Cloning and Characterization of Ferritin Mutant

Cloning and Expression of Ferritin Polypeptides—cDNAs
containing the sequence of human WT-FTL and human mutant
FTL498–499InsTC were introduced into the pET-28a(+) expression
vector (Novagen, EMD Chemicals Inc.). The cDNAs were cloned between the BamHI
and XhoI sites, downstream from and in-frame with the sequence encoding an
N-terminal His₆ tag. To eliminate the His₆ tag (included
in the expression vector), the sequence of the vector was modified by
introducing the recognition sequence for cleavage by factor Xa before the
coding sequence of the ferritin genes. PCR amplification of the ferritin cDNAs
was performed using the upstream primer F1 5′-TGG ATC CAT CGA AGG
TCG TAT GAG CTC CCA GAT T-3′ and the downstream primer R1
5′-TTA TGC CTC GAG CCC TAT TAC TTT GCA AGG-3′. F1 contains the
factor Xa sequence (underlined). pET-28a(+) carrying WT-FTL and MT-FTL cDNAs
was transformed into BL21 (DE3) Escherichia coli (Invitrogen).
Transformed cells were grown in Luria broth medium (LB) containing 30 μg/ml
kanamycin (Invitrogen) at 37 °C up to an absorbance of 0.9–1.0 at
600 nm. Bacteria were induced to overexpress recombinant proteins by adding 1
mm isopropyl thio-β-d-galactopyranoside (ICN
Biotechnologies) for 12 h at 25 °C.
Purification of Recombinant WT- and MT-FTL
Homopolymers—Cells were harvested by centrifugation and frozen at
-80 °C. The cell pellets were suspended in 50 mm sodium
phosphate, 500 mm NaCl (pH 7.4), 1 mg/ml lysozyme, and a protease
inhibitor mixture (Complete, Roche Applied Science) for 30 min. Bacteria were
disrupted by sonication, and the insoluble material was removed by
centrifugation at 21,000 × g for 30 min. The soluble fraction
was purified by nickel iminodiacetic acid affinity chromatography using an
AKTA purifier system (GE Healthcare). Purified protein was eluted with 250
mm imidazole in 50 mm sodium phosphate (pH 7.4), 0.5
m NaCl. Recombinant proteins were diluted with 50 mm Tris and 10% glycerol (v/v) down to an absorbance of 0.5 at 280 nm, and
ferritins were cleaved from the His tag by digestion with factor Xa protease
(GE Healthcare) (5 units/mg of protein). After being dialyzed against 50
mm Tris, pH 8.0, for 18 h, proteins were further purified by anion
exchange chromatography (Mono Q) using a linear NaCl elution gradient in 50
mm Tris (pH 8). Peak fractions were ∼95% pure based on SDS-12%
PAGE (Pierce) and Coomassie Blue staining. The efficiency of tag removal was
confirmed by N-terminal protein sequencing analysis, and the molecular weight
of the recombinant proteins was determined by matrix-assisted laser
desorption/ionization-time of flight mass spectrometry. Protein concentration
was determined using the BCA reagent (Pierce) with bovine serum albumin as
standard.
Gel Filtration Chromatography—Size exclusion chromatography
was performed on a Superose 6 10/300 GL column (GE Healthcare) equilibrated
with 50 mm Tris, 150 mm NaCl (pH 7.4) using an AKTA
purifier. The column was calibrated with gel filtration standards (GE
Healthcare). Fractions were detected photometrically, and peak areas and
k_av values were evaluated using the UNICORN 5.1 software
(GE Healthcare). All gel filtration experiments were run at room
temperature.
Transmission Electron Microscopy (TEM)—Ferritins were fixed
using the “single droplet” parafilm protocol. The specimens were
dropped onto a 400-mesh carbon/Formvar-coated grid (Nanoprobes) and allowed to
absorb to the Formvar for a minimum of 1 min. Excess fluid was removed using
filter paper, and the unbound protein was washed, and the grids were placed on
a 50-μl drop of Nanovan (Nanoprobes) with the section side downwards.
