Printed in Sweden Copyright Q 1975 by Academic Press, Inc. All rights of rcproducfion in any form reserved
Experimental
THE
EFFECT IRON
Cell Research 91 (1975) 87-94
OF CONCANAVALIN UPTAKE
A ON TRANSFERRIN
BY RABBIT
AND
RETICULOCYTES
T. T. LOHI and E. H. MORGAN Department
of Physiology,
University of Western Western Australia
Australia,
Nedlands,
SUMMARY The effect of concanavalin A on transferrin and iron uptake by reticulocytes was determined using rabbit reticulocytes and rabbit transferrin labelled with 58Fe and lzsI and concanavalin A (ConA) labelled with W. In concentrations of 5&200 pg/ml ConA markedly inhibited iron uptake but did not inhibit transferrin uptake or release from the cells. ConA was itself taken up by rabbit blood cells in a manner similar to that of transferrin except that the uptake was not specific for reticulocytes but occurred also with mature erythrocytes. The inhibition of iron uptake by concanavalin and the uptake of concanavalin by the cells were both inhibited by a-methyl-o-mannoside. It is concluded that the effects observed were due to the binding of concanavalin to glycoproteins of the cell membrane, either by a direct interaction with transferrin receptors or by the production of a non-specific change in the structure of the membrane.
Reticulocytes are able to take up iron from the plasma iron-binding protein, transferrin and incorporate it into haem [I]. During this process the transferrin molecules are themselves taken up by the reticulocytes [2-4], presumably being bound to specific receptors on the cell membrane. The nature of these receptors is unknown. It is possible that they are represented by one of the glycoproteins which are present in the red cell membrane [5-71. This is suggestedby the sensitivity of reticulocyte uptake of transferrin and iron to the action of proteolytic enzymes such as trypsin [2] which readily cause the releaseof glycoproteins from the red cell membrane [8]. Concanavalin A is a lectin or cell-agglutinating protein obtained from Canavalia ensijormis (jack bean). It specifically interacts -__
with polysaccharides which contain M-Dglucopyranosyl, ix-D-mannopyranosyl or cz-Dglucosaminyl residues[9]. As a result of such reactions it agglutinates various animal cells including erythrocytes [ 10, 1l] and stimulates blastic transformation and mitosis in lymphocytes [12]. The possibility that transferrin receptors of reticulocytes are glycoprotein in nature suggested that they may react with ConA. This reaction could affect the ability of reticulocytes to take up iron from transferrin. The aim of the present work was to investigate this question using rabbit reticulocytes, rabbit transferrin labelled with radioiodine and 5gFeand Con A.
’ Present address: Department of Physiology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia.
Rabbit transferrin was purified by ion-exchange chromatography on DEAE cellulose, gel-filtration on Sephadex G 200 (Pharmacia, Uppsala, Sweden) and
MATERIALS
AND METHODS
Proteins and protein-labelling
Exptl
Cell Res 91 (1975)
88
Loh and Morgan
crystallization from aqueous solution as previously described [13]. Rabbit y-globulin was obtained during the preparation of transferrin as a protein fraction which was eluted from the DEAE-cellulose column prior to the transferrin. Con A was purchased from Sigma Chemical Co., St Louis, USA. The proteins were labelled with radioiodine by the iodine monochloride method [14] using carrier-free lz61 or 1311 obtained from the Radiochemical Centre, Amersham, UK. Non-protein-bound radioiodine was removed by ion-exchange through a column of IRA 410 followed by dialysis against 0.15 M NaCl. Transferrin was also labelled with s9Fe by incubating apotransferrin with 69FeC1, (Radiochemical Centre) for 1 h at 37°C. The amount of iron used was always less then that required to achieve 50 % saturation of the iron-binding capacity of the transferrin. Phytohaemagglutmin (reagent grade) derived from PhkeoZus seeds was obtained from Wellcome Reagents Ltd., Beckenham, Kent, UK.
Reticulocytes Adult hybrid rabbits were bled from a marginal ear vein to produce a reticulocytosis. The blood used in the experiments was collected using heparin as an anticoagulant. The cells were washed three times at 4°C in 0.15 M NaCl and were then suspended in Hanks & Wallace balanced salt solution [15] to give an haematocrit of approx. 50%. The reticulocyte count of the reticulocyte-rich blood used varied from 15-45 %. No attempt was made to separate reticulocytes from mature erythrocytes. It has been shown that only reticulocytes take up transferrin and iron 12, 31.
