Britishjournql of Haematology, 1979, 42, 481483.
Short Commu.nica.tion FAILURE OF RETICULOCYTES TO TAKE UP IRON FROM LACTOFERRIN SATURATED BY VARIOUS METHODS Although it is well established that reticulocytes can take up iron from transferrin, there are contradictory data concerning the ability of lactoferrin, the iron binding protein of exocrine secretions and neutrophil granules, to donate iron to reticulocytes. Zapolski & Princiotto (1976) were unable to detect uptake of lactoferrin iron, and attributed their own earlier contrary results (unpublished, cited by Aisen & Leibman, 1973) to the use of ferric chloride for labelling the protein. It is now known that this procedure can cause nonspecific binding ofiron (Bates & Schlabach, 1973) which may be subsequently taken up by reticulocytes. However, Line et al(1976) have more recently reported that reticulocytes took up iron from lactoferrin, and also from a monoferric fragment of lactoferrin derived by limited proteolysis. These workers labelled their lactoferrin by equilibrating iron-saturated lactoferrin with 59Fein the presence ofO.1 M sodium citrate, pH 7.0. Zapolski & Princiotto (1976),however, added 59Fe(as ferrous ammonium sulphate ascorbic acid) to apolactoferrin, the preparation of which requires removal of bound iron by dialysis against citric acid at pH 2 (Masson & Heremans, 1968). This procedure has recently been shown to destroy the ability of lactoferrin to bind to macrophages (Van Snick & Masson, 1976) and it therefore seemed possible that lactoferrinreticulocyte interactions might also be affected by prior exposure of the protein to low pH. T o determine whether this factor could be a cause of conflicting results, we have investigated the ability of rabbit reticulocytes to take up iron from bovine lactoferrin labelled in three different ways. In the first method iron-saturated lactoferrin was prepared from bovine colostrum by the method of Reiter et al (1975), rendered iron-free by dialysis against citric acid as described by Masson & Heremans (1968) and after neutralization labelled by adding 59Fe-dinitrilotriacetate to saturation. In the second method the iron saturated lactoferrin was equilibrated with s9Fe in 0-1 M sodium citrate, pH 7.0, for 24 h as described by Line et al (1976). In the third method lactoferrin was prepared from colostrum in its partially-saturated native form by omitting the step ofadding iron to the colostrum, and this sample was then saturated by adding 59Fe-dinitrilotriacetateas for the first sample. A sample of bovine transferrin (labelled by adding 59Fe-dinitrilotriacetate to apotransferrin) served as control. All samples were then dialysed for 36 h against two changes of 0.15 M NaC1-0.005 M Tris-0.01 M glucose, pH 7.45, to remove any unbound iron and ensure that any nitrilotriacetate initially incorporated into the protein-Fe-anion complex was replaced by bicarbonate (Aisen et al, 1967). Uptake of iron by reticulocytes from the labelled proteins was then carried out as described by Brock et al(1978). As can be seen (Fig 1) none of the lactoferrin samples could donate iron to reticulocytes whereas iron was taken up from transferrin as reported previously (Brock et al, 1978). These results indicate that the failure of Zapolski & Princiotto (1976) to demonstrate iron uptake from lactoferrin cannot be ascribed to exposure of the protein to low pH to remove bound iron, and that lactoferrin cannot act as an iron donor to reticulocytes. The inability of rats to incorporate lactoferrin-bound iron in viva into red cells (Van Snick et al, 1975) also suggests an
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Short Communication
FIG 1. Uptake of iron by rabbit reticulocytes from bovine transferrin
(0) and from bovine lactoferrin labelled with 59Feby (i) addition of 59Fe-dinitr~lotriacetate to apolactoferrin (A); (ii) equilibration of Fez-lactoferrin with "Fe in 0.1 M citrate, pH 7.0 (A); and (iii) addition of SyFe-dinitrilotriacetate to native partially-saturated lactoferrin (0).
