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From: Terry S. Singeltary Sr. (
Subject: Re: New study shows how mad cow prions hitch a ride into intestine
Date: December 16, 2004 at 9:05 am PST

In Reply to: Re: New study shows how mad cow prions hitch a ride into intestine posted by TSS on December 15, 2004 at 12:20 pm:

-------- Original Message --------
Subject: Re: Protease-Resistant Human Prion Protein and Ferritin Are Cotransported across Caco-2 Epithelial Cells: Implications for Species Barrier in Prion Uptake from the Intestine
Date: Wed, 15 Dec 2004 16:33:52 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
References: <>

##################### Bovine Spongiform Encephalopathy #####################

Greetings list members,

THE full text is to long and complicated to send through the list
as html/text with all the bells and whistles attached, but thought
i might post the discussion below for some that might want to
comment on it. I would be most interested in comments on this

kind regards,


Here we provide insight into the pathway of PrPSc uptake and
transport across intestinal epithelial cells. In particular, our data
show that exposure of sCJD brain homogenate to DEs generates a
C-terminal PrPSc core of 2730 kDa that is transported across
Caco-2 cells in vesicular structures and that this process is not
influenced by the level of endogenous PrPC expression. Within
these vesicles, PrPSc is associated with ferritin, a major component
of the PrPScprotein complex, and remains associated with
ferritin after transcytosis. Because ferritin is normally absorbed
from food and is abundantly present in a typical meat dish, these
findings have important implications for prion uptake from contaminated
Using the well tested in vitro model for evaluating intestinal
uptake of selected food nutrients (Cereijido et al., 1978; Pinto et
al., 1983; Glahn et al., 1998), we show the resilience of PrPSc to
DEs and the facilitative effect of such treatment on PrPSc uptake
by Caco-2 cell monolayers. We noted that after treatment of
CJDH with stomach pepsin, PrPSc underwent limited proteolysis
and comigrated with the C-terminal PKresistant
core of PrPSc. Under similar conditions,
PrPC in the NH was completely
hydrolyzed. Much to our surprise, DEtreated
PrPSc was transported across
Caco-2 cells four times more efficiently
than PK-treated PrPSc. We believe that
this effect is attributable to the chaotropic
effect of bile salts that disperse PrPSccontaining
membrane phospholipds into
small micelles, preventing the aggregation
of PrPSc and facilitating its binding to epithelial
cells. This observation has significant
practical implications because there
could be qualitative and/or quantitative
differences in the digestive process between
individuals and certainly between
different species. Such differences, although
subtle and apparently trivial, may
influence host susceptibility to prion infection
from contaminated food.
Although purifying PrPSc from CJDH,
we noted that theHand L chains of ferritin
consistently cosediment with PrPSc. Resistance
of the PrPScferritin complex to elution
with low concentrations of salt and
coimmunoprecipitation with either anti-
PrP or anti-ferritin antibodies suggested
an association between the two proteins,
rather than coincidental sedimentation.
Remarkably, both the H and L chains of
ferritin resisted PK and DE treatment and
were associated with the protease-resistant
core of PrPSc. Electron microscopic examination
of the 8H4-immunoprecipitated
material revealed fibrils decorated with
ferritin aggregates. Although other proteins
were detected by silver staining of
8H4 and anti-ferritin immunoprecipitates
attesting to the remarkably sticky nature of
PrPSc, we believe that the association of
PrPSc with ferritin is stronger and is more
likely to be of biological significance. This
notion is based on the fact that after repeated
rounds of ultracentrifugation, only ferritin remained associated
with PrPSc, and the complex could be dissociated only
with 0.4 M NaCl. None of the other proteins copurified with
PrPSc, suggesting that their coimmunoprecipitation with PrPSc is
perhaps attributable to nonspecific interactions with the antibodies
or with PrPSc itself (Morel et al., 2004). Whether the association
of PrPSc and ferritin occurs in vivo or after homogenization
of brain tissue is unclear from our data. Nevertheless, this complex
is biologically significant because ingested PrPSc in contaminated
meat undergoes a process similar to homogenization and
DE treatment in the GI tract and is likely presented to the intestinal
epithelium in a complex with ferritin. Interestingly, the
-sheet-rich PrP peptide 106126 mixed with normal or CJD
homogenate was not transcytosed effectively, indicating that the
main determinant of PrPSc transport is not its -sheet-rich secondary
structure. Preincubation of PrP106126, NH, or CJDH
with exogenous purified ferritin did not facilitate the formation
of coimmunoprecipitable PrPferritin complexes, indicating
that the association of PrPSc with ferritin is more complex than a

