Structural organization of mammalian prions as probed by limited proteolysis

Elucidation of the structure of PrP(Sc) continues to be one major challenge in prion research. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. Given that high resolution techniques such as NMR or X-ray crystallography cannot be used, a number of lower resolution analytical approaches have been attempted. Thus, limited proteolysis has been successfully used to pinpoint flexible regions within prion multimers (PrP(Sc)). However, the presence of covalently attached sugar antennae and glycosylphosphatidylinositol (GPI) moieties makes mass spectrometry-based analysis impractical. In order to surmount these difficulties we analyzed PrP(Sc) from transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. Such animals produce prions that are devoid of the GPI anchor and sugar antennae, and, thereby, permit the detection and location of flexible, proteinase K (PK) susceptible regions by Western blot and mass spectrometry-based analysis. GPI-less PrP(Sc) samples were digested with PK. PK-resistant peptides were identified, and found to correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. The first 10 peptides (to position 153), match very well with PK cleavage sites we previously identified in wild type PrP(Sc). These results reinforce the hypothesis that the structure of PrP(Sc) consists of a series of highly PK-resistant β-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrP(Sc) is highly resistant to PK and therefore perhaps also contains β-sheet secondary structure

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

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

We employed a sensitive mass spectrometry-based method to deconstruct, confirm, and quantitate the prions present in elk naturally infected with chronic wasting disease and sheep naturally infected with scrapie. We used this approach to study the oxidation of a methionine at position 216 (Met216), because this oxidation (MetSO216) has been implicated in prion formation. Three polymorphisms (Ile218, Val218, and Thr218) of sheep recombinant prion protein were prepared. Our analysis showed the novel result that the proportion of MetSO216 was highly dependent upon the amino acid residue at position 218 (I > V > T), indicating that Ile218 in sheep and elk prion protein (PrP) renders the Met216 intrinsically more susceptible to oxidation than the Val218 or Thr218 analogue. We were able to quantitate the prions in the attomole range. The presence of prions was verified by the detection of two confirmatory peptides: GE­N­F­T­E­T­D­IK (sheep and elk) and ES­Q­A­Y­Y­QR (sheep) or ES­E­A­Y­Y­QR (elk). This approach required much smaller amounts of tissue (600 μg) than traditional methods of detection (enzyme-linked immunosorbent assay, Western blot, and immunohistochemical analysis) (60 mg). In sheep and elk, a normal cellular prion protein containing MetSO216 is not actively recruited and converted to prions, although we observed that this Met216 is intrinsically more susceptible to oxidation

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

Western blot analysis of PK-resistant GPI

<p>⁻<b>PrP^Sc.</b> Unpurified GPI^- PrP^Sc was treated with 25 µg/ml of PK and subsequently deglycosylated with PNGase F. Samples were resolved on Tricine-SDS-PAGE and probed with the monoclonal antibodies, #51 (lane 1), W226 (lane 2), and R1 (lane 3).</p

Kinetics of PK digestion of unpurified GPI

<p>⁻<b>PrP^Sc.</b> Samples were digested with PK (25 µg/ml) and the reaction stopped after 0, 30, 60, 120, 180, 240, 300 and 360 minutes. Samples were treated with PNGase F and subjected to Tricine-SDS-PAGE the blot was probed with R1 antibody.</p

Western blot of PK-digested series of GPI⁻ PrP^Sc samples following partial unfolding by guanidine HCl.

<p>After guanidine partial unfolding with 0 M, 0.5 M, 1 M, 2 M, 3 M and 4 M and PK treatment (25 µg/ml), the samples were treated with PNGase F and resolved on Tricine-SDS-PAGE. The WB was probed with the R1 antibody.</p

Characterization of GPI^- PrP^Sc. A.

<p>Western blot of brain homogenate from scrapie-infected GPI⁻ tg mouse before and after digestion with PK (25 µg/ml); WB probed with SAF83 antibody. <b>B.</b> Histopathological and immunohistochemical analyses of scrapie-infected GPI⁻ tg mouse brain. (a) Haematoxylin-eosin staining of the hippocampal formation. (b) IHC staining (antibody 6H4) of the hippocampal formation. <b>C.</b> Kaplan-Meier survival curves of wild-type mice (C57BL/6) inoculated with 2% of brain homogenate from scrapie-infected GPI⁻ PrP^Sc (green line) and a negative control inoculated with PBS (red line).</p

MALDI-TOF spectrum of PK-treated purified GPI

<p>⁻<b>PrP^Sc.</b> Doubly-charged ions from peptides with m/z 16371 and 17148 are indicated (*). Low resolution in the >16 kDa region precluded identifying unmarked peaks. A scheme of GPI^- PrP sequence with PK cleavage points (color coded) and secondary structure of PrP^C is included at the top: (octarepeats (□), β-sheets (▸), and α-helices (∥)); epitopes of the mAbs used are also indicated.</p

PK-sensitive PrP^Sc Is Infectious and Shares Basic Structural Features with PK-resistant PrP^Sc

<div><p>One of the main characteristics of the transmissible isoform of the prion protein (PrP^Sc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP^Sc following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrP^Sc that is sensitive to PK hydrolysis (sPrP^Sc). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrP^Sc versus the PK-resistant (rPrP^Sc) fractions of PrP^Sc and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrP^Sc and rPrP^Sc fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrP^Sc to the rPrP^Sc forms of PrP^Sc. Although the sPrP^Sc and rPrP^Sc fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.</p> </div