J. Biol. Chem., Vol. 281, Issue 46,
35245-35252, November 17, 2006
Ultra-efficient Replication of Infectious
Prions by Automated Protein Misfolding Cyclic Amplification*
1From the
George and Cynthia
Mitchell Center for Alzheimer Disease and Related Neurodegenerative
Disorders, Departments of Neurology, Neuroscience and Cell Biology, and
Biochemistry and Molecular Biology, University of Texas Medical Branch,
Galveston, Texas 77555 and Centro
de Biología Molecular, Universidad Autónoma de Madrid, Madrid 28049, Spain
Prions are the unconventional
infectious agents responsible for transmissible spongiform
encephalopathies, which appear to be composed mainly or
exclusively of the misfolded prion protein (PrPSc).
Prion replication involves the conversion of the normal prion
protein (PrPC) into the misfolded isoform, catalyzed
by tiny quantities of PrPSc present in the infectious
material. We have recently developed the protein misfolding
cyclic amplification (PMCA) technology to sustain the autocatalytic
replication of infectious prions in vitro. Here we show that
PMCA enables the specific and reproducible amplification of
exceptionally minute quantities of PrPSc. Indeed, after seven
rounds of PMCA, we were able to generate large amounts of PrPSc
starting from a 1 x 10-12 dilution of scrapie hamster
brain, which contains the equivalent of
26 molecules of protein monomers.
According to recent data, this quantity is similar to the minimum
number of molecules present in a single particle of infectious
PrPSc, indicating that PMCA may enable detection of as
little as one oligomeric PrPSc infectious particle.
Interestingly, the in vitro generated PrPSc was
infectious when injected in wild-type hamsters, producing a
disease identical to the one generated by inoculation of the
brain infectious material. The unprecedented amplification
efficiency of PMCA leads to a several billion-fold increase of
sensitivity for PrPSc detection as compared with
standard tests used to screen prion-infected cattle and at least
4000 times more sensitivity than the animal bioassay. Therefore,
PMCA offers great promise for the development of highly
sensitive, specific, and early diagnosis of transmissible
spongiform encephalopathy and to further understand the molecular
basis of prion propagation.
Received for publication, April 25, 2006 , and in revised form, August 21, 2006.
* This work was supported in part by National Institutes of Health Grants AG0224642 and NS049173. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains
supplemental Fig. 1.
1 To whom correspondence should be addressed: Dept. of Neurology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555. Tel.: 409-747-0017; Fax: 409-7470020; E-mail: clsoto@utmb.edu .
http://www.jbc.org/cgi/content/abstract/281/46/35245?ct