Iron-binding properties and amino acid composition of marsupial transferrins: Comparison with eutherian mammals and other vertebrates

Camp. Biochem. Physiol. Vol. 89A, No. 4, pp. 559-565, 1988 Printed in Great Britain

0300-9629/88 $3.00 + 0.00 0 1988 Pergamon Press plc

IRON-BINDING PROPERTIES AND AMINO ACID COMPOSITION OF MARSUPIAL TRANSFERRINS: COMPARISON WITH EUTHERIAN MAMMALS AND OTHER VERTEBRATES B. C. LIM,* T. PETERSJR? and E. H. MORGAN* The University of Western Australia, Nedlands, Western Australia 6009, Australia. Telephone: (09) 380 3320; and tThe Mary Imogene Bassett Hospital, Cooperstown, NY 13326, USA

*Department

of Physiology,

(Received Abstract-l. (Trachosurus

1 July 1987)

Some physicochemical properties of transferrin from three marsupials, viz a possum vulpecula), a kangaroo (Macropus fuliginosus) and the quokka (Seionix brachyurus) were

studied and compared with those of transferrins from mammalian and non-mammalian vertebrate species. 2. The molecular weight of the marsupial transferrins fell within the range of 76,00&79,000 daltons. 3. The marsupial transferrins were similar to the transferrins of eutherian mammals with respect to optical spectral properties, iron binding capacity and the pH-dependence of iron binding, and iron release mediated by 2,3-DPG. 4. The amino acid compositions of the marsupial transferrins were compared with each other and with the transferrins from the other vertebrate species. The compositions of the marsupial transferrin were closely related to each other, and also showed similarities with transferrins from eutherian mammals and chicken ovotransferrin.

INTRODUCTION

do they show a high degree of internal homology between the two halves within each protein but a pronounced homology was also found between the three proteins. The primary structure of other transferrins is yet to be investigated. However, the amino acid composition of transferrin from many species, including bovine and baboon (Bezkorovainy and Grohlich, 1974), rabbit, rat, frog, turtle (Parmour and Sutton, 1971) and brook trout (Hershberger, 1970), have been reported. Relatively little information regarding the properties and function of marsupial transferrins and nonmammalian transferrins is available. The present study is therefore aimed at investigating the properties of marsupial transferrins and comparing the results with those of the transferrins from other vertebrates. The species examined and compared in this work were a brush tail possum (Trachosurus vulpecula), western grey kangaroo (Macropus fuliginosus), quokka (Setonix brachyurus), chicken (Callus gullus), lizard (Tiliqua rugosu), toad (Bufo murinus) and a bony fish, the buffalo bream (Kyphosus sydneyanus). Samples of human, rabbit, rat and crocodile (Crocodylus siumensis) transferrin were also used in some of the studies.

Transferrin is the iron-transporting protein found in the plasma of all vertebrate species (Palmour and Sutton, 1971). It is involved in the delivery of iron from sites of storage and absorption to developing red cells for the synthesis of haemoglobin. The properties and functions of transferrin from many vertebrate species, especially the higher mammals, have been well characterized (for reviews, see Aisen and Listowsky, 1980; Morgan, 1980, 1981). In general, it is agreed that the protein consists of a single polypeptide of molecular weight approximately 80,000. It possesses two metal binding sites each of which can bind an atom of ferric iron together with a carbonate (or bicarbonate) ion. Iron-binding and release are pH-dependent processes. All the transferrins examined so far are glycoproteins and there are marked variations in the number and composition of the carbohydrate groups. On the contrary, the amino acid compositions of different transferrins investigated appear to be similar (Hudson et al., 1973). Recently, the complete amino acid sequences of human serum transferrin (MacGillivray et al., 1983), chicken conalbumin (Jeltsch and Chambon, 1982; Williams et al., 1982) and human lactoferrin (MetzBoutigue et al., 1984) have been reported. Not only

MATERIALS AND METHODS Transferrins Transferrins and apotransferrins were prepared from the plasma of the animals as previously reported (Lim and Morgan, 1984) and were stored at -20°C. Protein concentration was measured by the Lowry method (1951). Molecular weight was estimated bv SDS-PAGE performed in 8.5% poiyacrylamide according to Laemmli (i970) and the presence of carbohydrate was detected with periodic acidSchiff reagent stain.

