Biochemical Characterization of Prions

CHAPTER EIGHTEEN

Biochemical Characterization of Prions Michele Fiorini1, Matilde Bongianni, Salvatore Monaco, Gianluigi Zanusso University of Verona, Verona, Italy 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. 1D Analysis of Molecular Strains 2.1 Human Prion Disorders 2.2 Classical and Atypical BSE Forms 2.3 Scrapie 3. Molecular Analysis of Prion Strains by 2D-PAGE Analysis 3.1 2D-PAGE Analysis of PrPSc in sCJD Molecular Subtypes 3.2 Molecular Signature of PrPSc in P102L GSS Mutation 3.3 The Influence of GPI Anchor in PrPSc Migration in sCJD 3.4 Cooccurrence of PrPSc Types in sCJD 3.5 Molecular Signatures of PrPSc in BSE Forms 3.6 PrPSc GPI-Anchored and -Anchorless Forms in BSE Forms 3.7 Cooccurrence of PrPSc Types in Scrapie 4. Biochemical Assays to Characterize and Distinguish Prion Strains 4.1 Velocity Sedimentation in Sucrose Step Gradients 4.2 Conformational Stability in Increasing Concentration of GdnHCl 5. Molecular and Chemicophysical Similarities Between Human and Cattle Transmissible Spongiform Encephalopathies Forms 5.1 Comparison of Human and Cattle Molecular PrPSc by 1D Analysis 5.2 Comparison of Human and Cattle Molecular Signature by 2D Analysis of PrPSc 6. Conclusions References Further Reading

390 393 393 395 395 396 397 397 398 399 399 400 400 401 401 403 404 404 405 405 405 407

Abstract Prion disease or transmissible spongiform encephalopathies are characterized by the presence of the abnormal form of the prion protein (PrPSc). The pathological and transmissible properties of PrPSc are enciphered in its secondary and tertiary structures. Since it’s well established that different strains of prions are linked to different conformations

Progress in Molecular Biology and Translational Science, Volume 150 ISSN 1877-1173 http://dx.doi.org/10.1016/bs.pmbts.2017.06.012

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of PrPSc, biochemical characterization of prions seems a preliminary but reliable approach to detect, analyze, and compare prion strains. Experimental biochemical procedures might be helpful in distinguishing PrPSc physicochemical properties and include resistance to proteinase K (PK) digestion, insolubility in nonionic detergents, PK-resistance under denaturing conditions and sedimentation properties in sucrose gradients. This biochemical approach has been extensively applied in human prion disorders and subsequently expanded for PrPSc characterization in animals. In particular, in sporadic Creutzfedlt–Jakob disease (sCJD) PrPSc is characterized by two main glycotypes conventionally named Type 1 and Type 2, based on the apparent gel migration at 21 and 19 kDa of the PrPSc PK-resistant fragment. An additional PrPSc type was identified in sCJD characterized by an unglycosylated dominant glycoform pattern and in 2010 a variably protease-sensitive prionopathy (VPSPr) was reported showing a PrPSc with an electrophoretic ladder like pattern. Additionally, the presence of PrPSc truncated fragments completes the electrophoretic characterization of different prion strains. By two-dimensional (2D) electrophoretic analysis additional PrPSc pattern was identified, since this procedure provides information about the isoelectric point and the different peptides length related to PK cleavage, as well as to glycosylation extent or GPI anchor presence. We here provide and extensive review on PrPSc biochemical analysis in human and animal prion disorders. Further, we show that PrPSc glycotypes observed in CJD share similarities with PrPSc in bovine spongiform encephalopathy forms (BSE).

