Báo cáo khoa học: The soluble form of the membrane-bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein pdf

The soluble form of the membrane-bound transferrin homologue,
melanotransferrin, inefficiently donates iron to cells via nonspecific
internalization and degradation of the protein
Michael R. Food, Eric O. Sekyere and Des R. Richardson
The Heart Research Institute, Iron Metabolism and Chelation Group, Camperdown, Sydney, New South Wales, Australia
Melanotransferrin (MTf) is a membrane-bound transferrin
(Tf) homologue found particularly in melanoma cells. Apart
from membrane-bound MTf, a soluble form of the molecule
(sMTf) has been identified in vitro [Food, M.R., Rothen-
berger, S., Gabathuler, R., Haidl, I.D., Reid, G. & Jefferies,
W.A. (1994) J. Biol. Chem. 269, 3034–3040] and in vivo in
Alzheimer’s disease. However, nothing is known about the
function of sMTf or its role in Fe uptake. In this study, sMTf
labelled with
59
Fe and
125
I was used to examine its ability to
donate
59
Fe to SK-Mel-28 melanoma cells and other cell
types. sMTf donated
59
Fe to cells at 14% of the rate of Tf.
Analysis of sMTf binding showed that unlike Tf, sMTf did
not bind to a saturable Tf-binding site. Studies with Chinese
hamster ovary cells with and without specific Tf receptors
showed that unlike Tf, sMTf did not donate its
59
Fe via these
pathways. This was confirmed by experiments using lyso-

somotropic agents that markedly reduced
59
Fe uptake from
Tf, but had far less effect on
59
Fe uptake from sMTf. In
addition, an excess of
56
Fe-labelled Tf or sMTf had no effect
on
125
I-labelled sMTf uptake, suggesting a nonspecific
interaction of sMTf with cells. Protein-free
125
I determina-
tions demonstrated that in contrast with Tf, sMTf was
markedly degraded. We suggest that unlike the binding of Tf
to specific receptors, sMTf was donating Fe to cells via an
inefficient mechanism involving nonspecific internalization
and subsequent degradation.
Keywords: iron; iron uptake; melanotransferrin; transferrin;
transferrin receptor.
Melanotransferrin (MTf) is a homologue of the serum Fe-
binding protein, transferrin (Tf), that was first identified as
an oncofoetal antigen [1–3]. Initial studies suggested that
MTf was either not expressed, or expressed only slightly in
normal tissues, but was found in larger amounts in
neoplastic cells (especially malignant melanoma cells) and
foetal tissues [1–3]. However, in later reports MTf was
identified in a variety of normal tissues [4–9].

The MTf molecule has many properties in common
with Tf, including: (a) it has a 37–39% sequence
homology with human serum Tf, human lactoferrin,
and chicken Tf; (b) the MTf gene is on chromosome 3, as
are those for Tf and the Tf receptor 1 (TfR1); (c) many of
the disulphide bonds present in serum Tf and lactoferrin
are also present in MTf; (d) MTf has an N-terminal
Fe-binding site that is very similar to that found in serum
Tf; and (e) isolated and purified MTf can bind one Fe
atom/molecule from Fe(III) citrate [10–14]. This circum-
stantial evidence suggested that MTf played a role in Fe
transport (for a review see [15]).
In contrast with serum Tf, MTf is bound to the cell
membrane by a glycosyl phosphatidylinositol (GPI) anchor
[5,16], and can be removed using phosphatidylinositol-
specific phospholipase C [5,16,17]. Apart from the mem-
brane-bound form, it is known that a soluble form of MTf
(sMTf) exists in the serum of patients with melanoma [3],
arthritis [18], and Alzheimer’s disease
2
[19,20]. Furthermore,
several alternative transcripts from the originally identified
MTf gene (tentatively called MTf1) and a second melano-
transferrin gene (MTf2) have been identified [21].
Previously we endeavoured to assess the functional roles
of MTf compared to the TfR1 in Fe uptake by the
melanoma cell line SK-Mel-28 [22–26]. These cells were used
as they express the highest levels of MTf in all cell types
tested (3–3.8 · 10
5

MTf sites/cell [10]). Our studies showed
that SK-Mel-28 melanoma cells incorporated Fe from Tf by
two processes consistent with receptor-mediated endocyto-
sis (RME) and a nonspecific mechanism consistent with
pinocytosis of Tf [22,23]. Similar mechanisms of Fe uptake
from Tf were also reported by others using hepatocytes and
hepatoma cells [27,28]. In addition, melanoma cells could
take up Fe from low M
r
Fe complexes by a process
independent of the TfR1 [24]. Of interest, a membrane-
bound, pronase-sensitive, Fe-binding component was iden-
tified in SK-Mel-28 cells consistent with MTf [22,25].
However, while this membrane Fe-binding component
could bind Fe, it did not donate it to the cell [25].
Correspondence to D. R. Richardson, Children’s Cancer Institute
Australia, Iron Metabolism and Chelation Program, High St (PO Box
81), Randwick, Sydney, New South Wales, Australia.
Fax: +61 2 9382 1815, Tel.: +61 2 9382 1831,
E-mail:
Abbreviations: BSS, Hank’s balanced salt solution; CHO cells, Chinese
hamster ovary cells; GPI, glycosyl phosphatidylinositol; MEM,
Eagle’s modified minimum essential medium; MTf, melanotransfer-
rin; RME, receptor-mediated endocytosis; sMTf, soluble melano-
transferrin; TCA, trichloroacetic acid; Tf, transferrin; TfR1,
transferrin receptor 1; TfR2, transferrin receptor 2; TRVa, variant
Chinese hamster ovary cells without specific Tf-binding sites; WTB,
wild-type Chinese hamster ovary cells with specific Tf-binding sites;
TK, thymidine kinase.
1