Finally, the grids were dried, placed in the grid chamber, and stored in
desiccators before the samples were observed with a Tecnai G2 12 Bio Twin
(FEI) transmission electron microscope.
Preparation of Apoferritins—Recombinant FTL homopolymers
were treated for iron removal as described previously
(14 (link)). Briefly, recombinant
ferritins were incubated with 1% thioglycolic acid (pH 5.5) and
2,2′-bipyridine, followed by dialysis against 0.1 m phosphate
buffer (pH 7.4). We consistently achieve less than five atoms of iron per
ferritin 24-mer, as determined by the colorimetric ferrozine-based assay for
the quantitation of iron
(15 (link)).
Iron Loading of Apoferritins—Freshly prepared ferrous
ammonium sulfate (0.5–4.5 mm) in 10 mm HCl was
added to MT- and WT-FTL apoferritin homopolymers (1 μm) in 0.1
m Hepes buffer (pH 7.4) at room temperature
(16 (link)). After 2 h, the samples
were centrifuged at 14,000 × g for 15 min. Iron incorporation
was initially monitored by measuring absorbance of the supernatants at 310 nm
(14 (link),
17 (link)). Iron incorporation into
ferritin was more precisely determined by densitometric analysis of Prussian
blue staining of supernatants run on nondenaturing gel electrophoresis.
Pellets were analyzed by SDS-12% PAGE. Apoferritins were also incubated in a
molar ratio 1:3500 with ferrous ammonium sulfate and centrifuged at 14,000
× g for 15 min. Pellets were resuspended in a solution
containing 6 mm deferroxamine (DFX), 0.1 m Hepes (pH
7.4) and incubated for 2 h at 24 °C. After centrifugation, supernatants
were analyzed by nondenaturing gel electrophoresis.
Circular Dichroism Spectroscopy—CD spectra of recombinant
apoferritin homopolymers were obtained in 50 mm phosphate buffer
(pH 7.4) at 25 °C in a Jasco 810 spectropolarimeter (Jasco Corp.), using a
protein concentration of 0.12 and 1.5 μm for far-UV and near-UV,
respectively. Far-UV CD spectra were recorded in a 1.0-mm path length cell
from 250 to 190 nm with a step size of 0.1 nm and a bandwidth of 1.0 nm. Each
spectrum represents the mean of 15 scans. CD spectra of the buffer/cuvette
were recorded and subtracted from the protein spectra before averaging.
Secondary structure analyses were performed using DICHROWEB
(18 (link),
19 ), which allows secondary
structure analyses via the software package CDPro
(20 (link)). SELCON3
(21 (link)), CONTINLL
(22 (link)), and CDSSTR
(23 (link)) programs were used for
comparing variations in the amount of secondary structure between MT- and
WT-FTL homopolymers. Normalized root mean square deviation values of < 0.1
for the three methods meant that the experimental and simulated spectra were
in close agreement. Near-UV CD spectra were recorded in a 1.0-cm path length
cell from 400 to 250 nm with a step size of 1.0 nm and a bandwidth of 1.5 nm.
For all spectra, an average of five scans was obtained.
Intrinsic Protein Fluorescence and Thermal Stability Studies of
Homopolymers—Fluorescence spectra were recorded using a
spectrofluorimeter (PerkinElmer Life Sciences) equipped with a Selecta
Ultraterm water bath for temperature control. Apoferritin spectra were
obtained with excitation at 280 and 295 nm with 1.5 μm protein
in 1-cm path length cells and with 0.1 m phosphate (pH 7.4). Blanks
without protein were subtracted from the spectra. Thermal denaturation was
induced by increasing the temperature from 20 to 100 °C at a rate of 1
°C/min. To overcome the inherent difficulty in denaturing ferritin, these
experiments were performed in 0.1 m phosphate buffer (pH 7.4)
containing 4.0 m guanidine hydrochloride (GdnHCl). Homopolymer
stability was monitored using the ratio of intrinsic fluorescence emission of
355 over 330 nm with excitation at 295 nm
(24 (link),
25 (link)) with a maximum at 330 nm
signifying native ferritin (mt and WT) and 355 nm, denatured ferritin.
