In this study, sMTf labelled with59Fe and125I was used to examine its ability to donate59Fe to SK-Mel-28 melanoma cells and other cell types.. Fax: +61 2 9382 1815, Tel.: +61 2 9382 1831
Trang 1The 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 with59Fe and125I was used to examine its ability to
donate59Fe to SK-Mel-28 melanoma cells and other cell
types sMTf donated59Fe 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 its59Fe via these
pathways This was confirmed by experiments using lyso-somotropic agents that markedly reduced59Fe uptake from
Tf, but had far less effect on 59Fe uptake from sMTf In addition, an excess of56Fe-labelled Tf or sMTf had no effect
on 125I-labelled sMTf uptake, suggesting a nonspecific interaction of sMTf with cells Protein-free125I 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
several alternative transcripts from the originally identified MTfgene (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· 105MTf 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 conendocyto-sistent 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 Mr 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: d.richardson@ccia.org.au
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, melano-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)
Trang 2Studies 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
from59Fe-citrate complexes but not from Tf However, in
these transfected cells, the levels of MTf (1.2· 106sites/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 with59Fe-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 Mr (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 with59Fe and125I to
examine its ability to donate59Fe 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 with59Fe-Tf uptake,59Fe
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
M A T E R I A L S A N D M E T H O D S
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)1penicillin (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 37C in a humidified atmosphere
of 5% CO2/95% 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 cm2 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-lm filter 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 Mr obtained by SDS/PAGE suggests post-translational modification con-sistent with glycosylation 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 MSand 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 with59Fe (Dupont NEN) or nonradioactive 56Fe to produce holo-diferric transferrin (59Fe-Tf, 56Fe-Tf) or holo-monoferric sMTf (59Fe-sMTf,56Fe-sMTf) using procedures established in our laboratory [22,26] Free 59Fe or 56Fe was removed by exhaustive dialysis against a large excess of 0.15M NaCl buffered to pH 7.4 with 1.4% NaHCO3[22,26] Both sMTf and Tf were labelled with59Fe 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, the expected colour change from clear to salmon pink was observed The UV-visible absorption maximum of mono-ferric sMTf was 464 nm as described in previous studies [14] Native PAGE-59Fe-autoradiography studies (see below [39]) demonstrated that all59Fe was bound to the proteins
Trang 3To examine the uptake of the proteins by cells,
mono-ferric sMTf and dimono-ferric Tf were labelled with125I by using
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
125I in the protein sample was measured by trichloroacetic
acid (TCA) precipitation (see below) and was always
< 2.5% of the total 125I 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 of59Fe-125I-labelled sMTf or Tf
by cells Further, previous studies showed that molecules
labelled with59Fe and125I using the current methods
resul-ted in functional proteins [22,23,25–27,41,42] In all
experi-ments examining the uptake of the125I-sMTf or125I-Tf, the
proteins were also saturated with59Fe by the procedures
described above These dual labelling experiments enabled
examination of the uptake of both the59Fe label and the
125I-labelled protein
Protein-free125I assay
The amount of protein-free125I 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
4C followed by one freeze–thaw cycle Control
experi-ments demonstrated that this lysis procedure did not
influence the proportion of protein-free125I
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 37C or 3 h at
4C with 59Fe-125I-Tf (0.001–0.1 mgÆmL)1) or 59Fe-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 59Fe-125I-labelled proteins were determined by
incubating cells with the general protease, pronase
(1 mgÆmL)1; Boehringer Mannheim), for 30 min at 4C,
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 mM), chloroquine
(0.5 mM), or methylamine (15 mM), on59Fe uptake from
59Fe-125I-Tf or 59Fe-125I-sMTf were examined by
pre-incubating cells with these agents for 15 min at 37C
[23,41,42] This medium was then removed, and the cells
incubated for 3 h at 37C with medium containing the
lyso-somotropic agents and either59Fe-125I-Tf (0.05 mgÆmL)1)
or 59Fe-125I-sMTf (0.05 mgÆmL)1) The internalization of
59Fe 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 59Fe-125I-sMTf (0.05 mgÆmL)1) or
59Fe-125I-Tf (0.05 mgÆmL)1) in MEM containing BSA (10 mgÆmL)1) for 3 h or 24 h at 37C 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
37C 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-free125I
