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(50 pg) was labelled with '"I using 5 pg of tetrachloroglycoluri1 as the catalyst and a reaction time of 1 min (Regoeczi 1983). After removing the unbound '"I by dialysis, the preparation was treated with heparitinase as described above.

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Study of the effect o HSPG on iron release from rat Tf Iron release from e-labelled rat Tf was studied at pH 7.3 and 5.6 by the method of Bali et al. (1991), with minor modifications. Differic rat Tf was used routinely, except for one preparation in which 80% of the radioactive iron was directed to the binding site in the N-lobe; the N-lobe releases iron more readily than the binding site in the C-terminal domain (Aisen and Listowsky 1980). For the study at pH 7.3, rat Tf (125 pg/mL) and HSPG (equivalent to 80-90 pg/mL) were dissolved in 50 mM HEPES containing 0.1 M NaCl and 10 mM CHAPS, while the buffer at pH 5.6 was 50 mM MES with the same concentrations of NaCl and CHAPS as in the other buffer. Iron release was prompted by pyrophosphate (1 mM), an agent that plays a dual role in the release reaction: first, it destabilizes the Tf-Fe-carbonate ternary complex, probably by attacking the carbonate ion (Egyed 1975), and second, it chelates the nascent ferric ion (Pollack et al. 1977). Samples (20 pL) were removed at intervals, and after adding 600 pg of unlabelled rat Tf, the 5 9 ~still e bound to rat Tf was precipitated by polyethylene glrcol (M, 8000) at a final concentration of 20% (w/v). Tracer (I $1) studies showed that over 96% of the rat Tf was removed from samples under these conditions, irrespectively of whether HSPG was present.

Results Autoradiography (Fig. 1) showed that most of the [35~]sulphate label migrated in association with molecules whose mass was 205 kDa. A similarly smeary appearance of hepatic HSPG in the same high M, region was also reported by Lyon and Gallagher (1991). Incubation with heparan sulphate lyase converted the slowly moving radioactive material into small, fast migrating fragments of approximately 14 kDa. These fragments thus corresponded in size to the polysaccharide chains obtained from HSPG of rat liver membranes by Oldberg et al. (1979). Digestion with chondroitinase, on the other hand, brought about only a slight increase in film exposure close to 20 kDa. We conclude from these observations that our preparation consisted of HSPG, with chondroitin sulphate proteoglycan as a minor component. However, chondroitin sulphate does not bind to Tf (Omoto et al. 1990), and consequently, the phenomena described below are attributable to HSPG. Data pertaining to the affinity of intact HSPG for rat Tf are summarized in Fig. 2. At pH 7.3, HSPG bound comparably both to rat diferric and apo-Tf, so that close to 60% of the load was eluted by NaCl in the range of 0.2-0.3 M and the rest was eluted above 0.4 M. In sharp contrast, displacement of HSPG from rat apo-Tf at pH 5.6 required higher concentrations of NaCl. Thus, a mere 14% of HSPG was displaced by 0.4 M NaCl. A further 25% was recovered after raising the salt concentration to 0.5 M and 52% more was recovered by elution with 1 M NaCl. HSPG showed no affinity for Sepharose, to which ethanolamine was conjugated instead of rat Tf. Affinities for rat apo-Tf of the intact HSPG, its glycan chains, and the core protein at pH 6.0 are contrasted in Fig. 3. The whole molecule bound well at this pH, though less firmly than at pH 5.6 in Fig. 2. Most of the glycans liberated from the core protein also bound to rat apo-Tf, although with a reduced affinity, the reduction corresponding to a difference of approximately 0.15 M NaCl. Moreover, minor glycan subpopulations with little or no affinity

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FIG. 1. Autoradiograph of a HPG preparation. (A) Untreated HPG; (B) HPG after digestion with heparan sulphate lyase; (C) HPG after digestion with chondroitinase ABC. Approximate molecular masses (kDa) are based on a mixture of marker proteins (Sigma) were run in an adjacent track (not shown). calcium acetate. After 5 h at 37OC, the samples were electrophoresed (SDS-PAGE) in 7% gel under reducing conditions. The dried gel was sprayed with E N ~ H A N C E(New ~ ~ England Nuclear) and layered on a Kodak film (X-Omat AR) for 4 h. Protein was estimated by the method of Lowry et al. (1951).