Address correspondence to: B. C. Lim, Department of Physiology, The University of Western Australia, Nedlands, Western Australia 6009, Australia. Abbreviations used-DPG, diphosphoglycerate; HEPES, N2-hydroxyethylpiperazine; NTA, nitrilotriacetic acid; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; Tris, 2-amino-2(hydroxymethyl)propane-1,3-diol. c BP 89,4A--D

559

B. C. LIM et al.

560 Iron -binding capacity and absorpiion spectra

The iron-binding capacity of the apotransferrin samples dissolved in 0.1 M Tris buffer at pH 7.8 was determined by spectrophotometric titration at 470 nm in a Beckman DU8 spectrophotometer at 25°C for toad and fish transferrin, 33°C for lizard transferrin and 37°C for transferrin from the other species. Aliquots of Fe-NTA solution (1.78 mM Fe, 20 mM NTA) were added to a 1 ml cuvette containing 5&75 p M apotransferrin in 0.15 M NaCl. After each addition of iron solution the absorbance at 470 nm was read at 2 min intervals until a constant reading was reached. At the end of the reaction, the final concentration of the proteins was again estimated by the Lowry method, in order to correct for the dilution caused by the addition of the iron solution. pH-dependence

of iron -binding

The pH-dependence of iron-binding to transferrin was examined by measuring the absorbance of the transferrin solution at 470 nm. A series of 1 M sodium cacodylate buffers covering the pH range 2.1-7.5 was prepared. Diferric transferrin was added to 1 ml of each buffer to make up a final transferrin concentration of 5&60 p M. The mixture was thoroughly mixed and left to stand at 4°C for 3 days to reach equilibrium. The samples were then equilibrated to room temperature and the absorbance and pH were

from transferrin and the desferrioxamine as an acceptor of the iron released. Each reaction was allowed to proceed to completion, after which time the absorbance showed no further decrease. The final absorbance value was subtracted from the other absorbance values and the corrected values were graphed against time on semilogarithmic paper. The rate of change of absorbance was used as a measure of the rate of iron release from transferrin. Amino acid composition Amino acid analysis of the transferrins was performed by single-column Dowex 50 ion exchange chromatography, using citrate buffers and a ninhydrin detection system (Dionex 3 mm column). Proteins were hydrolysed for 24 hr at 110°C with 6 M HCl in uacuo. Results for threonine and serine were corrected for hydrolysis losses of 0.5 and 4.5%, respectively, observed for chicken albumin under 24 hr hydrolysis. Values for valine and leucine were similarly corrected for incomplete hydrolysis of 4.5 and 4.1%, respectively, and for methionine of 4.5%. Tryptophan content was assayed spectrophotometrically by the method of Edelhoch (1967) as modified by Bredderman (1974).

RESULTS

measured. Mediated iron release from transferrin The rate ‘of iron release from transferrin was measured by the decrease in absorbance at 295 nm of a solution of iron transferrin in 0.1 M HEPES buffer at various pH values, using a Beckman DU-8 spectrophotometer (Morgan, 1979). The solutions in the cuvettes contained transferrin (0.015-0.03 mM), 2,3-DPG (5 mM) and desferrioxamine (5 mM). The 2,3-DPG was used as a mediator of iron release

M,x

Molecular

weight

The transferrins from the three marsupial species and from the three eutherian mammals which were also studied (man, rabbit and rat) had similar electrophoretic mobilities on SDS-PAGE (Fig. 1). All the transferrins gave positive results when stained with the periodic-acid Schiff reagent for carbohydrate (results not shown). The molecular weights of the

1O-3

Fig. 1. SDS-PAGE of transferrins from some eutherin and marsupial animals. (1) Human, (2) rat, (3) rabbit, (4) possum, (5) quokka, (6) kangaroo. The smaller molecular weight component which appeared in kangaroo transferrin during storage at -20°C is indicated by the arrow.

Physicochemical properties of marsupial transferrins

561

Table 1. Molecularweight,extinction coefficientsand iron-binding capacity from 3 marsuoials

transferrins

Molecular weight

and 5 other vertebrate Extinction E(I%, 470 nm

animal

coefficient Icm) 280 nm

of

soecies

Iron-binding capacity mol Feimol Tf

Transferrin

(x 10-9

Possum Quokka Kangaroo Chicken Lizard

76 79 77 74 74

k 4.4 i 4.2 rfr 3.5 + 3.3 f 3.6

0.56 0.57 0.57 0.54 0.55

14.3 14.3 14.0 13.2 12.5

Crocodile

74 f 4.8

0.53

12.9

I .90

Toad Fish

70 f 3.4 70 f 3.3

0.56 0.52

14.3 11.5

I.91 1.90

I .90 I .90 2.06 I .95 2.05

The molecular weight values are the means f SD of 3-5 separate estimations. The other results are from single measurements. Iron saturated transferrin was used for the estimation of molecular weight and extinction coefficients.

