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Stucture and function of transferrin
146
STRUCTURE AND FUNCTION OF TRANSFERRIN
M CHING-MING CHUNG D e p a r t m e n t ' o f Biochemistry Faculty of Medicine National University of Singapore Republic of Singapore 0511
Introduction
The transferrins are a group of non-haem iron-binding glycoproteins widely distributed in the physiological fluids and cells of vertebrates. They comprise: (i) serum transferrin (also known as 'serotransferrin', 'siderophilin', and '13a metal-binding globulin'), the prototype found in blood serum, (ii) lactoferrin ('milk red protein', 'lactotransferrin'), the ironbinding protein first found in milk, but also since found in secretions (tears, saliva) and cells (neutrophil leukocytes): [lactoferrin should not be confused with the 'milk transferrin' found in the milk of several animal species (eg rabbit)], (iii) ovotransferrin ('ovoferrin', 'conalbumin'), the iron-binding protein isolated from avian egg white. The chief physiological function of serum transferrin is undoubtedly in iron transport within the circulatory system of the vertebrates, but a specific role in iron transport has not been established for ovotransferrin and lactoferrin. However, due to their very high affinity for iron and other trace metals, these two proteins could function to deprive microorganisms of essential metals for growth, thus acting to protect the egg and milk respectively against infection. The transferrins have been a subject of much intensive investigation in recent years and a great deal is known about their physical, chemical and biological properties. In this article, I will summarise only the more important aspects of the structure and function of human serum transferrin. Readers are directed to refer to several recent reviews ~-3 for a more detailed treatment of this subject.
Physical Properties
Human serum transferrin is a monomeric glycoprotein with a sedimentation coefficient of 5.1S and a molecular weight of about 80 000.1 It contains two similar but not identical metal-binding sites located in its N-terminal and C-terminal domains. Hydrodynamic studies suggest that it has a molecular shape best represented by a prolate ellipsoid with an axial ratio of 1:2 and 1:3 for the iron-saturated and iron-free protein respectively. However, a recent report based on small-angle neutron-scattering measurements, indicates that it is an oblate spheroid. 4 The tertiary structure of rabbit serum transferrin, as revealed by X-ray crystallography, is compatible with a bilobal model. The two lobes, which are nearly equal in size and possessing clefts near the region of the join, may correspond to the two domains formed from the monoferric N- and C-terminal halves of the polypeptide chain. 1
Chemical Properties
Amino acid composition The amino acid composition of human transferrin has been reported by several investigators. 2 It shows few unusual features except for a complete absence of free sulphydryl groups, and a high content of half-cystine corresponding to 19 intrachain disulphid e bonds. Transferrins of different species generally have fairly similar amino acid compositions. However, the minor differences manifested in their amino acid compositions coupled with the variation in carbohydrate contents result in differences in electrophoretic mobility. Transferrins exhibit extensive genetic polymorphism, and in cases where the genetic phenotypes are from the same species, variation in electrophoretic mobility is usually found to be a result of amino acid substitutions. For example, comparative studies on the chymotryptic digests of human transferrin C and D t indicate that an aspartic residue in transferrin C is probably replaced by a glycine residue in transferrin Da. 5 On the other hand, in a study on equine transferrin D and R, it was found that one aspartic acid and one glutamic acid residue in transferrin D are replaced by two glycine residues in transferrin R. 6 Amino acid sequence The partial amino acid sequence of human transferrin has been known for a number of years, but its complete sequence has only just been reported. 7 It consists of 678 amino acid residues, which together with the two glycan moieties, has an overall molecular weight of 79 550. The sequence shows extensive internal homology, with the N-terminal domain (residues 1-336) and C-terminal domain (residues 337-678)
BIOCHEMICAL EDUCATION 12(4) 1984
147 possessing 40% identical residues. This also seems to be the case for the sequence of ovotransferrin8 and partial sequence of lactoferrin. 9 This suggests that the transferrin molecule may have evolved from the structural gene of an ancestral protein possessing only one metal-binding site and about 340 amino acid residues by a process of gene duplication. 7 Williams et al l° proposed that this ancestral protein is not a serum protein but a membrane-bound metal-receptor protein, since the isolated half-molecule of ovotransferrin was rapidly lost from the blood stream via the kidneys. On the other hand, Mazurier et al li proposed that the transferrins may instead possess a 6-fold homology, and that the two regions with the highest homology are located in the two iron binding sites of the protein. Carbohydrate content All the transferrins are glycoproteins. When transferrins are compared on the basis of their carbohydrate composition, they appear to exhibit greater species variability than in their amino acid compositions. Thus the reported total carbohydrate content varies from 3.0 to 11.8% weight of protein and the number of carbohydrate chains per molecule from 1 to 4. The carbohydrate moiety of human serum transferrin represents about 6% of the weight of the protein. It manifests itself as two identical, branched heterosaccharide chains (glycans) that are attached to the amide groups of asparaginyi residues via 13-N-glycosidic linkages. However, it has been reported recently that there is in addition, a minor population of transferrin with solely tri-branched glycans. 12 The structure of these glycans has been carefully worked out by several investigators. Each glycan has been shown to consist of two sialic acid, two galactose, three mannose and four N-acetylglucosamine residues. 13 The sialic acid residues are located at the terminal position of the chain, and are susceptible to cleavage by neuraminidase. The complete carbohydrate sequence of the glycan can be represented by a 'biantennary' structure of the type shown in Fig. 1. From the known amino acid sequence of human serum transferrin the two glycans are believed to be attached to asparagine residues 413 and 610 in the C-terminal domain of the protein] NeuNAca ( 2 ~ 6 )
Gal,8 (I ~ 4 )
GLcNAcp(I - " 2) Mane ( 1 ~ 3 ) Man/9 { I ~ 4 } GLcNAc/£ ( I ~ 4 ) GLcNAc/~L -,-- Asn
NeuNAca ( 2 - - - 6 )
Figure 1
GaL/9 ( I - " 4 )
GLcNAc/S'{ I ~ 2) M a r i a ( I - ' 6 )
Glycan structure of human serum transferrin
The biological functions of these heterosaccharide chains have not been clearly established. They probably play a role in enhancing the solubility of the protein by virtue of their hydrophilic groups and increased charges? Enzymatic removal of sialic acid, on the other hand, did not bring about any gross conformational changes, nor was the carbohydrate required for uptake and exchange of iron. There has also been suggestion that the carbohydrate moiety may be responsible for the binding of transferrin to specific receptors on cell membranes during the physiological exchange of iron, but substantiative evidence for this concept however, is still lacking.14
Metal Binding
Iron(Ill) Transferrin combines with two Fe 3+ ions in the presence of bicarbonate (or carbonate) ions to form a pink-coloured complex with an absorption maximum at 465-470 nm. This reaction is found to be pH dependent: it is maximal between pH 7.5-10, but upon lowering the pH, partial dissociation occurs at pH 6.5, and complete dissociation at pH 4.5. Thus this property has been used extensively for the in vitro preparation of apotransferrin. It has also been shown that for every Fe 3÷ ion bound to the protein, one bicarbonate ion is concomitantly bound and three protons are released. Thus the overall reaction between Fe 3+ ions and transferrin can be represented by the following equations:
Fe 3+ + H6Tf + HCO3- ~- [Fe-H3Tf-HCO3]- + 3H +
(1)
Fe 3+ + [Fe-H3Tf-HCO3]- + HCO3- ~ [Fe2-Tf-(HCO3)2] 2- + 3H +
(2)
Although it is generally believed that the three protons released per Fe 3+ ion bound in the reaction are derived from the ionization of three tyrosine residues of the protein, 15 recent
BIOCHEMICAL EDUCATION 12(4) 1984
148 evidence however, suggests that probably only two tyrosines are involved in complexation, with the third proton being released from the water molecule already bound to Fe 3÷ ion. 16 As a consequence of this reaction, diferric transferrin gains two net negative charges, a fact readily demonstrated by electrophoretic mobility studies. 17 This 'charge balance' has in fact provided the main basis for suggesting that bicarbonate is the anion involved during the binding process. However, studies based on potentiometric titration, nuclear magnetic resonance spectroscopy and equilibrium binding implicate carbonate as the bound anion.1 Iron is absorbed from the intestine as Fe 2÷ and may therefore enter the circulation as Fe 2÷ ion. It has been proposed that the serum protein, caeruloplasmin (ferroxidase) may function to catalyse the oxidation of Fe 2÷ to Fe 3+ ion so that it may be bound by transferrin. However, from a physiological point of view, it is of interest to know if transferrin also binds Fe 2÷ ion. The bulk of evidence at present seems to suggest that it does not bind Fe 2÷ ion, and if at all, only very slightly. Bates and co-workers, TM on the other hand, have demonstrated that in the presence of carbonate ion and oxygen, apotransferrin can react with Fe 2+ ion to form first an intermediate ternary Fe2+-trans ferrin-CO~--complex, which is then subsequently oxidized by molecular oxygen to give the stable Fe3+-transferrin-CO~--complex.