Study of the affinity of HSPG for transferrin Affinity chromatography was used to test interaction between the various forms of transferrin (apo or differic) and HSPG. Rat Tf was prepared and immobilized on Sepharose 4B as described elsewhere (Rudolph and Regoeczi 1987). Columns containing 3 mL of gel and 10 mg of rat Tf were equilibrated at S°C with 50 mM NaCl and 0.1% Triton X-100, buffered either with 10 mM TrisHCl to pH 7.3 or 10 mM phosphate to pH 5.6. The HSPG sample to be chromatographed was dialyzed, in the cold, against the respective equilibrating buffer. In experiments with rat apo-Tf, iron was removed by washing the column with 0.1 M citrate buffer (pH 5.0) containing 50 pg/mL of the iron chelator desferrioxarnine mesylate (Ciba). The same concentration of the chelator was also included in all other solutions when working with rat apo-Tf. the routine HSPG load was in the range of 9-15 pg protein. Columns were eluted at room temperature by increasing (stepwise) the concentration of NaCl in the equilibrating buffer. A control column was made by activating gel in the usual way, but coupling ethanolamine instead of rat Tf. In addition to intact HSPG, the affinities for rat Tf of the cleaved HS glycan chains and of the enzymically deglycosylated HS core protein were investigated. Behaviour of the chains was followed by their "S activity. To study the HS core protein, intact HSPG

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HU AND REGOECZI

MINUTES FRACTION NUMBER FIG. 2. Chromatography of [ 3 5 ~ on] Tf-Sepharose. ~ ~ ~ ~ The chromatograms denote the following conditions with respect to column type and operating pH: - - -,diferric Tf, pH 7.3; 0 , apo-Tf, pH 7.3; A, apo-Tf, pH 5.6; a , ethanolamine-Sepharose, pH 7.3. Columns were eluted by changing the molarity of NaCl in the equilibrating buffer to the values appearing above the arrows. One millilitre fractions were collected.

FIG.4. Release of '9Fe from diferric Tf at pH 5.6 and 22°C without ( 0 ) or with (a) HSPG (equivalent to 5 1 pg pprtein/mL) present. The incubation mixture contained Tf (125 pg/mL) and 1 mM pyrophosphate in the buffer system given in Materials and methods.

5 9 ~ine the N-domain, discharged quantitatively the iron from the binding site at pH 5.6 in less than 3 min in the presence of HSPG (34 pg).

Discussion HSPG molecules isolated from different tissues vary considerably in structure, including the size of the core protein and the composition of side chains (Fransson 1985). As a possible reason for this, it is assumed that HSPG from various sources may engage in interactions with diverse macromolecules (Fransson et al. 1986). HSPG has been shown to bind Tf in preparations from cultured human embryonic skin fibroblasts (Fransson et al. 1984; Caster et al. 1986) and cultured hepatic endothelial cells (Omoto et al. 1990). However, no attempt was made in those studies to investigate the impact of HSPG binding on the physFRACTION NUMBER iological properties of transferrin. FIG. 3. Chromatography of intact [ 3 5 ~(- - ]-), cleaved ~ ~ ~ ~ In the light of the earlier studies referred to above, Tf [ 3 5 ~ glycan ] ~ ~ chains (-), and the [ ' Z S core ~ ] ~protein ~ (. . binding by hepatic HSPG comes less as a surprise, even on a column (1.5 x 3.5 cm) of apo-Tf-Sepharose at pH 6.0. The though the mechanism of attraction seems to vary with the column was eluted by changing the molarity of NaCl in the cell type. In the case of fibroblasts, Tf is bound by the core equilibrating buffer to the values given above the arrows. One millilitre fractions were collected. protein (Fransson et al. 1984). Data in Fig. 3, on the other hand, suggest that hepatic HSPG binds rat Tf via its glycan chains. Complete disruption of the HSPG-Tf interaction for rat apo-Tf were present as well. The implication is that by appropriate concentrations of NaCl suggests that ionic the hepatic HS glycan chains, like those of endothelial HS bonding is involved. The ineffectiveness of the hepatic HS (Lindblom et al. 1991), are heterogeneous. Of the deglycore protein as a direct rat Tf binder might be due to its cosylated core protein preparation, 81% passed through the small size relative to the fibroblast core protein (Fransson column with a minimum of interaction, while the remainder et al. 1986). Nevertheless, the hepatic HS core protein seems bound normally. We think that this bound fraction repreto play an indirect role in this event by anchoring and coorsented molecules which failed to deglycosylate. dinating the glycan chains. This is a likely explanation of HSPG did alter the kinetics of iron release from differic the weakened interaction of free chains with rat Tf in Fig. 3. rat Tf. At pH 5.6, HSPG augmented the early, rapid com- The type of interaction found here is thus analogous to lipoprotein lipase binding by endothelial HS (Lindblom ponent of the biphasic release reaction, while its effect on the slow component was less prominent (Fig. 4). Further et al. 1991). studies showed that the magnitude of the rapid component According to the present observations, HSPG-bound rat (representing release from the N-domain), but hardly that Tf exhibits several features that resemble those seen with of the slow one (release from the C-domain), depended on receptor-bound rat Tf. They may be, therefore, of physiological significance. One of these is the increased strength the concentration of HSPG. In the absence of pyrophosphate, HSPG did not release 5 9 ~from e rat Tf. At of interaction between rat Tf and HSPG at low pH (Fig. 2), pH 7.4, HSPG neither accelerated nor retarded iron release which is analogous to the situation when rat Tf is bound by pyrophosphate. Monoferric rat Tf (8 pg), with 80% of to a bonafide receptor (Ecarot-Charrier et al. 1990). As a )

