Recombinant Human Prion Protein Inhibits Prion Propagation in vitro
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Prion Propagation in vitro Jue Yuan,1, 3, 15 Yi-An Zhan,1, 7, 15 Romany
Abskharon,5, 6, 14, 15 Xiangzhu Xiao,1, 3 Manuel Camacho Martinez,1, 3 Xiaochen
Zhou,1, 7 Geoff Kneale,8 Jacqueline Mikol,9 Sylvain Lehmann,10 Witold K.
Surewicz,13 Joaquín Castilla,12 Jan Steyaert,5, 6 Shulin Zhang,1 Qingzhong
Kong,1, 2, 3 Robert B. Petersen,1, 2, 11 Alexandre Wohlkonig5, 6 & Wen-Quan
Zou1, 2, 3, 4, 7 Affiliations Contributions Corresponding authors Journal name:
Scientific Reports Volume: 3, Article number: 2911 DOI: doi:10.1038/srep02911
Received 31 May 2013 Accepted 24 September 2013 Published 09 October 2013
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Recombinant Human Prion Protein Inhibits Prion Propagation in vitro
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Prion diseases are associated with the conformational conversion of the
cellular prion protein (PrPC) into the pathological scrapie isoform (PrPSc) in
the brain. Both the in vivo and in vitro conversion of PrPC into PrPSc is
significantly inhibited by differences in amino acid sequence between the two
molecules. Using protein misfolding cyclic amplification (PMCA), we now report
that the recombinant full-length human PrP (rHuPrP23-231) (that is
unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong
inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits
mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds
to PrPSc, but not PrPC, suggesting that the inhibitory effect of recombinant PrP
results from blocking the interaction of brain PrPC with PrPSc. Our findings
suggest a new avenue for treating prion diseases, in which a patient's own
unglycosylated and anchorless PrP is used to inhibit PrPSc propagation without
inducing immune response side effects.
Subject terms: Neurodegeneration Medical research
snip...
Previous Figures index Discussion Introduction• Results• Discussion•
Methods• References• Acknowledgements• Author information• Supplementary
information The in vitro and in vivo conversion efficiency of PrPC into PrPSc
can be significantly affected by the presence of additional PrP molecules that
differ from the endogenous PrPC by as little as one residue6, 28, 11, 12. The
present study now demonstrates that a PrP molecule that shares the identical
amino acid sequence with the PrPC substrate and PrPSc template also causes
interference. It is worth noting that although the amino acid sequence is
identical, recombinant PrP does not contain N-linked glycans or a GPI anchor.
Our new results suggest that in addition to the amino acid sequence,
glycosylation and the GPI anchor are important in mediating the conversion of
PrPC into PrPSc. The unglycosylated and anchorless recombinant PrP appears to
act as an inhibitor of the conversion process by preferentially binding to
PrPSc.
PrPC is a glycoprotein with two non-obligatory, N-linked glycosylation
sites at residues 181 and 197 and a GPI anchor24, 29, 30. Binding of
heterologous PrPC to PrPSc can be influenced by PrPC glycosylation in a
species-specific manner31, 32. Moreover, using PMCA and a scrapie cell assay,
Nishina et al reported that the stoichiometry of host PrPC glycoforms modulates
the efficiency of PrPSc formation in vitro17. Specifically, their study
demonstrated that while unglycosylated PrPC is required to propagate mouse RML
prions, in a similar reaction, amplification of hamster Sc237 prions is
inhibited by substoichiometric levels of homologous unglycosylated PrPC. This
study provides direct in vitro evidence that changes in the PrP glycoform ratios
can affect the efficiency of PrPSc formation in a species-specific manner.