mere hydrophobic interaction during the
process of homogenization. Regardless of
the nature and site of PrPScferritin complex
formation, this phenomenon is likely
to influence the absorption of ingested
PrPSc significantly, especially because ferritin
in ingested food is known to undergo
active absorption by the human intestinal
epithelium (Murray-Kolb et al., 2003;
Theil, 2003).
Our results show that the PrPScferritin
complex is endocytosed by Caco-2
cells in vesicular structures that fuse to
form phagosomes within the cell. Some of
these vesicles are transcytosed intact to the
BL chamber, much like the reported release
of PrPSc-containing exosomes into
the extracellular environment by epithelial
cells (Fevrier et al., 2004). Sensitivity of the
PrPScferritin transport to incubation at
low temperature and treatment with
brefeldin A and nocodazole suggest the involvement
of an active transport process
(Klausner et al., 1992). Although Caco-2
cells are known to endocytose ferritin, the
mechanistic details of this process remain
elusive (Murray-Kolb et al., 2003). Specific
receptors for ferritin have been reported
on liver cells, lymphocytes, erythroblasts,
oligodendrocytes, and on various cell lines
(Mack et al., 1983, Harrison and Arosio,
1996; Hulet et al., 2000). Our data demonstrating
significant inhibition of PrPSc
ferritin uptake in the presence of excess
ferritin derived from human liver, spleen,
or brain suggests the presence of a ferritinspecific
receptor or a transporter on
Caco-2 cells. The presence of such a receptor
on epithelial cells and the close association
of PrPSc and ferritin in digested food
incriminate ferritin as a possible transporter
of PrPSc across the intestinal epithelial
cell barrier.

Our data show that 3040% of ferritin from NH is consistently
transcytosed across Caco-2 cells without degradation. In
CJDH, this amount varies with the size of PrPSc-ferritin aggregates.
Small, detergent soluble complexes are transcytosed intact,
whereas large, detergent insoluble aggregates remain on the
monolayer in the AP chamber (our unpublished observations).
These large aggregates may be internalized via M-cells, FDCs, or
dendritic cells as reported previously (Heppner et al., 2001;
Huang et al., 2002). It is conceivable that endocytosed ferritin is
packaged in distinct vesicles that are either targeted to lysosomes
or transcytosed to the BL surface. The associated PrPSc in CJDH
probably follows both routes, although the majority appears to be
transcytosed because very little PrPSc was detected in cell lysates
(our unpublished observations). This assumption is supported
by the fact that a significant proportion of the PrPScferritin
complex remains intact after transcytosis, as evidenced by coimmunostaining
of endocytosed aggregates in M17 cells cultured in
the BL chamber. PrPC from untreated NH did not show significant
association with ferritin and was not transported to the BL
chamber in several experiments. However, ferritin from untreated
NH was detected consistently in the BL chamber (our
unpublished observations). Thus, either PrPC is not endocytosed
at all or is degraded within Caco-2 cells. A small amount of PrPSc
was detected occasionally independent of associated ferritin. It is
unclear whether this fraction is associated with another protein,
is transported independently, or results from dissociation of the
PrPScferritin complex in an intracellular compartment.
The notion that PrPSc is cotransported with ferritin ignores
the key requirements of host susceptibility to prion infection,
such as the level of PrPC expression and the extent of homology
between host PrPC and incoming PrPSc (Prusiner et al., 1990;
Weissmann et al., 2002; Thackray et al., 2003). Although in apparent
contradiction, our data suggest that the uptake of PrPSc
and its subsequent replication are distinct processes. The former
is independent of host PrPC, whereas the latter requires PrPC as
substrate for additional replication. This hypothesis is supported
by our data that show no influence of PrPC overexpression on
PrPSc transport across Caco-2 cells and by a recent report demonstrating
PrPC expression below the tight junctions of polarized
epithelial cells, making it physically impossible for incoming
PrPSc to come in contact with host PrPC (E. Morel et al., 2004a).

