80
Biochimica et Biophysica Acta, 421 (1976) 80--86 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - Printed in The N e t h e r l a n d s
BBA 27788
F A I L U R E OF RABBIT RETICULOCYTES TO INCORPORATE CONALBUMIN OR L A C T O F E R R I N IRON
E.J. Z A P O L S K I and J.V. P R I N C I O T T O *
Department of Physiology and Biophysics, Schools of Medicine and Dentistry, Georgetown University, Washington D.C. 20007 (U.S.A.) (Received J u n e 20th, 1975)
Summary Despite the remarkable molecular similarity of human lactoferrin and human transferrin, the results of this investigation indicate that human lactoferrin was unable to furnish rabbit reticulocytes with iron for heme synthesis. Although conalbumin closely resembles transferrin in many of its properties, conalbumin iron-binding differs from human transferrin iron-binding. There are conflicting reports in the literature regarding conalbumin's ability to furnish iron to reticulocytes. In this study, small amounts of lactoferrin or conalbumin were adsorbed to mature and immature cell surfaces but neither of these ironbinding proteins surrendered iron intracellularly to reticulocytes for heme synthesis.
Introduction Lactoferrin is widely distributed in the human body but its precise physiologic role is still unknown. It is a bacteriostatic agent and may also play a role in glandular iron metabolism [1]. Lactoferrin and transferrin exhibit striking similarities in their primary structure [2]. Their optical and electron paramagnetic resonance spectra are virtually indistinguishable from each other [3]. Although conalbumin is not found in the human, it does resemble transferrin in many respects and has been traditionally compared to human transferrin with regard to virtually every physico-chemical property that has been studied [4-8]. Recent evidence now indicates that conalbumin iron-binding differs from transferrin [ 8,9]. Unlike transferrin, the iron-binding affinities of conalbumin's two ironbinding sites are not identical and this might influence its ability to surrender * To whom correspondence should be s e n t .
81 iron to reticulocytes. There are, however, conflicting reports in the literature regarding conalbumin's ability to fulfill this biological function. Jandl et al. [10] reported that iron bound to conalbumin was as effectively utilized by reticulocytes as was transferrin bound iron. Schade [11] reported that conalbumin was unable to furnish iron to reticulocytes. In light of recent findings that conalbumin iron-binding differs from transferrin iron-binding, the ability or inability of conalbumin to function in this capacity may be of future importance in our understanding of the interaction of iron-binding proteins with immature red cell membranes. We have investigated conalbumin's ability to donate iron to reticulocytes for heme synthesis. Since reticulocyte membrane receptors have been likened to other cell membrane receptors [ 10,12], we have also investigated the ability of lactoferrin to function in this capacity to determine if its similarity to transferrin can be extended to include the biological property of iron donation. Materials and Methods
Iron-binding proteins Human apolactoferrin was a generous gift of Dr Philip Aisen (Albert Einstein College of Medicine, Bronx, New York). The ratio of absorbancy at 470 nm to absorbancy at 410 nm for human transferrin (Behring Diagnostics, Woodbury, New York) and conalbumin (Sigma, Type I, St. Louis, Mo.) was less than 1.4. These proteins were purified by chromatography [8,14]. Absorbance ratios obtained after chromatographic purification were 1.40 for transferrin and 1.45 for conalbumin. Iron was removed by dialysis which included treatment with 0.1 M NaC104 [7,13]. Final dialysis was performed with the saline that we employed throughout these studies: 150 mM NaC1, 5 mM KC1, 1 mM NaHCO3, 0.5 mM HEPES (N-2-hydroxyethyl piperazine-N'-2 ethane sulfonic acid) pH 7.4. Protein concentration was estimated from absorbance at 280 nm and iron protein complexes were prepared with ferrous ammonium sulphate-ascorbic acid, tracer labeled with s 9 FeC13 [14]. An excess of 2 iron equivalents per transferrin was added to each protein solution. After incubation at 37°C for 30 min., each solution was passed through a small column of anion exchange resin (BioRad, AG 1X-4, 100-200 mesh C1- form, equilibrated with saline medium) to remove unbound iron (15). Iron which was added to buffer in the absence of protein was completely removed by resin. The iron-binding capacity of the protein and its molar concentration were then determined from the specific activity in the eluate and this data was used to prepare solutions for cell uptake studies. Fully saturated, diferric iron-binding protein solutions (0.010 mM) were prepared by adding a calculated 5 percent excess of iron to protein. Partially saturated solutions were prepared by adding the required a m o u n t of iron to protein. All solutions were passed through resin to remove unbound iron. Unlabeled iron protein solutions were prepared in the same manner, using an unlabeled iron solution. ~ 2 s I-labeled proteins (less than one g-atom 1/mol protein) were prepared from diferric proteins by the m e t h o d of McFarlane [16]. Unbound 12 s I was removed by anion exchange resin treatment and iron was removed by dialysis.
82 These labeled proteins were treated as we have described and they were finally resaturated with unlabeled iron. The 0.001 mM solutions used in our studies were prepared by dilution with saline.
Cell suspensions Heparinized, reticulocyte-rich cells, collected from previously bled rabbits, were washed three times with cold saline. Care was taken to remove the buffy coat and the cells were adjusted to give a 50 percent packed cell volume. Reticulocyte-poor cells were collected from unbled rabbits and were treated in the same manner. Reticulocyte counts, using New Methylene Blue, were performed on dried blood films. Reticulocyte-rich cell preparations contained 20--30% reticulocytes while mature cell suspensions contained less than 1% reticulocytes.