marsupial transferrins were estimated to vary from 76,000 to 79,000 (Table 1). These values are slightly higher than those of the submammalian species, chicken, lizard, toad and fish (Table 1). A feature of kangaroo transferrin which can be seen in Fig. 1 is the tendency to break down partially during storage at -20°C to yield a product which had a molecular weight approximately half that of the intact molecule. This was not present in the freshly prepared protein, nor was it observed with any of the other transferrins. Absorption spectra and ion -binding capacity

As is characteristic of the transferrins from other vertebrate species, the iron-saturated form of the marsupial transferrins showed an absorption maximum at 465-470nm (Fig. 2). The extinction coefficients of the proteins are recorded in Table 1. The change in absorbance at 470nm which occurred when iron was added to the transferrins was used to measure their iron-binding capacities. In all cases the proteins were found to bind two iron atoms per protein molecule (Table 1). pH-dependence

of iron -binding

When the pH of the transferrin

solutions

was

0.8-

Mediated iron release from transferrin

Two patterns of iron release mediated by 2,3-DPG were observed. The first was obtained with possum and kangaroo transferrins. In these cases, when the changes in absorbance were plotted on semilogarithmic paper a straight line was obtained, indicating a single rate of iron release from transferrin (Fig. 4A). This pattern was also observed with chicken and lizard transferrins and has been previously described for rabbit transferrin (Morgan et al., 1978). In the second pattern (quokka) the iron was released rapidly initially and then more slowly, thus forming a curvilinear graph when the results were plotted on semilogarithmic paper (Fig. 4B). This graph could be resolved into two exponentials, indicating two rates of iron release from the protein. This

A

0.6 -

0.4

I I 350

lowered the absorbance at 470 nm began to fall at a pH of 6.5-6.0. The pattern of change in absorbance with the three marsupial transferrins was similar to that shown for possum transferrin in Fig. 3A. The absorbance fell progressively to give a smooth curve over the pH range 6.0-4.0 and reached a low value by the latter pH. This was similar to the result observed with chicken transferrin but differed from that obtained with lizard, crocodile, toad and fish transferrins. With the latter four transferrins the absorbance curves showed a plateau between pH 5.7 and 4.7, as illustrated in Fig. 3B for lizard transferrin.

400

I I 450

1 I 500

r

8

Possum r

I 1 1 I 550

Lizard

0.4

7

654

32

600 PH

Wavelength

(nM)

Fig. 2. The absorption spectrum of purified quokka transferrin (0.1 mM in 0.15 M NaCI).

Fig. 3. The relationship between the absorbance at 470 nm and the pH of solutions of the iron-saturated transferrin from (A) possum and (B) lizard.

562

B. C. LIM et al. Ouokka

Possum 1.0

1.0

05

0.5 . .

s 5, 0.1

\ 0.1

R 4

t 0.05

0.05

0.0 1 I!!trY-b

‘> \

0.01 30

60

90

Incubation

Fig. and with (0) (0). into

a

40 Time

90

120

(mid

4. Iron release from diferric transferrin of (A) possum (B) quokka mediated by 2,3-DPG. The measurements possum transferrin were performed at pH 7.0 (A), 6.5 and 5.4 (w), and with quokka transferrin at pH 6.4 The curve for quokka transferrin has been resolved two exponentials, each representing approximately half of the total absorbance.

type of result was also found with toad transferrin and has been described for human and rat transferrins (Morgan, 1979). With all the proteins studied the rate of iron release increased as the pH was lowered (Fig. 4A). Amino acid composition Table 2 shows the amino acid compositions of the three marsupial transferrins, chicken ovotransferrin and the lizard, crocodile, toad and fish transferrins. The composition of each protein was calculated on the basis of 686 residues per molecule as is found in chicken ovotransferrin for which the complete amino acid sequence is established (Williams et al., 1982). Comparison of the amino acid values obtained for ovotransferrin in the present work (Table 2) with those derived from the sequence data shows little variation, indicating the reliability of the present results. The amino acid compositions of the marsupial transferrins were compared with each other and with transferrins from other vertebrate animals by the method of Cornish-Bowden (1977). In order to allow comparison with eutherian mammals and to broaden the scope of the study, published data for human (MacGillivary et al., 1983) rabbit (Baker ef al., 1968) and rat (Schreiber et aI., 1979) transferrins were included in the analysis. The transferrins were compared in pairs. The index, SAn, was calculated for transferrins of each pair, as half the sum of the squares of the differences between the proteins in the number of residues of each type of amino acid (Table 3). According to Cornish-Bowden (1977) it is an unbiased estimation of the number of differences between the sequences of the two proteins. This analysis showed that there was a low degree of dissimilarity between the marsupial transferrins, especially between those of the kangaroo and quokka and between the transferrins from the eutherian mammals. Overall, the differences between the transferrins of the marsupials and higher mammals were greater than between the transferrins of each of these groups. This was especially noticeable with the macropod

