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: 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, 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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Compositions of Compounds Conjugated to P97 and Their Methods of Use Inter7 national Publication no WO 02/13873 A2 51 Zak, O & Aisen, P (2002) A new method for obtaining human transferrin C-lobe in the native conformation: preparation and properties Biochemistry 41, 1647–1653 . The soluble form of the membrane-bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein Michael. 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. 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