Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis

Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133–134, 141, 152–153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.info:eu-repo/semantics/publishedVersio

Covalent Surface Modification of Prions: A Mass Spectrometry-Based Means of Detecting Distinctive Structural Features of Prion Strains

Prions (PrP^Sc) are molecular pathogens that are able to convert the isosequential normal cellular prion protein (PrP^C) into a prion. The only demonstrated difference between PrP^C and PrP^Sc 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 (<i>N</i>-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 strain-dependent 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

Mass Spectrometry-Based Method of Detecting and Distinguishing Type 1 and Type 2 Shiga-Like Toxins in Human Serum

Shiga-like toxins (verotoxins) are responsible for the virulence associated with a variety of foodborne bacterial pathogens. Direct detection of toxins requires a specific and sensitive technique. In this study, we describe a mass spectrometry-based method of analyzing the tryptic decapeptides derived from the non-toxic B subunits. A gene encoding a single protein that yields a set of relevant peptides upon digestion with trypsin was designed. The 15N-labeled protein was prepared by growing the expressing bacteria in minimal medium supplemented with 15NH4Cl. Trypsin digestion of the 15N-labeled protein yields a set of 15N-labeled peptides for use as internal standards to identify and quantify Shiga or Shiga-like toxins. We determined that this approach can be used to detect, quantify and distinguish among the known Shiga toxins (Stx) and Shiga-like toxins (Stx1 and Stx2) in the low attomole range (per injection) in complex media, including human serum. Furthermore, Stx1a could be detected and distinguished from the newly identified Stx1e in complex media. As new Shiga-like toxins are identified, this approach can be readily modified to detect them. Since intact toxins are digested with trypsin prior to analysis, the handling of intact Shiga toxins is minimized. The analysis can be accomplished within 5 h

Determining the Relative Susceptibility of Four Prion Protein Genotypes to Atypical Scrapie

Atypical scrapie is a sheep prion (PrP^Sc) disease whose epidemiology is consistent with a sporadic origin and is associated with specific polymorphisms of the normal cellular prion protein (PrP^C). To determine the relative amounts of PrP polymorphisms present in atypical scrapie, total PrP was digested with chymotrypsin to generate characteristic peptides spanning relevant polymorphisms at positions 136, 141, 154, 171, and 172 of sheep PrP^C. A multiple reaction monitoring method (MRM), employing ¹⁵N-labeled internal standards, was used to detect and quantify these polymorphisms present in both the PrP^Sc and PrP^C from heterozygous (ALRRY and ALHQY or ALRQD or AFRQY) atypical scrapie-infected or uninfected control sheep. Both polymorphisms of the full length and truncated (C1) natively expressed PrP^C are produced in equal amounts. The overall amount of PrP^C present in the infected or uninfected animals was similar. PrP^Sc isolated from heterozygotes was composed of significant amounts of both PrP polymorphisms, including the ALRRY polymorphism which is highly resistant to classical scrapie. Thus, an atypical scrapie infection does not result from an overexpression of sheep PrP^C. The replication of all atypical scrapie prions occurs at comparable rates, despite polymorphisms at positions 141, 154, 171, or 172

Quantitating PrP Polymorphisms Present in Prions from Heterozygous Scrapie-Infected Sheep

Scrapie is a prion (PrP^Sc) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep’s normal cellular prion protein (PrP^C). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic peptides [M₁₃₂LGS<b><u>X</u></b>MSRPL₁₄₁ (<b><u>X</u></b> = A or V), Y₁₅₃<b><u>X</u></b>ENMY₁₅₈ (<b><u>X</u></b>,= H or R), and Y₁₆₆RPVD<b><u>X</u></b>Y₁₇₂ (<b><u>X</u></b> = H, K, Q, or R)] that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrP^C or PrP^Sc. These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrP^Sc isolated from sheep heterozygous for their PrP^C proteins. The limit of detection for these peptides was less than 50 attomole. Spinal cord tissue from heterozygous (ARQ/VRQ or ARH/ARQ) scrapie-infected Rasa Aragonesa sheep was analyzed using this MRM method. Both sets of heterozygotes show the presence of both polymorphisms in PrP^Sc. This was true for samples containing both proteinase K (PK)-sensitive and PK-resistant PrP^Sc and samples containing only the PK-resistant PrP^Sc. These results show that heterozygous animals contain PrP^Sc that is composed of significant amounts of both PrP polymorphisms

Safe and Effective Means of Detecting and Quantitating Shiga-Like Toxins in Attomole Amounts

Shiga-like toxins (verotoxins) are a class of AB₅ holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (∼10 attomoles), which corresponded to a concentration of 1.7 femtomol/mL. A matrix effect was observed when dilute samples were digested in the buffer, Luria broth, or mouse plasma (LOD ∼ 30 attomol/injection = 5 femtomol/mL). In addition, we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins

Recombinant PrP^Sc shares structural features with brain-derived PrP^Sc: Insights from limited proteolysis

<div><p>Very solid evidence suggests that the core of full length PrP^Sc is a 4-rung β-solenoid, and that individual PrP^Sc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrP^Sc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133–134, 141, 152–153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrP^Sc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrP^Sc (recPrP^Sc). The term recPrP^Sc refers to <i>bona fide</i> recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrP^Sc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrP^Sc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrP^Sc preparations; similar fragments are characteristic of atypical strains of brain-derived PrP^Sc. Our results suggest a shared architecture of recPrP^Sc and brain PrP^Sc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrP^Sc. Furthermore, recombinant PrP^Sc offers exciting opportunities for structural studies unachievable with brain-derived PrP^Sc.</p></div

Analysis of PK-resistant fragments of recBVPrP^Sc.

<p>A) Coomassie-stained SDS-PAGE of recBVPrP^Sc treated with PK (see text for more details); numbered bands were excised, digested with trypsin in gel and analyzed as described in the Materials and Methods section. B) MALDI spectrum of the same PK-treated sample; the horizontal line in B indicates unassigned peaks perhaps corresponding to N-,C- doubly truncated fragments.</p

MALDI analysis of PK-resistant fragments in recBVPrP^Sc.

<p>MALDI analysis of PK-resistant fragments in recBVPrP^Sc.</p

Characterization of the infectivity of recPrP^Sc.

<p>A<b>)</b> Kaplan-Meier survival plots of Tga20 mice inoculated with recMoPrP^Sc. B) Histopathological and immunohistochemical analysis of brains fromTga20 mice inoculated with recMoPrP^Sc and uninoculated tga20 controls: left: haematoxylin-eosin (H&E) staining of the medulla oblongata, notice the spongiform lesion in the inoculated mice (bottom); right: PrP^Sc IHC staining (antibody 2G11, epitope: 151–159) of the medulla oblongata showing fine granular PrP^Sc deposits in the inoculated mice (bottom). C) WB showing the presence of PK-resistant PrP in the brains of Tga20 mice inoculated with recMoPrP^Sc.</p