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20% OptiMEM, and 70% FBS and stored m hqutd nitrogen
The potential mstabihty of the infection requires that Sc+MNB cultures rou-
tinely be monitored for the production of PrP-res. This can be done by lysing a
monolayer of cells m a lysmg buffer (LB) containing 0 5% Triton X-100 and
0.5% sodium deoxycholate (1 mL/25 cm* flask), clearing the lysate of debris
with a 5-min centrtfugation at 1OOOg, drgestmg the supernatant with 50 pg/mL
protemase K for 30 mm at 37OC, pelleting the PrP-res at 350,OOOg for 45 min,
and analyzing the pellet for PrP-res by immunoblot (10).
1.1 2. Metabolic Labeling and lmmunopreciprtation of PrP
Many types of analyses of PrP metabolism require labeling various forms of
PrP with [35S]methionme and/or [35S]cysteme m live cells followed by tmmu-
PrP Metabolism and PrP-res Formation 287

noprecipitation of the protein from cell lysates. Our usual procedure for MNB
cells involves preincubating the cells in methionine and cysteine-deficient
MEM (supplemented with 1% dialyzed FBS) for 1 h and incubating with
Expre35S35S(DuPont NEN, Boston, MA) label (0.1-I mCi/25 cm* flask of
60-80% confluent cells) for various time periods (typically, 2 h) in this same
medium. The cells can then be washed and lysed in ice-cold LB immediately
or after a chase incubation in full-methionine/cysteine medium.
The lysates are cleared by centrifugation for 5 min at 1OOOg 4°C and the
at
proteins precipitated with 4 vol of methanol at -20°C for 21 h. The precipi-
tated proteins are resuspended by cuphorn sonication into DLPC buffer (I I)
(1 mL/25 cm* flask equivalent of cell proteins). The appropriate PrP-specific
antibody is incubated with the suspension and antibody-antigen complexes plus
free antibodies are collected by binding to protein A sepharose beads. The
beads are washed prior to elution and SDS-PAGE/fluorography. The stringency
of the washes can be adjusted for different antibodies to minimize nonspecific
background without destabilizing the protein A-antibody-PrP complex. For
the selective immunoprecipitation of PrP-res, the cleared lysate can be treated
with PK as described earlier to eliminate PrP-sen prior to methanol precipi-
tation. Alternatively, the PrP-res can be isolated from the PrP-sen by ultra-
centrifugation of the cleared lysate and suspension of the pellet in DLPC
buffer (12).
1.1.3. PrP Metabolism
1.1.3.1, KINETICS OF BIOSYNTHESIS AND TURNOVER
The rates of biosynthesis and turnover of various PrP molecules can be com-
pared using standard pulse-chase metabolic labeling experiments (I I, 13,14).
The vast difference in the pulse labeling rates of PrP-sen and PrP-res provided
the first metabolic indication that PrP-res was derived from PrP-sen (I I, 24).
Similar experiments can also be used to determine if these rates are affected by
other non-PrP factors. For instance, pulse-chase labeling experiments showed
that PrP-res accumulation can be inhibited by Congo red and certain sulfated
glycans without affecting the formation and half-life of its precursor, PrP-sen
(vide infra) (I 0,15).
1 .1.3.2. ADDITION OF GLYCOPHOSPHATIDYLINOSITOL(GPI)
The addition of a GPI moiety is one of the earliest events in PrP biosynthesis
(13,16). Several observations can be used as evidence for the presence of the
GPI moiety on PrP (13,16-19):
I. Direct chemicalanalysis.
2. Metabolic labeling with radioactive precursorsof the GPI moiety.
Caughey et al.
288
3. Shifts in SDS-PAGE migration (approx 1 kDa) of PrP molecules on treatment
with phosphatidylinositol-specific phospholipase C (PI-PLC).
4. Release of PrP from cells or membranes by PI-PLC.
In the last case, it should be emphasized that the lack of release cannot be
taken as evidence that the GPI moiety is missing since certain GPI-linked PrP
forms (e.g., PrP-res) are known to be resistant to release by PI-PLC (I 7-19).
1.1.3.3. N-LINKED GLYCOSYLATION
PrP is variably glycosylated at two Asn-linked glycosylation sites (I 3,20-24)
and there is evidence that the extent of glycosylation can affect the efficiency
of PrP-sen conversion to PrP-res (14). The N-linked glycosylation of PrP
forms can be monitored by specific metabolic labeling with radioactive
glycan precursors, such as N-acetylglucosamine or fucose, or by changes in
SDS-PAGE migration on treatment of the PrP molecules with endogly-
cosidases (23,25). Lectins and endoglycosidases with varying specificities can
be used to discriminate between various types of glycans (20,21,26,27). For
instance, endoglycosidase H will remove only the high mannose glycans
present on early PrP precursors produced, whereas endoglycosidase F and
peptidyl-N-glycanase can remove the more mature complex and hybrid gly-
cans as well. The glycosylation of PrP can be manipulated metabolically by
using specific inhibitors (28). Tunicamycin blocks the formation of the pre-
cursor of N-linked glycans, and, when applied to cells making PrP, leads to
the formation of only the 25-26 kDa unglycosylated PrP-sen and 19 kDa
PrP-res (13,25).
1 .1.3.4. TRANSPORT OF PRP TO AND FROM THE CELL SURFACE
Studies with a variety of cells have indicated that PrP-sen can be labeled on the
cell surface by membrane immunofluorescence (2,Z I, 16,Z7,29), biotinylation
(I I), radioiodination (14,29), and immunogold (30-33). Cell surface PrP-sen
is also susceptible to treatments of intact cells by trypsin, proteinase K, dispase,
and PI-PLC (.5,13,16,17,29,34). Although trypsin and proteinase K have the
added effect of releasing the tissue culture cells from their vessels, PI-PLC and
dispase can remove PrP-sen from the plasma membrane without dislodging the
cells. The sensitivity of PrP-sen in intact cells to PI-PLC varies even between
laboratories working with MNB cells (I 1,14,17,34), but the reason for this
variability is not clear.
The rate at which PrP-sen is translocated to the plasma membrane can be
tested by monitoring its exposure to extracellular proteases or PI-PLC in pulse-
chase metabolic labeling experiments (13,34). The transport of newly synthe-
sized PrP-sen through the Golgi apparatus and to the plasma membrane can be
disrupted by brefeldin A (35).
PrP Metabolism and PrP-res Formation 289