(Received 16 May 2002, revised 2 July 2002, accepted 23 July 2002)
Eur. J. Biochem. 269, 4435–4445 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03140.x
Studies in our laboratory using Chinese hamster ovary
(CHO) cells transfected with the MTf1 gene [17], showed
that membrane-bound MTf could transport Fe into cells
from
59
Fe-citrate complexes but not from Tf. However, in
these transfected cells, the levels of MTf (1.2 · 10
6
sites/cell
[17]) were much greater than that found on SK-Mel-28
melanoma cells [10]. As Fe uptake by MTf-transfected
CHO cells after a 4 h incubation with
59
Fe-citrate was only
2.4-fold of that seen with control CHO cells [17], these data
questioned the role of MTf in Fe uptake by melanoma cells
where it is expressed at much lower levels [10]. Recent
studies [9] using SK-Mel-28 melanoma cells have shown
that MTf expression, unlike that of the TfR1 [26], is not
regulated by Fe. Moreover, in melanoma cells, MTf does
not actively internalize Fe from Fe-citrate [9], casting serious
doubt on the role of this molecule in Fe transport.
In contrast with membrane-bound MTf, nothing is
known concerning the function of sMTf or its role in Fe
uptake. Considering the high sequence homology of sMTf
to Tf, sMTf may bind to Tf-binding sites, namely the high
affinity TfR1 [29,30] or the lower affinity TfR2 [31].
Alternatively, as Fe can be taken up from Tf via a process

consistent with nonspecific pinocytosis in melanoma cells
[23] and other normal [27,32,33] and neoplastic cell types
[28], this Fe uptake pathway may be functional for sMTf.
Moreover, it was important to assess whether sMTf could
bind to cells via a high-affinity binding site. Previous studies
using surface labelling of SK-Mel-28 cells demonstrated that
partitioning of MTf from the cell surface was unlikely, and
that active secretion of a high M
r
(95–97 kDa) form of
sMTf occurred [16]. Hypothetically, in vivo, sMTf could
bind Fe released from the liver, and then donate it back to
the cell via a sMTf receptor. This autocrine mode of action
suggested for other Fe-binding molecules [34,35] may be
vital for the biological role of sMTf.
In this study, we used sMTf labelled with
59
Fe and
125
Ito
examine its ability to donate
59
Fe to SK-Mel-28 melanoma
cells. This cell type was initially used because its Fe
metabolism is well characterized [9,22–26] and we showed
that sMTf can be released from these cells [16]. Therefore,
sMTf may have relevance to the biology of melanoma cells.
In our current investigation, sMTf was shown to donate Fe
to cells but at a much lower efficiency than Tf. The sMTf
does not bind to a saturable high affinity receptor and its

internalization occurred via a nonspecific process e.g.
pinocytosis. Further, in contrast with
59
Fe-Tf uptake,
59
Fe
uptake from sMTf was less sensitive to the effects of
lysosomotropic agents, suggesting a different intracellular
trafficking route than Tf. In fact, sMTf was markedly
degraded by the cell.
MATERIALS AND METHODS
Cell culture
Human SK-Mel-28 melanoma cells, SK-N-MC neuroepi-
thelioma cells, MRC-5 fibroblasts and MCF-7 breast cancer
cells, were obtained from the American Type Culture
Collection. Mouse LMTK
–
fibroblasts were obtained from
the European Collection of Cell Cultures. The CHO cells
with (wild-type B; WTB) and without specific Tf-binding
sites (variant A; TRVa) [36,37] were from F.R. Maxfield
(Department of Biochemistry, Weill Medical College of
Cornell University, New York). All cell lines were grown in
Eagle’s minimum essential medium (MEM; Gibco) con-
taining 10% foetal calf serum (Gibco), 1% (v/v) nonessen-
tial amino acids (Gibco), 100 lgÆmL
)1
streptomycin
(Gibco), 100 UÆmL
)1

penicillin (Gibco), and 0.28 lgÆmL
)1
fungizone (Squibb Pharmaceuticals, Montre
´
al, Canada).
Cells were grown in an incubator (Forma Scientific,
Marietta,
3
Ohio, USA) at 37 °C in a humidified atmosphere
of 5% CO
2
/95% air and subcultured as described previ-
ously [22]. Cellular growth and viability were assessed by
phase contrast microscopy, cell adherence to the culture
substratum, and Trypan blue staining. Cells were routinely
cultured in bulk in 75 cm
2
flasks and subcultured to
35 · 10 mm Petri dishes for experiments.
Protein preparation, purification and labelling
Apo-Tf was from Sigma Chemical Co. and apo-sMTf was
kindly provided by M. Kennard, Synapse Technologies Inc,
Vancouver, Canada. The sMTf was genetically engineered
to lack the 27 C-terminal amino acids, thus abolishing the
GPI-attachment signal sequence and insertion into the
membrane [38]. The appropriate constructs were prepared
using pNUT and the recombinant vector was then stably
transfected into baby hamster kidney (BHK) thymidine
kinase
4

(TK)
–
cells [38]. The media obtained from these cells
was concentrated and the sMTf purified by immunoaffinity
chromatography using anti-MTf mAb L235 (HB8446;
ATCC). This solution was sterilized using a 0.2-lmfilter
and the purity confirmed by SDS/PAGE [38] which yielded
one band at 95 kDa. The protein sequence of sMTf predicts
a molecule of 77 600 Da, and thus, the M
r
obtained by
SDS/PAGE suggests post-translational modification con-
sistent withglycosylation. Furthermore, comparison of endo-
glycosidase H resistance between sMTf from SK-Mel-28
cells [16] and that secreted from the BHK TK
–
cell line [38],
suggested that the two proteins were glycosylated in a
similar fashion. MS and N-terminal sequence analysis
(Australian Proteome Analysis Facility, Macquarie Uni-
versity, Sydney, NSW) performed on sMTf derived from
BHK TK
–
cells demonstrated that it was the correct size
and sequence. Previous studies have shown that this form of
sMTf reacted with a panel of anti-MTf mAbs (L235,
HybC, 2C7, 9B6) in the same way as sMTf released from
SK-Mel-28 melanoma cells [38]. These results indicated that
the tertiary structure of these molecules were very similar,
and the fact that sMTf bound Fe (see below) suggested that

this protein was folded correctly.
Apo-Tf and apo-sMTf were labelled with
59
Fe (Dupont
NEN) or nonradioactive
56
Fe to produce holo-diferric
transferrin (
59
Fe-Tf,
56
Fe-Tf) or holo-monoferric sMTf
(
59
Fe-sMTf,
56
Fe-sMTf) using procedures established in our
laboratory [22,26]. Free
59
Fe or
56
Fe was removed by
exhaustive dialysis against a large excess of 0.15
M
NaCl
bufferedtopH7.4with1.4%NaHCO
3
[22,26]. Both sMTf
and Tf were labelled with
59