Incubation procedures Equal volumes of temperature equilibrated cell suspension and test solution were mixed and incubated in small open Erlenmeyer flasks. In some experiments two different iron-binding proteins were present (one labeled with s 9 Fe and the other unlabeled). These solutions were mixed just prior to cell incubation. Aliquots of incubation mixtures were delivered into 10 vol. of ice-cold saline media for cell uptake determinations and were also lysed in ice-cold 0.5 mM HEPES buffer (pH 7.4) for stromal determinations. The samples were centrifuged, washed three times with corresponding media and resuspended before counting. Cell stroma preparations yielded a buff colored pellet. Hemin was isolated directly from one hour incubation samples following the procedure of Labbe and Nishada [ 1 7 ] . Counting was performed in a well-type scintillation counter and the percentage of cell isotope uptake was calculated. Cell stroma values were calculated as the fraction of whole cell uptake that was recovered with stroma. ..." ...""
....I,
59Fe
.....""
%
~."
%
..~
,.o
I
3~
A C T
... ~.: .."
T Y
......
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...
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.,'
:'
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.....~ 1251 ~.: ~. ,W . . . . . . . . -~ . . . . . . . . . . . • ........... ~ . . . . . . . . . . . . . . . . . . . . . . . ~
;,~..*, ~ - -' ~ - - - ~ - - - ~ ~
1 2 5 1 & 59Fe
.~
~ ~n
~
:.
................
/
:.~•
/
:' .."
...
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//
V
:.~
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,,"
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.......... ! 59Fe
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...
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...........~.. . . . . . . . . . . . . .
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~mmmm
LI 1125 I & 5gFe )
m
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m
& 59Fe )
/
:. ..~
/,~ . . . . .-' -- -- -- ,,I,, -- -- --~, ~i¢4,~ ~ ,~.-,,~ . ,~,,,i ~ ~ ,-,~ ~ - " " ~ ................. ~'........... ':" ............ ~'. . . . . . . . . . . . . . . . . . . . . . . . . 3'0
6"0
~
125, 9"0
rain
Fig. 1. P e r c e n t a g e 59 Fe a n d 1 2 5 i . l a b e l e d iron binding p r o t e i n in w a s h e d cells. Tr, Transferrin; L f , L a c t o f e r r i n ; CA, c o n a l b u m i n . I n c u b a t i o n m i x t u r e : cells, 24% r e t i c u l o c y t e s , 0 . 0 1 0 m M p r o t e i n s o l u t i o n . Fig. 2. S a m e as Fig. 1, e x c e p t i n c u b a t i o n m i x t u r e : cells, 20% r e t i c u l o c y t e s , 0 . 0 0 1 m M protein solution.
83
Results
Re tic~locy te up take from diferric pro teins T h e p e r c e n t a g e of 59Fe a n d 1: s I-labeled p r o t e i n t a k e n up by reticuloc y t e s f r o m 0 . 0 1 0 m M diferric transferrin, l a c t o f e r r i n , a n d c o n a l b u m i n are illus t r a t e d in Fig. 1. All s o l u t i o n s were i n c u b a t e d with the s a m e r e t i c u l o c y t e suspension. Less t h a n 2 p e r c e n t o f the labeled iron p r e s e n t as l a c t o f e r r i n or conalb u m i n was t a k e n u p b y the cells. In c o n t r a s t , a high p e r c e n t a g e u p t a k e was o b s e r v e d f o r t r a n s f e r r i n b o u n d iron-59. Cell u p t a k e o f 125 I-labeled p r o t e i n was l o w f o r all t h r e e p r o t e i n s . This s t u d y was r e p e a t e d at a l o w e r c o n c e n t r a t i o n (0.001 raM) to determ i n e w h e t h e r t h e r e were a n y a p p a r e n t d i f f e r e n c e s b e t w e e n u p t a k e of e i t h e r i s o t o p e f o r l a c t o f e r r i n or c o n a l b u m i n . T h e s e results are s h o w n in Fig. 2. A f t e r 10-fold dilution, u p t a k e o f labeled l a c t o f e r r i n or c o n a l b u m i n increased to nearly 10 p e r c e n t . H o w e v e r , it is e v i d e n t t h a t t h e r e was no d i f f e r e n c e in p e r c e n t u p t a k e o f e i t h e r s 9 Fe or 1 : 5 I-labeled l a c t o f e r r i n or c o n a l b u m i n .
