxide. Arch. Biochem. Biophys. 432, 188−195. (32) Pamplona, R., Naudi, A., Gavin, R., Pastrana, M. A., Sajnani, G., Ilieva, E. V., Del Rio, J. A., Portero-Otin, M., Ferrer, I., and Requena, J. R. (2008) Increased oxidation, glycoxidation, and lipoxidation of brain proteins in prion disease. Free Radical Biol. Med. 45, 1159−1166. (33) Gong, B., Ramos, A., Vazquez-Fernandez, E., Silva, C. J., Alonso, J., Liu, Z., and Requena, J. R. (2011) Probing structural differences between PrP(C) and PrP(Sc) by surface nitration and acetylation:
principle, covalent modification coupled with mass spectrometry-based analysis of these proteins may reveal the structural features that make these misfolded proteins pathogenic.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01068. Experimental procedures described in greater detail (PDF)
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AUTHOR INFORMATION
Corresponding Author
*USDA, ARS, 800 Buchanan St., Albany, CA 94710. E-mail:
[email protected]. Telephone: (510) 559-6135. Fax: (510) 559-6429. Notes
The authors declare no competing financial interest.
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REFERENCES
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Biochemistry
Article
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DOI: 10.1021/acs.biochem.5b01068 Biochemistry 2016, 55, 894−902