Fe based upon the fact that there
are one [14] and two [29] high affinity Fe-binding sites per
molecule, respectively. For both proteins, upon Fe-loading,
theexpectedcolourchangefromcleartosalmonpinkwas
observed. The UV-visible absorption maximum of mono-
ferric sMTf was 464 nm as described in previous studies
[14]. Native PAGE-
59
Fe-autoradiography studies (see below
[39]) demonstrated that all
59
Fe was bound to the proteins.
4436 M. R. Food et al. (Eur. J. Biochem. 269) Ó FEBS 2002
To examine the uptake of the proteins by cells, mono-
ferric sMTf and diferric Tf were labelled with
125
Ibyusing
the iodine monochloride method [40] or the chloramine T
procedure [9,10]. The results obtained using the two
methods were very similar, but the iodine monochloride
method was implemented because of its more gentle
labelling conditions. In these studies, the amount of free
125
I in the protein sample was measured by trichloroacetic
acid (TCA) precipitation (see below) and was always
<2.5% of the total
125
I. The functional integrity of the
protein after labelling was ensured by competition studies
where the labelled or nonlabelled protein acted in the same

manner to block the uptake of
59
Fe-
125
I-labelled sMTf or Tf
by cells. Further, previous studies showed that molecules
labelled with
59
Fe and
125
I using the current methods resul-
ted in functional proteins [22,23,25–27,41,42]. In all experi-
ments examining the uptake of the
125
I-sMTf or
125
I-Tf, the
proteins were also saturated with
59
Fe by the procedures
described above. These dual labelling experiments enabled
examination of the uptake of both the
59
Fe label and the
125
I-labelled protein.
Protein-free
125
I assay
The amount of protein-free

125
I was measured in lysed cells
and media using TCA precipitation [43]. The cells were lysed
by removal from the Petri dish using a plastic spatula at
4 °C followed by one freeze–thaw cycle. Control experi-
ments demonstrated that this lysis procedure did not
influence the proportion of protein-free
125
I.
Uptake of labelled sMTf or Tf and the use
of lysosomotropic agents
The uptake of radioactively labelled proteins was analysed
using standard techniques [22,23,26]. Briefly, cells in Petri
dishes were incubated for 30 min to 30 h at 37 °Cor3hat
4 °Cwith
59
Fe-
125
I-Tf (0.001–0.1 mgÆmL
)1
)or
59
Fe-
125
I-
sMTf (0.001–0.1 mgÆmL
)1
) in MEM containing BSA
(10 mgÆmL
)1

). The cells were then placed on a tray of ice
and washed four times with ice-cold Hank’s balanced salt
solution (BSS; Gibco). The internalized and membrane
uptake of
59
Fe-
125
I-labelled proteins were determined by
incubating cells with the general protease, pronase
(1 mgÆmL
)1
; Boehringer Mannheim), for 30 min at 4 °C,
as described previously [22,26,30]. Control experiments in
previous investigations have found that this technique is
valid for measuring membrane-bound and internalized
radioactivity [22,30]. The cells were then removed from the
Petri dishes using a plastic spatula and transferred to
c-counting tubes. Radioactivity was measured using a
c-scintillation counter (LKB Wallace 1282 Compugamma).
The effects of the well-characterized lysosomotropic
agents, ammonium chloride (15 m
M
), chloroquine
(0.5 m
M
), or methylamine (15 m
M
), on
59
Fe uptake from

59
Fe-
125
I-Tf or
59
Fe-
125
I-sMTf were examined by pre-
incubating cells with these agents for 15 min at 37 °C
[23,41,42]. This medium was then removed, and the cells
incubated for 3 h at 37 °C with medium containing the lyso-
somotropic agents and either
59
Fe-
125
I-Tf (0.05 mgÆmL
)1
)
or
59
Fe-
125
I-sMTf (0.05 mgÆmL
)1
). The internalization of
59
Fe was then determined using pronase as described
above.
Efflux of labelled sMTf and Tf by cells
The release of sMTf or Tf by pre-labelled cells was examined

using standard procedures [25,44,45]. Cells in Petri dishes
were labelled with
59
Fe-
125
I-sMTf (0.05 mgÆmL
)1
)or
59
Fe-
125
I-Tf (0.05 mgÆmL
)1
) in MEM containing BSA
(10 mgÆmL
)1
) for 3 h or 24 h at 37 °C. The Petri dishes
were subsequently placed on a tray of ice and washed four
times with ice-cold BSS. The cells were then reincubated with
warm MEM for incubation periods from 1 to 120 min at
37 °C. The overlying medium was then removed and placed
into c-counting tubes. The cells were removed from the Petri
dishes in 1 mL of BSS using a plastic spatula and transferred
to a separate set of c-counting tubes. Both media and lysed
cells were subjected to TCA precipitation to determine the
proportion of protein-free
125
I.
Determination of intracellular iron distribution
using native-PAGE-

59
Fe-autoradiography
Native-PAGE-
59
Fe-autoradiography was performed using
standard techniques in our laboratory [39] after incubation
of cells with
59
Fe-sMTf (0.05 mgÆmL
)1
)or
59
Fe-Tf
(0.05 mgÆmL
)1
)for24hat37°C. Bands on X-ray film
were quantified by scanning densitometry using a laser
densitometer and analysed by Kodak Biomax I Software
(Kodak Ltd).
Statistics
Experimental data were compared using Student’s t-test.
Results were considered statistically significant when
P < 0.05. Results are expressed as mean ± SD (three
determinations) in a typical experiment of at least three
performed.
RESULTS
Iron uptake from sMTf as a function of time is far less
efficient than iron uptake from Tf
In all of the studies reported below, we have examined the
uptake of