Determination of intracellular iron distribution using native-PAGE-59Fe-autoradiography Native-PAGE-59Fe-autoradiography was performed using standard techniques in our laboratory [39] after incubation
of cells with 59Fe-sMTf (0.05 mgÆmL)1) or 59Fe-Tf (0.05 mgÆmL)1) for 24 h at 37C 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
R E S U L T S 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 59Fe-125I-sMTf by SK-Mel-28 melanoma cells and a number of other cell types These results have been compared to the uptake of 59Fe-125I-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
59Fe to cells as a linear function of incubation time up to
30 h at 37C (Fig 1A) However, sMTf donates its59Fe to cells at 14% the rate of Tf (Fig 1A) Similar results were also found when the uptake of59Fe 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-59Fe-autoradiography showed that both Tf and sMTf donated59Fe to cells, and this could label the Fe-storage protein, ferritin (see inset Fig 1A) However, densitometric analysis of 59Fe incor-poration into ferritin from sMTf demonstrated that it was about 10% of that found using Tf As shown previously, the ferritin-59Fe band comigrated with horse spleen ferritin and
Trang 4can be supershifted using an antiferritin polyclonal antibody
[46]
As found for Tf [22,29], the internalization of59Fe from
sMTf was markedly temperature dependent, there being
little internalized 59Fe uptake at 4C (data not shown)
Examining the total amount of radioactivity added to each
Petri dish of cells (approximately 500 000 cpm), the
proportion of59Fe radioactivity taken up by cells at 37C was equal to 0.09% for sMTf and 1.51% for Tf, a significant (P < 0.0001) 16-fold difference Hence, the59Fe uptake from sMTf by cells was much less efficient than Tf
Internalization of125I-sMTf as a function of time
is less marked than that of125I-Tf
To determine whether SK-Mel-28 melanoma cells could internalize sMTf, experiments were performed to assess the uptake of125I-sMTf compared to125I-Tf as a function of time up to 30 h at 37C 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 125I-sMTf was internal-ized than 125I-Tf in SK-Mel-28 melanoma cells For instance, after labelling for 3 h at 37C with 125I-sMTf (0.05 mgÆmL)1) or125I-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 125I-sMTf (0.05 mgÆmL)1) internalized after labelling for 3 h varied between 11 and 17% In contrast, the internalization of125I-Tf (0.05 mgÆmL)1) was much greater, ranging between 47 and 69% (data not shown)
Uptake of125I-sMTf and125I-Tf as a function of ligand concentration
To assess if a saturable binding site for 59Fe-125I-sMTf occurred on the cell membrane, experiments were designed to investigate the uptake of 125I-sMTf as a function of ligand concentration using SK-Mel-28 melan-oma cells (Fig 2A) In the same experiment, the binding
of 125I-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
125I-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
125I-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 of125I-Tf was biphasic, with saturation occurring at a Tf concentration of
0.01 mgÆmL)1, as we showed previously [22,23,26] Furthermore, unlike 125I-sMTf uptake, the internalized
125I-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,125I-Tf was taken up by a saturable binding site as found previously [22,23,26], whereas the binding of 125I-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 from59Fe-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 with59Fe-125I-sMTf (0.05 mgÆmL)1) for up to 30 h
at 37 C The cells were then washed and incubated with pronase
(1 mgÆmL)1) for 30 min at 4 C to separate internalized from
mem-brane-bound59Fe and125I Native PAGE-59Fe-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).
Trang 5Uptake of59Fe from sMTf and Tf as a function
of ligand concentration
The uptake of59Fe 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 59Fe
uptake from59Fe-sMTf was linear, the r for each being 0.97,
0.99 and 0.99, respectively (Fig 3A) Higher concentrations
of sMTf, up to 0.5 mgÆmL)1, also resulted in linear uptake of
Fe as a function of ligand concentration (data not shown)
The internalized59Fe uptake from sMTf varied from 26 to
44% of the total59Fe uptake, there being more59Fe uptake
by the membrane than that internalized at all ligand concentrations (Fig 3A) In contrast,59Fe uptake from Tf was biphasic as a function of ligand concentration with saturation of 59Fe uptake occurring at approximately 0.01 mgÆmL)1(Fig 3B), as found in our previous studies [22,23] The internalized59Fe uptake from Tf ranged from 84
to 92% of the total59Fe 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)125I-Tf, by SK-Mel-28 melanoma cells The cells were
incubated with59Fe-125I-sMTf (0.005–0.1 mgÆmL)1) or 59Fe-125I-Tf
(0.005–0.1 mgÆmL)1) for 3 h at 37 C The cells were then washed and
incubated with pronase (1 mgÆmL)1) for 30 min at 4 C to separate
internalized from membrane-bound 125I 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 on59Fe uptake from: (A)59 Fe-sMTf, or (B)59Fe-Tf, by SK-Mel-28 melanoma cells The cells were incubated with 59Fe-125I-sMTf (0.005–0.1 mgÆmL)1) or 59Fe-125I-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) for 30 min at
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.