Other metals In addition to Fe 3+ ion, transferrin can also bind a variety of divalent, trivalent and tetravalent metal ions. These include metals of the transition, lanthanide and actinide series 3'1sA6 (see Table 1). The mechanism of reaction of these metals with transferrin seems to parallel that for Fe 3÷ ion binding. However, the number of protons released and the number of metal ions bound in the overall reaction is dependent on the hydrolytic tendencies and ionic radii of the metal ions respectively. 15'~6 For example, proton release studies showed that the binding of two A13+ ions by transferrin releases six protons, while a similar reaction with Cu 2+ ion displaces only four protons. This observation can be rationalized on the basis that for every metal ion bound, two protons are derived from the ionization of two co-ordinated tyrosine residues, while the remaining protons (if any) are from the hydrolysis of the metal ion itself. On the other hand, the ionic radii of the metal ions seem to influence the stoichiometry of the metal binding reaction. While it is fairly well established that transferrin binds, for example, two Cu 2+ and A13+ ions, recent experiments suggest that metal ions with ionic radii greater than that of europium (0.095 nm) can bind to only one of the metal binding sites of transferrin. This larger site is located in the C-terminal domain of transferrin. 16 Table 1
Metal Binding Ions of Transferrin 16 Metal ion Cu 2+ Zn 2+ Fe 3+ Eu 3+ Th 4÷ Nd 3+ Pr 3+
Ionic radius (nm)
No. of metals bound
No. of tyrosine residues
0.073 0.074 0.0645 0.095 0.094 0.0983 0.099
2 2 2 2 2 1 1
4 3.7 4.2 4.2 2.9 2.2 1.8
Nature of the iron binding sites One of the most important aspects of the study of transferrin has been concerned with the elucidation of the nature of the two iron binding sites of the protein. Thus, considerable attention has been directed to determining whether (i) there is any difference in binding affinity and interaction between the two sites during iron binding, and (ii) the two sites are structurally and functionally equivalent. Initial studies by Warner and Weber 17 showed that metal binding by transferrin was highly cooperative, and therefore implicated a pairwise mode of binding. However, later work by Aasa et a119indicated that the association constants for the binding of the two iron atoms were approximately equal. Therefore it was concluded that the two sites are equivalent and independent, and that the iron atoms will bind at random. Recent data, on
BIOCHEMICAL EDUCATION 12(4) 1984
149 the other hand, suggest that the sites are non-equivalent and that the binding process is neither pairwise nor random, but sequential: Tf + Fe K1 ~- FeTf + Fe K2 ~ FeTfFe Support for this conclusion comes mainly from the following spectroscopic and chemical data: (i) EPR spectroscopy showed that the two sites are clearly distinguishable when occupied by the vanadyl ion, VO 2+ and Cr 3+ ion. 1,3 This spectroscopic inequivalence may be due to a charge difference of the liganding groups at the two sites. (ii) pH dependence of iron binding: the N-terminal site does not bind iron below pH 5.7 while the C-terminal site, in the absence of chelating agents remains occupied down to pH 4.8. 20 In addition, the stoichiometric binding constant of the C-terminal site is 5 times greater than the N-terminal site at pH 7.4, which increases to a factor of 33 at pH 6.7.1 The sites are, however, virtually independent since their affinities for iron do not change appreciably either with or without occupancy of the other. (iii) Chelate specificity in which Fem(nitrilotriacetate)2 directs iron to the C-terminal site at low pH while Fern(citrate)4 directs iron to the N-terminal site at neutral pH. 1 Based on this observation, C-terminal and N-terminal monoferric transferrins (designated as TfFec and FeNTf respectively) have been prepared for kinetic studies 21 and for producing the respective C-terminal and N-terminal iron-binding fragments of human transferrin by proteolytic cleavage. 1 (iv) In fresh human serum the two sites are unequally occupied: there is a preferential occupation of the N-terminal site, and upon incubation at 37°C, the preference becomes even more marked. However, on storage of serum at - 15°C, the preference is shifted to the C-terminal site. 22
Structure of metal-binding sites Recent advances have given us a clearer picture of the structure of the metal-binding sites of transferrin. For example, it has been reported that the metal-binding sites are located less than 1.7 nm below the surface of the protein. 23'24 In addition, based on the results obtained by numerous physicochemical techniques, it is now generally accepted that the iron binding-site ligands of transferrin comprise of two tyrosines, two histidines, a bicarbonate/carbonate ion, and a hydroxide ion (from water), which together form a six-coordinate complex with Fe 3÷ ion.16 With the recent publication of the complete sequence of human transferrin 7 and ovotransferrin, ~ Chasteen 25 proposed that the binding sites for Fe 3+ and bicarbonate ions are probably located near the junction of two peptide fragments joined by Cys-117 to Cys194 in the N-terminal domain of human transferrin. Tyr-185, Tyr-188, and two of the three histidines, 119,207, and 249 probably serve as ligands to the metal, while Arg-124 (and/or the cluster Lys-ll5, Lys-ll6, His-ll9) is electrostatically bound to the carbonate anion binding site. The arrangement of amino acids is similar in the C-terminal domain. 25 Confirmation of the assignment of these amino acid residues will, however, have to await for X-ray crystallographic studies of the protein at high resolution. Anion Binding by Transferrin
Anions It is now generally accepted that for the specific binding of Fe 3+ ions by transferrin to occur under physiological conditions, an anion such as carbonate or bicarbonate must also be bound. In fact, due to this absolute anion requirement for metal ion binding, the term 'synergistic' has been used to describe the anion binding process. In the absence of carbonate or bicarbonate, other anions such as oxalate, pyruvate, thioglycolate, nitrilotriacetate (NTA), glycine, phenylalanine, etc will also facilitate iron binding 26 (see Table 2). It is interesting to note that all these anions possess a carboxyl group and a second electron-withdrawing functional group (typically another carboxyl, an amino, or a sulphydryl group), within 0.63 nm of the first carboxyl group, and are capable of adopting a 'carbonate-like' configuration. These anions can be represented
R
I by the general formula, L - - C - - C O 2 - , in which L is the proximal electron-withdrawing
f
H
BIOCHEMICAL EDUCATION 12(4) 1984
150 Table 2
Synergistic Anions of Transferrin 26 Anion
Structure
Anion
Structure
1. Carbonate
....~_. o e o-- ( =-
5. Nitrilotriacetate
N,~,2,oo'-:,,
2. Oxalate
° G o--! !