ANS Fluorescence and Binding Studies—Extrinsic fluorescence
spectra were recorded using a spectrofluorimeter (PerkinElmer Life Sciences)
in 1.0-cm cuvettes at 25 °C. ANS binding to apoferritin homopolymers was
monitored through fluorescence enhancement with ANS excitation at 360 nm and
emission recorded from 600 to 400 nm. MT-FTL apoferritins were prepared by
diluting stock solutions to 1.5 μm in 0.05 m phosphate buffer (pH 7.4). Stock solutions of ANS (Invitrogen) were prepared
in water, and the concentration was determined optically at 350 nm using an
extinction coefficient of 4950 m^-1 cm^-1. ANS
was added to the diluted ferritin samples and equilibrated for 30 min prior to
the measurements, and spectra were background corrected. Binding of ANS to
ferritin was quantitated by Scatchard analysis
(26 ).
Thermolysin Treatment of WT- and MT-FTL Apoferritin
Homopolymers—Proteolysis of recombinant MT- and WT-FTL homopolymers
was initiated by adding to 10 μg of ferritin a 10-fold concentrated stock
solution (36.5 units/mg) of thermolysin (Fluka) in Hepes (0.1 m)
(pH 7.0), 10 mm CaCl₂ to a final concentration of 0.2
mg/ml. The reaction was stopped by the addition of EDTA (50 mm) and
Laemmli sample buffer. Samples treated with thermolysin and controls without
thermolysin were boiled and loaded onto SDS-polyacrylamide gels (4–20%)
(Pierce). Gels were stained with Coomassie Blue (Total protein) or blotted
against the C-terminal antibodies (MT-1283 or WT-1278)
(9 (link)) or against the N-terminal
antibody D18 (Santa Cruz Biotechnology, Inc), which recognized both
polypeptides.
Astrocyte Cell Cultures and Iron/Chelator Treatment—Primary
cortical astrocyte cultures were prepared from 1-day-old mouse pups according
to the procedures of Saneto and De Vellis
(27 ) and Cassina et
al. (28 (link)), with minor
modifications. Pups were obtained from transgenic dams homozygous for the
FTL498–499InsTC mutation in C57BL/6J genetic background
(29 (link)). Briefly, cerebral
cortices were removed, and the tissue was minced and dissociated in 0.25%
trypsin (Invitrogen) for 15 min at 37 °C. Cells were collected by
centrifugation and plated at a density of 2.0 × 10⁶ cells in
25-cm² flasks (Corning Glass) in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, Hepes (25 mm), penicillin
(100 IU/ml), and streptomycin (100 μg/ml) (Invitrogen). When confluent,
cultures were shaken for 48 h at 250 rpm at 37 °C, incubated for another
48 h with 10 μm cytosine arabinoside, and then amplified to 2.5
× 10⁴ cells/cm² in 75-cm² flasks
(Corning Glass). The astrocyte monolayers were >98% pure as determined by
GFAP immunoreactivity. Confluent astrocyte monolayers were changed to
Dulbecco's modified Eagle's media devoid of serum prior to treatment. Stock
solutions (20 mm) of ferric ammonium citrate (FAC) (Sigma), and
1,10-phenanthroline (Phen) (Sigma) were prepared in distilled water and
directly applied to the monolayer at the indicated final concentrations. Each
flask was treated with either of the following: (a) vehicle (water)
as control group; (b) Phen at 100 μm during 48 h
followed by 24 h at 50 μm;(c) FAC 50 μm during 4 days; (d) FAC treatment as in c followed by Phen
treatment as in b in the absence of iron.