Stromal, low temperature and mature cell uptake T a b l e I lists d a t a o b t a i n e d f r o m studies with r e t i c u l o c y t e - r i c h cell p r e p a r a tions ( i n c u b a t e d at 3 7 ° C and at l o w t e m p e r a t u r e s ) a n d m a t u r e cells ( i n c u b a t e d at 37 °C). T h e s e d a t a were o b t a i n e d a f t e r one h o u r o f i n c u b a t i o n with 0.001 m M s o l u t i o n s a n d in each case the f r a c t i o n o f w a s h e d cell r a d i o a c t i v i t y which was r e c o v e r e d w i t h t h e cell s t r o m a is listed. R e t i c u l o c y t e - r i c h cells i n c u b a t e d at 3 7 ° C t o o k u p 92% o f the diferric t r a n s f e r r i n iron a n d less t h a n 3% o f this u p t a k e was r e c o v e r e d with cell s t r o m a . Cell u p t a k e o f t r a n s f e r r i n 59 Fe was m a r k e d l y r e d u c e d w h e n r e t i c u l o c y t e s were i n c u b a t e d at l o w t e m p e r a t u r e . T h e s t r o m a iron f r a c t i o n was c o n s i d e r a b l y higher f o r r e t i c u l o c y t e s i n c u b a t e d at l o w t e m p e r a t u r e s (0.99) and f o r m a t u r e cells i n c u b a t e d at 3 7 ° C (0.16). T h e f r a c t i o n of 125i.labele d transferrin recovered with cell s t r o m a u n d e r all these c o n d i t i o n s was u n i f o r m l y high for e i t h e r r e t i c u l o c y t e s or m a t u r e cells (close to 90% o f the t o t a l cell u p t a k e ) . In c o n t r a s t , f o r e i t h e r l a c t o f e r r i n or c o n a l b u m i n t h e r e was n o d i f f e r e n c e in r e t i c u l o c y t e p e r c e n t u p t a k e o f 5 ~ Fe or 12 5 I, and a high f r a c t i o n of this u p t a k e
TABLE I RETICULOCYTE
AND MATURE Label
Transferrin Transferrin Lactoferrin Lactoferrin Conalbumin Conalbumin
59Fe 125I 59Fe 125I 59Fe 125I
CELL UPTAKE
R e t i c u l o c y t e s (at 3 7 ° C ) ( W a s h e d cells)
Reticulocytes (at 0°C) ( W a s h e d cells)
M a t u r e cells (at 3 7 ° C ) (Washed cells)
% Activity -+ S E M
Stroma*
% Activity +- S E M
Stroma
% Activity + SEM
Stroma
92.0 3.4 7.6 7.4 5.9 4.2
0.03 0.91 0.60 0.72 0.45 0.98
7.1 + 1.2 7.4-+ 1.8 7.6 +- 1.4 7.1 + 1.0 5.2 + 0 . 9 4.9 + 1 . 0
0.99 0.94 0.88 0.85 0.96 0.82
9.7 3.1 6.3 4.0 5.8 3.9
0.16 0.89 0.68 0.80 0.65 0.99
-+ 2 . 0 + 1.2 -+ 1 . 2 + 2.4 + 0.8 + 0.5
+ 1.1 + 1.0 + 1.3 +- 1.8 +- 0 . 9 +- 0 . 8
* A l l s t r o m a v a l u e s r e p o r t e d as f r a c t i o n o f w a s h e d c e l l a c t i v i t y r e c o v e r e d w i t h s t r o m a .
84 TABLE
II
RETICULOCYTES: % Saturation
25 50 75 100
%59Fe
UPTAKE
FROM
PARTIALLY
SATURATED
SOLUTIONS
% 59 F e Lacto ferrin
Conalbumin
Transferrin
1 3 8 9
0 1 4 6
62 78 83 91
was recovered b o und to cell stroma. Uptake was the same with reticulocytes or mature cells. Reticulocyte SgFe uptake was unaltered when 0.010 or 0.001 mM labeled conalbumin or lactoferrin was mixed with an equal concentration of unlabeled transferrin and then incubated with cells. More significantly, addition o f unlabeled lactoferrin or conalbumin had no effect on reticulocyte utilization of s 9Fe from transferrin, suggesting that these proteins were nonspecifically adsorbed to the cell surface rather than specifically bound to reticulocyte receptor sites. Virtually no s 9 Fe activity was recovered with hemin isolated from reticulocytes incubated at 37°C with lactoferrin or conalbumin (less than 1% of the total cell uptake). In contrast 55--65% of the cell S~ Fe uptake from transferrin was recovered with isolated hemin.
Reticulocyte uptake from partially saturated solutions s 9 Fe reticulocyte uptake data from partially saturated protein preparations are listed in Table II. Uptake of lactoferrin or conalbumin bound iron, even at this low concentration (0.001 mM) was abolished when the protein was less than half saturated with iron. Discussion Reticulocytes have the ability to take up iron from albumin, gamma globulin, ferric chloride, and various iron chelate solutions [ 1 0 , 1 8 ] . This may involve exchange of the metal to transferrin bound to the cells [ 1 9 ] , so that precautions must be taken to assure that one is studying the direct effect of the specific iron c o m p o u n d under investigation. The A4 ~ o/A41 o absorbance ratios obtained after chr om a t ogr aphy of commercial transferrin and conalbumin indicate that they were of a high degree of purity. Human lactoferrin used in our investigation was the subject of an earlier study [ 3]. Prior to reports by Cavill [20] and by Bates and Schlabach [21] we, like other investigators, assumed that iron-binding proteins completely bound iron at neutral pH. As a result, in preliminary studies with iron-binding proteins that were formed using ferric chloride as the iron source, we observed that both lactoferrin and conalbumin delivered iron to reticulocytes (as much as 50% from " s a t u r a t e d " conalbumin). On the basis of these observations, Aisen and Leibman reported that the reticulocyte is capable of incorporating iron which is complexed to lactoferrin [ 2 2 ] . Considerably less than two iron atoms/mol of transferrin actually occupy
85 iron binding sites when ferric chloride is added to apotransferrin. The remainder is n o t b o u n d at the specific iron-binding sites. This non-specifically b o u n d iron is even eluted with transferrin from Sephadex G-25 [ 2 1 ] , so our initial observation, that conalbumin and lactoferrin surrendered iron to reticulocytes, did n o t reflect uptake of iron that was specifically bound at the protein iron-binding sites. In the present study, we utilized an anion exchange resin to remove all non-specifically bound iron from transferrin solutions [ 15,20]. Our results for conalbumin confirm Schade's findings [11]. Although Schade emp lo y ed crude egg white in his study, he concluded that neither h u man nor rabbit reticulocytes t o o k up iron bound to conalbumin. In our study, less than 2% of the s 9Fe from 0.01 mM conalbumin was taken up by the cells in comparison to 50% uptake of transferrin bound iron (Fig. 1). When the c o n c e n t r a t i o n of conalbumin was reduced ten-fold, percentage uptake (but n o t absolute uptake) was enhanced and it is evident that