59
Fe-
125
I-sMTf by SK-Mel-28 melanoma cells
and a number of other cell types. These results have been
compared to the uptake of
59
Fe-
125
I-Tf that has been
extensively characterized in our laboratory [22–26] and
provides an appropriate positive control.
Our experiments show that like Tf [22], sMTf donates
59
Fe to cells as a linear function of incubation time up to
30 h at 37 °C (Fig. 1A). However, sMTf donates its
59
Fe to
cells at 14% the rate of Tf (Fig. 1A). Similar results were
also found when the uptake of
59
Fe from Tf and sMTf was
examined in a range of cell lines commonly used in our
laboratory, including human SK-N-MC neuroepithelioma
cells, human MRC-5 fibroblasts, human MCF-7 breast
cancer cells and mouse LMTK
–
fibroblasts (data not
shown). Studies using native-PAGE-
59

Fe-autoradiography
showed that both Tf and sMTf donated
59
Fe to cells, and
this could label the Fe-storage protein, ferritin (see inset
Fig. 1A). However, densitometric analysis of
59
Fe incor-
poration into ferritin from sMTf demonstrated that it was
about 10% of that found using Tf. As shown previously, the
ferritin-
59
Fe band comigrated with horse spleen ferritin and
Ó FEBS 2002 Soluble melanotransferrin inefficiently donates iron to cells (Eur. J. Biochem. 269) 4437
can be supershifted using an antiferritin polyclonal antibody
[46].
As found for Tf [22,29], the internalization of
59
Fe from
sMTf was markedly temperature dependent, there being
little internalized
59
Fe uptake at 4 °C (data not shown).
Examining the total amount of radioactivity added to each
Petri dish of cells (approximately 500 000 cpm), the
proportion of
59
Fe radioactivity taken up by cells at 37 °C
was equal to 0.09% for sMTf and 1.51% for Tf, a
significant (P < 0.0001) 16-fold difference. Hence, the

59
Fe
uptake from sMTf by cells was much less efficient than Tf.
Internalization of
125
I-sMTf as a function of time
is less marked than that of
125
I-Tf
To determine whether SK-Mel-28 melanoma cells could
internalize sMTf, experiments were performed to assess the
uptake of
125
I-sMTf compared to
125
I-Tf as a function of
time up to 30 h at 37 °C. As shown in Fig. 1B, the kinetics
of sMTf and Tf uptake were clearly different. The internal-
ization of sMTf by the cell occurred as a linear function of
incubation time [correlation coefficient (r) ¼ 0.97]. In
contrast, the internalization of Tf occurred by a biphasic
process consistent with RME (Fig. 1B), as shown in our
previous investigation [22].
Significantly (P < 0.001) less
125
I-sMTf was internal-
ized than
125
I-Tf in SK-Mel-28 melanoma cells. For
instance, after labelling for 3 h at 37 °Cwith

125
I-sMTf
(0.05 mgÆmL
)1
)or
125
I-Tf (0.05 mgÆmL
)1
), 13% and 66%
was internalized, respectively. Examining MCF-7 breast
cancer cells, MRC-5 fibroblasts, and LMTK
–
fibroblasts,
the percentage of
125
I-sMTf (0.05 mgÆmL
)1
) internalized
after labelling for 3 h varied between 11 and 17%. In
contrast, the internalization of
125
I-Tf (0.05 mgÆmL
)1
)was
much greater, ranging between 47 and 69% (data not
shown).
Uptake of
125
I-sMTf and
125

I-Tf as a function of ligand
concentration
To assess if a saturable binding site for
59
Fe-
125
I-sMTf
occurred on the cell membrane, experiments were
designed to investigate the uptake of
125
I-sMTf as a
function of ligand concentration using SK-Mel-28 melan-
oma cells (Fig. 2A). In the same experiment, the binding
of
125
I-Tf was assessed (Fig. 2B) and acted as a positive
control, as we had previously demonstrated a high affinity
Tf-binding site in this cell type [22,23,26]. It is obvious
from a comparison of Fig. 2A and B that there was a
marked difference in the mechanism of ligand uptake.
Internalized, membrane, and therefore total uptake of
125
I-sMTf was linear as a function of concentration, the r
for each being 0.99, 0.99 and 0.97, respectively (Fig. 2A).
Higher concentrations of ligand, up to 0.5 mgÆmL
)1
,also
resulted in linear uptake of sMTf by cells as a function of
concentration (data not shown). Most (76–88%) of the
125

I-sMTf bound to the cell was present on the membrane
whereas only 12–24% of the molecule was internalized
(Fig. 2A). In contrast, the uptake of
125
I-Tf was biphasic,
with saturation occurring at a Tf concentration of
 0.01 mgÆmL
)1
, as we showed previously [22,23,26].
Furthermore, unlike
125
I-sMTf uptake, the internalized
125
I-Tf formed the largest proportion (60–76%) of the
total uptake of this ligand, while 24–40% was bound to
the membrane (Fig. 2B). Hence,
125
I-Tf was taken up by a
saturable binding site as found previously [22,23,26],
whereas the binding of
125
I-sMTf increased linearly with
concentration. This was consistent with nonspecific bind-
ing of sMTf to the membrane and its subsequent
internalization.
Fig. 1. (A) Iron uptake from
59
Fe-sMTf was far less than that from
59
Fe-Tf as a function of time. The inset shows intracellular