I. Abscissa: incubation time (min); ordinate: iron uptake @g/ml cells). Effect of ConA on iron uptake by rabbit reticulocvtes. The iron concentration was 1.4 ,ug/ml; transferrin concentration: 3.1 mg/ml; reticulocyte count: 26 %. O-O, control; a-m, ConA, 200 pg/ml plus u-methyl-n-mannoside, 60 mM; O-O, ConA, 50 pg/ml; A-A, ConA, 100 fig/ml; A-A, ConA, 200 pug/ml. Fig.
RESULTS
The effect of ConA on transferrin and iron uptake by reticulocytes was initially deterIncubation procedure mined at concentrations varying from 50Transferrin and iron uptake by reticulocytes and 300 ,ug/ml. Considerable haemolysis occurred transferrin release from the cells were measured as described previously [3, 41. In some experiments 1311 when concentrations of 250-300 pg/ml ConA labelled y-globulin or ConA were used in addition were used. Therefore concentrations above to ‘2SI-tr&ferrin or instead of transferrin. The effect of Con A or phytohaemagglutinin on transferrin and 200 pg/ml were not used in later experiments. iron uptake was determined by first incubating reOnly slight haemolysis occurred at this conticulocytes at 37°C with the desired concentration of the lectin for 5 min, then cooling the cells in an icecentration and the 5gFe values of cells treated bath followed by addition of labelled transferrin with Con A were corrected for loss of 5gFe (and in one experiment y-globulin) and reincubation at 37°C. by measuring the haemoglobin concentration of control and test samples and assuming Analytical methods that the loss of 5sFe in the test samples were proportional to their loss of haemoglobin. The haematocrit was measured by a microhaematocrit procedure. Haemoglobin was determined as oxyAlthough agglutination of the cells was nohaemoglobin. Reticulocytes were counted after stainticed towards the end of periods of incubaing with new methylene blue. Iron concentration was determined by the method recommended by the Intertion this did not interfere with sampling of national Hadmatology Society 1161 and -total ironthe cells or with subsequent washing when binding capacity as described by Morgan & Carter [17]. Protein was measured by a biuret procedure [18] the cells were easily dispersed in the 0.15 M using bovine serum albumin as a standard. RadioNaCl used for this purpose. activity was counted in a well-type y-detector. Exptl
Cell Res 91 (1975)
Concanavalin and transferrin
.A A’ /
c
10 0’ {. .
0
Experimental
THE
EFFECT IRON
Cell Research 91 (1975) 87-94
OF CONCANAVALIN UPTAKE
A ON TRANSFERRIN
BY RABBIT
AND
RETICULOCYTES
T. T. LOHI and E. H. MORGAN Department
of Physiology,
University of Western Western Australia
Australia,
Nedlands,
SUMMARY The effect of concanavalin A on transferrin and iron uptake by reticulocytes was determined using rabbit reticulocytes and rabbit transferrin labelled with 58Fe and lzsI and concanavalin A (ConA) labelled with W. In concentrations of 5&200 pg/ml ConA markedly inhibited iron uptake but did not inhibit transferrin uptake or release from the cells. ConA was itself taken up by rabbit blood cells in a manner similar to that of transferrin except that the uptake was not specific for reticulocytes but occurred also with mature erythrocytes. The inhibition of iron uptake by concanavalin and the uptake of concanavalin by the cells were both inhibited by a-methyl-o-mannoside. It is concluded that the effects observed were due to the binding of concanavalin to glycoproteins of the cell membrane, either by a direct interaction with transferrin receptors or by the production of a non-specific change in the structure of the membrane.
Reticulocytes are able to take up iron from the plasma iron-binding protein, transferrin and incorporate it into haem [I]. During this process the transferrin molecules are themselves taken up by the reticulocytes [2-4], presumably being bound to specific receptors on the cell membrane. The nature of these receptors is unknown. It is possible that they are represented by one of the glycoproteins which are present in the red cell membrane [5-71. This is suggestedby the sensitivity of reticulocyte uptake of transferrin and iron to the action of proteolytic enzymes such as trypsin [2] which readily cause the releaseof glycoproteins from the red cell membrane [8]. Concanavalin A is a lectin or cell-agglutinating protein obtained from Canavalia ensijormis (jack bean). It specifically interacts -__
with polysaccharides which contain M-Dglucopyranosyl, ix-D-mannopyranosyl or cz-Dglucosaminyl residues[9]. As a result of such reactions it agglutinates various animal cells including erythrocytes [ 10, 1l] and stimulates blastic transformation and mitosis in lymphocytes [12]. The possibility that transferrin receptors of reticulocytes are glycoprotein in nature suggested that they may react with ConA. This reaction could affect the ability of reticulocytes to take up iron from transferrin. The aim of the present work was to investigate this question using rabbit reticulocytes, rabbit transferrin labelled with radioiodine and 5gFeand Con A.
