Prion Disease Pathogenesis - Recent Advances in Microbiology - page 60

The identity of iron labeled bands in Figure 2 was further confi rmed by cutting
each band from fractionated PrP C lysates and re-fractionating electro-eluted proteins
on SDS-PAGE followed by immunoblotting. Lane 1 represents proteins eluted from
the loading well that did not enter the running native gel. Lanes 2, 3, and 5 represent
iron labeled bands that resolve adequately on native gels, and lane 4 represents unla-
beled section of the gel that serves as a negative control. Sequential immunoreaction
with specifi c antibodies confi rms the presence of PrP in band 1, TfR in bands 1 and 2,
ferritin in band 3, and Tf in band 5. Band 4 does not react with antibodies to known
iron binding proteins. Silver staining shows co-migration of a few other un-identifi ed
proteins with bands 1-3, and almost none with bands 4 and 5.
To determine if PrP C mediates iron uptake directly, a modifi ed non-denaturing gel
system with a 3-9% gradient was used to separate 59 Fe-labeled PrP effectively. Ac-
cordingly, M17 and PrP C cells were radiolabeled with 59 FeCl 3 -citrate complex for 4 hr
as above, and lysates were fractionated in duplicate under non-denaturing conditions.
One part was dried and exposed to an X-ray fi lm, while the other was transferred to a
PVDF membrane and probed for PrP, ferritin, TfR, and Tf. As in Figure 2, the amount
of 59 Fe incorporated by ferritin in PrP C cells is signifi cantly higher than M17 cells
(Figure 3A, lanes 1 and 2, black arrow). A slower migrating 59 Fe labeled band corre-
sponding to Tf/TfR complex is detected in M17 lysates (Figure 3A, lanes 1, 7, and 9,
open arrow). Unlike Figure 2, PrP is resolved on this less concentrated gel system and
is detected by PrP specifi c antibody 3F4 (Figure 3A, lane 4, arrow-head). However,
a corresponding 59 Fe labeled band is not detected in lane 2, though pure 59 Fe-labeled
recombinant PrP is readily detected by this method as demonstrated previously [17].
Evaluation of iron modulating proteins shows higher levels of ferritin and lower levels
of TfR and Tf in PrP C lysates relative to M17 as in Figure 1 above (Figure 3A, lanes
5-10, arrow-head). Ferritin and Tf/TfR complex show corresponding iron labeled
bands as expected (Figure 3A, compare lanes 1, 2 with 5, 6, 9, 10). Fractionation
of the same samples by SDS-PAGE followed by immunoblotting confi rms increased
levels of ferritin and decreased levels of Tf and TfR in PrP C lysates compared to M17
controls (Figure 3B, lanes 1 and 2). Together, these results demonstrate that PrPC
increases total cellular iron, ferritin iron, and ferritin levels, and decreases Tf and TfR
levels. However, the absence of 59 Fe-labeled PrP C indicates that either the association
of PrP with 59 Fe is transient or relatively weak and disrupted after cell lysis, or alterna-
tively, PrP facilitates the incorporation of 59 Fe into ferritin by an indirect mechanism
that does not involve the formation of a PrP-iron complex.
To evaluate if expression of PrP C on the cell surface is required for iron uptake, a
similar evaluation was carried out in cells expressing PrP 231stop that lacks the GPI an-
chor and is secreted into the medium. Radiolabeling of M17, PrP C , and PrP 231stop cells
with 59 FeCl 3 -citrate complex for 4 hr shows signifi cantly more 59 Fe-ferritin in PrP C
cells compared to M17 as above, and minimal change in PrP 231stop samples (Figure
3C, lanes 1-3, black arrow). Western blotting of M17, PrP C , and PrP 231stop lysates and
medium sample from PrP 231stop cells cultured overnight in serum-free medium with
3F4 shows the expected glycoforms of PrP in PrP C lysates, and undetectable reactivity
in M17 and PrP 231stop lysates as expected (Figure 3D, lanes 1-3). However, signifi cant
reactivity is detected in the medium of PrP 231stop cells, demonstrating adequate expression