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already pointed out, this is a key prerequisite for Tf to avoid degradation while moving through cells. The other feature is the effect on iron release. Bali et al. (1991) found recently that receptor binding alters the kinetics of iron release from Tf, in such a way that removal is facilitated at low p H and hindered at extracellular pH. As an explanation they suggest that a conformational change takes place during binding; the change forces open the ironbinding cleft(s) (Anderson et al. 1990) at endosomal pH, with the result that iron release becomes accelerated. If this proposition is correct, then our data imply that HSPG binding also affects Tf conformation. However, while receptor binding mainly affects the cleft in the C-domain with little impact o n that in the N-domain (Bali and Aisen 1991), HSPG binding elicits the converse effect. Furthermore, HSPG binding, unlike receptor binding (Bali et al. 1991), does not stabilize the Fe-Tf complex at p H 7.4, and HSPG, in contrast to the receptor (Young and Aisen 1981), does not distinguish between the differic and apo forms of Tf. These differences notwithstanding, it now seems that those unique features which make Tf a reusable iron transporter (i.e., pH-dependent binding strength and binding-dependent iron release) are vested in the Tf molecule itself, rather than in any particular acceptor counterpart. HSPG is a membrane-intercalated proteoglycan of the hepatocyte (Hook et al. 1984), and as just explained, it interacts with Tf in a receptor-like manner. Further studies will have to show whether this interaction translates into a function in vivo. Several arguments are in favour of such a possibility. First, the liver is relatively rich in HSPG (Oldberg et al. 1979), thus explaining the large number of secondary binding sites for Tf (cf. Introduction). Second, liver cells are highly pinocytic (Hoffenberg et al. 1970; Rudolph and Regoeczi 1991), whereby the HSPG-Tf adduct is expected to be internalized. Indeed, as known from work o n macrophages, cell-surface HSPG is endocytosed (Owens and Wagner 1991). Lastly, a large dose of lactoferrin, another protein with affinity for HSPG but not for the Tf receptor, significantly reduces the hepatic'acquisition of iron from Tf in vivo (Hu and Regoeczi 1991).

Acknowledgements We thank P.A. Chindemi for his assistance. This work was supported by the Medical Research Council of Canada. Aisen. P., and Listowsky, I. 1980. Iron transport and storage proteins. Annu. Rev. Biochem. 49: 357-393. Anderson, B.F., Baker, H.M., Norris, G.E., et 01. 1990. Apolactoferrin structure demonstrates ligand-induced conformational change in transferrins. Nature (London), 344: 784-787. Bali, P., and Aisen, P. 1991. Receptor-modulated iron release from transferrin: differential effects on N and C-terminal sites. Biochemistry, 30: 9947-9952. Bali, P.K., Zak, O., and Aisen, P. 1991. A new role for the transferrin receptor in the release of iron from transferrin. Biochemistry, 30: 324-328. Cole, E.S., and Glass, J. 1983. Transferrin biding and iron uptake in mouse hepatocytes. Biochim. Biophys. Acta, 762: 102-1 10. Coster, L., Carlstedt, I., Kendall, S., et al. 1986. Structure of proteoheparan sulfates from fibroblasts. J. Biol. Chem. 261: 12 079 - 12 088. Dautry-Varsat, A. 1986. Receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor. Biochimie, 68: 375-381. Ecarot-Charrier, B., Grey, V.L., Wilczynska, A., and Schulman,