Characterization of Detergent-insoluble MT-FTL Ferritin from Astrocyte
Cultures—Cerebral cortical astrocytes cultures were homogenized in
lysis buffer (3 ml of 50 mm Tris-HCl (pH 7.4), 1% SDS, 30 units/ml
benzonase, 2 mm MgCl₂) containing Complete protease
inhibitor mixture (Roche Applied Science) and incubated for 15 min at room
temperature. Lysates containing equal amounts of protein were ultracentrifuged
at 46,000 rpm (TLA 110, Beckman) for 25 min at 4 °C. The supernatant
(SDS-soluble) was removed, and the SDS-insoluble pellet was resuspended in
lysis buffer and then subjected to another step of centrifugation in the same
conditions. The final pellet was resuspended in 5× Laemmli sample buffer
and heated for 10 min at 95 °C. The SDS-soluble, -insoluble, and total
cell lysates (before SDS extraction) were resolved on 4–20% gradient
SDS-PAGE (Pierce) and transferred to nitrocellulose membranes (Amersham
Biosciences). Membranes were blocked for1 h in 70 mm Tris-buffered
saline, 0.1% Tween 20, and 5% nonfat dry milk, followed by an overnight
incubation with polyclonal antibodies (1283) against the MT-FTL polypeptide,
as described previously (9 (link),
29 (link)) at 1:10,000. After
washing, membranes were incubated with peroxidase-conjugated secondary
antibody (GE Healthcare) for 1 h, washed, and developed using the ECL
chemiluminescent detection system (GE Healthcare). MT-FTL recombinant
polypeptides were loaded and used as positive control.
FIGURE 1.

Sequence comparison between WT- and MT-FTL polypeptide. The wild
type FTL polypeptide (WT-FTL) consists of 175 amino acids. The
p.Phe167SerfsX26 mutant polypeptide (MT-FTL) has 191 amino acids and
a different C-terminal sequence (underlined). The boxes indicate the five α-helical domains in the WT-FTL polypeptide according
to Protein Data Bank accession number 2FG4. The mutant C-terminal sequence
contains both metal-binding and hydrophobic groups.

FIGURE 2.

MT-FTL polypeptides assemble into 24-mer homopolymers. A, elution profiles of purified WT- and MT-FTL apoferritin homopolymers from a
Superose 6 column at pH 7.4 in 0.05 m Tris, 0.15 m NaCl.
Retention times for both proteins are shown. Arrows indicate the
elution time for the molecular weight markers. B, ultrastructural
characterization of WT- and MT-FTL homopolymers by TEM. The dark cores most
likely represent Nanovan that has penetrated in some cases the interior of the
24-mers. Bars, 10 nm. C, native PAGE (3–8% (pH7.4)) of
0.5 μm WT- and MT-FTL proteins loaded before the removal of iron
and stained with Coomassie Blue (protein staining) and with Prussian blue
(iron staining).

Immunofluorescence of Cultured Cells—Astrocyte cultures in
Lab-Tek chambered coverglass slides (Nunc) were fixed for 15 min with 4%
paraformaldehyde in PBS at 4 °C. Briefly, the slides were washed
successively with PBS, permeabilized with 0.1% Triton X-100 for 15 min, and
incubated for 1 h at room temperature in blocking solution (0.1% Triton X-100,
2% bovine serum albumin in PBS). The cultures were incubated overnight at 4
°C with the primary antibodies diluted in blocking solution, washed with
PBS, and further incubated for 1 h at room temperature with the secondary
antibodies diluted in blocking solution. The slides were then washed with PBS,
rinsed with distilled water, and mounted with the Prolong Gold antifade
mounting reagent (Molecular Probes). Primary antibodies used were monoclonal
antibody to GFAP (1:400; Sigma) and polyclonal antibody against MT-FTL (1283).
Secondary antibodies used were Alexa 488 Fluor-conjugated goat anti-rabbit and
Alexa Fluor 594-conjugated goat anti-mouse (4 μg/ml; Molecular Probes).
Images were captured with a Zeiss LSM-510 confocal scanner attached to a Zeiss
Axiovert 100 M inverted microscope.