1. INTRODUCTION Prion diseases are fatal neurodegenerative disorders affecting humans and animals with a sporadic, inherited, or infectious etiology.1,2 The common feature of these disorders is the accumulation of a pathologic prion protein (PrPSc) in the nervous tissue. The crucial event in the pathogenesis of the disease is the conversion of the normal host-encoded cellular prion protein (PrPC), to a misfolded pathogenic isoform named PrPSc.3 In fact, PrPC and PrPSc share the same primary structure but, while PrPC secondary structure is rich in α-helices, PrPSc is characterized by increased β-sheet content and decreased α-helices. As consequence of conformational change PrPSc acquires tertiary and quaternary different feature leading to new physicochemical properties: PrPSc is insoluble in nondenaturing detergents and partially resistant to proteolysis. PrPSc is also infective since it can propagate abnormal conformation to normal PrPC in a self-propagating mechanism.4 Mature PrPC is a 209 aminoacids glycoprotein, with two potential N-linked glycosylation sites at codon 181 and 197, anchored to the plasma

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membrane by a glycosylphosphatidylinositol molecule. PrPC N-terminal is characterized by an unstructured domain, composed by four copies of an octapeptide sequence (PHGGGWGQ) between residues 40 and 91, while C-terminal region is composed by two short β-sheet structures of four residues each and three α-helical regions.4 The octarepeat sequence is a Cu2+ binding motif. Western blot analysis of prion protein is characterized by three bands corresponding to the three glycoforms: di-, mono-, and unglycosylated; after proteinase-K (PK) treatment electrophoretic pattern of the three bands shifts to a lower apparent molecular weight due to the cleavage of the N-terminal PK-sensitive sequence of the protein. PrPSc PK-resistant glycoforms are also named PrP27–305 (Fig. 1). For simplicity, along this chapter we use PrPSc to define pathological PrP, including PrP27–30 and the truncated fragments. Since the same prion protein can be the cause of several different disease phenotypes, variations in prions were referred to as strains. Prion strains are characterized by experimental transmission and distinguished according to differences in incubation time and regional distribution of cerebral lesions. Biochemically it is well established that these different disease phenotypes are associated with distinct PrPSc conformations, reflected in the pattern of PK-induced proteolytic cleavage of PrPSc.6–8 Early evidence of the connection between prion conformation and strain features was shown by molecular analysis of transmissible mink encephalopathy by Bessen and Marsh.9 They studied the two known strains, called

PK

PrPC

PrPSc

–

+

Fig. 1 Schematic representation of human western blot profile of PrP before ( ) and after (+) PK treatment.

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hyper (HY) and drowsy (DY), showing different clinical signs, incubation period, brain titer, brain lesion profile, and pathogenicity after transmission to laboratory animals. In particular, they found that the degradation rate of PrPTME by PK was peculiar for each strain and correlated with inactivation of the TME titer; after PK digestion and sequence determination, HY PrPTME was at least 10 aminoacids longer than DY PrPTME at the N-terminal PK-resistant end of the protein. Finally differences in biochemical and physical properties were easily resumed by western blot after PK treatment showing two peculiar electrophoretic pattern in terms of molecular weight: HY with a core fragment at about 21 kDa and DY at about 19 kDa9 (Fig. 2). In human sporadic CJD (sCJD) six molecular phenotypes are defined, considering the molecular weight of the PK-resistant core fragment (Type 1 at 21 kDa and Type 2 at 19 kDA) and the polymorphism at codon 129 (MM, MV, and VV), reflecting the clinicopathological features of the disease.8 Western blot analysis shows peculiar migration pattern associated to different prion strains: evidence for PK cleavage at different sites in the N-terminal region of PrPSc both in animal and human prion diseases. In addition, based on the occupancy of the N-glycosylation sites or the ratio of the three PK-resistant PrPSc glycoforms (diglycosylated, monoglycosylated, and unglycosylated), further biochemical types of PrPSc can be classified.8 In the last few years, we introduced two-dimensional (2D) analysis of prions representing an important step forward in biochemical characterization of prion strain. In particular, 2D analysis allows to recognize specific biochemical fingerprints of different prion strains otherwise undetectable by conventional western blot analysis.5 Additional biochemical procedures can help in differentiating prion strains such as testing resistance to denaturing conditions or investigating the size and the solubility properties of aggregates.10,11 Hyper

Drowsy Molecular weight (kDa)

21 kDa 19 kDa

Fig. 2 Schematic representation of transmissible mink encephalopathy western blot profile drowsy and hyper molecular types.