59
Fe uptake
into ferritin from sMTf and Tf using native PAGE-
59
Fe-autoradio-
graphy. (B) The uptake of
125
I-sMTf as a function of time was much
less than that from
125
I-Tf. The SK-Mel-28 malignant melanoma cell
line was incubated with
59
Fe-
125
I-sMTf (0.05 mgÆmL
)1
)forupto30h
at 37 °C. The cells were then washed and incubated with pronase
(1 mgÆmL
)1
)for30minat4°C to separate internalized from mem-
brane-bound
59
Fe and
125
I. Native PAGE-
59
Fe-autoradiography was
performed using standard procedures after a 24 h incubation at 37 °C

with
59
Fe-sMTf (0.05 mgÆmL
)1
)or
59
Fe-Tf (0.05 mgÆmL
)1
)(see
Materials and methods). The results are a typical experiment from
three performed and are expressed as the mean ± SD (three deter-
minations).
4438 M. R. Food et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Uptake of
59
Fe from sMTf and Tf as a function
of ligand concentration
The uptake of
59
Fe from Tf and sMTf was also investigated
as a function of ligand concentration (0.001–0.1 mgÆmL
)1
)
(Fig. 3A and B). Internalized, membrane and total
59
Fe
uptake from
59
Fe-sMTf was linear, the r for each being 0.97,
0.99 and 0.99, respectively (Fig. 3A). Higher concentrations

ofsMTf,upto0.5mgÆmL
)1
,alsoresultedinlinearuptakeof
Fe as a function of ligand concentration (data not shown).
The internalized
59
Fe uptake from sMTf varied from 26 to
44% of the total
59
Fe uptake, there being more
59
Fe uptake
by the membrane than that internalized at all ligand
concentrations (Fig. 3A). In contrast,
59
Fe uptake from Tf
was biphasic as a function of ligand concentration with
saturation of
59
Fe uptake occurring at approximately
0.01 mgÆmL
)1
(Fig. 3B), as found in our previous studies
[22,23]. The internalized
59
Fe uptake from Tf ranged from 84
to 92% of the total
59
Fe uptake (Fig. 3B), which was far
greater than that found for sMTf. The biphasic kinetics of Fe

uptake and extent of Fe internalization were consistent with
the binding of Tf to a specific and saturable binding site
[22,23].
Fig. 2. The effect of ligand concentration on the uptake of: (A)
125
I-
sMTf, or (B)
125
I-Tf, by SK-Mel-28 melanoma cells. The cells were
incubated with
59
Fe-
125
I-sMTf (0.005–0.1 mgÆmL
)1
)or
59
Fe-
125
I-Tf
(0.005–0.1 mgÆmL
)1
)for3hat37°C. The cells were then washed and
incubated with pronase (1 mgÆmL
)1
)for30minat4°Ctoseparate
internalized from membrane-bound
125
I. The results are a typical
experiment from three performed and are expressed as the means of

two determinations.
Fig. 3. The effect of ligand concentration on
59
Fe uptake from: (A)
59
Fe-
sMTf, or (B)
59
Fe-Tf, by SK-Mel-28 melanoma cells. The cells were
incubated with
59
Fe-
125
I-sMTf (0.005–0.1 mgÆmL
)1
)or
59
Fe-
125
I-Tf
(0.001–0.1 mgÆmL
)1
) for 3 h at 37 °C. After this incubation the cells
were washed and incubated with pronase (1 mgÆmL
)1
)for30minat
4 °C to separate internalized from membrane-bound
59
Fe. The results
are a typical experiment from three performed and are expressed as the

means of two determinations.
Ó FEBS 2002 Soluble melanotransferrin inefficiently donates iron to cells (Eur. J. Biochem. 269) 4439
Competition studies between sMTf and Tf
Further studies were performed to assess whether sMTf
could be donating Fe to cells through the same or a similar
pathway as Tf. This was done using competition experi-
ments where SK-Mel-28 cells were incubated with an
excess of sMTf over Tf or vice versa and the effect on
uptake of
59
Fe- or the
125
I-labelled protein was assessed.
Coincubation of cells with
59
Fe-
125
I-sMTf (0.05 mgÆmL
)1
)
and an excess of nonradioactive
56
Fe-Tf (1 mgÆmL
)1
)
inhibited
59
Fe uptake to 12 ± 1% of that found for sMTf
alone, but had no effect on
125

I-MTf uptake (data not
shown). These former results indicated that Fe donated by
sMTf and Tf appears to compete for a common carrier,
and that Fe donated from Tf is a good competitive
inhibitor of sMTf-Fe uptake. However, interestingly, the
fact that an excess of
56
Fe-Tf had no effect on the uptake
of
125
I-sMTf indicated no competition between the
proteins in terms of their binding and uptake by the cell.
Moreover, these results suggest that the uptake of each
protein (in contrast with their bound Fe) was mediated
independently.
We showed that incubation of
59
Fe-Tf (0.01 mgÆmL
)1
)
with an excess of nonradioactive
56
Fe-sMTf (0.1 mgÆmL
)1
),
did not significantly affect
59
Fe uptake (101 ± 6% of
that found for
59

Fe-Tf). These results suggest that sMTf
is not a good competitive inhibitor of Fe uptake from Tf.
Incubation of cells with
59
Fe-sMTf (0.05 mgÆmL
)1
)anda
twofold excess of nonradioactive
56
Fe-sMTf (0.1 mgÆmL
)1
)
decreased
59
Fe uptake to 29 ± 7% of that found with
59
Fe-sMTf alone. This latter experiment was important to
determine whether competition occurred between
59
Fe-
labelled sMTf and its
56
Fe-labelled counterpart, and indi-
cated the functional integrity of the radioactively labelled
molecule.
Unlike Tf, sMTf does not donate iron by specific
transferrin-binding sites
To further assess the role of specific Tf-binding sites in Fe
uptake from sMTf, we used the well-characterized CHO cell
lines with functional Tf-binding sites (known as WTB) or

without these molecules (known as TRVa) [36,37] (Fig. 4).
Previous studies have shown that the TRVa cell line does
not express any specific Tf-binding sites [36,37]. Experi-
ments compared the uptake of
59
Fe from
59
Fe-
125
I-Tf
(0.05 mgÆmL
)1
)and
59
Fe-
125
I-sMTf (0.05 mgÆmL
)1
)over
24 h at 37 °C. As expected, the WTB cells efficiently inter-
nalized
59
Fe from Tf, while
59
Fe uptake from this molecule
by the TRVa cells was sixfold lower (Fig. 4). In contrast,
there was no significant difference in
59
Fe uptake from
sMTf by WTB and TRVa (Fig. 4). Hence, sMTf did neither