Recently, we observed glycoform-selective prion formation in unique sporadic and
inherited forms of Creutzfeldt-Jakob disease (CJD) including variably
protease-sensitive prionopathy (VPSPr) and familial CJD linked to a valine to
isoleucine mutation at residue 180 (fCJDV180I)16. Although all four glycoforms
are present, including di-, monoglycosylated at residue 181 (mono-181),
monoglycosylated at residue 197 (mono-197), and unglycosylated PrP forms in the
brain of VPSPr and fCJDV180I, only the mono-197 and unglycosylated PrP species
were converted into PrPSc. The mono-181 and diglycosylated PrP species were not
converted into PrPSc in the cerebral cortical brain areas examined. Moreover,
the level of the classic PK-resistant PrPSc probed with the 3F4 antibody was
significantly decreased compared to typical sporadic CJD. Instead, a unique
five-step ladder-like electrophoretic profile of PK-resistant PrPSc was detected
in both diseases by the 1E4 antibody16. In contrast to the threonine to alanine
mutation at residue 183 of PrP (PrPT183A), the PrPV180I mutation exhibits a
typical PrP glycosylation profile, although there is no detectable mono-181 and
diglycosylated PrPSc33, 16. However, using the N-linked glycosylation prediction
algorithm NetNGlyc 1.0 at http://www.cbs.dtu.dk/services/NetNGlyc/34,
we predicted a slight decrease in the glycosylation potential at N181 in
PrPV180I compared to PrPWt (0.597 vs 0.664) while no potential change was
predicted at all for N181 in PrPT183A16. The prediction data suggests that
although the T183A mutation completely eliminates the N181 glycosylation site,
the V180I mutation may merely alter the glycan composition at N181, which
modifies the ratio of the four PrP glycoforms in the PrP mixture. Further
investigation into the mechanism, by which altered glycosylation affects both
conversion efficiency of PrPC into PrPSc and PrPSc conformation, is warranted.
Unglycosylated and anchorless recombinant human PrP may have greater affinity
for PrPSc seeds compared to the brain PrPC, but it is a poor substrate for
conversion into PrPres by the standard PMCA protocol or in ScN2a cells. Indeed,
both recombinant hamster and mouse PrP are not converted into PK-resistant PrP
by serial PMCA in the presence of hamster prion Sc237 or mouse prion RML,
respectively17. It is worth noting, however, that the recombinant PrP could be
converted into PrPres using a modified PMCA protocol in which the conversion
buffer contained 0.1% SDS and the normal brain-derived PrPC was replaced by
recombinant hamster PrP as a substrate35, 36 and the product of this reaction
was proved to be infectious in animal bioassays37. Furthermore, in the absence
of brain homogenates, recombinant PrP was converted by PMCA to highly infectious
prions in the presence of additional cofactors such as phosphatidylglycerol and
RNA38 or phosphatidylethanolamine39. Prion infectivity was also produced in
Syrian hamsters by inoculating full-length recombinant hamster PrP that was
converted into a cross-β-sheet amyloid conformation and subjected to an
annealing procedure40.
We cannot rule out the possibility that the inhibition of human PrPSc
amplification by recombinant human PrP results from the lack of the GPI anchor,
although the GPI anchor of PrPC is believed to have little or no effect on the
formation of PK-resistant PrP31, 32. Anchorless PrP generated in either cultured
mammalian cells or E. coli are converted to PK-resistant PrP by a cell-free
conversion approach41, 42, 43, 44. Moreover, it has been reported that
anchorless prion protein induced an infectious amyloid disease in transgenic
animals, although the animal themselves were asymptomatic45. However,
amplification of hamster PrPSc using a standard PMCA protocol is inhibited when
the substrate of normal hamster brain PrPC was pretreated with
phosphatidylinositol-specific phospholipase C (PIPLC) to remove the GPI
anchor19. Furthermore, recombinant hamster PrP was previously shown to inhibit
PMCA of hamster PrPSc using the normal hamster brain homogenate as a
substrate18. Kim et al proposed that both of these effects are due to the lack
of the GPI anchor in PIPLC-treated PrPC or recombinant PrP19. On the other hand,
the unglycosylated hamster PrPC purified from brains and containing an intact
GPI anchor likewise inhibits amplification of Sc237 prions17. Therefore, the
role of GPI anchor in the inhibition of human and mouse PrPSc propagation by
recombinant human PrP observed in our study remains to be determined.
We demonstrated that recombinant human and other PrP that exhibited 50% or
greater inhibition of PrPSc formation in a PMCA reaction bind to human PrPSc but
not to PrPC. Although full-length, N- or C-terminally truncated recombinant PrP
all bind to PrPSc efficiently, the full-length rHuPrP23-231 exhibits the highest
inhibition efficiency compared to the two truncated forms, suggesting that the
inhibition involves both N- and C-terminal domains. Moreover, antibodies
including SAF32, 3F4, and 6H4 directed against PrP regions covering residues 59
to 152 showed less than 10% inhibition. The 8H4 antibody against human
PrP175-185 exhibited virtually no inhibition. These results are in good
agreement with a previous report by Horiuchi and Caughey1. In addition, it has
been shown that the 3F4 and 6H4 antibodies preferentially bind to native PrPC,
although they also detect denatured PrPSc on Western blots46, 47. Therefore, the
interaction of inhibitors with PrPSc may be required for the inhibition of PrPC
conversion. We observed that recombinant mouse PrP is also able to bind to human
PrPSc (Figure S2), although it caused significantly less inhibition compared to
recombinant human PrP. Moreover, the anti-DNA antibody that specifically
captures PrPSc but not PrPC showed less than 10% inhibition while g5p caused
more than 50% inhibition. This suggests that the different inhibitors have
distinct binding sites on PrPSc: one class of sites is specifically associated
with recruiting PrPC while the other is not. Recombinant human PrP is likely to
compete with brain PrPC for the same site on the PrPSc molecule. Moreover, its
affinity for PrPSc seems to be greater than that of brain-derived PrPC.