6. Glycine
I ...~-- ° .~N--C--<~ ] "~'00
-
3. Pyruvate
~"~ "~0
-
II
('ti~
o~---c.--c: ::~ "~'o
7. Phenylalanine
('H~
H
4. Thioglycolate
9
,s--c--c: II
II
functional group. However, carbonate or bicarbonate is the anion bound most tightly by transferrin, so that it will displace other anions from the Fe23+-transferrin-anion ternary complex when it is present in the medium. Structure of anion binding sites In a detailed investigation of the structure and binding relationships of a set of 40 possible synergistic anions, Schlabach and Bates 26 concluded that the anion binding site is (a) at least 0.3 nm deep, approximately 0.6 nm wide and between 0.4 and 0.65 nm in length, and (b) asymmetric and located near the surface of the protein. These workers also proposed a model to account for the synergistic binding of Fe 3+ ion and 'carbonate-like' anion by transferrin. This model, depicted in Fig. 2, shows that the anion interacts with the positive charges of arginyl or lysyl residues on the protein via two of its oxygens, and forms a coordinate bond with the Fe 3+ ion via the proximal ligand, L, while the large substituent group 'R' is directed away from the surface of the protein. This 'interlocking metal sites' model seems to have found fairly wide acceptance. For example, that the carbonate ion (or other anion) is directly bound to Fe 3+ ion (or other metal ion) has been confirmed by 13C-NMR and by EPR spectroscopic studies. In addition, there is evidence that the anion binds to transferrin before the metal ion so that anion binding may be a preliminary step for metal binding. 24 Interestingly, recent work has also shown that the two anion binding sites may have different properties. Only the N-terminal site for instance, will accept oxalate as the synergistic anion for Cu 2+ ion binding.l In addition, for cobalt transferrin complexes, it has been shown that the synergistic anion is carbonate in the N-terminal site, while it is bicarbonate in the C-terminal site. 24 Thus, the anion binding ligand in the N-terminal site is most probably the guanidino group of arginine, and as for the C-terminal site, the anion binding ligand appears to be either a guanidino group of arginine or an t-amino group of lysine. Non-synergistic anions Folajtar and Chasteen 2v have recently reported that human serum transferrin has additional binding sites for a class of non-synergistic anions that seems to affect the metal-binding sites of the protein. The interaction of these anions with the protein is very unusual in that the anions bind pairwise and with strong cooperativity in
l Fe~L__
t\ Figure 2
--C-l
C .<" ~
~'o
@
®
A hypothetical scheme depicting the binding of a synergistic anion and Fe s+ to transferrin. The organic anion is shown as interacting with positive charges on the protein and donating a coordinate bond to Fe3+ via a proximal functional group. Possible nucleophilic attack on the carbonyl carbon by an amino acid residue is also suggested. - ..... restrictions at the site based on molecule model studies (From ref 26)
BIOCHEMICAL EDUCATION 12(4) 1984
151 each of the domains of transferrin. The overall apparent association constant for anion binding follows the sequence of thiocyanate > perchlorate > pyrophosphate > adenosine triphosphate > chloride > > tetrafiuoroborate, orthophosphate, adenosine monophosphate, fluoride, sulphate and bicarbonate. This order seems to parallel the lyotropic series for the strength of anion interaction with proteins. 27 The presence of these anion binding sites naturally raises the question as to where they are located in the transferrin molecule. Existing evidence precludes the possibility that they are located in the synergistic anion binding site, as well as they being directly ligated to the metal ion. It is more likely, however, for them to be bound directly to positively charged amino acid residues or amide dipoles of the protein. It is unclear, at present, what is the exact role played by these non-synergistic anions, but since they were found to have a pronounced effect on the kinetics of iron release in vitro, 21 they could play an important role in influencing the iron binding properties of transferrin.
Biological Function of Transferrin
Iron usually exists in the ferric (Fe 3+) state under physiological conditions. Unfortunately, ferric salts are highly susceptible to hydrolysis at neutral pH to give insoluble ferric hydroxide, so that the equilibrium concentration of free Fe 3+ ion in physiological fluids must be maintained below 2.5 x 10-18M. However, since the daily turnover of haemoglobin iron in man is about 30 mg (= 5.4 x 10-4M), the need for a high-affinity, iron-binding protein, transferrin, is apparent. In serum, transferrin is only 30% saturated so that it has the capacity to bind excess iron, and thus help in controlling the build-up of toxic amounts of excess iron. In addition, the presence of a latent iron-binding capacity of transferrin may also be of value in the defence against infection. Perhaps the most important role of transferrin is in the transport of iron among the sites of absorption (eg, intestinal mucosal cells), sites of utilization (eg, immature erythroid cells), sites of storage (eg the liver) and sites of haemoglobin degradation. In doing this, transferrin plays a vital and central role in iron metabolism (see Fig. 3). It is a true carrier molecule in that it is conserved for many cycles of iron transport in its interaction with target tissues. It has a relatively long half-life of 8-10 days in vivo. Recently, it has been reported that transferrin may also play a role in zinc(II) transport, 28 and that plasma aluminium, when bound to transferrin, may be a cause for anaemia since aluminium would enter the pathways of iron distribution and metabolism.29
Transfer of Iron from Transferrin to Cells
Under physiological conditions, iron(III) is very tightly bound to transferrin. It has been shown, for instance, that the apparent equilibrium constant (Ka) for the reaction between the transferrin and one iron atom is approximately 1024M-1, so that it would take about 10 000 years for an iron atom to dissociate spontaneously from transferrin in blood. 3° It is therefore apparent that special mechanisms must be invoked to effect the transfer of iron from transferrin to active growing cells. It is now generally accepted that the first stage in this delivery of iron involves the binding of transferrin to specific iron receptors on the cell membrane, and that this interaction is a time-, temperature- and energy-dependent process. The mechanism by which iron is transferred into the interior of the cell is, Excretion of unabsorbed ICe in stool
rngested food i'on (~e 2~ and Fe 3.) (20 mg/d) Fe ~" Fe~,*-ferritin in mucosaL cell
/ d
Excretion of Fe through urine and skin ( < 0 I°/o)
intestinal
Fractional iron (Feb+or Fe
(eg,,enzymes)
3*
)
. -
3*
Fe2 -tronsferrin . Jgnd Fe3. in P t a s m c l [ -
F e ~ ' - f e r r i t i n or hemoslderin (storage forms in RE system)
E system Myoglobin(3_ 5 °/o)(Fe2. )
Hemotopo~et ic o r g a n s / z / Hemoglobin (Fe *) (65 -70%
)
Loss of Fe due to bleeding
Figure 3
Pathways of iron [From 'Fundamentals of Clinical Chemistry' (Tietz, NW editor) second edition, p 922, W B Saunders Co, Philadelphia (1976)]
BIOCHEMICAL EDUCATION 12(4) 1984
152 however, plagued with controversy, but recent evidence has been accumulating which suggests that it entails the receptor-mediated endocytosis of transferrin. 31-33
Receptor-mediated endocytosis of transferrin A wide variety of proteins and peptides, including hormones, low density lipoprotein (LDL) and toxins, enter cells by receptormediated endocytosis. 34 In this cellular pathway, the molecules (ligands) are first bound to surface-bound receptors before the ligand-receptor complex is rapidly internalised by the cell. In many instances, rapid internalization is achieved by clustering of receptors in specialised regions of the surface membrane called coated pits that invaginate rapidly into the cell during endocytosis to form coated vesicles. 34 Although the exact pathway followed by the protein-receptor complex is not known, it is now generally believed that the protein usually dissociates from its receptor in a prelysosomal acid vesicle (endosome), after which the protein is transported to the lysosomes where degradation to amino acids takes place. The receptor, on the other hand, is usually not broken down, but is recycled back to the cell surface to be re-utilized. However, a notable exception to this generalized picture of receptor-mediated endocytosis is exhibited by transferrin. Here, diferric transferrin enters the cell bound to transferrin receptor, but internalization of the transferrin-transferrin receptor complex does not result in the degradation of transferrin. 31'32 Instead, after releasing the iron to cells (which is eventually shuttled to ferritin), apotransferrin together with its receptor is recycled back to the plasma membrane to be released and re-utilized. 35-37. That transferrin remains intact after endocytosis is explained by the fact that the molecule is delivered to a nonlysosomal acidic compartment (pH 5.4) rather than to the lysosome. 33 In this acidic nonlysosomal compartment, the low pH reduces the affinity of transferrin for iron by destabilizing the Fe-transferrin-complex, thus allowing for iron removal. 3s Iron is then transported across the vesicular membrane and delivered to cytosolic ferritin. 33 However, the question as to why transferrin does not reach the lysosome and become degraded is unclear. It is likely, perhaps, that even in this low pH micro-environment of the nonlysosomal compartment, the transferrin molecule, in contrast to other proteins, is still bound to its receptor. Recent evidence seems to come out strongly in favour of this hypothesis. 39-41 For example, it has been shown that at pH 5.4, apotransferrin binds to transferrin receptors to the same extent and with the same affinity as diferric transferrin does at pH 7.0 so that in the acidic nonlysosomal compartment, transferrin remains bound to its receptor both before and after iron removal. On the other hand, apotransferrin is rapidly dissociated from its receptor at pH 7.0, so that when the apotransferrin-receptor complex is recycled to the cell surface, where it is now exposed to a physiological pH of 7.0-7.4, the apotransferrin molecule is released from the receptor and leaves the cell to be reused for iron binding/transport again. This cycle of iron transport in plasma and iron release in the cell is repeated with the net result that the transferrin molecule is conserved in the process. A schematic representation of the 'transferrin cycle' is depicted in Fig. 4.