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Aim of this chapter is to describe how biochemical characterization of prions results of crucial importance in finding and discriminating strains, not only in describing prion strains but also in large-scale epidemiological studies and surveillance.

2. 1D ANALYSIS OF MOLECULAR STRAINS 2.1 Human Prion Disorders 2.1.1 Sporadic Forms In human prion disorders, several different PrPSc types have been described based on the electrophoretic migration and the glycosylation profile of PrP27–30. In human sCJD, PrPSc separates as two main glycotypes: PrPSc Type 1 and PrPSc Type 2A with an apparent gel migration of the unglycosylated isoform at 21 and 19 kDa, respectively. Types 1 and 2A show similar glycosylation profile9 (Figs. 3 and 4). In 2007, a novel PrPSc glycotype was reported, mainly characterized by the unglycosylated isoform and slightly by the monoglycosylated isoform, but completely lacking the diglycosylated one; this new strain was named Type U. For this prion strain genetic or inherited origins were excluded12 (Figs. 3 and 4). In 2010, an additional sporadic form of prion disease has been described, called variably protease-sensitive prionopathy due to relative sensitivity to protease digestion of disease associated PrPSc (VPSPr). VPSPr is characterized by a peculiar ladder-like electrophoretic profile of the PK-resistant PrP fragments. The electrophoretic profile after western blot was detected sCJD MW (kDa)

Type 1

Type 2A

vCJD Type U

Type 2B

fCJD E200k 129M

E200K 129V

FFI B178N

GSS

VPSPr

P102L

24 20

12 8

Fig. 3 Schematic representation of human western blot profile of all CJD molecular types.

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Glycoform relative percentage

100 Diglycosylated Monoglycosylated Unglycosylated

50

Type 1

Type 2A

Type U

Type 2B

Fig. 4 Relative glycoform profile of four sCJD molecular strains.

exclusively by the use of unique anti-PrP antibody (clone 1E4) and consisted of five major bands migrating at approximately 26, 23, 20, 17, and 7 kDa13 (Fig. 3). 2.1.2 Genetic Forms Genetic prion disorders include familial Creutzfeldt–Jakob Disease (fCJD), fatal familial insomnia (FFI), and Gerstmann–Str€aussler–Scheinker syndrome (GSS), each form characterized by distinct disease phenotypes, neuropathological hallmarks, and pattern of PrP deposition, respectively. Western blot analysis in fCJD and FFI shows the typical triplet of PrP27–30 differentially PK cleaved at residues 82 or at 97 with variable pattern of glycosylation.14,15 In GSS, the PK-digested western blot pattern is defined by the enrichment of constitutive internal fragments of 8 and 11 kDa. P102L GSS is an exception presenting hybrid behavior between GSS and other genetic forms: its peculiar pattern is composed by both PrP27–30 and the 8 kDa internal fragment7,16 (Fig. 3). 2.1.3 Infectious Forms Early known cases of iatrogenic CJD (iCJD), except cannibalistic kuru, have been caused by medical procedures such as implantation of dura mater grafts and treatment with growth hormone derived from cadaveric pituitary glands obtained from patients with CJD. Less frequent incidences of human prion disease have resulted from iatrogenic transmission of CJD during corneal transplantation, contaminated electroencephalographic (EEG) electrode implantation, and surgical operations using contaminated instruments or apparatus. In iCJD, PrP27–30 is almost identical to that of sCJD in terms of molecular weight and glycoform profile.17 In contrast, variant CJD

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(vCJD),18 followed by bovine spongiform encephalopathy (BSE) exposure, shows a PrPSc with an unglycosylated band of 19 kDa (comigrating with Type 2A) and a diglycosylated dominant pattern, similar to PrP27–30 observed in cattle with BSE.19