Physicochemical properties of marsupial transferrins

563

Table 3. Index of difference (SAn) in amino acid compositions of the transferrins of 10 different animal species. Figures show the values of SAn, calculated according to Comisb-Bowden (1977). as described in the Materials and Methods section

Human Rabbit Rat Possum Kangaroo Quokka Chicken Crocodile

Lzard Toad Fish

Rabbit

Rat

Possum

Kanearoo

Ouokka

Chicken

Crocodile

Lizard

152

-

218

296

-

250

306

360

-

428

531

554

285

317

377

350

295

II3

299

504

349

220

292

345

-

436 637

772 835

300 446

275 666

508 496

529 527

247 356

385

-

643

803

482

538

741

716

364

I60

407

843

1156

637

999

905

933

548

440

267

marsupials, kangaroo and quokka, in which the transferrins showed less dissimilarity with chicken ovotransferrin than with the transferrins of eutherian mammals. Generally the SAn values for the pairs of

proteins increased, the further apart the animals were on the evolution scale, proceeding from mammals to fish. The data for the amino acid analyses of the proteins were analysed in a different way, as illustrated in Fig. 5. The mol/mol figures for the individual amino acids (ordinate) were plotted against the animal species arranged as indicated along the abscissa. The sequence of this arrangement was derived from the analysis shown in Table 3, while the intervals between the species were determined arbitrarily on the basis of evolutionary groups. A significant trend (P < 0.05) was found with only four of the 18 amino acids. With two, aspartic acid/asparagine and leucine, the relative abundance of the amino acid increased with ascent of the evolutionary scale,

Aspartic

and with the other two, threonine decreased.

478

Fish

-

and alanine,

it

DISCUSSION

The transferrin from the three marsupials which were investigated in this study have similar physicochemical properties to those previously described for eutherian transferrins and chicken ovotransferrin (Aisen and Listowsky, 1980; Morgan, 1980, 1981). This is true for molecular weight, spectral properties, number of iron-binding sites and pH-dependence of iron-binding and release. The pattern of pHdependence of iron-binding by the marsupial transferrins (Fig. 3A) is similar to that of rabbit transferrin (Baker et al., 1968). However, not all mammalian transferrins display this pattern. With human transferrin a result similar to that obtained in the present work with lizard, crocodile, toad, and fish transferrins is obtained, i.e. biphasic pH-dependent dis-

Acid

8 Asparagine . .‘/

Toad

50

r

Threonine

Leucine

I

.

Fig. 5. Relationships between amino acid content of transferrins (mol/mol protein) and evolutionary

level

of vertebrate animals. The species are fish (F), toad (T), lizard (L), crocodile (C), chicken (Ch), kangaroo (K), quokka (Q), possum (P), human (H), rat (R) and rabbit (Rb). Significant correlations (P < 0.05) were found with the four amino acids shown, with correlation coefficient r of 0.835 (aspartic acid/asparagine), 0.936 (threonine), 0.673 (alanine) and 0.850 (leucine).