Once PrP-sen is at the cell surface, its reinternalization and cycling through
endocytic processes can be monitored after surface iodination, immunofluo-
rescence, and immunogold labeling (29,31). PrP-res is formed on the plasma
membrane or along an endocytic pathway to the lysosomes in Sc+MNB cells
(12,14,34,35). The N-terminal truncation of PrP-res by cellular acid proteases
that are sensitive to lysosomotropic amines can be used as a marker for the
transport of PrP-res to an acidic endosomal or lysosomal compartment (12,35).
Immunochemical staining has indicated that PrP-res accumulates in secondary
lysosomes in Sc+MNB cells (36).
1 .1.3.5. PRECURSOR-PRODUCT RELATIONSHIPS

As noted, pulse-chase labeling experiments can be used to help establish
precursor-product relationships between various forms of PrP. More definitive
evidence that PrP-res is derived from PrP-senin Sc+MNB cells has been obtained
by combining the pulse-labeling approach with the ability to selectively remove
PrP-sen from the cell surface enzymatically (14,34). When PrP-sen was labeled
with a short pulse-chase and then selectively removed from the cell surface by
PI-PLC or proteases, no subsequent labeling of PrP-res occurred. This estab-
lished that PrP-res is derived from a protease- and phospholipase-sensitive pre-
cursor that is at least transiently exposed on the cell surface. Similar approaches
might be useful in determining whether this sequence of events varies in other
TSE-infected cell types or in cells expressing mutant PrP molecules.
1.1.4. Inhibitors of PrP-res Accumulation and Scrapie Agent Replication
One useful approach to dissecting the mechanism of PrP-res formation that
may also lead to effective drug treatments for the TSEs is the identification of
inhibitors of PrP-res formation. Sc+MNB cell cultures can be used to screen for
such inhibitors (10,15). Typically, potential inhibitors are added to the medium
of cultures seeded at low density. The cultures are grown up to confluence and
assayed for effects on the accumulation of PrP-res by immunoblot. Compounds
such as Congo red and certain sulfated glycans have been shown to block PrP-res
accumulation and scrapie agent replication at concentrations in the nanomolar
or subnanomolar range and may act by blocking PrP-glycosaminoglycan inter-
actions (I0,2.5,37,38). These inhibitors also are known to prolong the lives of
animals inoculated with scrapie (39-43).
The selectivity of an inhibitor for PrP-res formation, as opposed to PrP bio-
synthesis generally, can be tested using the described pulse-chase metabolic
labeling experiments to look for effects on PrP-sen labeling, transport to the
cell surface, and turnover. For instance, Congo red and the sulfated glycan
inhibitors block PrP-res accumulation without apparent effects on PrP-sen
metabolism (20,15).
290 Caughey et al.
1.1.5. Expressron of Recombinant PrP Molecules in MA@ Cells
Recombmant PrP molecules have been expressed m a vartety of eukaryottc
vector systems (2,44-47). We have used a retrovtral expression vector, pSFF,
which IS derived from the murme spleen focus-forming retrovnus (45,46)
Recombmant PrP is cloned into the pSFF polylinker using standard clonmg
protocols The PrP-pSFF clone is transfected mto the retrovtral packagmg cells
Y2 and PA3 17 (4.5). We mamtam Y2 cells m 10% calfbovme serum m DMEM