Trang 6Competition 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 of59Fe- or the 125I-labelled protein was assessed
Coincubation of cells with59Fe-125I-sMTf (0.05 mgÆmL)1)
and an excess of nonradioactive 56Fe-Tf (1 mgÆmL)1)
inhibited59Fe uptake to 12 ± 1% of that found for sMTf
alone, but had no effect on 125I-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 of56Fe-Tf had no effect on the uptake
of 125I-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 59Fe-Tf (0.01 mgÆmL)1)
with an excess of nonradioactive56Fe-sMTf (0.1 mgÆmL)1),
did not significantly affect 59Fe uptake (101 ± 6% of
that found for59Fe-Tf) These results suggest that sMTf
is not a good competitive inhibitor of Fe uptake from Tf
Incubation of cells with59Fe-sMTf (0.05 mgÆmL)1) and a
twofold excess of nonradioactive56Fe-sMTf (0.1 mgÆmL)1)
decreased 59Fe uptake to 29 ± 7% of that found with
59Fe-sMTf alone This latter experiment was important to
determine whether competition occurred between 59
Fe-labelled sMTf and its56Fe-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 59Fe from 59Fe-125I-Tf
(0.05 mgÆmL)1) and 59Fe-125I-sMTf (0.05 mgÆmL)1) over
24 h at 37C As expected, the WTB cells efficiently
inter-nalized59Fe from Tf, while59Fe uptake from this molecule
by the TRVa cells was sixfold lower (Fig 4) In contrast,
there was no significant difference in 59Fe 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
equivalent59Fe 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 on59Fe Uptake from59Fe-Tf and59Fe-sMTf
To further examine whether sMTf could be donating59Fe 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 affect59Fe 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 59Fe uptake from 59Fe-125I-Tf to 15–27% of the relevant control, while they had far less effect on 59Fe uptake from 59Fe-125I-sMTf (Fig 5) In fact, ammonium chloride and methylamine had no significant effect on 59Fe uptake from 59Fe-125I-sMTf, while chloroquine reduced 59Fe uptake to 59% of the control (Fig 5) These results may suggest that the intracellular trafficking route leading to59Fe release from sMTf could be different to that of Tf It is unclear why chloroquine decreased59Fe uptake from sMTf compared
to ammonium chloride and methylamine, although it is clear its efficiency at doing this was about fourfold less than59Fe uptake from Tf (Fig 5)
Fig 4 sMTf does not donate59Fe 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 and TRVa CHO cells were incubated for 24 h at 37 C with 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) for 30 min at
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.