little if any intracellular utilization of conalbumin iron had occurred (Fig. 2, Table I). The percentage s 9 Fe or 12 s I-labeled conalbumin present in washed cells was identical for each isotope, at low temperature, at 37°C and, in studies with mature cells. A major fraction of each isotope was recovered still bound to the cell stroma. Heroin, isolated from reticulocytes, indicated that no s 9Fe was incorporated into heme. Similar results were also observed for lactoferrin. The findings that neither conalbumin or lactoferrin bound iron was utilized for heme synthesis directly contrasted results obtained with transferrin. Jandl et al. [10] r e por t ed that reticulocytes readily utilized conalbumin iron. This observation was based on experiments in which they em pl oyed ironconalbumin prepared from FeCla and no a t t e m p t was made to remove any u n b o u n d iron. Their results are similar to our earlier observations and can be explained if a por t i on of the iron in their conalbumin preparation was not specifically b o u n d at the conalbumin iron-binding sites and instead this source of iron was utilized by the cells. There have been several studies (reviewed in ref. 23) of reticulocyte and marrow cell acquisition of iron from heterogeneous cell-transferrin systems such as we have e m p l o y e d in our study. In no report was cell uptake ever abolished; rather, the rate of iron accumulation from heterogeneous transferrin-reticulocyte systems was slower than the rate of uptake from homogeneous systems. Verhoeff et al. [ 23] demonstrated that the subcellular distribution of metal was the same in all of the transferrin-cell systems that they investigated. The failure of rabbit reticulocytes to utilize hum an lactoferrin iron cannot be totally ascribed to a p r o p e r t y of the rabbit reticulocyte. These cells do incorporate iron from human transferrin, hence the inability of lactoferrin to serve in this capacity illustrates that there is a functional difference between human lactoferrin and h u m a n transferrin. Despite its close similarity to transferrin, lactoferrin may not even interact with reticulocyte transferrin r ecept or sites. We did observe that a small a m o u n t of lactoferrin or conalbumin was bound by cell suspensions but this observation was n o t restricted to reticulocytes since it was also observed with mature cells. However, reticulocytes did not utilize any iron bound to these proteins. Secondly, neither lactoferrin or conalbumin interfered with reticulocyte uptake of transferrin iron. If lactoferrin or conalbumin was bound at the specific
86 membrane receptor sites their presence should have inhibited uptake of transferrin iron. It has been suggested that the release of transferrin bound iron to the reticulocyte is the result of enzymatic cleavage of carbonate (or bicarbonate) from the ternary iron-protein-anion complex [22,24,25]. Possibly lactoferrin or conalbumin complexes cannot serve as substrate for the anion-detaching enzyme of rabbit reticulocytes and this could also account for the failure of the cell to utilize this source of iron for heme synthesis. In either case the failure of human lactoferrin to mimic the reticulocyte iron donating role of human transferrin implies that this iron-binding protein is not involved in iron metabolism at the erythroid cell and its physiologic role remains unknown. Williams [ 26] reported findings which might be misinterpreted that conalbumin and fragments of conalbumin (isolated after trypsin digestion) are both effective rabbit reticulocyte iron donors. He reported reticulocyte uptake values that ranged from 5--50 cpm/0.1 ml. On the basis of the 0.2 mCiS9Fe he used to prepare his solutions, we calculate that this cell uptake is no more than 0.5--5% of the total activity per samples. This value is in keeping with results that we have reported in this study.
Acknowledgements This investigation was supported in part by the National Institutes of Health, Research Grant RO1 AM 15553 from the National Institute of Arthritis, Metabolism, and Digestive Disease and a grant from the Washington Heart Association.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Masson, P.L. a n d H e r e m a n s , J . F . ( 1 9 7 1 ) C o m p . B i o c h e m . Physiol. 3 9 B , 1 1 9 - - 1 2 9 Q u e r i n i e a n , P., M a s s o n , P.L. a n d H e r e m a n s , J . F . ( 1 9 7 1 ) Eur. J. B i o c h e m . 20, 4 2 0 - - 4 2 5 Aisen, P. a n d L e i b m a n , A. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 5 7 , 3 1 4 - - 3 2 3 Aisen, P. a n d L e i b m a n , A. ( 1 9 6 8 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 30, 4 0 7 - - 4 1 3 Windle, J . J . , W i e r s e m a , A . K . , Clark, J . R . a n d F e e n e y , R . E . ( 1 9 6 3 ) B i o c h e m i s t r y 2, 1 3 4 1 - - 1 3 4 5 A a s a , R., M a l m s t r o m , B.G., S a l t m a n , P. a n d V a n n g a r d , T, ( 1 9 6 3 ) B i o c h i m . B i o p h y s . A c t a 75, 2O3--222 Aisen, P., L e i b m a n , A. a n d R e i c h , H . A . ( 1 9 6 6 ) J. Biol. C h e m . 2 4 1 , 1 6 6 6 - - 1 6 7 1 Price, E.M. a n d G i b s o n , J . F . ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 8 0 3 1 - - 8 0 3 5 Aisen, P., L a n g , G. a n d W o o d w o r t h , R.C. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 6 4 9 - - 6 5 3 J a n d l , J . H . , I n m a n , J . K . , S i m m o n s , R . L . a n d Allen, D.W. ( 1 9 5 9 ) J. Clin. Invest. 38, 1 6 1 - - 1 8 5 S c h a d e , A . L . ( 1 9 6 4 ) I1 F a r m i c a 19, 1 8 5 - - 2 0 2 F l e t c h e r , J. a n d H u e h n s , E.I~. ( 1 9 6 8 ) N a t u r e 2 1 8 , 1 2 1 1 - - 1 2 1 4 Williams, J. ( 1 9 6 8 ) B i o c h e m . J. 1 0 8 , 5 7 - - 6 7 M a s s o n , P.L. a n d H e r e m a n s , J . F . ( 1 9 6 8 ) Eur. J. B i o c h e m . 6, 5 7 9 - - 5 8 4 L e h m a n n , H.P. a n d K a p l a n , A. ( 1 9 7 1 ) Clin. C h e m . 17, 9 4 1 - - 9 4 7 M c F a r l a n e , A.S. ( 1 9 5 8 ) N a t u r e 1 8 2 , 53 L~bbe, R . F . a n d N i s h a d a , G. ( 1 9 5 7 ) B i o c h i m . B i o p h y s . A c t a 26, 4 3 7 P r i n c i o t t o , J . V . , R u b i n , M., S h a s h a t y , G.C. a n d Z a p o l s k i , E.J. ( 1 9 6 4 ) J. Clin. Invest. 43, 8 2 5 - - 8 3 3 H e m m a p l a r d h , D. a n d M o r g a n , E . H . ( 1 9 7 4 ) . B i o c h i m . B i o p h y s . A c t a 3 7 3 , 8 4 - - 9 9 Cavill, I. ( 1 9 7 1 ) . J. Clin. P a t h o l . 24, 4 7 2 - - 4 7 4 Bates, G.W. a n d S c h l a b a c h , M.R. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 3 2 2 8 - - 3 2 3 2 Aisen, P. a n d L e i b m a n , A. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 0 4 , 7 9 7 - - 8 0 4 V e r h o e f f , N . J . , K r e m e r s , J.H.W. a n d Leijnse, B. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 0 4 , 1 1 4 - - 1 2 2 M a r t i n e z - M e d e l l i n , J. a n d S c h u l m a n , J. ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 53, 3 2 - - 3 8 E g y e d , A. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 0 4 , 8 0 5 - - 8 1 3 Williams, J. ( 1 9 7 4 ) B i o c h e m . J. 1 4 1 , 7 4 5 - - 7 5 2