Transferrin receptors Transferrin receptors have a relatively long half-life of approximately 60 hours. They have been found in a variety of growing cell types, including fibroblasts, lymphocytes, kidney tissue, tumour cells, but they are present most abundantly in tissues that are very active in iron uptake such as reticulocytes and placental cells. For example, it has been reported that there are about 300 000 transferrin receptors in each reticulocyte cell, so that if active reticulocytes can incorporate over 1 million iron
Figure 4
The transferrin cycle (From ref 39) * CURL = Compartment of uncoupling of receptor and ligand
BIOCHEMICAL EDUCATION 12(4) 1984
153 atoms per minute, the transferrin cycle must be completed within 15-30 seconds. 42 In addition, the isolated transferrin-receptor complex42 has been shown to possess a stability constant of near 107M -1, and that the controlling genes for the receptor in human have been mapped to chromosome number three. 43 Several groups of workers have reported the isolation of transferrin receptors from reticulocytes, placenta, and tumour cells. 43 There is general agreement that the receptor has an apparent molecular weight of 170 000-200 000, and a disulphide-linked subunit structure that consists of two identical 90 000-dalton subunits. 43 Each of these subunits binds one transferrin molecule so that there exists the possibility that transferrin and its receptor may form linear polymers. The transferrin receptor also appears to be a transmembrane glycoprotein. The major portion of each subunit, with Mr 70 000 is exposed to the extracellular environment while the cytoplasmic 'tail' has only Mr 5000 (see Fig. 5). The 70 000-dalton fragment has also been shown to retain the antigenic sites and the ability to bind iron after tryptic cleavage. 43
Functional Heterogeneity/Homogeneity
Although the two iron binding sites of transferrin have been shown recently to exhibit well-established physicochemical differences, the question as to whether they also function differently in vivo remains unclear. Fletcher and H e h n s 44 first proposed that each site has a specific function: one site (A-site) preferentially directs its iron to immature red cells and placenta, while the other site (B-site) deposits its iron in the liver and intestinal mucosal cells. Much of the evidence in favour of functional heterogeneity of the site has come from experiments in which human transferrin was used as the iron donor for rabbit or rat cells (ie, heterologous system), a However, in experiments using homologous systems of human protein and human cells, and rabbit reticulocytes and rabbit transferrin, no difference in iron uptake from the two sites is detected. ~ Based on these lines of evidence, it is more probable that there is no functional difference between the two sites of transferrin under physiological (ie, homologous) conditions. The functional differences that were reported using heterologous systems could be a result of the species variation in transferrin-receptor interaction. 45 It was reported recently, however, that there may exist, in vivo, a functional difference between the two iron binding sites of rabbit transferrin at the level of iron loading rather than iron release. 46 This conclusion is based on the observation that absorbed iron was found to bind preferentially to the acid-labile site in the N-terminal domain of rabbit transferrin.
Conclusions
This short review has highlighted the major advances that have been made in recent years in the elucidation of the structure and function of transferrins. For instance, the completion of the amino acid sequence of human serum transferrin and ovotransferrin is significant because it has enabled us to have a better understanding of the overall structure of the protein, especially with regard to the possible arrangement and assignment of the amino acid residues (ligands) at the active sites. However, to facilitate the unequivocal identification of amino acid residues and a complete description of the three-dimensional structure, we need to await the completion of the X-ray crystallographic structure of transferrin. From the functional point of view, there is now a general consensus among workers in the field that the most plausible mechanism of the transfer of iron from
..~--.,,.
..--~-,
I
70K
$--S
!90K
Key: ~ High mannose oligosaccharide chain 0 Complex type chain vw Covalently bound fatty acid
11 Figure 5
Schematic representation of the cell surface transferrin receptor (From ref 43)
BIOCHEMICAL EDUCATION 12(4) 1984
154 transferrin to cells involves the receptor-mediated endocytosis of transferrin. However, information on how the released iron is transported across the vesicular membrane into
the cytoplasm is, at present, still lacking. Acknowledgement
Part of this review was written while the author was a Research Fellow in the Department of Physical Biochemistry, John Curtin School of Medical Research. The Australian National University, Canberra, Australia.
References
a Aisen, P and Listowsky, I (1980) Ann Rev Biochem 49, 357-393 2 Putman, F W (1975) in The Plasma Proteins (Putnam, F W, editor) second edition, Volume 1, pp 265-315, Academic Press, New York 3 Chasteen, N D (1977) Coord Chem Rev 22, 1-36 4 Martel, P, Kim, S M and Powell, B M (1980) Biophys. J 31,371-380 5 Wang, A-C and Sutton, H E (1965) Science 149, 435-437 6 Chung, M C-M and McKenzie, H A (1983) in Proc. 8th Annual Lorne Conference on Protein Structure and Function, Feb. 7-11, Victoria, Australia, p 59 7 MacGillivray, R T A, Mendez, E, Simha, S K, Sutton, M R, Lineback-Zins, J and Brew, K (1982) Proc Natl Acad Sci USA 79, 2504-2508 8 Williams, J, Elleman, T C, Kingston, I. B, Wilkins, A. G and Kuhn, K A (1982) Europ J Biochem 122, 297-303 9 Metz-Boutigue, M-H, Mazurier, J, Jolles, J, Spik, G, Montreuil, J and Jolles, P (1981) Biochim Biophys Acta 670, 243-254 10 Williams, J, Grace, S A and Williams, J M (1982) Biochem J 201,417-419 11 Mazurier, J, Metz-Boutigue, M-H, Jolles, J, Spik, G, Montreuil, J and Jolles, P (1983) Experientia 39, 135-141 12 Kerckaert, J-P and Bayard, B (1982) Biochem Biophys Res Commun 105, 1023-1030 13 Dorland, L, Hoverkamp, J, Schut, B L, Vliegenart, J F G, Spik, G, Strecker, G, Fournet, B and Montreuil, J (1977) FEBS Lett 77, 15-20 14 Hemmaplardh, D and Morgan, E H (1976) Biochim Biophys Acta 426,385-398 15 Gelb, M H and Harris, D C (1980) Arch Biochem Biophys 200, 93-98 16 Pecoraro, V L, Harris, W R, Carrano, C J and Raymond, K N (1981) Biochemistry 20, 7033-7039 17 Warner, R C and Weber, I (1953) J A m Chem Soc 75, 5094-5101 18 Kojima, N and Bates, G W (1981) J Biol Chem 256, 12034-12039 ~9 Aasa, R, Malmstrom, B G, Saltman, P and Vanngard, T (1963) Biochim Biophys Acta 75,202-223 2o Princiotto, J V and Zapolski, E J (1975) Nature 255, 87-88 2x Baldwin, D A and de Sousa, D M R (1981) Biochem Biophys Res Commun 99, 1101-1107 22 Williams, J and Moreton, K (1980) Biochem J 185,483-488 23 Yeh, S M and Meares, C F (1980) Biochemistry 19, 5057-5062 24 Zweier, J L, Wooten, J B and Cohen, J S (1981) Biochemistry 20, 3503-3510 25 Chasteen, N D (1983) Trends in Biochem Sci 8, 272-275 26 Schlabach, M R and Bates, G W (1975) J Biol Chem 250, 2182-2188 27 Folajtar, D A and Chasteen, N D (1982) J Am Chem Soc 104, 5775-5780 28 Harris, W R (1983) Biochemistry 22, 3920-3926 29 Trapp, G A (1983) Life Sciences 33,311-316 3~)Aisen, P and Liebman, A (1968) Biochem Biophys Res Commun 32,220-226 31 Karin, M and Mintz B (1981) J Biol Chem 256, 3245-3252 32 Octave, J-N, Schneider, Y-J, Crichton, R R and Trouet, A (1981) Eur J Biochem 115, 611-618 33 van Renswoude, J, Bridges, K R, Harford, J B and Klausner, R D (1982) Proc Natl Acad Sci USA 79, 6186-6190 34 Goldstein, J L, Anderson, R G W and Brown, M S (1979) Nature 279, 679-685 35 Harding, C, Heuser, J and Stahl, P (1983) J Cell Biol 97,329-339 30 Hopkins, C R and Trowbridge, I S (1983) J Cell Biol 97, 508-521 37 Enns, C A, Jarrick, J W, Suomalainen, H, Schroder, J and Sussman, H H (1983) J Cell Biol 97,579-585 38 Rao, K, van Renswoude, J, Kempf, C and Klausner, R D (1983) FEBS Lett 160, 213-216 39 Dautry-Varsat, A, Ciechanover, A and Lodish, H F (1983) Proc Natl Acad Sci USA 80, 2258-2262 40 Klausner, R D, Ashwell, G, van Renswoude, J, Harford, J B and Bridges, K R (1983) Proc Natl Acad Sci USA 80, 2263-2266 41 Harding, C and Stahl, P (1983) Biochem Biophys Res Commun 113, 650-658 42 van Bockxmeer, F M and Morgan, E H (1979) Biochim Biophys Acta 584, 76-83 43 Newman, R, Schneider, C, Sutherland, R, Vodinelich, L and Greaves, M (1982) Trends in Biochem Sci 7, 397-400 44 Fletcher, J and Huehns, E R (1968) Nature 218, 1211-1214 45 van Bockxmeer, F M and Morgan, E H (1982) Comp Biochem Physiol 71A, 211-218 46 Marx, J J M, Gebbink, J A G K, Nishisato, T and Aisen, P (1982) Brit J Haematol 52, 105-110