2.2 Classical and Atypical BSE Forms BSE forms have always been considered defined by a single strain characterized by a Western blot profile with a diglycosylated pattern and a 20 kDa core PK-resistant fragment. In 2003, Casalone et al. discovered a new disease phenotype in two Italian old cows showing a cerebral PrP amyloidosis, sparing the brainstem. Western blot pattern showed a lower molecular weight of the prion core fragment and a PrPSc monoglycosylated dominant pattern distinct from that observed in classical BSE.20 Contemporary, other BSE cases where reported in France showing a PrPSc with glycosylation profile similar to that of classical BSE but with a higher molecular weight.21 These new atypical molecular types where named L-Type BSE or BASE (bovine amyloidotic spongiform encephalopathy) and H-Type BSE on the basis of the lower or higher migration of PrPSc at western blot analysis compared to classical BSE. These forms of BSE associated with distinct molecular profiles reflect different clinicopathological features, indicating that each biochemical signature is associated with a distinct form of BSE (Fig. 5).

2.3 Scrapie Natural scrapie is characterized by the presence of multiple circulating prion strains. However, molecular analysis of PrPSc is mainly characterized by a biochemical pattern, resembling to classical BSE in terms of molecular weight (20 kDa core fragment) and glycoform profile (diglycosylated)22 (Fig. 5). BSE C

L

Scrapie H

Natural

SSBP/1

BSE CH1641 in sheep

Nor98

Italian Italian Italian MW iatrogenic iatrogenic iatrogenic (kDa) 20kDA 17kDA 17-20kDA

24 20

12 8

Fig. 5 Schematic representation of human western blot profile of sporadic, genetic, and acquired CJD molecular types.

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A second form of scrapie was described in 1998, as an unprecedented form, named Nor98, from the year of its description. This form of scrapie was defined “atypical” based on several criteria: clinical presentation, molecular characteristics of PrPSc, distribution of PrPSc within infected sheep, genotypes of affected sheep and epidemiology. In particular, from a molecular view, Nor98 associate PrPSc was characterized by a faster migration of PrP27–30, by a smaller N-terminally truncated fragment of approximately 12/14 kDa and by an internal fragment of about 7 kDa23 (Fig. 5). In 2002, we described a molecular typing of PrPSc in sheep and goats belonging to a single flock accidentally exposed to an infected vaccination against Mycoplasma agalactiae in 1997 and 1998. A group of animals showed a PrPSc with a 20 kDa core fragment with a diglycosylated dominant pattern while other group showed the cooccurrence of two PrPSc types one, overlapping that observed in the first group and a second PrPSc with a highly glycosylated pattern and a PrPSc protease-resistant backbone of 17 kDa. Additional PrPSc conformational studies indicated that the 20 kDa isoform showed physicochemical properties indistinguishable from PrPSc in sheep with natural field scrapie24 (Fig. 5). However, in UK several experimental scrapie strains were isolated and used to compare novel emerging field prion strains. In particular, SSBP/1 was obtained from a pool of brain homogenates of three different sheeps suffering from scrapie and CH1641, isolated in 1999 from a natural scrapie.25 Since PrPSc fragment size observed in the brain tissue of sheep experimentally infected with BSE was identical to that of CH 1641 it was hypothesized the BSE strain of agent might circulate in sheep,26 however neuropathological pattern and transmission studies in rodents indicated that represented two distinct strains.27,28 The same conclusion was raised by SSBP/1 scrapie strain, which appeared to be very close from that found in natural scrapie cases.29

3. MOLECULAR ANALYSIS OF PRION STRAINS BY 2D-PAGE ANALYSIS 2D-PAGE analysis is an electrophoretic analysis that couples two principles of proteins electrophoretic separation, in particular the isoelectric point and the molecular mass. This analysis gives the possibility to investigate posttranslational modifications of proteins such as the extent of glycosylation, phosphorylation, the presence of GPI anchor and differences in

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peptides sequence, based on their polarity. In particular, following limited PK proteolysis and glycans removal by PNG-ase treatment, unglycosylated PrP peptides separate as trains of spots showing PrP peptides with distinct N-ragged ends. Additional C-terminal PrPSc truncated fragments can be revealed, providing additional information on PrPSc molecular signature.