Biochimica et Biophysica Acta, 421 (1976) 80--86 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - Printed in The N e t h e r l a n d s
BBA 27788
F A I L U R E OF RABBIT RETICULOCYTES TO INCORPORATE CONALBUMIN OR L A C T O F E R R I N IRON
E.J. Z A P O L S K I and J.V. P R I N C I O T T O *
Department of Physiology and Biophysics, Schools of Medicine and Dentistry, Georgetown University, Washington D.C. 20007 (U.S.A.) (Received J u n e 20th, 1975)
Summary Despite the remarkable molecular similarity of human lactoferrin and human transferrin, the results of this investigation indicate that human lactoferrin was unable to furnish rabbit reticulocytes with iron for heme synthesis. Although conalbumin closely resembles transferrin in many of its properties, conalbumin iron-binding differs from human transferrin iron-binding. There are conflicting reports in the literature regarding conalbumin's ability to furnish iron to reticulocytes. In this study, small amounts of lactoferrin or conalbumin were adsorbed to mature and immature cell surfaces but neither of these ironbinding proteins surrendered iron intracellularly to reticulocytes for heme synthesis.
Introduction Lactoferrin is widely distributed in the human body but its precise physiologic role is still unknown. It is a bacteriostatic agent and may also play a role in glandular iron metabolism [1]. Lactoferrin and transferrin exhibit striking similarities in their primary structure [2]. Their optical and electron paramagnetic resonance spectra are virtually indistinguishable from each other [3]. Although conalbumin is not found in the human, it does resemble transferrin in many respects and has been traditionally compared to human transferrin with regard to virtually every physico-chemical property that has been studied [4-8]. Recent evidence now indicates that conalbumin iron-binding differs from transferrin [ 8,9]. Unlike transferrin, the iron-binding affinities of conalbumin's two ironbinding sites are not identical and this might influence its ability to surrender * To whom correspondence should be s e n t .
81 iron to reticulocytes. There are, however, conflicting reports in the literature regarding conalbumin's ability to fulfill this biological function. Jandl et al. [10] reported that iron bound to conalbumin was as effectively utilized by reticulocytes as was transferrin bound iron. Schade [11] reported that conalbumin was unable to furnish iron to reticulocytes. In light of recent findings that conalbumin iron-binding differs from transferrin iron-binding, the ability or inability of conalbumin to function in this capacity may be of future importance in our understanding of the interaction of iron-binding proteins with immature red cell membranes. We have investigated conalbumin's ability to donate iron to reticulocytes for heme synthesis. Since reticulocyte membrane receptors have been likened to other cell membrane receptors [ 10,12], we have also investigated the ability of lactoferrin to function in this capacity to determine if its similarity to transferrin can be extended to include the biological property of iron donation. Materials and Methods
Iron-binding proteins Human apolactoferrin was a generous gift of Dr Philip Aisen (Albert Einstein College of Medicine, Bronx, New York). The ratio of absorbancy at 470 nm to absorbancy at 410 nm for human transferrin (Behring Diagnostics, Woodbury, New York) and conalbumin (Sigma, Type I, St. Louis, Mo.) was less than 1.4. These proteins were purified by chromatography [8,14]. Absorbance ratios obtained after chromatographic purification were 1.40 for transferrin and 1.45 for conalbumin. Iron was removed by dialysis which included treatment with 0.1 M NaC104 [7,13]. Final dialysis was performed with the saline that we employed throughout these studies: 150 mM NaC1, 5 mM KC1, 1 mM NaHCO3, 0.5 mM HEPES (N-2-hydroxyethyl piperazine-N'-2 ethane sulfonic acid) pH 7.4. Protein concentration was estimated from absorbance at 280 nm and iron protein complexes were prepared with ferrous ammonium sulphate-ascorbic acid, tracer labeled with s 9 FeC13 [14]. An excess of 2 iron equivalents per transferrin was added to each protein solution. After incubation at 37°C for 30 min., each solution was passed through a small column of anion exchange resin (BioRad, AG 1X-4, 100-200 mesh C1- form, equilibrated with saline medium) to remove unbound iron (15). Iron which was added to buffer in the absence of protein was completely removed by resin. The iron-binding capacity of the protein and its molar concentration were then determined from the specific activity in the eluate and this data was used to prepare solutions for cell uptake studies. Fully saturated, diferric iron-binding protein solutions (0.010 mM) were prepared by adding a calculated 5 percent excess of iron to protein. Partially saturated solutions were prepared by adding the required a m o u n t of iron to protein. All solutions were passed through resin to remove unbound iron. Unlabeled iron protein solutions were prepared in the same manner, using an unlabeled iron solution. ~ 2 s I-labeled proteins (less than one g-atom 1/mol protein) were prepared from diferric proteins by the m e t h o d of McFarlane [16]. Unbound 12 s I was removed by anion exchange resin treatment and iron was removed by dialysis.