Interestingly, a two-site model has been proposed by Horiuchi and co-workers to
explain the molecular mechanism for sequence-difference interference28.
According to this model, PrPSc has two types of PrPC binding sites: one is able
to induce conversion to PrPSc while the other is not.
A recombinant mouse PrP with a substitution of lysine for glutamine at
mouse codon 218 (rPrP-Q218K), corresponding to human PrPE219K, an Asian-specific
polymorphism believed to be resistant to CJD infection, considerably prolonged
incubation time of prion infection in an iatrogenic mouse model48. Recombinant
mouse PrP was delivered into the mouse brain for 7 days by
intracerebroventricular administration using an indwelling catheter connected to
an implanted osmotic pump. The same group also found that rPrP-Q218K reduced
PrPSc formation in ScN2a cells. However, using wild-type mouse PrP did not cause
inhibition, which is different than our findings. This discrepancy may be due to
different experimental conditions between the studies. We showed that murine
PrPSc amplification was inhibited in both PMCA and ScN2a by unglycosylated and
anchorless recombinant human PrP. Most importantly, since the amino acid
sequence of recombinant human PrP is identical to that of human brain PrPC, it
is expected that this protein would not elicit an immune response after
intracerebroventricular administration while it inhibits PrPSc propagation.
Therefore, our findings suggest a new therapeutic strategy for treating human
prion diseases.
snip...see full text ;
Recombinant Human Prion Protein Inhibits Prion Propagation
Case Western Reserve Researchers Discover New Avenue for Preventing and
Treating Fatal Brain Disease
News Release: October 9, 2013
Jessica Studeny
216-368-4692
Jessica.studeny@case.edu
Case Western Reserve University researchers today published findings that
point to a promising discovery for the treatment and prevention of prion
diseases, rare neurodegenerative disorders that are always fatal. The
researchers discovered that recombinant human prion protein stops the
propagation of prions, the infectious pathogens that cause the diseases.
“This is the very first time recombinant protein has been shown to inhibit
diseased human prions,” said Wen-Quan Zou, MD, PhD, senior author of the study
and associate professor of pathology and neurology at Case Western Reserve
School of Medicine.
Recombinant human prion protein is generated in E. coli bacteria and it
has the same protein sequence as normal human brain protein. But different in
that, the recombinant protein lacks attached sugars and lipids. In the study,
published online in Scientific Reports, researchers used a method called protein
misfolding cyclic amplification which, in a test-tube, mimics the prions’
replication within the human brain. The propagation of human prions was
completely inhibited when the recombinant protein was added into the test-tube.
The researchers found that the inhibition is dose-dependent and highly specific
in responding to the human-form of the recombinant protein, as compared to
recombinant mouse and bovine prion proteins. They demonstrated that the
recombinant protein works not only in the cell-free model but also in cultured
cells, which are the first steps of translational research. Further, since the
recombinant protein has an identical sequence to the brain protein, the
application of the recombinant protein is less likely to cause side effects.
Prion diseases are a group of fatal transmissible brain diseases affecting
both humans and animals. Prions are formed through a structural change of a
normal prion protein that resides in all humans. Once formed, they continue to
recruit other normal prion protein and finally cause spongiform-like damage in
the brain. Currently, the diseases have no cure.
Previous outbreaks of mad cow disease and subsequent occurrences of the
human form, variant Creutzfeldt–Jakob disease, have garnered a great deal of
public attention. The fear of future outbreaks makes the search for successful
interventions all the more urgent.
Zou, who also serves as the associate director of the National Prion
Disease Pathology Surveillance Center at Case Western Reserve, and collaborators
hope to extend their finding using transgenic mice expressing the human prion
protein and patient-specific induced pluripotent stem cells (iPSCs)-derived
neurons because they are made from human cells, offering an additional level of
authenticity. The new animal models were generated in collaboration with Case
Western Reserve School of Medicine faculty members, Robert Petersen, PhD, and
Qingzhong Kong, PhD, who are the co-authors in this study. Further,
patient-specific iPSCs-derived neurons have also just been generated in
collaboration with fellow faculty, Paul Tesar, PhD, and Xin Qi, PhD.