3.1 2D-PAGE Analysis of PrPSc in sCJD Molecular Subtypes In sCJD, we have identified by 2D-PAGE analysis two major patterns of C-terminal protease-resistant PrP fragments among different molecular phenotypes. In all sCJD cases with a Type 1 PrPSc we detected the presence of C-terminal fragments (CTFs) of 16–17 and 12–14 kDa. This CTFs pattern was also observed in subjects with MM at codon 129 with a Type 2 PrPSc. Conversely, VV and MV patients with a Type 2 PrPSc have CTFs migrating at 17.5–18 kDa. Taken together, these findings indicate that the mechanism involved in the formation of CTFs is not influenced by codon 129 and by PrPSc glycotype. Finally, the biochemical patterns of PrPSc following 2D-PAGE analysis resulting from the combination of PrPSc glycotype and PrP27–30 lower truncated PrP species, defined three distinct PrPSc fingerprints in sCJD; a first group with Type 1 PrPSc, regardless codon 129, a second group of MM2 subjects, and a third group of MV2/VV2 cases.8 Interestingly, this biochemical distinction had a correlation with CJD strains obtained following transmission in transgenic humanized mice30 (Fig. 6).

3.2 Molecular Signature of PrPSc in P102L GSS Mutation PrPSc molecular signature in GSS cases carrying a P102L mutation is characterized by a train of spots with an isoelectric point between 6.3 and 8.3 similar to that observed in PrPSc Type 2 in sCJD (Fig. 7A and B) but with CTFs pattern similar to Type 1 PrPSc (compare Figs. 6 and 7B). The GSS specific 8 kDa fragment, corresponding to 82–146 aminoacidic sequence, PrPSc Type 2

PrPSc Type 1 MM MV VV

MV VV

MM PrP27–30

6

PrP27–30

16 kDa set

16 kDa set

12–14 kDa set

12–14 kDa set

pI

8

6

pI

PrP27–30 18.5 kDa set 8

6

pI

8

Fig. 6 Schematic 2D pattern of PrP27–30 and C-terminal PrP fragments in sCJD subtypes.

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Anti internal region antibody

Anti-C terminal antibody

PrP27–30 PrP27–30 CTFs

8 kDa fragment

3

pl

10

3

pl

10

Fig. 7 2D pattern of PrP27–30, internal 8 kDa fragment, and CTFs in a GSS P102L case.

3

pl

10 MW (kDa)

23–231 90–231

16 14.5

105–231

6.6

7.9

23

9.4

Fig. 8 Scheme of 2D mapping of synthetic PrP peptides.

can be identified by western blot with 3F4 antibody (PrP epitope between 109 and 112) (Fig. 7A) and shows a measured isoelectric point around 9. Since this fragment lacks posttranslational modifications, as expected, the 2D-PAGE migration of PrPSc corresponds to the pI of the PrP peptide sharing the same sequence at 9.0.

3.3 The Influence of GPI Anchor in PrPSc Migration in sCJD Recombinant prion protein peptides with known aminoacidic sequence were used to determine molecular coordinates in the 2D map in terms of molecular weight and isoelectric point. Since 2D-migration of the synthetic peptides exactly correspond to theoretical coordinates, it confirms the reliability of 2D-PAGE analysis (Fig. 8 and Table 1). To establish the pI and molecular weight contributions of GPI anchor, we compared PrP migration by 2D-PAGE in wild-type mice and GPI-anchorless mice showing that the

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Table 1 Theoretical Molecular Weight and Isoelectric Point of Recombinant PrP Synthetic Peptides PrP Peptides Molecular Mass (kDa) Isoelectric Point

23–231

22.9

9.39

90–231

16.2

7.95

105–231

14.7

6.56

PrPSc Type 1

PrPSc Type 2 MM

PrPSc Type 2 MV – VV

7.2 7.2

20 kDA

20 kDA

6.0

7.2

7.9 18 kDA

18 kDA

Fig. 9 Schematic representation of anchored and anchorless PK-resistant PrP core fragments in sCJD subtypes.

