Biology Reference
In-Depth Information
The effect of increased endocytosis of PrP
C
on ferritin iron content was evaluated
by radiolabeling cells cultured in the presence of 1 μg/ml of 3F4 with
59
FeCl
3
for the
last 4 hr of the incubation, and analyzing radiolabeled lysates as in Figure 1 above.
Fractionation by non-denaturing page shows a signifi cant increase in ferritin iron in
the 3F4 exposed lysate compared to untreated control (Figure 7A, lanes 1 and 2, open
arrow). Analysis by SDS-PAGE and immunoblotting shows 2-3 fold increase in reac-
tivity for all PrP glycoforms with anti-PrP antibodies 3F4 and 8H4 (Figure 7A, lanes
3-6). However, the 18 kDa fragment that results from recycling of PrPC from the plas-
ma membrane is not increased in 3F4 exposed lysates, indicating stimulation of PrPC
internalization and possible intracellular accumulation by 3F4 binding rather than in-
creased recycling from the plasma membrane (Figure 7A, lanes 5 and 6) [28]. The 50
kDa band represents internalized 3F4 (Figure 7A, lanes 4 and 6). Immunobloting for
ferritin, Tf, and TfR shows an increase in TfR, and minimal change in ferritin and Tf
levels (Figure 7A, lanes 7 and 8). Quantifi cation by densitometry shows an increase
in ferritin iron to 271%, and insignifi cant change in ferritin and Tf levels by 3F4 treat-
ment. The increase in TfR levels to 175% is probably due to co-endocytosis with PrP-
antibody complex (Figure 7B). Measurement of cellular LIP revealed insignifi cant
difference between 3F4 exposed and untreated controls after 24 hr (data not shown)
or 5 days of treatment, indicating effi cient transport of iron to ferritin within this time
frame (Figure 7C). PrP
C
cells treated with anti-Thy-1 antibody, however, demonstrated
a signifi cant decrease in LIP after 5 days of incubation with 3F4 (Figure 7C).
A similar evaluation of cells exposed to 12 μg/ml of 3F4 for 4 hr shows signifi -
cantly less increase in ferritin iron compared to untreated controls (Figure 8A, lanes 1
and 2, open arrow). Separation by SDS-PAGE and immunoblotting shows increase in
PrP reactivity (Figure 8A, lane 4) and an increase in the levels of ferritin, Tf, and TfR
(Figure 8A, lanes 5 and 6). Quantifi cation shows an increase in ferritin iron to 148%,
and an increase in the levels of ferritin and TfR to 153 and 146% respectively. Tf levels
show insignifi cant change by this treatment (Figure 8B). A similar increase in ferritin
iron is observed when M17 cells expressing endogenous levels of PrP are exposed to
3F4, ruling out the effect of over-expression of PrP
C
on these observations. Exposure
to equivalent amounts of anti-Thy-1 does not alter ferritin iron content signifi cantly.
Measurement of intracellular LIP after 4 hr of exposure to 12 μg/ml of 3F4 shows an
increase to 170% in treated cells compared to untreated controls. Exposure to similar
concentrations of anti-Thy-1 shows a decrease to 70% (Figure 8C), an unexpected ef-
fect that requires further evaluation.
The above results indicate that stimulation of PrP
C
endocytosis over a prolonged
period increases iron incorporation into ferritin, whereas cross-linking of PrP
C
that
is likely to result in its degradation following endocytosis has relatively less effect
on ferritin iron. The increase in intracellular LIP by cross-linking PrP without any
increase in ferritin iron probably refl ects ineffi cient transport of iron to ferritin in the
absence of PrP, as observed for certain mutant forms of PrP. The levels of ferritin, Tf,
and TfR probably refl ect an artifactual change due to membrane perturbation by anti-
body treatment rather than a response to intracellular LIP.