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H.M. 1980. Reticulocyte membrane transferrin receptors. Can. J. Biochem. 58: 418-426. Egyed, A. 1975. Effect of adenine nucleotides and pyrophosphate on the exchange of transferrin-bound carbonate. Biochim. Biophys. Acta, 411: 349-356. Fransson, L.-A. 1985. Mammalian glycosaminoglycans. In The polysaccharides. Vol. 3. Edited by G.O. Aspinall. Academic Press, New York. pp. 337-415. Fransson, L.-A., Carlstedt, I., Coster, L., and Malmstrom, A. 1984. Binding of transferrin to the core protein of fibroblast proteoheparan sulfate. Proc. Natl. Acad. Sci. U.S.A. 81: 5657-5661. Fransson, L.-A., Carlstedt, I.. Coster, L., and Malmstrom, A. 1986. The functions of the heparan sulphate proteoglycans. Ciba Found. 124: 125-142. Hoffenberg, R., Gordon, A.H., Black, E.G., and Louis, L.N. 1970. Plasma albumin catabolism by the perfused rat liver. The effect of alteration of albumin concentration and dietary protein depletion. Biochem. J. 118: 401-404. Hook, M., Kjellkn, L., Johansson, S., and Robinson, J. 1984. Cellsurface glycosaminoglycans. Annu. Rev. Biochem. 53: 847-869. Hu, W.-L., and Regoeczi, E. 1991. Effect of lactoferrin on transferrin-mediated iron uptake by rat liver. In Proceedings of the 10th International Conference on Iron and Iron Proteins, Oxford University, Oxford, U.K. July 27-31. p. 13. Jandl, J.H., and Katz, J.H. 1963. The plasma-to-cell cycle of transferrin. J. Clin. Invest. 42: 314-326. Klausner, R.D., Ashwell, G., Van Renswounde, J., et al. 1983. Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc. Natl. Acad. Sci. U.S.A. 80: 2263-2266. Lindblom, A., Bengtsson-Olivecrona, G., and Fransson, L.-A. 1991. Domain structure of endothelial heparan sulphate. Biochem. J. 279: 821-829. Lowry, O.H., Rosebrough, M. J., Farr, A.L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Lyon, M., and Gallagher, J.T. 1991. Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver. Biochem. J. 273: 415-422. Morgan, E.H. 1983. Effect of pH and iron content of transferrin onits binding to reticulocyte receptors. Biochim. Biophys. Acta, 762: 498-502. Oldberg, A., Kjellkn, L., and Hook, M. 1979. Cell-surface heparan sulfate. Isolation and characterization of a proteoglycan from rat liver membranes. J. Biol. Chem. 254: 8505-8510. Omoto, E., Minguell, J. J., and Tavassoli, M. 1990. Proteoglycan synthesis by cultured liver endothelium: the role of membraneassociated heparan sulfate in transferrin binding. Exp. Cell Res. 187: 85-90. Owens, R.T., and Wagner, W.D. 1991. Metabolism and turnover of cell surface-associated heparan sulfate proteoglycan and chondroitin sulfate proteoglycan in normal and cholesterol-enriched macrophages. Arterioscler. Thromb. 11: 1752- 1758. Page, M.A., Baker, E., and Morgan, E.H. 1984. Transferrin and iron uptake by rat hepatocytes in culture. Am. J. Physiol. 246: G26-G33. Pollack, S., Vanderhoff, G., and Lasky, F. 1977. Iron removal from transferrin. An experimental study. Biochim. Biophys. Acta, 497: 481-487. Regoeczi, E. 1983. Iodogen-catalyzed iodination of transferrin. Int. J. Pept. Protein Res. 22: 422-433. Rudolph, J.R., and Regoeczi, E. 1987. Isolation of the rat transferrin receptor by affinity chromatography. J. Chromatogr. 396: 369-373. Rudolph, J .R., and Regoeczi, E. 1991. Relationship between pinocytic rate and uptake of transferrin by suspended rat hepatocytes. Biol. Met. 4: 166-172. Young, S.P., and Aisen, P. 1981. Transferrin receptors and the uptake and release of iron by isolated hepatocytes. Hepatology (Baltimore), 1: 114-1 19.

Hepatic heparan sulphate proteoglycan and the recycling of transferrin.

Heparan sulphate proteoglycan, labelled with [35S]sulphate, was prepared from rat livers for studies of its interaction with purified rat transferrin...
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