and PA3 17 cells m 10% FBS m RPMI. The PrP-pSFF vector 1spackaged mto
an mfecttous retroviral parttcle and a “ping-pang” effect leads to the spread of
the mfectlous retrovtrus throughout the culture (48). Vtral spread of the recom-
binant PrP is momtored at each cell passage by membrane immunofluores-
cence usmg an antibody specific to the recombmant PrP or by cytoplasmic
immunofluorescence using an antibody to a retrovtral gag protein expressedfrom
the pSFF vector (17,45). Depending on the efficiency of the mmal transfec-
non, the PrP-containing retrovirus ˜111spread to 100% of the cells within l-2 wk
Occasionally, when <30-40% of the cells are positive for the recombmant PrP,
retrovtral spread will stop. This is a result of interference of viral spread by
infectious helper vtrus that has been “rescued” from the Y2/PA3 17 culture
(48) If thts occurs, the cultures should be discarded, replaced wrth early pas-
sage Y2 and PA3 17 cells, and the transfection repeated.
When 100% of the cells express the recombinant PrP protein, viral superna-
tants are harvested. The cells are split and when the culture is approx 70%
confluent, the media 1sreplaced with fresh media and the cells are incubated a
further 24 h. The media is collected the next day, cell debris 1scentrtfuged out
at low speed, and the cleared supernatant is stored m aliquots at -70°C.
Depending on cell vtabtltty, one to three vnal supernatants can be harvested
over consecutive days. The supernatants can be used directly to infect the
desired cell type. Alternatively, the Y2/PA317 cells expressing the recombi-
nant PrP can be cloned by standard limiting dtlution cell cloning to derive a
clonal cell line expressmg the PrP gene of interest
The recombinant PrP-pSFF retroviral supernatants can be used to mfect any
cell type susceptible to infection by an amphotroptc or ecotroptc murme
retrovnus. We have used both mouse neuroblastoma cells (46,49,.50) and
monkey Vero cells. Briefly, cells are split into a six-well tissue culture plate
at 3-5 x lo5 cells/well and incubated overnight at 37°C. The cells should be
70-80% confluent by the next day. Original medium is replaced with fresh
medium containmg 1 mg/mL polybrene, and a 1-mL aliquot of the retroviral
supernatant is added directly to the cells. The culture is incubated overnight at
37OC, split the next day at 1:5 mto a new six-well tissue culture plate, and
assayed l-4 d later for expression of the recombinant PrP by tmmunofluores-
PrP Metabolism and PrP-res Formation 291

cence (17,451. Typically, from 50-100% of the cells will be posmve for the
recombinant PrP protein. The cells are cloned by llmttmg dtlutron to derive a
clonal cell line expressing high levels of the recombinant PrP protein. One or
more coptes of the recombinant PrP will be integrated into the cellular DNA,
but delettons in the recombinant PrP gene occur with variable frequency m this
system and tt IS important to insure that full-length recombinant PrP protem 1s
expressed in the clonal cell line. The size of the clonal insert(s) can be con-
firmed by genomic Southern blot while the recombinant PrP protein 1sassayed
by radiotmmunoprecipttatton as described prevrously (see Section 1.2.). The
mouse neuroblastoma clonal cell lines can stably express recombinant PrP pro-
tem and be maintained indefinitely in culture or frozen in liquid nitrogen by
standard procedures.