###
About Case Western Reserve University School of Medicine Founded in 1843,
Case Western Reserve University School of Medicine is the largest medical
research institution in Ohio and is among the nation's top medical schools for
research funding from the National Institutes of Health. The School of Medicine
is recognized throughout the international medical community for outstanding
achievements in teaching. The School's innovative and pioneering Western
Reserve2 curriculum interweaves four themes--research and scholarship, clinical
mastery, leadership, and civic professionalism--to prepare students for the
practice of evidence-based medicine in the rapidly changing health care
environment of the 21st century. Eleven Nobel Laureates have been affiliated
with the school.
Annually, the School of Medicine trains more than 800 M.D. and M.D./Ph.D.
students and ranks in the top 25 among U.S. research-oriented medical schools as
designated by U.S. News & World Report's "Guide to Graduate Education."
The School of Medicine's primary affiliate is University Hospitals Case
Medical Center and is additionally affiliated with MetroHealth Medical Center,
the Louis Stokes Cleveland Department of Veterans Affairs Medical Center, and
the Cleveland Clinic, with which it established the Cleveland Clinic Lerner
College of Medicine of Case Western Reserve University in 2002.
Saturday, March 5, 2011
MAD COW ATYPICAL CJD PRION TSE CASES WITH CLASSIFICATIONS PENDING ON THE
RISE IN NORTH AMERICA
Sunday, February 12, 2012
National Prion Disease Pathology Surveillance Center Cases Examined1
(August 19, 2011) including Texas
Tuesday, June 26, 2012
Creutzfeldt Jakob Disease Human TSE report update North America, Canada,
Mexico, and USDA PRION UNIT as of May 18, 2012
type determination pending Creutzfeldt Jakob Disease (tdpCJD), is on the
rise in Canada and the USA
Sunday, August 11, 2013
Creutzfeldt-Jakob Disease CJD cases rising North America updated report
August 2013
*** Creutzfeldt-Jakob Disease CJD cases rising North America with Canada
seeing an extreme increase of 48% between 2008 and 2010
Monday, August 26, 2013
***The Presence of Disease-Associated Prion Protein in Skeletal Muscle of
Cattle Infected with Classical Bovine Spongiform Encephalopathy
Monday, September 02, 2013
Atypical BSE: role of the E211K prion polymorphism
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF
TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES
Location: Virus and Prion Research Unit
Sunday, September 1, 2013
Evaluation of the Zoonotic Potential of Transmissible Mink Encephalopathy
We previously described the biochemical similarities between PrPres derived
from L-BSE infected macaque and cortical MM2 sporadic CJD: those observations
suggest a link between these two uncommon prion phenotypes in a primate model
(it is to note that such a link has not been observed in other models less
relevant from the human situation as hamsters or transgenic mice overexpressing
ovine PrP [28]). We speculate that a group of related animal prion strains
(L-BSE, c-BSE and TME) would have a zoonotic potential and lead to prion
diseases in humans with a type 2 PrPres molecular signature (and more
specifically type 2B for vCJD)
snip...
Together with previous experiments performed in ovinized and bovinized
transgenic mice and hamsters [8,9] indicating similarities between TME and
L-BSE, the data support the hypothesis that L-BSE could be the origin of the TME
outbreaks in North America and Europe during the mid-1900s.
Tuesday, September 24, 2013
NORDION (US), INC., AND BIOAXONE BIOSCIENCES, INC., Settles $90M Mad Cow
TSE prion Contamination Suit Cethrin(R)
Case 0:12-cv-60739-RNS Document 1 Entered on FLSD Docket 04/26/2012 Page 1
of 15
Wednesday, September 25, 2013
Inspections, Compliance, Enforcement, and Criminal Investigations BSE TSE
PRION 2013
Tuesday, July 2, 2013
APHIS USDA Administrator Message to Stakeholders: Agency Vision and Goals
Eliminating ALL remaining BSE barriers to export market
Friday, August 16, 2013
*** Creutzfeldt-Jakob disease (CJD) biannual update August 2013 U.K. and
Contaminated blood products induce a highly atypical prion disease devoid of
PrPres in primates
Wednesday, October 09, 2013
WHY THE UKBSEnvCJD ONLY THEORY IS SO POPULAR IN IT'S FALLACY, £41,078,281
in compensation
WHO WILL WATCH THE CHILDREN !
Saturday, September 21, 2013
Westland/Hallmark: 2008 Beef Recall A Case Study by The Food Industry
Center January 2010 THE FLIM-FLAM REPORT
TSS
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