bind to Tf-binding sites nor donate its Fe via this pathway.
Interestingly, while TRVa cells do not have any func-
tional Tf-binding sites [36,37], there was slight and almost
equivalent
59
Fe uptake from Tf and sMTf (Fig. 4). These
data could be explained by the presence of a nonspecific,
nonsaturable process of Fe uptake from Tf that was
previously characterized in TRVa cells [37]. This mechanism
is functionally comparable to that seen in melanoma cells
[22,23], hepatoma cells [28] and hepatocytes [33]. Hence,
limited Fe uptake from Tf or sMTf may occur by a
nonspecific mechanism.
Effect of Lysosomotropic Agents on
59
Fe Uptake
from
59
Fe-Tf and
59
Fe-sMTf
To further examine whether sMTf could be donating
59
Fe
through the same pathway as Tf (i.e. via TfR1-mediated
endocytosis and endosomal acidification [29]), experiments
were designed to assess if the well-characterized lysosomo-
tropic agents, ammonium chloride, chloroquine, or meth-
ylamine [23,41,42], could affect
59

Fe uptake (Fig. 5). It has
been well characterized in previous studies that these agents
inhibit acidification of the endosome which prevents Fe
uptake from Tf via RME [23,41,42]. These experiments
would provide further information on whether sMTf could
donate Fe through the RME pathway that is involved in Fe
uptake from Tf.
Interestingly, the lysosomotropic agents significantly
(P < 0.0001) reduced
59
Fe uptake from
59
Fe-
125
I-Tf to
15–27% of the relevant control, while they had far less
effect on
59
Fe uptake from
59
Fe-
125
I-sMTf (Fig. 5). In
fact, ammonium chloride and methylamine had no
significant effect on
59
Fe uptake from
59
Fe-
125

I-sMTf,
while chloroquine reduced
59
Fe uptake to 59% of the
control (Fig. 5). These results may suggest that the
intracellular trafficking route leading to
59
Fe release from
sMTf could be different to that of Tf. It is unclear why
chloroquine decreased
59
Fe uptake from sMTf compared
to ammonium chloride and methylamine, although it is
clear its efficiency at doing this was about fourfold less
than
59
Fe uptake from Tf (Fig. 5).
Fig. 4. sMTf does not donate
59
Fe to cells via specific transferrin-binding
sites. Comparison with Tf using WTB CHO cells with Tf-binding sites
and variant A CHO cells (TRVa) lacking Tf-binding sites. The WTB
andTRVaCHOcellswereincubatedfor24hat37°Cwith
59
Fe-
125
I-
sMTf (0.05 mgÆmL
)1
)or

59
Fe-
125
I-Tf (0.05 mgÆmL
)1
). The cells were
then washed and incubated with pronase (1 mgÆmL
)1
)for30minat
4 °C to separate internalized from membrane-bound radioactivity.
The results are a typical experiment from three performed and are
expressed as the mean ± SD of three determinations.
4440 M. R. Food et al. (Eur. J. Biochem. 269) Ó FEBS 2002
In contrast with
125
I-Tf,
125
I-sMTf is markedly degraded
by cells
Considering the results described above showing that sMTf
may be internalized by a nonspecific mechanism, we
explored the possibility that sMTf may be taken up and
degraded by the cell. This was examined using TCA
precipitation studies examining the proportion of protein-
free
125
I in the reincubation (efflux) media (Fig. 6A and B)
and the cells (Fig. 6C and D).
Protein-free
125

I in the reincubation (efflux) medium. In
contrast with
59
Fe-
125
I-Tf, marked degradation of
59
Fe-
125
I-
sMTf occurred after incubation with SK-Mel-28 melanoma
cells (Fig. 6). In all experiments, the
59
Fe-
125
I-sMTf or
59
Fe-
125
I-Tf initially added to cells contained < 2.5% free
125
I. However, after cells were labelled with
59
Fe-
125
I-MTf
for 3 h, washed, and then reincubated in efflux medium for
5–120 min, the percentage of protein-free
125
Iinthis

medium varied from 32 to 38% of the total (Fig. 6A).
More strikingly, after labelling for 24 h with
59
Fe-
125
I-MTf,
and the same reincubation time, the percentage of protein-
free
125
I in the efflux medium varied between 66 and 70%
(Fig. 6A). In contrast, after a 3 h incubation of cells with
59
Fe-
125
I-Tf followed by a reincubation for 5–120 min, the
percentage of protein-free
125
I in the efflux medium varied
from 2 to 3% of the total (Fig. 6B). This latter data using Tf
was in good agreement with our previous experiments with
SK-Mel-28 cells showing no significant degradation of this
molecule after an incubation of 2 h [23]. After incubating
cells with
59
Fe-
125
I-Tf for 24 h at 37 °C, more protein-free
125
I was found in the medium than that found after 3 h i.e.
9–19% (Fig. 6B), although this was still significantly

(P < 0.0001) less than that found for
59
Fe-
125
I-sMTf (66–
70%). It is relevant to note that our previous investigations
using this cell type also showed that longer labelling times
with
125
I-Tf (24 h) compared to shorter intervals (15 min to
2 h) resulted in accumulation into a noncycling compart-
ment where degradation may occur [25].
Protein-free
125
I in the cells. Assessment of protein-free
125
I was also performed on the cells from the experiments
described above, with significant (P < 0.0001) differences
being observed between
125
I-sMTf (Fig. 6C) and
125
I-Tf
(Fig. 6D). However, in contrast with the efflux media where
amarkeddifferencewasobservedinprotein-free
125
I
between a 3 h and 24 h incubation with
125
I-sMTf

(Fig. 6A), no significant difference was found between these
time points in the cells (Fig. 6C). This may be because once
protein-free
125
I is generated, most of it is released from the
cell into the efflux medium. Hence, we propose that a
steady-state level may be achieved between intracellular
breakdown of the
125
I-labelled protein and efflux of protein-
free
125
I.
The release of
125
I-Tf and
125
I-sMTf from melanoma cells
To examine whether sMTf could be internalized and then
released from the cell like Tf, cells were incubated with
59
Fe-
125
I-sMTf or
59
Fe-
125
I-Tffor3hor24hat37°C. The
cells were then washed and reincubated with new medium
forupto2hat37°C. The release of the