The cotransport of PrPSc with ferritin raises important questions
regarding prion uptake from contaminated food. Although
this report uses a homologous experimental setup, ferritinHand
L chains are known to share significant homology across species
(Harrison and Arosio, 1996) and may facilitate the transport of
PrPSc from distant species across the intestine. Because PrPSc is
notorious for its sticky nature, ferritin may be only one such
carrier protein. The identification and functional role of other
proteins associated with DE-treated PrPSc is important for fully
understanding the mechanism of PrPSc uptake from ingested
food and preventing a carrier state across species. Heterologous
PrPSc in such carriers may be transported to sites where it may
undergo conformational adaptation with time (Hill et al., 2000;
Race et al., 2001), or in the case of livestock, lie dormant until
ingested by a susceptible host. Such apparently healthy carriers
would disseminate PrPSc through a variety of means, posing a
potential threat to the general population...END

Figure 7. sCJDPrP Sc remains associated with ferritin after
transcytosis. A, Caco-2 cell monolayers were placed in a 12-well
culture dish containing M17 neuroblastoma cells cultured on coverslips
in the BL chamber, and biotinylated CJDH-DE was added
to the AP chamber (see Fig. 6 A). After an overnight incubation, Caco-2
cells on filters and M17 cells on coverslips were processed
for immunostaining. Immunoreaction of filters with anti-ZO-1 shows the
presence of tight junctions in all monolayers (green;
panels 1, 5). Immunostaining of M17 cells in the BL chamber for PrP
(green) and ferritin (red) shows colocalization of the two
proteins, indicating the presence of intact PrP Scferritin complexes
after transcytosis (panels2 4, arrows). Coimmunostaining of
M17 cells for PrP (green) and streptavidin (red) (panels 68) confirms
that the PrP signal is derived from biotinylated CJDH-DE in
the AP chamber. Scale bar, 10 m. B, Electron microscopic analysis of
the PrP Scferritin complex immunoprecipitated with 8H4
shows fibrillar structures and membranes decorated with ferritin
aggregates (top inset; arrows). When added to Caco-2 cells, the
complex is internalized in relatively large phagosome-like structures
surrounded by a single membrane (top; arrowheads). Some
of these vesicular structures are extruded out from the BL surface of
Caco-2 cells and are seen within the pore of the membrane
filter (bottom; arrowhead). (The internalized material in phagosomes
shows similar structures as observed in the immunoprecipitated
material.) Scale bar: 0.25 m; inset,1.5. N, Nucleus; T, tight junction.