intrinsic inability of lactoferrin to react with erythrocyte precursors. The contrary observations of Line et al(1976) may perhaps be explained by the fact that they gave no indication as to whether excess 59Feand citrate was removed prior to incubation with reticulocytes. Failure to do so would result in reticulocyte uptake of 59Fe-citrate (Morgan, 1971), which could be misinterpreted as uptake from lactoferrin. A difference in results due to the use of bovine rather than human lactoferrin in the present study seems unlikely in view of the widespread species cross-reactivity observed in the transferrin-reticulocyte system (Verhoef et al, 1973; Esparza & Brock, in preparation). ACKNOWLEDGMENT
We thank Dr M. Lozano of the Centro Regional de Oncologia, Zaragoza, for assistance with the radioisotope studies. Fundacio'n F . Cuenca Villoro, Instituto de Investigacio'n ' D .Juan Carlos I , , Gasco'n de Gotor 4 , Zaragoza-6, Spain
J. H. BROCK* INMACULADA ESPARZA
* Present address and offprints: Department of Bacteriology and Immunology, University of Glasgow, Western Infirmary, Glasgow G11 6NT, Scotland.
Short Communication
483
REFERENCES AISEN, P., AASA,R. M A L M S T R ~ B.G. M , & VANNCARD, T . (1967) Bicarbonate and the binding of iron to transferrin. journal of Biological Chemistry, 242, 2484-2490. AISEN,P. & LEIBMAN, A. (1973) The role ofthe anionbinding site of transferrin in its interaction with the reticulocyte. Biochiniica et Biophysica Acra, 304, w7-804. BATES, G.W. & SCHLABACH, M.R. (1973) Thereaction of ferric salts with transferrin. Journal of Biological Chemistry , 248, 3228-3232. BROCK,J.H., ARZABE,F.R., RICHARDSON, N.E. & DEVERSON, E.V. (1978) Characterization of monoferric fragments obtained by tryptic cleavage of bovine transferrin. Biochemical journal, 171, 73-78. LINE,W.F., SLY,D.A. & BEZKOROVAINY, A . (1976) Limited cleavage of human lactoferrin with pepsin. International journal qf Bio chemistry, 7, 203-208. MASSON, P.L. & HEREMANS, J. (1968) Metal-combining properties of human lactoferrin (red milk protein). 1. The involvement of bicarbonate in the reaction. European Journal of Biochemistry, 6, 579-584. MORGAN, E.H. (1971) A study of iron transfer from rabbit transferrin to reticulocytes using synthetic
chelating agents. Biochimica et Biophysica Acta, 244, 103-116. REITER,B., BROCK,].H. & STEEL, E.D. (1975) Inhibition of Escherichia coli by bovine colostrum and post-colostral bovine milk. 11. The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli. Immunology, 28, 83-95. VANSNICK, J.L. & MASSON, P.L. (1976) The binding of lactoferrin to mouse peritoneal cells. joirrnal of Experimental Medicine, 144. 1568-1580. VAN SNICK,J.L., MASSON,P.L. & HEREMANS, J.F. (1975) The affinity of lactoferrin for the reticuloendothelial system (RES) as the molecular basis for the hyposideraemia of inflammation. Proteins of Iron Storage in Biochemistry and Medicine (ed. by R. R. Crichton), pp. 433-438. North Holland, Amsterdam. VERHOEF, N.J., KREMERS,J.H.W. & LEIJNSE, B. (1973) The effect ofheterologous transferrin on the uptake of iron and haem synthesis by bone marrow cells. Biochimica et Biophysica Acta, 304, 114-122. ZAPOLSKI, Ed. & PRINCIOITO, J.V. (1976) Failure of rabbit reticulocytes to incorporate conalbumin or lactoferrin iron. Biochimica et Biophysica Acta, 421, 8&86.