BIOCHEMICAL EDUCATION 12(4) 1984
STRUCTURE AND FUNCTION OF TRANSFERRIN
M CHING-MING CHUNG D e p a r t m e n t ' o f Biochemistry Faculty of Medicine National University of Singapore Republic of Singapore 0511
Introduction
The transferrins are a group of non-haem iron-binding glycoproteins widely distributed in the physiological fluids and cells of vertebrates. They comprise: (i) serum transferrin (also known as 'serotransferrin', 'siderophilin', and '13a metal-binding globulin'), the prototype found in blood serum, (ii) lactoferrin ('milk red protein', 'lactotransferrin'), the ironbinding protein first found in milk, but also since found in secretions (tears, saliva) and cells (neutrophil leukocytes): [lactoferrin should not be confused with the 'milk transferrin' found in the milk of several animal species (eg rabbit)], (iii) ovotransferrin ('ovoferrin', 'conalbumin'), the iron-binding protein isolated from avian egg white. The chief physiological function of serum transferrin is undoubtedly in iron transport within the circulatory system of the vertebrates, but a specific role in iron transport has not been established for ovotransferrin and lactoferrin. However, due to their very high affinity for iron and other trace metals, these two proteins could function to deprive microorganisms of essential metals for growth, thus acting to protect the egg and milk respectively against infection. The transferrins have been a subject of much intensive investigation in recent years and a great deal is known about their physical, chemical and biological properties. In this article, I will summarise only the more important aspects of the structure and function of human serum transferrin. Readers are directed to refer to several recent reviews ~-3 for a more detailed treatment of this subject.
Physical Properties
Human serum transferrin is a monomeric glycoprotein with a sedimentation coefficient of 5.1S and a molecular weight of about 80 000.1 It contains two similar but not identical metal-binding sites located in its N-terminal and C-terminal domains. Hydrodynamic studies suggest that it has a molecular shape best represented by a prolate ellipsoid with an axial ratio of 1:2 and 1:3 for the iron-saturated and iron-free protein respectively. However, a recent report based on small-angle neutron-scattering measurements, indicates that it is an oblate spheroid. 4 The tertiary structure of rabbit serum transferrin, as revealed by X-ray crystallography, is compatible with a bilobal model. The two lobes, which are nearly equal in size and possessing clefts near the region of the join, may correspond to the two domains formed from the monoferric N- and C-terminal halves of the polypeptide chain. 1
Chemical Properties
Amino acid composition The amino acid composition of human transferrin has been reported by several investigators. 2 It shows few unusual features except for a complete absence of free sulphydryl groups, and a high content of half-cystine corresponding to 19 intrachain disulphid e bonds. Transferrins of different species generally have fairly similar amino acid compositions. However, the minor differences manifested in their amino acid compositions coupled with the variation in carbohydrate contents result in differences in electrophoretic mobility. Transferrins exhibit extensive genetic polymorphism, and in cases where the genetic phenotypes are from the same species, variation in electrophoretic mobility is usually found to be a result of amino acid substitutions. For example, comparative studies on the chymotryptic digests of human transferrin C and D t indicate that an aspartic residue in transferrin C is probably replaced by a glycine residue in transferrin Da. 5 On the other hand, in a study on equine transferrin D and R, it was found that one aspartic acid and one glutamic acid residue in transferrin D are replaced by two glycine residues in transferrin R. 6 Amino acid sequence The partial amino acid sequence of human transferrin has been known for a number of years, but its complete sequence has only just been reported. 7 It consists of 678 amino acid residues, which together with the two glycan moieties, has an overall molecular weight of 79 550. The sequence shows extensive internal homology, with the N-terminal domain (residues 1-336) and C-terminal domain (residues 337-678)
BIOCHEMICAL EDUCATION 12(4) 1984
147 possessing 40% identical residues. This also seems to be the case for the sequence of ovotransferrin8 and partial sequence of lactoferrin. 9 This suggests that the transferrin molecule may have evolved from the structural gene of an ancestral protein possessing only one metal-binding site and about 340 amino acid residues by a process of gene duplication. 7 Williams et al l° proposed that this ancestral protein is not a serum protein but a membrane-bound metal-receptor protein, since the isolated half-molecule of ovotransferrin was rapidly lost from the blood stream via the kidneys. On the other hand, Mazurier et al li proposed that the transferrins may instead possess a 6-fold homology, and that the two regions with the highest homology are located in the two iron binding sites of the protein. Carbohydrate content All the transferrins are glycoproteins. When transferrins are compared on the basis of their carbohydrate composition, they appear to exhibit greater species variability than in their amino acid compositions. Thus the reported total carbohydrate content varies from 3.0 to 11.8% weight of protein and the number of carbohydrate chains per molecule from 1 to 4. The carbohydrate moiety of human serum transferrin represents about 6% of the weight of the protein. It manifests itself as two identical, branched heterosaccharide chains (glycans) that are attached to the amide groups of asparaginyi residues via 13-N-glycosidic linkages. However, it has been reported recently that there is in addition, a minor population of transferrin with solely tri-branched glycans. 12 The structure of these glycans has been carefully worked out by several investigators. Each glycan has been shown to consist of two sialic acid, two galactose, three mannose and four N-acetylglucosamine residues. 13 The sialic acid residues are located at the terminal position of the chain, and are susceptible to cleavage by neuraminidase. The complete carbohydrate sequence of the glycan can be represented by a 'biantennary' structure of the type shown in Fig. 1. From the known amino acid sequence of human serum transferrin the two glycans are believed to be attached to asparagine residues 413 and 610 in the C-terminal domain of the protein] NeuNAca ( 2 ~ 6 )
Gal,8 (I ~ 4 )
GLcNAcp(I - " 2) Mane ( 1 ~ 3 ) Man/9 { I ~ 4 } GLcNAc/£ ( I ~ 4 ) GLcNAc/~L -,-- Asn
NeuNAca ( 2 - - - 6 )
Figure 1
GaL/9 ( I - " 4 )
GLcNAc/S'{ I ~ 2) M a r i a ( I - ' 6 )
Glycan structure of human serum transferrin
The biological functions of these heterosaccharide chains have not been clearly established. They probably play a role in enhancing the solubility of the protein by virtue of their hydrophilic groups and increased charges? Enzymatic removal of sialic acid, on the other hand, did not bring about any gross conformational changes, nor was the carbohydrate required for uptake and exchange of iron. There has also been suggestion that the carbohydrate moiety may be responsible for the binding of transferrin to specific receptors on cell membranes during the physiological exchange of iron, but substantiative evidence for this concept however, is still lacking.14
Metal Binding
Iron(Ill) Transferrin combines with two Fe 3+ ions in the presence of bicarbonate (or carbonate) ions to form a pink-coloured complex with an absorption maximum at 465-470 nm. This reaction is found to be pH dependent: it is maximal between pH 7.5-10, but upon lowering the pH, partial dissociation occurs at pH 6.5, and complete dissociation at pH 4.5. Thus this property has been used extensively for the in vitro preparation of apotransferrin. It has also been shown that for every Fe 3÷ ion bound to the protein, one bicarbonate ion is concomitantly bound and three protons are released. Thus the overall reaction between Fe 3+ ions and transferrin can be represented by the following equations:
Fe 3+ + H6Tf + HCO3- ~- [Fe-H3Tf-HCO3]- + 3H +
(1)
Fe 3+ + [Fe-H3Tf-HCO3]- + HCO3- ~ [Fe2-Tf-(HCO3)2] 2- + 3H +
