Cell-free conversion

Kocisko DA, Priola SA, Raymond GJ, Chesebro B, Lansbury PT Jr, Caughey B. Species specificity in the cell-free conversion of prion protein to protease-resistant forms a model for the scrapie species barrier. Proc Natl Acad USA 1995 92 3923-3927. 

The ability of PrPres to induce the conversion of PrPc to PrPres was initially demonstrated in cell-free reactions in which brain-derived PrPres was incubated with radioactively labeled PrPc, which, under suitable conditions, bound to the PrPres and became similarly partially protease-resistant [2], These first generation cell-free conversion (CFC) reactions were shown to be highly specific in ways that correlated with prion transmission barriers [3-7] and strains [8]. However, the newly generated PrPres was usually substoichiometric relative to the initial PrPres seed, and was not demonstrably associated with new infectivity [9]. As a result, these CFC reactions (reviewed in [10]) were not suitable for sensitive detection of PrPres or prions. 

Early efforts to recapitulate PrPSc formation and mammalian prion propagation in vitro included cell-free conversion reactions in which PrPc was incubated in the presence of PrPSc from TSE-affected animals [85, 172], Such simple incubation resulted in species- and strain-specific conversion of substrate PrPc to a PrPSc-like conformation (as judged by very similar PK resistance) [172], The yields of these conversion reactions were, however, very low, typically substoichiometric with respect to the input PrPSc template. Furthermore, no infectivity could be attributed to the newly converted material [173], indicating that not every PK resistant form of the prion protein is necessarily associated with prion infectivity. 

Fig. 4. PrP-res isolated from hamsters infected with different scrapie strains can determine the final conformation of the protease-resistant product. Radiolabeled hamster PrP-sen without the GPI anchor was mixed with hamster PrP-res isolated from the brains of hamsters infected with either hyper (Hy) or drowsy (Dy) scrapie in a cell-free conversion assay (Kocisko et al, 1994). The PrP-res was either pre-treated (+) or not (-) with proteinase K to remove any contaminating proteins (Bessen et al, 1995). The left side panel shows the radiolabeled PrP-sen in the reaction after a 2-day incubation and before digestion with proteinase K (-PK) and represents 10% of the total reaction. The right side panel shows the radiolabeled, protease-resistant product remaining after digestion of the reaction with proteinase K (+PK) and represents the remaining 90% of the reaction. Note the characteristic size shift of 1 to 2 kDa which is used to distinguish PrP-res from the hyper and drowsy scrapie strains (Bessen and Marsh, 1992a). There is no significant difference in the amount of protease-resistant product formed when PK pretreated or untreated PrP-res is used (53% vs. 48% for hyper PrP-res and 47% vs. 40% for drowsy PrP-res). Molecular mass markers are shown in kilodaltons on the right.

The products of cell-free conversion reactions have not yet been shown to be infectious (Hill et al, 1999 C.J. Raymond, D. Kocisko and... 

Fig. 6. In situ conversion reaction in brain slices on glass slides (Bessen et al., 1997). A distinct autoradiographic image is seen with scrapie-infected, but not uninfected control, brains. If the brain slices are solubilized after the conversion reaction and analyzed by SDS-PAGE/autoradiography, then PK-resistant S-PrP conversion products similar to those produced in cell-free conversion reactions (see Fig. 5) are observed. Higher magnification images (bottom panels) show that the pattern of in situ conversion product closely matches that of immunohistochemical staining for PrP-res in regions known to contain either amyloid plaques or diffuse apparently nonamyloid deposits.

Fig. 7. Stimulation of cell-free conversion reaction by heparan sulfate. In the absence of input (unlabeled) PrP-res there is no formation of newly formed S-PrP-res (lane 1). In the presence of input PrP-res, there is an 8-fold stimulation of S-PrP-res formation by 100 pg/ml heparan sulfate (compare lanes 2 and 3). S-PrP-sen representing 20% of the input radioactivity is shown in lane 4 without PK digestion. Adapted from Wong et al, (2001).

Evidence that the two strain-associated conformations of hamster PrP-res could propagate themselves from the same hamster PrP-sen precursor was obtained using a cell-free conversion reaction (Fig. IB) (Bessen et al, 1995). In these studies, HY and DY PrP-res were each incubated with hamster PrP-sen, and the PrP-sen was converted to PK-resistant PrP products with the same l-2 kDa difference in molecular mass that distinguishes the PK-treated HY and DY PrP-res molecules. This finding suggested that the strain-specific forms of PrP-res are faithfully propagated through direct PrP-sen-PrP-res interactions both in vitro and in vivo as had been proposed earlier (Bolton and Bendheim, 1988 Prusiner, 1991 Bessen and Marsh, 1994). 