’ Present address: Department of Physiology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia.
Rabbit transferrin was purified by ion-exchange chromatography on DEAE cellulose, gel-filtration on Sephadex G 200 (Pharmacia, Uppsala, Sweden) and
MATERIALS
AND METHODS
Proteins and protein-labelling
Exptl
Cell Res 91 (1975)
88
Loh and Morgan
crystallization from aqueous solution as previously described [13]. Rabbit y-globulin was obtained during the preparation of transferrin as a protein fraction which was eluted from the DEAE-cellulose column prior to the transferrin. Con A was purchased from Sigma Chemical Co., St Louis, USA. The proteins were labelled with radioiodine by the iodine monochloride method [14] using carrier-free lz61 or 1311 obtained from the Radiochemical Centre, Amersham, UK. Non-protein-bound radioiodine was removed by ion-exchange through a column of IRA 410 followed by dialysis against 0.15 M NaCl. Transferrin was also labelled with s9Fe by incubating apotransferrin with 69FeC1, (Radiochemical Centre) for 1 h at 37°C. The amount of iron used was always less then that required to achieve 50 % saturation of the iron-binding capacity of the transferrin. Phytohaemagglutmin (reagent grade) derived from PhkeoZus seeds was obtained from Wellcome Reagents Ltd., Beckenham, Kent, UK.
Reticulocytes Adult hybrid rabbits were bled from a marginal ear vein to produce a reticulocytosis. The blood used in the experiments was collected using heparin as an anticoagulant. The cells were washed three times at 4°C in 0.15 M NaCl and were then suspended in Hanks & Wallace balanced salt solution [15] to give an haematocrit of approx. 50%. The reticulocyte count of the reticulocyte-rich blood used varied from 15-45 %. No attempt was made to separate reticulocytes from mature erythrocytes. It has been shown that only reticulocytes take up transferrin and iron 12, 31.
I. Abscissa: incubation time (min); ordinate: iron uptake @g/ml cells). Effect of ConA on iron uptake by rabbit reticulocvtes. The iron concentration was 1.4 ,ug/ml; transferrin concentration: 3.1 mg/ml; reticulocyte count: 26 %. O-O, control; a-m, ConA, 200 pg/ml plus u-methyl-n-mannoside, 60 mM; O-O, ConA, 50 pg/ml; A-A, ConA, 100 fig/ml; A-A, ConA, 200 pug/ml. Fig.
RESULTS
The effect of ConA on transferrin and iron uptake by reticulocytes was initially deterIncubation procedure mined at concentrations varying from 50Transferrin and iron uptake by reticulocytes and 300 ,ug/ml. Considerable haemolysis occurred transferrin release from the cells were measured as described previously [3, 41. In some experiments 1311 when concentrations of 250-300 pg/ml ConA labelled y-globulin or ConA were used in addition were used. Therefore concentrations above to ‘2SI-tr&ferrin or instead of transferrin. The effect of Con A or phytohaemagglutinin on transferrin and 200 pg/ml were not used in later experiments. iron uptake was determined by first incubating reOnly slight haemolysis occurred at this conticulocytes at 37°C with the desired concentration of the lectin for 5 min, then cooling the cells in an icecentration and the 5gFe values of cells treated bath followed by addition of labelled transferrin with Con A were corrected for loss of 5gFe (and in one experiment y-globulin) and reincubation at 37°C. by measuring the haemoglobin concentration of control and test samples and assuming Analytical methods that the loss of 5sFe in the test samples were proportional to their loss of haemoglobin. The haematocrit was measured by a microhaematocrit procedure. Haemoglobin was determined as oxyAlthough agglutination of the cells was nohaemoglobin. Reticulocytes were counted after stainticed towards the end of periods of incubaing with new methylene blue. Iron concentration was determined by the method recommended by the Intertion this did not interfere with sampling of national Hadmatology Society 1161 and -total ironthe cells or with subsequent washing when binding capacity as described by Morgan & Carter [17]. Protein was measured by a biuret procedure [18] the cells were easily dispersed in the 0.15 M using bovine serum albumin as a standard. RadioNaCl used for this purpose. activity was counted in a well-type y-detector. Exptl
Cell Res 91 (1975)
Concanavalin and transferrin
.A A’ /
c
10 0’ {. .
0