(2)
Although it is generally believed that the three protons released per Fe 3+ ion bound in the reaction are derived from the ionization of three tyrosine residues of the protein, 15 recent
BIOCHEMICAL EDUCATION 12(4) 1984
148 evidence however, suggests that probably only two tyrosines are involved in complexation, with the third proton being released from the water molecule already bound to Fe 3÷ ion. 16 As a consequence of this reaction, diferric transferrin gains two net negative charges, a fact readily demonstrated by electrophoretic mobility studies. 17 This 'charge balance' has in fact provided the main basis for suggesting that bicarbonate is the anion involved during the binding process. However, studies based on potentiometric titration, nuclear magnetic resonance spectroscopy and equilibrium binding implicate carbonate as the bound anion.1 Iron is absorbed from the intestine as Fe 2÷ and may therefore enter the circulation as Fe 2÷ ion. It has been proposed that the serum protein, caeruloplasmin (ferroxidase) may function to catalyse the oxidation of Fe 2÷ to Fe 3+ ion so that it may be bound by transferrin. However, from a physiological point of view, it is of interest to know if transferrin also binds Fe 2÷ ion. The bulk of evidence at present seems to suggest that it does not bind Fe 2÷ ion, and if at all, only very slightly. Bates and co-workers, TM on the other hand, have demonstrated that in the presence of carbonate ion and oxygen, apotransferrin can react with Fe 2+ ion to form first an intermediate ternary Fe2+-trans ferrin-CO~--complex, which is then subsequently oxidized by molecular oxygen to give the stable Fe3+-transferrin-CO~--complex.
Other metals In addition to Fe 3+ ion, transferrin can also bind a variety of divalent, trivalent and tetravalent metal ions. These include metals of the transition, lanthanide and actinide series 3'1sA6 (see Table 1). The mechanism of reaction of these metals with transferrin seems to parallel that for Fe 3÷ ion binding. However, the number of protons released and the number of metal ions bound in the overall reaction is dependent on the hydrolytic tendencies and ionic radii of the metal ions respectively. 15'~6 For example, proton release studies showed that the binding of two A13+ ions by transferrin releases six protons, while a similar reaction with Cu 2+ ion displaces only four protons. This observation can be rationalized on the basis that for every metal ion bound, two protons are derived from the ionization of two co-ordinated tyrosine residues, while the remaining protons (if any) are from the hydrolysis of the metal ion itself. On the other hand, the ionic radii of the metal ions seem to influence the stoichiometry of the metal binding reaction. While it is fairly well established that transferrin binds, for example, two Cu 2+ and A13+ ions, recent experiments suggest that metal ions with ionic radii greater than that of europium (0.095 nm) can bind to only one of the metal binding sites of transferrin. This larger site is located in the C-terminal domain of transferrin. 16 Table 1
Metal Binding Ions of Transferrin 16 Metal ion Cu 2+ Zn 2+ Fe 3+ Eu 3+ Th 4÷ Nd 3+ Pr 3+
Ionic radius (nm)
No. of metals bound
No. of tyrosine residues
0.073 0.074 0.0645 0.095 0.094 0.0983 0.099
2 2 2 2 2 1 1
4 3.7 4.2 4.2 2.9 2.2 1.8
Nature of the iron binding sites One of the most important aspects of the study of transferrin has been concerned with the elucidation of the nature of the two iron binding sites of the protein. Thus, considerable attention has been directed to determining whether (i) there is any difference in binding affinity and interaction between the two sites during iron binding, and (ii) the two sites are structurally and functionally equivalent. Initial studies by Warner and Weber 17 showed that metal binding by transferrin was highly cooperative, and therefore implicated a pairwise mode of binding. However, later work by Aasa et a119indicated that the association constants for the binding of the two iron atoms were approximately equal. Therefore it was concluded that the two sites are equivalent and independent, and that the iron atoms will bind at random. Recent data, on
BIOCHEMICAL EDUCATION 12(4) 1984
149 the other hand, suggest that the sites are non-equivalent and that the binding process is neither pairwise nor random, but sequential: Tf + Fe K1 ~- FeTf + Fe K2 ~ FeTfFe Support for this conclusion comes mainly from the following spectroscopic and chemical data: (i) EPR spectroscopy showed that the two sites are clearly distinguishable when occupied by the vanadyl ion, VO 2+ and Cr 3+ ion. 1,3 This spectroscopic inequivalence may be due to a charge difference of the liganding groups at the two sites. (ii) pH dependence of iron binding: the N-terminal site does not bind iron below pH 5.7 while the C-terminal site, in the absence of chelating agents remains occupied down to pH 4.8. 20 In addition, the stoichiometric binding constant of the C-terminal site is 5 times greater than the N-terminal site at pH 7.4, which increases to a factor of 33 at pH 6.7.1 The sites are, however, virtually independent since their affinities for iron do not change appreciably either with or without occupancy of the other. (iii) Chelate specificity in which Fem(nitrilotriacetate)2 directs iron to the C-terminal site at low pH while Fern(citrate)4 directs iron to the N-terminal site at neutral pH. 1 Based on this observation, C-terminal and N-terminal monoferric transferrins (designated as TfFec and FeNTf respectively) have been prepared for kinetic studies 21 and for producing the respective C-terminal and N-terminal iron-binding fragments of human transferrin by proteolytic cleavage. 1 (iv) In fresh human serum the two sites are unequally occupied: there is a preferential occupation of the N-terminal site, and upon incubation at 37°C, the preference becomes even more marked. However, on storage of serum at - 15°C, the preference is shifted to the C-terminal site. 22
Structure of metal-binding sites Recent advances have given us a clearer picture of the structure of the metal-binding sites of transferrin. For example, it has been reported that the metal-binding sites are located less than 1.7 nm below the surface of the protein. 23'24 In addition, based on the results obtained by numerous physicochemical techniques, it is now generally accepted that the iron binding-site ligands of transferrin comprise of two tyrosines, two histidines, a bicarbonate/carbonate ion, and a hydroxide ion (from water), which together form a six-coordinate complex with Fe 3÷ ion.16 With the recent publication of the complete sequence of human transferrin 7 and ovotransferrin, ~ Chasteen 25 proposed that the binding sites for Fe 3+ and bicarbonate ions are probably located near the junction of two peptide fragments joined by Cys-117 to Cys194 in the N-terminal domain of human transferrin. Tyr-185, Tyr-188, and two of the three histidines, 119,207, and 249 probably serve as ligands to the metal, while Arg-124 (and/or the cluster Lys-ll5, Lys-ll6, His-ll9) is electrostatically bound to the carbonate anion binding site. The arrangement of amino acids is similar in the C-terminal domain. 25 Confirmation of the assignment of these amino acid residues will, however, have to await for X-ray crystallographic studies of the protein at high resolution. Anion Binding by Transferrin
Anions It is now generally accepted that for the specific binding of Fe 3+ ions by transferrin to occur under physiological conditions, an anion such as carbonate or bicarbonate must also be bound. In fact, due to this absolute anion requirement for metal ion binding, the term 'synergistic' has been used to describe the anion binding process. In the absence of carbonate or bicarbonate, other anions such as oxalate, pyruvate, thioglycolate, nitrilotriacetate (NTA), glycine, phenylalanine, etc will also facilitate iron binding 26 (see Table 2). It is interesting to note that all these anions possess a carboxyl group and a second electron-withdrawing functional group (typically another carboxyl, an amino, or a sulphydryl group), within 0.63 nm of the first carboxyl group, and are capable of adopting a 'carbonate-like' configuration. These anions can be represented
R
I by the general formula, L - - C - - C O 2 - , in which L is the proximal electron-withdrawing
f
H
BIOCHEMICAL EDUCATION 12(4) 1984
150 Table 2
Synergistic Anions of Transferrin 26 Anion
Structure
Anion
Structure
1. Carbonate
....~_. o e o-- ( =-
5. Nitrilotriacetate
N,~,2,oo'-:,,
2. Oxalate
° G o--! !