Short Commu.nica.tion FAILURE OF RETICULOCYTES TO TAKE UP IRON FROM LACTOFERRIN SATURATED BY VARIOUS METHODS Although it is well established that reticulocytes can take up iron from transferrin, there are contradictory data concerning the ability of lactoferrin, the iron binding protein of exocrine secretions and neutrophil granules, to donate iron to reticulocytes. Zapolski & Princiotto (1976) were unable to detect uptake of lactoferrin iron, and attributed their own earlier contrary results (unpublished, cited by Aisen & Leibman, 1973) to the use of ferric chloride for labelling the protein. It is now known that this procedure can cause nonspecific binding ofiron (Bates & Schlabach, 1973) which may be subsequently taken up by reticulocytes. However, Line et al(1976) have more recently reported that reticulocytes took up iron from lactoferrin, and also from a monoferric fragment of lactoferrin derived by limited proteolysis. These workers labelled their lactoferrin by equilibrating iron-saturated lactoferrin with 59Fein the presence ofO.1 M sodium citrate, pH 7.0. Zapolski & Princiotto (1976),however, added 59Fe(as ferrous ammonium sulphate ascorbic acid) to apolactoferrin, the preparation of which requires removal of bound iron by dialysis against citric acid at pH 2 (Masson & Heremans, 1968). This procedure has recently been shown to destroy the ability of lactoferrin to bind to macrophages (Van Snick & Masson, 1976) and it therefore seemed possible that lactoferrinreticulocyte interactions might also be affected by prior exposure of the protein to low pH. T o determine whether this factor could be a cause of conflicting results, we have investigated the ability of rabbit reticulocytes to take up iron from bovine lactoferrin labelled in three different ways. In the first method iron-saturated lactoferrin was prepared from bovine colostrum by the method of Reiter et al (1975), rendered iron-free by dialysis against citric acid as described by Masson & Heremans (1968) and after neutralization labelled by adding 59Fe-dinitrilotriacetate to saturation. In the second method the iron saturated lactoferrin was equilibrated with s9Fe in 0-1 M sodium citrate, pH 7.0, for 24 h as described by Line et al (1976). In the third method lactoferrin was prepared from colostrum in its partially-saturated native form by omitting the step ofadding iron to the colostrum, and this sample was then saturated by adding 59Fe-dinitrilotriacetateas for the first sample. A sample of bovine transferrin (labelled by adding 59Fe-dinitrilotriacetate to apotransferrin) served as control. All samples were then dialysed for 36 h against two changes of 0.15 M NaC1-0.005 M Tris-0.01 M glucose, pH 7.45, to remove any unbound iron and ensure that any nitrilotriacetate initially incorporated into the protein-Fe-anion complex was replaced by bicarbonate (Aisen et al, 1967). Uptake of iron by reticulocytes from the labelled proteins was then carried out as described by Brock et al(1978). As can be seen (Fig 1) none of the lactoferrin samples could donate iron to reticulocytes whereas iron was taken up from transferrin as reported previously (Brock et al, 1978). These results indicate that the failure of Zapolski & Princiotto (1976) to demonstrate iron uptake from lactoferrin cannot be ascribed to exposure of the protein to low pH to remove bound iron, and that lactoferrin cannot act as an iron donor to reticulocytes. The inability of rats to incorporate lactoferrin-bound iron in viva into red cells (Van Snick et al, 1975) also suggests an
+
0007-1048/79/07oo-O481$02.00
01979 Blackwell Scientific Publications 48 1
482
Short Communication
FIG 1. Uptake of iron by rabbit reticulocytes from bovine transferrin
(0) and from bovine lactoferrin labelled with 59Feby (i) addition of 59Fe-dinitr~lotriacetate to apolactoferrin (A); (ii) equilibration of Fez-lactoferrin with "Fe in 0.1 M citrate, pH 7.0 (A); and (iii) addition of SyFe-dinitrilotriacetate to native partially-saturated lactoferrin (0).