125
I-label into the
efflux medium from the cells was then assessed (Fig. 7).
The release of both sMTf and Tf from cells was
quantitatively similar comparing a 3 h and 24 h pre-
labelling time, with approximately 70–80% of the total
125
I-label being released within a 2 h reincubation at 37 °C.
Hence, only the release of
125
I-label after a 24 h incubation
is shown (Fig. 7).
Kinetic analysis of the efflux of the
125
I-label revealed
that after a pre-labelling period of 24 h with
59
Fe-
125
I-Tf
or
59
Fe-
125
I-sMTf, the release of
125
Ifromthecellafter
incubation with sMTf (Fig. 7A) was much more rapid
than when the incubation was with
125

I-Tf (Fig. 7B). In
fact, the time taken to release 50% of
125
I-label was 4 min
and 49 min when cells were labelled with
59
Fe-
125
I-sMTf
and
59
Fe-
125
I-Tf, respectively. The rapid release of
125
I
from the cell after incubation with
59
Fe-
125
I-sMTf was
consistent with release of the ligand from the cell surface
to the overlying medium and/or alternatively the release
of free (nonprotein-bound)
125
I from the cell. Considering
that 66–70% of
125
I in the efflux medium was not protein-
bound after labelling for 24 h with

125
I-sMTf (Fig. 6A), it
can be suggested that a large proportion of the
125
I
released (Fig. 7A) may be derived from the diffusion of
low M
r
125
I from the cells. In contrast, the slower release
of
125
I-Tf (Fig. 7B) was consistent with the efflux of the
Fig. 5. Lysosomotropic agents have far less effect on
59
Fe uptake from
59
Fe-
125
I-sMTf than
59
Fe-
125
I-Tf by SK-Mel-28 melanoma cells. Cells
were preincubated for 15 min at 37 °C with the lysosomotropic agents,
ammonium chloride (15 m
M
), chloroquine (0.5 m
M
) or methylamine

(15 m
M
). Then
59
Fe-
125
I-sMTf (0.05 mgÆmL
)1
)or
59
Fe-
125
I-Tf
(0.05 mgÆmL
)1
) was added and incubated with the cells for 3 h at
37 °C. The cell monolayer was washed and incubated with pronase
(1 mgÆmL
)1
)for30minat4°Ctodetermine
59
Fe internalization. The
results are a typical experiment from three performed and are
expressed as the mean ± SD of three determinations.
Ó FEBS 2002 Soluble melanotransferrin inefficiently donates iron to cells (Eur. J. Biochem. 269) 4441
intact
125
I-Tf molecule from the internalized compartment
by exocytosis, as seen in our previous investigation with
this cell type [25].

DISCUSSION
Previous investigations have shown that membrane-bound
MTf does not act as an efficient transporter of Fe into
human melanoma cells despite marked expression of the
molecule [9,24]. However, considering that sMTf has been
identified in the serum of patients with melanoma [3] and
Alzheimer’s disease [19,20], this form of the molecule
could donate Fe to cells via binding to specific Tf-binding
sites [15]. At present nothing is known concerning the
biological function of MTf or sMTf, and this is the first
study to assess the ability of sMTf to bind to cells and
donate its bound Fe. It is possible that sMTf could be
released from cells by the action of enzymes that cleave
the GPI-anchor or the protein itself [47,48]. However,
previous investigations have demonstrated that, at least in
culture, sMTf was secreted from SK-Mel-28 melanoma
cells [16]. It was vital to assess whether sMTf could bind
to cells via a high-affinity binding site, as this could be
critical in terms of its biological function. Indeed, MTf
has a high homology to Tf [11,12], and this could result in
binding to specific Tf-binding sites [29–31]. In addition,
this was important as membrane-bound MTf may act as
a potential intercellular adhesion molecule by binding to
the TfR1 on adjacent cells [15].
Our investigation shows that sMTf can donate
59
Fe to
melanoma cells but at a much lower efficiency than Tf
(Fig. 1A) and without binding to a saturable high affinity
receptor (Fig. 2A). Experiments with CHO cells with and

without specific Tf-binding sites [36,37] demonstrated that
in marked contrast with Tf, sMTf could not donate its
59
Fe
to cells via this pathway (Fig. 4). In addition, in contrast
with Tf (Fig. 2B), saturable uptake of sMTf by cells was not
observed (Fig. 2A). Further, membrane-binding of
125
I-
sMTf as a function of concentration was linear and
quantitatively far greater than internalization (Fig. 2A),
indicating nonspecific adsorption to the cell membrane.
Together, these observations indicate that the uptake of
59
Fe-
125
I-sMTf was not mediated by a saturable high
affinity-binding site in melanoma cells. In addition, as
125
I-
sMTf internalization increased linearly as a function of
concentration (Fig. 2A), this may be due to a nonspecific
uptake process e.g. adsorption to the membrane followed by
pinocytosis. Indeed, a second Fe uptake pathway from Tf
mediated by this latter pathway has been described in the
same cell type i.e. SK-Mel-28 melanoma cells [23].
The existence of another pathway of Fe uptake from
sMTf that was independent of RME was also suggested by
our studies using lysosomotropic agents. These studies
showed that

59
Fe uptake from sMTf was much less affected
than that from Tf (Fig. 5). Further evidence that sMTf was
taken up along another intracellular trafficking route was
the extent of
125
I-sMTf internalization which was signifi-
cantly (P < 0.0001) lower than that found for
125
I-Tf
(compare Fig. 2A with Fig. 2B). In addition, while
125
I-Tf
remained largely intact during its route through the
melanoma cell, particularly after short incubations (Fig. 6B
and D and [23]), sMTf was markedly degraded (Fig. 6A
and C). Thus, as a working model of sMTf uptake, we
propose that the molecule is internalized by nonspecific
Fig. 6. sMTf, in contrast with Tf, is markedly
degraded by SK-Mel-28 cells. The SK-Mel-28
melanoma cells were incubated with
59
Fe-
125
I-
sMTf (0.05 mgÆmL
)1
)or
59
Fe-