Figure 8. The binding of sCJDPrP Scferritin to Caco-2 cells is
competitively inhibited by excess ferritin. A, Western blotting of
NH Pellet and CJDH Pellet fractions with 3F4 reveals no reactivity with
the NH sample but strong reactivity with N-terminally
truncated PrP Sc bands from the CJDH Pellet sample (lanes 1, 2).
Reblotting with anti-ferritin shows the presence of ferritin in both
NH and CJDH samples (lanes 3, 4). Longer exposure reveals high molecular
weight bands of PrP and ferritin that appear to
comigrate (lanes 5 8, arrow). B, Caco-2 cell monolayers were incubated
with biotin-tagged NH Pellet or CJDH Pelletbrain lipid
mixture and processed for staining with Texas Redstreptavidin. Both NH
Pellet and CJDH Pellet bind to the cell surface (panels 1, 2,
Ferritin), and the binding is inhibited by preincubation of the cells
with 1.5 g/ml human spleen ferritin (panels 3, 4,
C, Caco-2 cells were incubated with 25 l of biotinylated CJDH-DE with
no previous exposure to ferritin (Ferritin) or after
preincubation with 1.5 g/ml human spleen ferritin (
Ferritin) and processed for staining with Texas Redstreptavidin and
8H4 anti-mouse FITC (panels 1 6). Mock-treated cells show PrP-specific
immunoreactivity (green) that colocalizes with
streptavidin (red) (panels 13). However, preincubation with ferritin
abolishes PrP-specific staining significantly (panels 46).D,
Caco-2 cells with no previous treatment (panel 1,NH Pellet) or after
preincubation with NH Pelletbrain lipid mixture containing
human brain-derived ferritin (panel 2,
NH Pellet) were exposed to 25 l of CJDH-DE and immunostained with 8H4
FITC. Mock-treated cells show strong PrP-specific immunoreactivity
(panel 1), which is lost significantly after pretreatment of the
cells with NH Pellet (panel 2). Scale bar, 10 m.
10 " J. Neurosci., January 5, 2005 " 25(5):?? Mishra et al. " Transport
of Protease-Resistant PrP across Epithelial Cells
balt6/zns-neusci/zns-neusci/zns-orig/zns9802-05a simmsl S4 11/16/04
13:18 Art: 1192112 Input-YY(v)

Terry S. Singeltary Sr. wrote:

> ##################### Bovine Spongiform Encephalopathy
> #####################
> Neurobiology of Disease
> Protease-Resistant Human Prion Protein and Ferritin Are Cotransported
> across Caco-2 Epithelial Cells: Implications for Species Barrier in
> Prion Uptake from the Intestine
> Ravi Shankar Mishra, * Subhabrata Basu, * Yaping Gu, Xiu Luo, Wen-Quan
> Zou, Richa Mishra, Ruliang Li, Shu G. Chen, Pierluigi Gambetti,
> Hisashi Fujioka, and Neena Singh
> Institute of Pathology, Case Western Reserve University, Cleveland,
> Ohio 44106
> Foodborne transmission of bovine spongiform encephalopathy (BSE) to
> humans as variant Creutzfeldt-Jakob disease (CJD) has affected over
> 100 individuals, and probably millions of others have been exposed to
> BSE-contaminated food substances. Despite these obvious public health
> concerns, surprisingly little is known about the mechanism by which
> PrP-scrapie (PrPSc), the most reliable surrogate marker of infection
> in BSE-contaminated food, crosses the human intestinal epithelial cell
> barrier. Here we show that digestive enzyme (DE) treatment of sporadic
> CJD brain homogenate generates a C-terminal fragment similar to the
> proteinase K-resistant PrPSc core of 27-30 kDa implicated in prion
> disease transmission and pathogenesis. Notably, DE treatment results
> in a PrPSc-protein complex that is avidly transcytosed in vesicular
> structures across an in vitro model of the human intestinal epithelial
> cell barrier, regardless of the amount of endogenous PrPC expression.
> Unexpectedly, PrPSc is cotransported with ferritin, a prominent
> component of the DE-treated PrPSc-protein complex. The transport of
> PrPSc-ferritin is sensitive to low temperature, brefeldin-A, and
> nocodazole treatment and is inhibited by excess free ferritin,
> implicating a receptor- or transporter-mediated pathway. Because
> ferritin shares considerable homology across species, these data
> suggest that PrPSc-associated proteins, in particular ferritin, may
> facilitate PrPSc uptake in the intestine from distant species, leading
> to a carrier state in humans.
> Key words: prion infection; subclinical infection; PrP transport; new
> variant CJD; ferritin; epithelial cell barrier; Caco-2
> ------------------------------------------------------------------------
> Received July 15, 2004; revised October 27, 2004; accepted November 2,
> 2004.
> ##############
> ##############

############## ##############

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