GPI anchor improves molecular weight of about 2 kDa and shifts measured pI to the basic pole of about 1 pH unit.7 Therefore we extended 2D-PAGE analysis to all sCJD molecular Type 1 and 2 subtypes showing that PrPSc migrated one unit of pH more acidic than expected from the theoretical pH of PrP peptides indicating that both PrPSc are GPI anchored. However, an extra isoform at pI about eight was detected in MV-2/VV-2 subtypes indicating that only in these forms PrPSc might undergo to GPI-anchor cleavage (Fig. 9).

3.4 Cooccurrence of PrPSc Types in sCJD Cooccurrence of PrPSc Types 1 and 2 have been previously reported within a single individual, evidenced by the copresence of two bands at 19 and 21 kDa by western blot31 (Fig. 10A). By 2D analysis it is possible to distinguish PrPSc types based on the different pI, as reported in Fig. 10B.

3.5 Molecular Signatures of PrPSc in BSE Forms 2D-PAGE analysis of PrPSc in classical and atypical BSEs shows distinct biochemical signatures in the three disease phenotypes. As mentioned earlier, in H-Type BSE PrPSc is characterized by a slower migration compare to the other forms and by a consistent presence of truncated fragments at

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A

B

6

pl

8.5

MW kDa

PrPSc Type 1

PrPSc Type 1

21 19

PrPSc Type 2

PrPSc Type 2 PK + PNGase

PK PNGase

–

+

Fig. 10 Representation of human western blot profile of PrPSc cooccurrence in sCJD before and after PNGase treatment (A); 2D pattern of PrPSc cooccurrence in sCJD before PK and PNGase treatment (B). H-BSE

L-BSE PrP27–30

PrP27–30

pl

8.5 6

pl

PrP27–30

16 kDa set

17 kDa set 16 kDa set

12 kDa set

6

C-BSE

8.5 6

pl

8.5

Fig. 11 Schematic 2D pattern of PrP27–30 and C-terminal PrP fragments in BSE subtypes.

12 kDa. In contrast, in C-T and L-Type, except the distinct migration of PrPSc, additional truncated fragments were not observed (Fig. 11).

3.6 PrPSc GPI-Anchored and -Anchorless Forms in BSE Forms A schematic 2D pattern of deglycosylated PrPSc is reported in Fig. 12. PrPSc core fragments are characterized by two sets of spots shifted of about 1.0 unit of pI and 2.0 kDa. Since 2D-PAGE migration of the lower basic spots correspond to PrP peptides theoretical coordinates, these correspond to GPI-anchorless forms, while the higher molecular weight spots are GPI anchored. 2D-PAGE analysis indicates that classical BSE shows only a single PrPSc anchorless isoform, while L-BSE and H-BSE atypical forms show anchorless isoforms.

3.7 Cooccurrence of PrPSc Types in Scrapie 2D-PAGE analysis of brain tissue from scrapie cases following iatrogenic exposure in Italy in 2002, clearly shows the overlap of 2D-PAGE pattern of PrPSc, as reported in Fig. 13. Each single PrPSc isoform of 17 kDa strain is distinctly identified and separated from the 20 kDa strain.24

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H-BSE

C-BSE

GPI-

6

pl

L-BSE

GPI-

GPI10

6

pl

10

6

pl

10

Fig. 12 Schematic representation of anchored and anchorless PK-resistant PrP core fragments in BSE molecular strains. 20 kDa scrapie

17 kDa scrapie

17–20 kDa cooccurrence scrapie

Fig. 13 Schematic 2D pattern of iatrogenic Italian scrapie molecular types described by Zanusso et al. (2003).