Trang 7In contrast with125I-Tf,125I-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-free125I in the reincubation (efflux) media (Fig 6A and B)
and the cells (Fig 6C and D)
Protein-free125I in the reincubation (efflux) medium.In
contrast with59Fe-125I-Tf, marked degradation of59Fe-125
I-sMTf occurred after incubation with SK-Mel-28 melanoma
cells (Fig 6) In all experiments, the 59Fe-125I-sMTf or
59Fe-125I-Tf initially added to cells contained < 2.5% free
125I However, after cells were labelled with59Fe-125I-MTf
for 3 h, washed, and then reincubated in efflux medium for
5–120 min, the percentage of protein-free 125I in this
medium varied from 32 to 38% of the total (Fig 6A)
More strikingly, after labelling for 24 h with59Fe-125I-MTf,
and the same reincubation time, the percentage of
protein-free125I in the efflux medium varied between 66 and 70%
(Fig 6A) In contrast, after a 3 h incubation of cells with
59Fe-125I-Tf followed by a reincubation for 5–120 min, the percentage of protein-free125I 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 with59Fe-125I-Tf for 24 h at 37C, more protein-free
125I 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 for59Fe-125I-sMTf (66– 70%) It is relevant to note that our previous investigations using this cell type also showed that longer labelling times with125I-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 125I in the cells Assessment of protein-free
125I was also performed on the cells from the experiments described above, with significant (P < 0.0001) differences being observed between 125I-sMTf (Fig 6C) and 125I-Tf (Fig 6D) However, in contrast with the efflux media where
a marked difference was observed in protein-free 125I between a 3 h and 24 h incubation with 125I-sMTf (Fig 6A), no significant difference was found between these time points in the cells (Fig 6C) This may be because once protein-free125I 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 the125I-labelled protein and efflux of protein-free125I
The release of125I-Tf and125I-sMTf from melanoma cells
To examine whether sMTf could be internalized and then released from the cell like Tf, cells were incubated with
59Fe-125I-sMTf or59Fe-125I-Tf for 3 h or 24 h at 37C The cells were then washed and reincubated with new medium for up to 2 h at 37C The release of the125I-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
125I-label being released within a 2 h reincubation at 37C Hence, only the release of125I-label after a 24 h incubation
is shown (Fig 7)
Kinetic analysis of the efflux of the 125I-label revealed that after a pre-labelling period of 24 h with 59Fe-125I-Tf
or 59Fe-125I-sMTf, the release of 125I from the cell after incubation with sMTf (Fig 7A) was much more rapid than when the incubation was with 125I-Tf (Fig 7B) In fact, the time taken to release 50% of125I-label was 4 min and 49 min when cells were labelled with 59Fe-125I-sMTf and 59Fe-125I-Tf, respectively The rapid release of 125I from the cell after incubation with 59Fe-125I-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)125I from the cell Considering that 66–70% of125I in the efflux medium was not protein-bound after labelling for 24 h with125I-sMTf (Fig 6A), it can be suggested that a large proportion of the 125I released (Fig 7A) may be derived from the diffusion of low Mr125I from the cells In contrast, the slower release
of 125I-Tf (Fig 7B) was consistent with the efflux of the
Fig 5 Lysosomotropicagents have far less effect on 59 Fe uptake from
59
Fe-125I-sMTf than59Fe-125I-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 59Fe-125I-sMTf (0.05 mgÆmL)1) or 59Fe-125I-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) for 30 min at 4 C to determine 59
Fe internalization The results are a typical experiment from three performed and are
expressed as the mean ± SD of three determinations.
Trang 8intact125I-Tf molecule from the internalized compartment
by exocytosis, as seen in our previous investigation with
this cell type [25]
D I S C U S S I O N
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 donate59Fe 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 its59Fe
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
59Fe-125I-sMTf was not mediated by a saturable high affinity-binding site in melanoma cells In addition, as125 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 that59Fe 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 of125I-sMTf internalization which was signifi-cantly (P < 0.0001) lower than that found for 125I-Tf (compare Fig 2A with Fig 2B) In addition, while125I-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) for 3 or 24 h at 37 C Cells were then washed and reincubated with fresh medium for up to 120 min at 37 C The overlying medium (efflux medium) and cells were separated at the reincubation times indicated and examined for radioactivity The proportion of125I 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 with59Fe-125I-sMTf (B) Percentage of free 125 I in the efflux medium after incubation
of cells with 59 Fe- 125 I-Tf (C) Percentage of free125I in the cells after incubation with
59 Fe- 125 I-sMTf (D) Percentage of free 125 I in 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).
Trang 9process 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 59Fe from sMTf was inhibited by nonradioactive 56Fe-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
of125I-sMTf cannot be inhibited by an excess of nonradio-active 56Fe-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 the59Fe 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 of125I-Tf or125I-sMTf from SK-Mel-28 cells after
labelling for 24 h The cells were labelled with59Fe- 125 I-Tf or 59 Fe- 125
I-sMTf (0.05 mgÆmL)1) for 24 h at 37 C Cells were then washed and
reincubated with fresh medium for up to 120 min at 37 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.
Trang 10A C K N O W L E D G E M E N T S
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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