82 These labeled proteins were treated as we have described and they were finally resaturated with unlabeled iron. The 0.001 mM solutions used in our studies were prepared by dilution with saline.
Cell suspensions Heparinized, reticulocyte-rich cells, collected from previously bled rabbits, were washed three times with cold saline. Care was taken to remove the buffy coat and the cells were adjusted to give a 50 percent packed cell volume. Reticulocyte-poor cells were collected from unbled rabbits and were treated in the same manner. Reticulocyte counts, using New Methylene Blue, were performed on dried blood films. Reticulocyte-rich cell preparations contained 20--30% reticulocytes while mature cell suspensions contained less than 1% reticulocytes.
Incubation procedures Equal volumes of temperature equilibrated cell suspension and test solution were mixed and incubated in small open Erlenmeyer flasks. In some experiments two different iron-binding proteins were present (one labeled with s 9 Fe and the other unlabeled). These solutions were mixed just prior to cell incubation. Aliquots of incubation mixtures were delivered into 10 vol. of ice-cold saline media for cell uptake determinations and were also lysed in ice-cold 0.5 mM HEPES buffer (pH 7.4) for stromal determinations. The samples were centrifuged, washed three times with corresponding media and resuspended before counting. Cell stroma preparations yielded a buff colored pellet. Hemin was isolated directly from one hour incubation samples following the procedure of Labbe and Nishada [ 1 7 ] . Counting was performed in a well-type scintillation counter and the percentage of cell isotope uptake was calculated. Cell stroma values were calculated as the fraction of whole cell uptake that was recovered with stroma. ..." ...""
....I,
59Fe
.....""
%
~."
%
..~
,.o
I
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... ~.: .."
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......
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...
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:'
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.....~ 1251 ~.: ~. ,W . . . . . . . . -~ . . . . . . . . . . . • ........... ~ . . . . . . . . . . . . . . . . . . . . . . . ~
;,~..*, ~ - -' ~ - - - ~ - - - ~ ~
1 2 5 1 & 59Fe
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~ ~n
~
:.
................
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...
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:.~
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.......... ! 59Fe
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~mmmm
LI 1125 I & 5gFe )
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& 59Fe )
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/,~ . . . . .-' -- -- -- ,,I,, -- -- --~, ~i¢4,~ ~ ,~.-,,~ . ,~,,,i ~ ~ ,-,~ ~ - " " ~ ................. ~'........... ':" ............ ~'. . . . . . . . . . . . . . . . . . . . . . . . . 3'0
6"0
~
125, 9"0
rain
Fig. 1. P e r c e n t a g e 59 Fe a n d 1 2 5 i . l a b e l e d iron binding p r o t e i n in w a s h e d cells. Tr, Transferrin; L f , L a c t o f e r r i n ; CA, c o n a l b u m i n . I n c u b a t i o n m i x t u r e : cells, 24% r e t i c u l o c y t e s , 0 . 0 1 0 m M p r o t e i n s o l u t i o n . Fig. 2. S a m e as Fig. 1, e x c e p t i n c u b a t i o n m i x t u r e : cells, 20% r e t i c u l o c y t e s , 0 . 0 0 1 m M protein solution.
83
Results
Re tic~locy te up take from diferric pro teins T h e p e r c e n t a g e of 59Fe a n d 1: s I-labeled p r o t e i n t a k e n up by reticuloc y t e s f r o m 0 . 0 1 0 m M diferric transferrin, l a c t o f e r r i n , a n d c o n a l b u m i n are illus t r a t e d in Fig. 1. All s o l u t i o n s were i n c u b a t e d with the s a m e r e t i c u l o c y t e suspension. Less t h a n 2 p e r c e n t o f the labeled iron p r e s e n t as l a c t o f e r r i n or conalb u m i n was t a k e n u p b y the cells. In c o n t r a s t , a high p e r c e n t a g e u p t a k e was o b s e r v e d f o r t r a n s f e r r i n b o u n d iron-59. Cell u p t a k e o f 125 I-labeled p r o t e i n was l o w f o r all t h r e e p r o t e i n s . This s t u d y was r e p e a t e d at a l o w e r c o n c e n t r a t i o n (0.001 raM) to determ i n e w h e t h e r t h e r e were a n y a p p a r e n t d i f f e r e n c e s b e t w e e n u p t a k e of e i t h e r i s o t o p e f o r l a c t o f e r r i n or c o n a l b u m i n . T h e s e results are s h o w n in Fig. 2. A f t e r 10-fold dilution, u p t a k e o f labeled l a c t o f e r r i n or c o n a l b u m i n increased to nearly 10 p e r c e n t . H o w e v e r , it is e v i d e n t t h a t t h e r e was no d i f f e r e n c e in p e r c e n t u p t a k e o f e i t h e r s 9 Fe or 1 : 5 I-labeled l a c t o f e r r i n or c o n a l b u m i n .