The influence of chemical chaperones has also been demonstrated in cell-free conversion assays (DebBurman etal, 1997). The conversion of hamster PrP using partially denatured PrP was only inhibited by DMSO. Glycerol and cyclodextrin compounds had no effect, whereas molecular chaperones (Hspl04) were able to block the conversion process. Chemical chaperones such as glycerol and cyclodextrin, acting as co-chaperones, might have an influence on molecular chaperones that are lacking in a cell-free system. 

In order to demonstrate cell-free conversion of futalosine (8) to the expected hypoxanthine (36), an SC04327 (MqnB) recombinant was prepared and assayed for enzymatic activity. However, no enzymatic activity could be detected and the failure was attributed to the inherent instability of the enzyme. Hence, these authors expressed and assayed an ortholog of SC04327 (MqnB) from the thermophilic bacterium... 

T. thermophilus (TTHA1092) failed to convert DHFL to cyclic DHFL. The lack of success in demonstrating cell-free conversions has been attributed to the lack of optimization of assay condition or requirement for additional enzymes or cofactors. ... 

Figure 13 Cell-free conversion to the alkaloids with sarpagan bridge (C-5/C-16 bond) which is typical of all sarpagan- and ajmalan-type alkaloids.

Rueckert PW. Wiley PF, McGovern jP, Marshall VP. Mammalian and microbial cell-free conversion of anthracycline antibiotics and analogs. J Antibiot 1979 32 141-147. 

Saini, M.S. and Anderson, J.A. (1978) Cell-free conversion of 4-y, y-dimethylallyltryptophan to 4-[4-hydroxy-methyl-A butenyl]tryptophan in Claviceps purpurea PRL 1980. Phytochemistry, 17, 799-800. 

Cell-Free Conversion of 8-(L-a-Aminoadipyl)-L-cysteinyl-D-valine into an Antibiotic with the Properties of Isopenicillin N in Cephalosporium acremonium. Biochem. J. 184, 427 (1979). 

Baldwin, J. E., B. L. Johnson, J. J. Usher, E. P. Abraham, J. A. Huddleston, and R. L. White Direct N.M.R. Observation of Cell-Free Conversion of (L-a-Amino-8-adipyl)-L-cysteinyl-D-valine into Isopenicillin N. J. C. S. Chem. Commun. 1980, 1271. 

Anderson and Saini (1974) isolated 8 from Claviceps. The compound was found to be identical by mass spectroscopy and chromatography with reference to -8. Cell-free conversion of 7 to 8 has been obtained by Petroski and Kelleher (1977) and by Saini and Anderson (in press). The above results indicate that there probably is a pathway in Claviceps 7 8 -> elymoclavine... 

Cell-free conversion of the precursors into the tetracyclic clavines and chanoclavine I and II was obtained by Cavender and Anderson (1970). Incubation of [ " Cjtryptophan, isopentenyl pyrophosphate, methionine, ATP, and liver concentrate (mixture of cofactors) with the 60-80% ammonium sulfate fraction from C. purpurea PRL 1970 for 12 hr gave radioactive chanoclavine I and II, agroclavine, and elymoclavine. Conversion was 0.15% for chanoclavine I and II. 

E, Cell-Free Conversion of Dimethylallyltryptophan to Clavicipitic Acid... 

With slant cultures of an Elymus-type Claviceps strain, Ohashi and Abe (1970) obtained cell-free conversion of agroclavine and elymoclavine to peptide alkaloids. There have been no reports so far of cell-free peptide alkaloid synthesis with shake cultures. Groger s laboratory (Maier et al., 1972) studied the activation reactions that could be involved in peptide alkaloid biosynthesis. They observed cell-free synthesis of lysergyl-CoA and activation of valine, serine, leucine, and proline. Activation was found in nonproducing, clavine alkaloid-producing, and peptide alkaloid-producing strains. The connection between these activities and peptide alkaloid synthesis is therefore uncertain. 

Cephaloaporim aaremonium.- After much effort, the cell-free conversion of 6-(L-a-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV-tripep-tide) to isopenicillin N has been achieved. This transform-... 

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