564

B. C.

sociation of iron from transferrin (Princiotto and Zapolski, 1975, 1978; Lestas, 1976). This is interpreted as evidence for a difference between the ironbinding properties of the two sites on human transferrin. A similar interpretation is applicable to the results obtained with the lizard, toad and fish transferrins studied in this investigation. However, the presence of a monophasic pH-dependent dissociation curve, as found with the marsupial transferrins, should not be used as evidence that the sites are identical, since there are differences in the ironbinding sites of rabbit transferrin even though it displays a monophasic curve (Delany et al., 1982). Also, the fact that rabbit and human transferrins have different types of pH-dependent dissociation curves indicates that the results obtained in the present study cannot be used as evidence that this property is based on phylogenetically determined differences between marsupial and chicken transferrins on the one hand and the lizard, crocodile, toad and fish transferrins on the other. The marsupial transferrins resembled those of eutherian mammals, with respect to iron release mediated by 2,3-DPG. This compound leads to rapid release of iron from the transferrins of both groups of mammals and the rate of release increases as the pH is lowered. Moreover, the results with the marsupial transferrins illustrated the two patterns of release which have been observed with eutherian transferrins, either one (possum, kangaroo, rabbit) or two (quokka, human, rat) rates of release being observed. In the case of quokka transferrin, as well as human and rat transferrins, this may be interpreted as evidence for a difference between the two iron-binding sites on the molecule. The physiological significance of the pHdependence of iron-binding and release from transferrins, probably lies in the fact that, in uivo, iron release from transferrin occurs within acidic endocytotic vesicles (Morgan, 1981). Evidence that iron uptake by marsupial erythroid cells occurs by receptor-mediated endocytosis has been obtained in recent studies with quokka reticulocytes (Lim, McArdle and Morgan, 1987). Whether or not the ability of a wide range of phosphate compounds, such as 2,3-DPG. to mediate iron release from transferrin (Morgan, 1979) has any functional significance is yet to be determined. The similarity in the structure and properties of the eutherian and marsupial transferrins is confirmed by analysis of their amino acid compositions by the method of Cornish-Bowden (Table 3). The more closely related are the amino acid sequences of two proteins and the lower is the value of SAn (CornishBowden, 1977, 1981). Hence, the present results conform to the known evolutionary relations of the two groups of mammals. In addition, they are consistent with the concept of a closer relationship between the members of the eutherian group on the one hand and of the marsupial group on the other, than between the two groups of mammals.. Thus, the results are supportive of the concept of divergent evolution of marsupials and eutherian mammals (Paterson, 1956; Paterson and Pascal, 1972; TyndaleBiscoe, 1973). Looked at another way, if current ideas of mammalian evolution are correct, the present

LIMetal. results confirm the validity of techniques of assessing the degree of similarity between the amino acid sequences of proteins by analyses such as that described by Cornish-Bowden. This is further supported by the comparison between the mammalian and non-mammalian species in Table 3. In general, the values for SAn became greater as the evolutionary distance between the species increased. Moreover, when the transferrins were arranged in order of the values for SAn, significant trends for the relative abundance of four of the amino acids were observed (Fig. 5). This suggests that changes have occurred during evolution in the utilization of certain amino acids in the synthesis of transferrins. Possibly, the only surprise in the results is the evidence for a relatively low degree of dissimilarity between chicken ovotransferrin and the mammalian transferrins. However, this has been confirmed in the case of human transferrin by amino acid sequence data (MacGillivray et al., 1983; Williams et al., 1982). The transferrins contain two domains consisting of the N-terminal and the C-terminal halves of the molecule and each containing one iron-binding site (Williams et al., 1982; MacGillivray et al., 1983). The breakdown of kangaroo transferrin during storage led to the appearance of a component with approximately half the molecular weight of the intact molecule. This suggests the presence of a labile region in the peptide chain at the junction between the two domains. If so, this would not be a feature peculiar only to kangaroo transferrin, since bovine transferrin (Brock et al., 1978) and chicken ovotransferrin (Keung et al., 1982) may be split into their two halves by mild proteolytic digestions. Possibly, the breakdown of kangaroo transferrin resulted from proteolytic activity of micro-organisms contaminating the transferrin solution. A similar explanation may apply to the reported subunit structure of human transferrin (Jeppsson, 1967) and the low molecular weight of hagfish transferrin (Palmour and Sutton, 197lt_claims which were subsequently shown to be incorrect (Mann et al., 1970; Aisen et al., 1972). It is of interest to consider functional implications of the similarity between marsupial and eutherian transferrins. Using quokka, possum, rabbit and rat reticulocytes and the transferrins examined in the present paper, we have found that the marsupial transferrins cannot react with membrane receptors or donate iron to the eutherian reticulocytes, and vice versa, yet within the eutherian or the marsupial there is a high degree of reactivity between proteins and cells (Lim et al., 1987). This suggests that the relatively small differences between marsupial and eutherian transferrins are sufficient to significantly alter the conformation of the receptor-binding region of the proteins, even though one might have predicted that the structure of this part of the protein would be conserved during evolution. Of course, it is not possible to rule out the possibility that the lack of reactivity is a consequence of differences in structure of the transferrin receptor, rather than in transferrin itself. Acknowledgements-This research was supported by the Australian Research Grants Scheme. The authors are grateful

to Dr H. J. McArdle

for his interest.

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