intrinsic inability of lactoferrin to react with erythrocyte precursors. The contrary observations of Line et al(1976) may perhaps be explained by the fact that they gave no indication as to whether excess 59Feand citrate was removed prior to incubation with reticulocytes. Failure to do so would result in reticulocyte uptake of 59Fe-citrate (Morgan, 1971), which could be misinterpreted as uptake from lactoferrin. A difference in results due to the use of bovine rather than human lactoferrin in the present study seems unlikely in view of the widespread species cross-reactivity observed in the transferrin-reticulocyte system (Verhoef et al, 1973; Esparza & Brock, in preparation). ACKNOWLEDGMENT
We thank Dr M. Lozano of the Centro Regional de Oncologia, Zaragoza, for assistance with the radioisotope studies. Fundacio'n F . Cuenca Villoro, Instituto de Investigacio'n ' D .Juan Carlos I , , Gasco'n de Gotor 4 , Zaragoza-6, Spain
J. H. BROCK* INMACULADA ESPARZA
* Present address and offprints: Department of Bacteriology and Immunology, University of Glasgow, Western Infirmary, Glasgow G11 6NT, Scotland.
Short Communication
483
REFERENCES AISEN, P., AASA,R. M A L M S T R ~ B.G. M , & VANNCARD, T . (1967) Bicarbonate and the binding of iron to transferrin. journal of Biological Chemistry, 242, 2484-2490. AISEN,P. & LEIBMAN, A. (1973) The role ofthe anionbinding site of transferrin in its interaction with the reticulocyte. Biochiniica et Biophysica Acra, 304, w7-804. BATES, G.W. & SCHLABACH, M.R. (1973) Thereaction of ferric salts with transferrin. Journal of Biological Chemistry , 248, 3228-3232. BROCK,J.H., ARZABE,F.R., RICHARDSON, N.E. & DEVERSON, E.V. (1978) Characterization of monoferric fragments obtained by tryptic cleavage of bovine transferrin. Biochemical journal, 171, 73-78. LINE,W.F., SLY,D.A. & BEZKOROVAINY, A . (1976) Limited cleavage of human lactoferrin with pepsin. International journal qf Bio chemistry, 7, 203-208. MASSON, P.L. & HEREMANS, J. (1968) Metal-combining properties of human lactoferrin (red milk protein). 1. The involvement of bicarbonate in the reaction. European Journal of Biochemistry, 6, 579-584. MORGAN, E.H. (1971) A study of iron transfer from rabbit transferrin to reticulocytes using synthetic
chelating agents. Biochimica et Biophysica Acta, 244, 103-116. REITER,B., BROCK,].H. & STEEL, E.D. (1975) Inhibition of Escherichia coli by bovine colostrum and post-colostral bovine milk. 11. The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli. Immunology, 28, 83-95. VANSNICK, J.L. & MASSON, P.L. (1976) The binding of lactoferrin to mouse peritoneal cells. joirrnal of Experimental Medicine, 144. 1568-1580. VAN SNICK,J.L., MASSON,P.L. & HEREMANS, J.F. (1975) The affinity of lactoferrin for the reticuloendothelial system (RES) as the molecular basis for the hyposideraemia of inflammation. Proteins of Iron Storage in Biochemistry and Medicine (ed. by R. R. Crichton), pp. 433-438. North Holland, Amsterdam. VERHOEF, N.J., KREMERS,J.H.W. & LEIJNSE, B. (1973) The effect ofheterologous transferrin on the uptake of iron and haem synthesis by bone marrow cells. Biochimica et Biophysica Acta, 304, 114-122. ZAPOLSKI, Ed. & PRINCIOITO, J.V. (1976) Failure of rabbit reticulocytes to incorporate conalbumin or lactoferrin iron. Biochimica et Biophysica Acta, 421, 8&86.