125
I-Tf
(0.05 mgÆmL
)1
)for3or24hat37°C. Cells
were then washed and reincubated with fresh
mediumforupto120minat37°C. The
overlying medium (efflux medium) and cells
were separated at the reincubation times
indicated and examined for radioactivity. The
proportion of
125
I that was free and protein-
bound in these two fractions was determined
by TCA precipitation. (A) Percentage of free
125
I in the efflux medium after incubation of
cells with
59
Fe-
125
I-sMTf. (B) Percentage of
free
125
I in the efflux medium after incubation
of cells with
59
Fe-
125
I-Tf. (C) Percentage of

free
125
I in the cells after incubation with
59
Fe-
125
I-sMTf. (D) Percentage of free
125
Iin
the cells after incubation with
59
Fe-
125
I-Tf.
The results are a typical experiment from three
performed and are expressed as the
mean ± SD (three determinations).
4442 M. R. Food et al. (Eur. J. Biochem. 269) Ó FEBS 2002
process e.g. adsorptive pinocytosis and then routed towards
the lysosome for proteolysis.
The results of the current study have implications for the
suggested use of sMTf as a vehicle to deliver chemothera-
peutic agents [49,50]. These latter investigators have specu-
lated that sMTf could be conjugated to drugs such as
doxorubicin, and preliminary data suggests increased effi-
cacy of conjugates compared to free agents [49,50]. It is
possible that the use of the conjugate may enable the
delivery of the agent to the lysosomal compartment that
then results in degradation of sMTf and sustained release of
the chemotherapeutic drug. Further studies assessing the

specific targeting to tumour cells will be required, as many
normal cell types are known to internalize molecules via
nonspecific processes such as pinocytosis, e.g. hepatocytes
and macrophages. Relevant to this, it is probable that sMTf
found in the serum of patients with melanoma and
Alzheimer’s disease [3,19,20] may be cleared from the
plasma by cell types with high pinocytotic activity. Indeed,
experiments examining the clearance of sMTf by rats
indicate marked uptake by the liver (E.H. Morgan and D.R.
Richardson, unpublished data).
The uptake of
59
Fe from sMTf was inhibited by
nonradioactive
56
Fe-Tf, indicating that during the Fe
uptake process, sMTf can donate Fe through a similar
pathway as Tf. Considering the discussion above, this
pathway was not consistent with RME, but has character-
istics concordant with the nonspecific Fe uptake mechanism
from Tf (e.g. pinocytosis) identified in melanoma cells
[22,23]. For instance, we demonstrated that internalization
of
125
I-sMTf cannot be inhibited by an excess of nonradio-
active
56
Fe-Tf. Indeed, a nonspecific process such as
pinocytosis has a large capacity for ligand uptake and will
not be inhibited by an excess of unlabelled ligand. However,

paradoxically, Fe uptake was inhibited from sMTf by an
excess of Tf. We suggest this may be because internalization
of both Tf and sMTf into a pinosome may result in
competition of the
59
Fe released from these molecules for an
Fe transporter (e.g. Nramp2).
Considering the results above, it is intriguing to note that
the N-terminal lobe of Tf does not bind to the hepatocyte
TfR1 but can donate Fe to these cells by a nonreceptor-
mediated mechanism [33] similar to the nonspecific process
identified in melanoma cells [22,23,26]. This is because the
TfR1 recognition site(s) appear to be on the C-terminal lobe
of Tf [51]. While the N-terminal lobe of sMTf has high
homology to the C-terminal of the Tf molecule [12], the
specific sites required for recognition by the TfR1 appear to
be absent [51]. Hence, it is remarkable that very similar
results can be found by comparing the N-terminal lobe of Tf
in hepatocytes [33] and sMTf in melanoma cells. These data
may indicate a similar mechanism of nonspecific Fe uptake
from these molecules in the two cell types.
We previously suggested that membrane-bound MTf
may act as an intercellular adhesion molecule by binding to
the high affinity TfR1 on adjacent cells [15]. Our current
investigation shows that sMTf does not bind to specific and
saturable Tf-binding sites or any other high affinity
receptor, which argues against this hypothesis. Further the
lack of a specific receptor for sMTf does not support the role
of this molecule as an autocrine-like growth factor. At this
point, it should also be mentioned that the role of TfR2 in

sMTf binding is not likely, as no saturable receptor binding
was identified.
In summary, our experiments demonstrate that sMTf can
donate Fe to cells but with much lower efficiency than Tf.
This Fe uptake pathway was not mediated by a specific Tf-
binding site or any other high affinity receptor, as there was
no saturable binding of sMTf to the cell. Further evidence
that sMTf was internalized by a pathway other than RME
is the fact that Fe uptake from sMTf was less sensitive to
lysosomotropic agents than Tf-bound Fe uptake. More-
over, in contrast with Tf, sMTf was markedly degraded by
the cell. These experiments have relevance to the clearance
of sMTf from the circulation in conditions such as
melanoma and Alzheimer’s disease [19,20] and the possible
use of this molecule as a carrier for chemotherapeutic agents
[49,50].
Fig. 7. The release of
125
I-Tf or
125
I-sMTf from SK-Mel-28 cells after
labelling for 24 h. The cells were labelled with
59
Fe-
125
I-Tf or
59
Fe-
125
I-

sMTf (0.05 mgÆmL
)1
)for24hat37°C. Cells were then washed and
reincubatedwithfreshmediumforupto120minat37°C. The
overlying medium and cells were separated at the reincubation times
indicated and examined for radioactivity (see Materials and methods).
The results are a typical experiment from three performed and are
expressed as the means of duplicate determinations.
Ó FEBS 2002 Soluble melanotransferrin inefficiently donates iron to cells (Eur. J. Biochem. 269) 4443
ACKNOWLEDGEMENTS
This work was supported by a Ph.D Scholarship (to M.F.) from the
Natural Sciences and Engineering Research Council of Canada
(NSERC) and by fellowship and grant support from the National
Health and Medical Research Council of Australia and an Australian
Research Council Large Grant (D.R.R.). We also thank R. Watts for
assistance in preparing the figures and the Heart Research Institute for
financial support.
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