4. BIOCHEMICAL ASSAYS TO CHARACTERIZE AND DISTINGUISH PRION STRAINS Since the chemicophysical properties of PrPSc are related to distinct conformations, by testing conformational stability to denaturing agents or sedimentation properties and size of aggregates we might be able to discriminate different prion strains.

4.1 Velocity Sedimentation in Sucrose Step Gradients Ultracentrifugation in Sucrose Step Gradients coupled to the ionic surfactant sarkosyl determines the sizes of PrP aggregates based on solubility properties and consequently to prion strains conformational properties.32 4.1.1 Size Aggregates in sCJD PrPSc shows three distinct patterns of sedimentation, before and after PK treatment (Fig. 14). In MV1 sCJD subtype, PrP is composed by soluble forms of small size and by large insoluble aggregates, before and after PK

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MV-1

50

1

2

3

4

5

6

7

8

100 Relative PrP amount

Relative PrP amount

Relative PrP amount

+ PK – PK

MV-U

MV-2

100

100

+ PK – PK 50

9 10 11

1

2

3

Fraction number

4

5

6

7

8

+ PK – PK 50

9 10 11

1

2

3

Fraction number

4

5

6

7

8

9 10 11

Fraction number

Fig. 14 Fractionation of sCJD PrP aggregates. Brain homogenates from frontal cortexes of MV-1 and MV-2 and MV-U were sedimented in a 10% (fraction 1) to 60% (fraction 11) sucrose gradient. After sedimentation, half samples were digested with PK. Relative cellular (gray) and PK-resistant (black) percentage of fraction.

50

1

2

3

4 5 6 7 8 9 10 11 Fraction number

L-BSE 100 Relative PrP amount

Relative PrP amount

+ PK – PK

+ PK – PK

50

1

2

3

4 5 6 7 8 9 10 11 Fraction number

C-BSE 100 Relative PrP amount

H-BSE 100

+ PK – PK 50

1

2

3

4 5 6 7 8 9 10 11 Fraction number

Fig. 15 Fractionation of BSE PrP aggregates. Brain homogenates from frontal cortexes of H-BSE and L-BS and C-BSE were sedimented in a 10% (fraction 1) to 60% (fraction 11) sucrose gradient. After sedimentation, half samples were digested with PK. Relative cellular (gray) and PK-resistant (black) percentage of fraction distribution.

treatment. In MV2 sCJD subtype, PrP is widely distributed all over the gradient, while after PK digestion PrPSc is detected mainly at the bottom of the gradient as insoluble aggregates of large size. Disease-associated PrP in MV-U sCJD subtype shows a sedimentation pattern sharing properties with both PrPSc Types 1 and 2. 4.1.2 PrP Sedimentation Aggregates in Different Forms of BSE BSE forms molecular subtypes of PrP show a sedimentation pattern characterized by both small soluble and large insoluble aggregates (Fig. 15). In L-Type BSE, the relative amount of insoluble forms is consistently higher than other BSEs. After PK treatment, all BSE brain samples show the predominant presence of insoluble species except in C-Type BSE which shows also an additional small amount of soluble forms (Fig. 15). It’s interesting to note that from the comparison between human and cattle sedimentation patterns a similarity between MV-2 and L-BSE can be observed.

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4.2 Conformational Stability in Increasing Concentration of GdnHCl Conformational stability can be tested by PK digestion after incubation in increasing concentration of chaotropic agents: each PrPSc conformer can maintain its properties and its PK resistance below a guanidine concentration limit, then can be completely digested.10 The strength of conformational state is related to the GdnHCl concentration exposure needed to denature the protein and giving the possibility to PK to digest it completely. 4.2.1 Conformational Stability Assay in sCJD In sCJD, Types 1 and 2, PrPSc are more resistant to guanidine denaturation than Type U: an exposure to 2 M guanidine is enough to disrupt conformational resistance to PK degradation in Type U, while in Types 1 and 2, PrPSc digestion is observed at 2.5 M guanidine concentration. The optical density of prion electrophoretic bands after incubation in increasing guanidine concentrations and PK treatment is representative of the distinct kinetics of PrP proteolysis (Fig. 16). Type U western blot shows the presence of two conformers after incubation in 1.5 M guanidine and PK digestion, indicating that two molecular strains are hidden in the 3D conformation.33 Inoculation experiments in bank voles of the same MV-U case showed it contains two different strains with different incubation time and different lesion profile associated with two different molecular electrophoretic patterns after transmission.33 MV-1 1 Relative otpical density