Stromal, low temperature and mature cell uptake T a b l e I lists d a t a o b t a i n e d f r o m studies with r e t i c u l o c y t e - r i c h cell p r e p a r a tions ( i n c u b a t e d at 3 7 ° C and at l o w t e m p e r a t u r e s ) a n d m a t u r e cells ( i n c u b a t e d at 37 °C). T h e s e d a t a were o b t a i n e d a f t e r one h o u r o f i n c u b a t i o n with 0.001 m M s o l u t i o n s a n d in each case the f r a c t i o n o f w a s h e d cell r a d i o a c t i v i t y which was r e c o v e r e d w i t h t h e cell s t r o m a is listed. R e t i c u l o c y t e - r i c h cells i n c u b a t e d at 3 7 ° C t o o k u p 92% o f the diferric t r a n s f e r r i n iron a n d less t h a n 3% o f this u p t a k e was r e c o v e r e d with cell s t r o m a . Cell u p t a k e o f t r a n s f e r r i n 59 Fe was m a r k e d l y r e d u c e d w h e n r e t i c u l o c y t e s were i n c u b a t e d at l o w t e m p e r a t u r e . T h e s t r o m a iron f r a c t i o n was c o n s i d e r a b l y higher f o r r e t i c u l o c y t e s i n c u b a t e d at l o w t e m p e r a t u r e s (0.99) and f o r m a t u r e cells i n c u b a t e d at 3 7 ° C (0.16). T h e f r a c t i o n of 125i.labele d transferrin recovered with cell s t r o m a u n d e r all these c o n d i t i o n s was u n i f o r m l y high for e i t h e r r e t i c u l o c y t e s or m a t u r e cells (close to 90% o f the t o t a l cell u p t a k e ) . In c o n t r a s t , f o r e i t h e r l a c t o f e r r i n or c o n a l b u m i n t h e r e was n o d i f f e r e n c e in r e t i c u l o c y t e p e r c e n t u p t a k e o f 5 ~ Fe or 12 5 I, and a high f r a c t i o n of this u p t a k e
TABLE I RETICULOCYTE
AND MATURE Label
Transferrin Transferrin Lactoferrin Lactoferrin Conalbumin Conalbumin
59Fe 125I 59Fe 125I 59Fe 125I
CELL UPTAKE
R e t i c u l o c y t e s (at 3 7 ° C ) ( W a s h e d cells)
Reticulocytes (at 0°C) ( W a s h e d cells)
M a t u r e cells (at 3 7 ° C ) (Washed cells)
% Activity -+ S E M
Stroma*
% Activity +- S E M
Stroma
% Activity + SEM
Stroma
92.0 3.4 7.6 7.4 5.9 4.2
0.03 0.91 0.60 0.72 0.45 0.98
7.1 + 1.2 7.4-+ 1.8 7.6 +- 1.4 7.1 + 1.0 5.2 + 0 . 9 4.9 + 1 . 0
0.99 0.94 0.88 0.85 0.96 0.82
9.7 3.1 6.3 4.0 5.8 3.9
0.16 0.89 0.68 0.80 0.65 0.99
-+ 2 . 0 + 1.2 -+ 1 . 2 + 2.4 + 0.8 + 0.5
+ 1.1 + 1.0 + 1.3 +- 1.8 +- 0 . 9 +- 0 . 8
* A l l s t r o m a v a l u e s r e p o r t e d as f r a c t i o n o f w a s h e d c e l l a c t i v i t y r e c o v e r e d w i t h s t r o m a .
84 TABLE
II
RETICULOCYTES: % Saturation
25 50 75 100
%59Fe
UPTAKE
FROM
PARTIALLY
SATURATED
SOLUTIONS
% 59 F e Lacto ferrin
Conalbumin
Transferrin
1 3 8 9
0 1 4 6
62 78 83 91
was recovered b o und to cell stroma. Uptake was the same with reticulocytes or mature cells. Reticulocyte SgFe uptake was unaltered when 0.010 or 0.001 mM labeled conalbumin or lactoferrin was mixed with an equal concentration of unlabeled transferrin and then incubated with cells. More significantly, addition o f unlabeled lactoferrin or conalbumin had no effect on reticulocyte utilization of s 9Fe from transferrin, suggesting that these proteins were nonspecifically adsorbed to the cell surface rather than specifically bound to reticulocyte receptor sites. Virtually no s 9 Fe activity was recovered with hemin isolated from reticulocytes incubated at 37°C with lactoferrin or conalbumin (less than 1% of the total cell uptake). In contrast 55--65% of the cell S~ Fe uptake from transferrin was recovered with isolated hemin.