6. Glycine
I ...~-- ° .~N--C--<~ ] "~'00
-
3. Pyruvate
~"~ "~0
-
II
('ti~
o~---c.--c: ::~ "~'o
7. Phenylalanine
('H~
H
4. Thioglycolate
9
,s--c--c: II
II
functional group. However, carbonate or bicarbonate is the anion bound most tightly by transferrin, so that it will displace other anions from the Fe23+-transferrin-anion ternary complex when it is present in the medium. Structure of anion binding sites In a detailed investigation of the structure and binding relationships of a set of 40 possible synergistic anions, Schlabach and Bates 26 concluded that the anion binding site is (a) at least 0.3 nm deep, approximately 0.6 nm wide and between 0.4 and 0.65 nm in length, and (b) asymmetric and located near the surface of the protein. These workers also proposed a model to account for the synergistic binding of Fe 3+ ion and 'carbonate-like' anion by transferrin. This model, depicted in Fig. 2, shows that the anion interacts with the positive charges of arginyl or lysyl residues on the protein via two of its oxygens, and forms a coordinate bond with the Fe 3+ ion via the proximal ligand, L, while the large substituent group 'R' is directed away from the surface of the protein. This 'interlocking metal sites' model seems to have found fairly wide acceptance. For example, that the carbonate ion (or other anion) is directly bound to Fe 3+ ion (or other metal ion) has been confirmed by 13C-NMR and by EPR spectroscopic studies. In addition, there is evidence that the anion binds to transferrin before the metal ion so that anion binding may be a preliminary step for metal binding. 24 Interestingly, recent work has also shown that the two anion binding sites may have different properties. Only the N-terminal site for instance, will accept oxalate as the synergistic anion for Cu 2+ ion binding.l In addition, for cobalt transferrin complexes, it has been shown that the synergistic anion is carbonate in the N-terminal site, while it is bicarbonate in the C-terminal site. 24 Thus, the anion binding ligand in the N-terminal site is most probably the guanidino group of arginine, and as for the C-terminal site, the anion binding ligand appears to be either a guanidino group of arginine or an t-amino group of lysine. Non-synergistic anions Folajtar and Chasteen 2v have recently reported that human serum transferrin has additional binding sites for a class of non-synergistic anions that seems to affect the metal-binding sites of the protein. The interaction of these anions with the protein is very unusual in that the anions bind pairwise and with strong cooperativity in
l Fe~L__
t\ Figure 2
--C-l
C .<" ~
~'o
@
®
A hypothetical scheme depicting the binding of a synergistic anion and Fe s+ to transferrin. The organic anion is shown as interacting with positive charges on the protein and donating a coordinate bond to Fe3+ via a proximal functional group. Possible nucleophilic attack on the carbonyl carbon by an amino acid residue is also suggested. - ..... restrictions at the site based on molecule model studies (From ref 26)
BIOCHEMICAL EDUCATION 12(4) 1984
151 each of the domains of transferrin. The overall apparent association constant for anion binding follows the sequence of thiocyanate > perchlorate > pyrophosphate > adenosine triphosphate > chloride > > tetrafiuoroborate, orthophosphate, adenosine monophosphate, fluoride, sulphate and bicarbonate. This order seems to parallel the lyotropic series for the strength of anion interaction with proteins. 27 The presence of these anion binding sites naturally raises the question as to where they are located in the transferrin molecule. Existing evidence precludes the possibility that they are located in the synergistic anion binding site, as well as they being directly ligated to the metal ion. It is more likely, however, for them to be bound directly to positively charged amino acid residues or amide dipoles of the protein. It is unclear, at present, what is the exact role played by these non-synergistic anions, but since they were found to have a pronounced effect on the kinetics of iron release in vitro, 21 they could play an important role in influencing the iron binding properties of transferrin.