MV-2 MV-U

0 0

0.5

1

1.5

2

2.5

3

GND-HCl (M)

Fig. 16 Relative quantification of denaturation transitions for the MV-1, MV-2, and MV-U types.

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H-BSE C-BSE

1 Relative otpical density

L-BSE

0 0

0.5

1

1.5

2

2.5

3

GND-HCl (M)

Fig. 17 Relative quantification of denaturation transitions for the H-BSE, C-BSE, and L-BSE.

4.2.2 Conformational Stability Assay in BSEs Classical BSE can be distinguished from atypical forms of BSE since its conformational stability is maintained until 2 M guanidine concentration, while atypical forms show a higher resistance to 2.5 M guanidine. However, all BSE forms show a lesser extent of PrPSc with proteolysis resistance forms until 3 M guanidine (Fig. 17).

5. MOLECULAR AND CHEMICOPHYSICAL SIMILARITIES BETWEEN HUMAN AND CATTLE TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES FORMS Several similarities between human and cattle PrPSc forms can be found at molecular level in terms of both electrophoretic pattern of separation following 1D- and/or 2D-PAGE analysis, and chemicophysical properties of different prion strains conformation.

5.1 Comparison of Human and Cattle Molecular PrPSc by 1D Analysis A comparative analysis between PrPSc types in human CJD indicates that PrPSc Type 1, PrPSc Type 2A, MV at codon 129, and PrPSc Type 2B, associated with vCJD, show biochemical similarities to H-Type, L-Type, and C-Type BSEs in terms of molecular weight or glycoform ratio. PrPSc Type 1 and H-Type BSE share the same molecular weight; PrPSc Type 2A and L-Type BSE show similar molecular weight and glycosylation profile;

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finally, PrPSc Type 2B shows a similar diglycosylated profile with that observed in classical BSE (compare Figs. 3 and 5). However, the link between CJD and BSE has been extensively studied by several transmission experiments in rodents and transgenic mice.34–36

5.2 Comparison of Human and Cattle Molecular Signature by 2D Analysis of PrPSc 2D-PAGE analysis confirms qualitative similarities between human and cattle PrPSc molecular types, described by 1D. The molecular signature of PrPSc, following 2D-PAGE analysis, of H-BSE and Type 1 sCJD shows an abundance of CTFs, as specific finding. Type 2A sCJD and L-BSE show the same pattern of the CTFs at 18 kDa. C-BSE and vCJD share the same CTFs pattern of the upper set of spots at 16 kDa, while the lower one at 12–14 kDa is present only in vCJD (compare Figs. 6 and 11).

6. CONCLUSIONS In this chapter, we have described several applications of molecular analysis for prions characterization, showing a panel of different biochemical approaches able to detect molecular differences and similarities among prion conformers. In particular, we have shown by both 1D and 2D electrophoretic separations PrPSc patterns in human and animal prion disorders based on electrophoretic migration of PrP27–30 and on CTFs. By 2D analysis, we improved the identification of molecular prion strains showing distinct molecular signatures reflecting clinicopathological phenotypes. Furthermore, we enhanced similarities between human and cattle prions in terms of molecular and physicochemical properties, giving, in some cases, possible clues about similar origin of the strain. Finally, such a rapid and informative biochemical analysis should be very helpful for large-scale epidemiological studies and surveillance in finding and discriminating strains (sCJD, vCJD, atypical BSEs).

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