Reticulocyte uptake from partially saturated solutions s 9 Fe reticulocyte uptake data from partially saturated protein preparations are listed in Table II. Uptake of lactoferrin or conalbumin bound iron, even at this low concentration (0.001 mM) was abolished when the protein was less than half saturated with iron. Discussion Reticulocytes have the ability to take up iron from albumin, gamma globulin, ferric chloride, and various iron chelate solutions [ 1 0 , 1 8 ] . This may involve exchange of the metal to transferrin bound to the cells [ 1 9 ] , so that precautions must be taken to assure that one is studying the direct effect of the specific iron c o m p o u n d under investigation. The A4 ~ o/A41 o absorbance ratios obtained after chr om a t ogr aphy of commercial transferrin and conalbumin indicate that they were of a high degree of purity. Human lactoferrin used in our investigation was the subject of an earlier study [ 3]. Prior to reports by Cavill [20] and by Bates and Schlabach [21] we, like other investigators, assumed that iron-binding proteins completely bound iron at neutral pH. As a result, in preliminary studies with iron-binding proteins that were formed using ferric chloride as the iron source, we observed that both lactoferrin and conalbumin delivered iron to reticulocytes (as much as 50% from " s a t u r a t e d " conalbumin). On the basis of these observations, Aisen and Leibman reported that the reticulocyte is capable of incorporating iron which is complexed to lactoferrin [ 2 2 ] . Considerably less than two iron atoms/mol of transferrin actually occupy
85 iron binding sites when ferric chloride is added to apotransferrin. The remainder is n o t b o u n d at the specific iron-binding sites. This non-specifically b o u n d iron is even eluted with transferrin from Sephadex G-25 [ 2 1 ] , so our initial observation, that conalbumin and lactoferrin surrendered iron to reticulocytes, did n o t reflect uptake of iron that was specifically bound at the protein iron-binding sites. In the present study, we utilized an anion exchange resin to remove all non-specifically bound iron from transferrin solutions [ 15,20]. Our results for conalbumin confirm Schade's findings [11]. Although Schade emp lo y ed crude egg white in his study, he concluded that neither h u man nor rabbit reticulocytes t o o k up iron bound to conalbumin. In our study, less than 2% of the s 9Fe from 0.01 mM conalbumin was taken up by the cells in comparison to 50% uptake of transferrin bound iron (Fig. 1). When the c o n c e n t r a t i o n of conalbumin was reduced ten-fold, percentage uptake (but n o t absolute uptake) was enhanced and it is evident that little if any intracellular utilization of conalbumin iron had occurred (Fig. 2, Table I). The percentage s 9 Fe or 12 s I-labeled conalbumin present in washed cells was identical for each isotope, at low temperature, at 37°C and, in studies with mature cells. A major fraction of each isotope was recovered still bound to the cell stroma. Heroin, isolated from reticulocytes, indicated that no s 9Fe was incorporated into heme. Similar results were also observed for lactoferrin. The findings that neither conalbumin or lactoferrin bound iron was utilized for heme synthesis directly contrasted results obtained with transferrin. Jandl et al. [10] r e por t ed that reticulocytes readily utilized conalbumin iron. This observation was based on experiments in which they em pl oyed ironconalbumin prepared from FeCla and no a t t e m p t was made to remove any u n b o u n d iron. Their results are similar to our earlier observations and can be explained if a por t i on of the iron in their conalbumin preparation was not specifically b o u n d at the conalbumin iron-binding sites and instead this source of iron was utilized by the cells. There have been several studies (reviewed in ref. 23) of reticulocyte and marrow cell acquisition of iron from heterogeneous cell-transferrin systems such as we have e m p l o y e d in our study. In no report was cell uptake ever abolished; rather, the rate of iron accumulation from heterogeneous transferrin-reticulocyte systems was slower than the rate of uptake from homogeneous systems. Verhoeff et al. [ 23] demonstrated that the subcellular distribution of metal was the same in all of the transferrin-cell systems that they investigated. The failure of rabbit reticulocytes to utilize hum an lactoferrin iron cannot be totally ascribed to a p r o p e r t y of the rabbit reticulocyte. These cells do incorporate iron from human transferrin, hence the inability of lactoferrin to serve in this capacity illustrates that there is a functional difference between human lactoferrin and h u m a n transferrin. Despite its close similarity to transferrin, lactoferrin may not even interact with reticulocyte transferrin r ecept or sites. We did observe that a small a m o u n t of lactoferrin or conalbumin was bound by cell suspensions but this observation was n o t restricted to reticulocytes since it was also observed with mature cells. However, reticulocytes did not utilize any iron bound to these proteins. Secondly, neither lactoferrin or conalbumin interfered with reticulocyte uptake of transferrin iron. If lactoferrin or conalbumin was bound at the specific
86 membrane receptor sites their presence should have inhibited uptake of transferrin iron. It has been suggested that the release of transferrin bound iron to the reticulocyte is the result of enzymatic cleavage of carbonate (or bicarbonate) from the ternary iron-protein-anion complex [22,24,25]. Possibly lactoferrin or conalbumin complexes cannot serve as substrate for the anion-detaching enzyme of rabbit reticulocytes and this could also account for the failure of the cell to utilize this source of iron for heme synthesis. In either case the failure of human lactoferrin to mimic the reticulocyte iron donating role of human transferrin implies that this iron-binding protein is not involved in iron metabolism at the erythroid cell and its physiologic role remains unknown. Williams [ 26] reported findings which might be misinterpreted that conalbumin and fragments of conalbumin (isolated after trypsin digestion) are both effective rabbit reticulocyte iron donors. He reported reticulocyte uptake values that ranged from 5--50 cpm/0.1 ml. On the basis of the 0.2 mCiS9Fe he used to prepare his solutions, we calculate that this cell uptake is no more than 0.5--5% of the total activity per samples. This value is in keeping with results that we have reported in this study.
Acknowledgements This investigation was supported in part by the National Institutes of Health, Research Grant RO1 AM 15553 from the National Institute of Arthritis, Metabolism, and Digestive Disease and a grant from the Washington Heart Association.
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