Biological Function of Transferrin
Iron usually exists in the ferric (Fe 3+) state under physiological conditions. Unfortunately, ferric salts are highly susceptible to hydrolysis at neutral pH to give insoluble ferric hydroxide, so that the equilibrium concentration of free Fe 3+ ion in physiological fluids must be maintained below 2.5 x 10-18M. However, since the daily turnover of haemoglobin iron in man is about 30 mg (= 5.4 x 10-4M), the need for a high-affinity, iron-binding protein, transferrin, is apparent. In serum, transferrin is only 30% saturated so that it has the capacity to bind excess iron, and thus help in controlling the build-up of toxic amounts of excess iron. In addition, the presence of a latent iron-binding capacity of transferrin may also be of value in the defence against infection. Perhaps the most important role of transferrin is in the transport of iron among the sites of absorption (eg, intestinal mucosal cells), sites of utilization (eg, immature erythroid cells), sites of storage (eg the liver) and sites of haemoglobin degradation. In doing this, transferrin plays a vital and central role in iron metabolism (see Fig. 3). It is a true carrier molecule in that it is conserved for many cycles of iron transport in its interaction with target tissues. It has a relatively long half-life of 8-10 days in vivo. Recently, it has been reported that transferrin may also play a role in zinc(II) transport, 28 and that plasma aluminium, when bound to transferrin, may be a cause for anaemia since aluminium would enter the pathways of iron distribution and metabolism.29
Transfer of Iron from Transferrin to Cells
Under physiological conditions, iron(III) is very tightly bound to transferrin. It has been shown, for instance, that the apparent equilibrium constant (Ka) for the reaction between the transferrin and one iron atom is approximately 1024M-1, so that it would take about 10 000 years for an iron atom to dissociate spontaneously from transferrin in blood. 3° It is therefore apparent that special mechanisms must be invoked to effect the transfer of iron from transferrin to active growing cells. It is now generally accepted that the first stage in this delivery of iron involves the binding of transferrin to specific iron receptors on the cell membrane, and that this interaction is a time-, temperature- and energy-dependent process. The mechanism by which iron is transferred into the interior of the cell is, Excretion of unabsorbed ICe in stool
rngested food i'on (~e 2~ and Fe 3.) (20 mg/d) Fe ~" Fe~,*-ferritin in mucosaL cell
/ d
Excretion of Fe through urine and skin ( < 0 I°/o)
intestinal
Fractional iron (Feb+or Fe
(eg,,enzymes)
3*
)
. -
3*
Fe2 -tronsferrin . Jgnd Fe3. in P t a s m c l [ -
F e ~ ' - f e r r i t i n or hemoslderin (storage forms in RE system)
E system Myoglobin(3_ 5 °/o)(Fe2. )
Hemotopo~et ic o r g a n s / z / Hemoglobin (Fe *) (65 -70%
)
Loss of Fe due to bleeding
Figure 3
Pathways of iron [From 'Fundamentals of Clinical Chemistry' (Tietz, NW editor) second edition, p 922, W B Saunders Co, Philadelphia (1976)]
BIOCHEMICAL EDUCATION 12(4) 1984
152 however, plagued with controversy, but recent evidence has been accumulating which suggests that it entails the receptor-mediated endocytosis of transferrin. 31-33
Receptor-mediated endocytosis of transferrin A wide variety of proteins and peptides, including hormones, low density lipoprotein (LDL) and toxins, enter cells by receptormediated endocytosis. 34 In this cellular pathway, the molecules (ligands) are first bound to surface-bound receptors before the ligand-receptor complex is rapidly internalised by the cell. In many instances, rapid internalization is achieved by clustering of receptors in specialised regions of the surface membrane called coated pits that invaginate rapidly into the cell during endocytosis to form coated vesicles. 34 Although the exact pathway followed by the protein-receptor complex is not known, it is now generally believed that the protein usually dissociates from its receptor in a prelysosomal acid vesicle (endosome), after which the protein is transported to the lysosomes where degradation to amino acids takes place. The receptor, on the other hand, is usually not broken down, but is recycled back to the cell surface to be re-utilized. However, a notable exception to this generalized picture of receptor-mediated endocytosis is exhibited by transferrin. Here, diferric transferrin enters the cell bound to transferrin receptor, but internalization of the transferrin-transferrin receptor complex does not result in the degradation of transferrin. 31'32 Instead, after releasing the iron to cells (which is eventually shuttled to ferritin), apotransferrin together with its receptor is recycled back to the plasma membrane to be released and re-utilized. 35-37. That transferrin remains intact after endocytosis is explained by the fact that the molecule is delivered to a nonlysosomal acidic compartment (pH 5.4) rather than to the lysosome. 33 In this acidic nonlysosomal compartment, the low pH reduces the affinity of transferrin for iron by destabilizing the Fe-transferrin-complex, thus allowing for iron removal. 3s Iron is then transported across the vesicular membrane and delivered to cytosolic ferritin. 33 However, the question as to why transferrin does not reach the lysosome and become degraded is unclear. It is likely, perhaps, that even in this low pH micro-environment of the nonlysosomal compartment, the transferrin molecule, in contrast to other proteins, is still bound to its receptor. Recent evidence seems to come out strongly in favour of this hypothesis. 39-41 For example, it has been shown that at pH 5.4, apotransferrin binds to transferrin receptors to the same extent and with the same affinity as diferric transferrin does at pH 7.0 so that in the acidic nonlysosomal compartment, transferrin remains bound to its receptor both before and after iron removal. On the other hand, apotransferrin is rapidly dissociated from its receptor at pH 7.0, so that when the apotransferrin-receptor complex is recycled to the cell surface, where it is now exposed to a physiological pH of 7.0-7.4, the apotransferrin molecule is released from the receptor and leaves the cell to be reused for iron binding/transport again. This cycle of iron transport in plasma and iron release in the cell is repeated with the net result that the transferrin molecule is conserved in the process. A schematic representation of the 'transferrin cycle' is depicted in Fig. 4.
Transferrin receptors Transferrin receptors have a relatively long half-life of approximately 60 hours. They have been found in a variety of growing cell types, including fibroblasts, lymphocytes, kidney tissue, tumour cells, but they are present most abundantly in tissues that are very active in iron uptake such as reticulocytes and placental cells. For example, it has been reported that there are about 300 000 transferrin receptors in each reticulocyte cell, so that if active reticulocytes can incorporate over 1 million iron
Figure 4
The transferrin cycle (From ref 39) * CURL = Compartment of uncoupling of receptor and ligand
BIOCHEMICAL EDUCATION 12(4) 1984
153 atoms per minute, the transferrin cycle must be completed within 15-30 seconds. 42 In addition, the isolated transferrin-receptor complex42 has been shown to possess a stability constant of near 107M -1, and that the controlling genes for the receptor in human have been mapped to chromosome number three. 43 Several groups of workers have reported the isolation of transferrin receptors from reticulocytes, placenta, and tumour cells. 43 There is general agreement that the receptor has an apparent molecular weight of 170 000-200 000, and a disulphide-linked subunit structure that consists of two identical 90 000-dalton subunits. 43 Each of these subunits binds one transferrin molecule so that there exists the possibility that transferrin and its receptor may form linear polymers. The transferrin receptor also appears to be a transmembrane glycoprotein. The major portion of each subunit, with Mr 70 000 is exposed to the extracellular environment while the cytoplasmic 'tail' has only Mr 5000 (see Fig. 5). The 70 000-dalton fragment has also been shown to retain the antigenic sites and the ability to bind iron after tryptic cleavage. 43
Functional Heterogeneity/Homogeneity
Although the two iron binding sites of transferrin have been shown recently to exhibit well-established physicochemical differences, the question as to whether they also function differently in vivo remains unclear. Fletcher and H e h n s 44 first proposed that each site has a specific function: one site (A-site) preferentially directs its iron to immature red cells and placenta, while the other site (B-site) deposits its iron in the liver and intestinal mucosal cells. Much of the evidence in favour of functional heterogeneity of the site has come from experiments in which human transferrin was used as the iron donor for rabbit or rat cells (ie, heterologous system), a However, in experiments using homologous systems of human protein and human cells, and rabbit reticulocytes and rabbit transferrin, no difference in iron uptake from the two sites is detected. ~ Based on these lines of evidence, it is more probable that there is no functional difference between the two sites of transferrin under physiological (ie, homologous) conditions. The functional differences that were reported using heterologous systems could be a result of the species variation in transferrin-receptor interaction. 45 It was reported recently, however, that there may exist, in vivo, a functional difference between the two iron binding sites of rabbit transferrin at the level of iron loading rather than iron release. 46 This conclusion is based on the observation that absorbed iron was found to bind preferentially to the acid-labile site in the N-terminal domain of rabbit transferrin.
Conclusions
This short review has highlighted the major advances that have been made in recent years in the elucidation of the structure and function of transferrins. For instance, the completion of the amino acid sequence of human serum transferrin and ovotransferrin is significant because it has enabled us to have a better understanding of the overall structure of the protein, especially with regard to the possible arrangement and assignment of the amino acid residues (ligands) at the active sites. However, to facilitate the unequivocal identification of amino acid residues and a complete description of the three-dimensional structure, we need to await the completion of the X-ray crystallographic structure of transferrin. From the functional point of view, there is now a general consensus among workers in the field that the most plausible mechanism of the transfer of iron from
..~--.,,.
..--~-,
I
70K
$--S
!90K
Key: ~ High mannose oligosaccharide chain 0 Complex type chain vw Covalently bound fatty acid
11 Figure 5
Schematic representation of the cell surface transferrin receptor (From ref 43)
BIOCHEMICAL EDUCATION 12(4) 1984
154 transferrin to cells involves the receptor-mediated endocytosis of transferrin. However, information on how the released iron is transported across the vesicular membrane into
the cytoplasm is, at present, still lacking. Acknowledgement
Part of this review was written while the author was a Research Fellow in the Department of Physical Biochemistry, John Curtin School of Medical Research. The Australian National University, Canberra, Australia.
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