2. Materials
2.7. Reagents
1. TEND* (10 mMTrts-HCl, pH 8.3, 1 mA4EDTA, 130 mMNaCI, 1 mM dtthto-
threttol, supplemented wtth 0 5 pg/mL leupeptm, 1 0 pg/mL aprotinin, 0 7 pg/mL
pepstatrn, 0 1 mA4 Petabloc SC, Boehringer, Mannheim (Mannhetm, Germany)
2 10% Sarkosyl (N-lauryl-sarcosine) m TEND
3. 10% NaCl, 1% sulfobetame (SB) m TEND
4. TMCS. (10 mM Trrs-HCl, 5 tnA4MgCl*, 5 mM CaC12,100 mM NaCl, pH 7.4).
5 SNSB: ( 1M sucrose, 100 mM NaCl, 0.5% sulfobetame)
6 Stock3M guanrdmehydrochlorrde (GdnHCl)--to be diluted as appropriate
7. TN (130 m&I NaCI, 50 mM Trts-HCl, pH 7.4 at 20°C)
8 Cetylpyrtdinium chlortde (CPC)

3. Methods
3.1. Cell-Free Conversion of PrP-Sen to Protease-Resistant Forms
Until recently, the simplest system capable of generating PK-resistant PrP
was an intact scrapie-infected tissue culture cell. Efforts to observe the con-
version of PrP-sen to PrP-res m subfractions of such cells have failed (51). To
simplify the analysis of PrP-res formation, we recently developed a cell-free
system that converts PrP-sen to PK-resistant PrP (52-B). This system factll-
tates more defined studies of the conversion reaction. The main strategy that
we used in developing this PrP-sen-to-PrP-res conversion was to keep the
reactants as concentrated as possible to help drive what might otherwise be a
slow or inefficient reaction. The major components of the cell-free conver-
sion reaction are PrP-res purified from scrapie-infected brain tissue and
metabolically labeled PrP-sen immunoprecipltated from uninfected tissue
culture cells.
292 Caughey et a/.

3.1.1. Isolation of the Cell-free Conversion Reaction Components
3 1 1 .l PRP-RES

The purification method we have used is a modification of the procedure of
Bolton and coworkers (55,56,. We will present this protocol m some detail
because this particular preparation of PrP-res may be important m getting the
conversion reaction to work as we have described (52-54).

1. Thaw brains from scraple-infected hamsters or mice and rinse in phosphate-buf-
fered saline.
2. Using a Dounce homogenizer, make a 10% (w/v) homogenate m 10% sarkosyi
(N-lauryl-sarcosme) m TEND.
3 Centrifuge the homogenate m a Beckman (Fullerton, CA) T150.2 rotor at 28,OOOg
at 4™C for 30 min and keep the pellet
4 Centrifuge the supernatant at 180,OOOgat 4™C for 2 5 h in the same rotor Discard
the supematant and keep the pellet.
5 Combme the pellets, rinse them, and resuspend m 10% NaCl and 1% sulfobetame
(SB) in TEND using a Dounce homogenizer Centrifuge the suspension at
250,OOOg for 90 min at 20°C (TL 50 2 rotor). Discard the supematant
6 Rinse pellet and resuspend by Dounce homogenization in TMCS Add DNase I
and RNase A to concentrations of 20 and 100 pg/mL, respectively, and agitate
the suspensionovernight at 4™C
7. To the suspensionadd EDTA to 20 mM, NaCl to 10% w/v, and SB to 1% and
layer the suspensionabove a cushion of SNSB Centrifuge at 250,OOOg 90 for
mm at 20°C
8. Discardthe supematant,rinsethe pellet, and somcatemto 0 5% sulfobetame PBS.
m

The purity of the preparation ts checked using SDS-PAGE with silver stain-
ing. The yield can be determined by a BCA protein assay (Pierce, Rockford,
IL) if it is sufficiently pure and, if not, by quantitative immunoblottmg. The
PrP-res preparations can be stored frozen indefinitely, but repeated freeze-thaw
cycles may affect their behavior.
3 1.1.2. PRP-SEN
The PrP-sen that is used as a substrate in the cell-free reaction is labeled for
90-120 min with 35S m uninfected tissue culture cells (e.g., MNB, Y2, or
PA3 17 cells) and mununopreclpltated as described earlier (see Section 1.2.),
except that after a final water wash of the PrP-antibody-protein A Sepharose
complex, the residual liquid is drawn off the bead pellet with a Hamilton
syringe and the PrP 1s eluted in a minimal volume of 3-7.5M GdnHCl (e.g.,
25 vL/5 mg dry wt equivalent of beads). The beads are agitated in the eluant
for 15 min at 37°C. The eluate is drawn off the beads, and the elution step is
repeated with fresh eluant. Both eluates usually have enough cpm of 35S-PrP-sen
293
PrP Metabolism and PrP-res Formation

Tissue culture cells (scrapie-negative) Scrapie-positive brain
1 1
3%PrPc (proteass-sensitive) PrPSc (protease-resistant)
1 1

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