- Email: [email protected]
Molecular Mechanism of Cellular Uptake and Intracellular Translocation of α-Tocopherol: Role of Tocopherol-binding Proteins
Food and Chemical Toxicology 37 (1999) 967±971
www.elsevier.com/locate/foodchemtox
Molecular Mechanism of Cellular Uptake and Intracellular Translocation of a-Tocopherol: Role of Tocopherol-binding Proteins A. K. DUTTA-ROY Rowett Research Institute, Aberdeen, Scotland, UK SummaryÐVitamin E (a-tocopherol) is a lipid-soluble antioxidant which is present in cellular membranes where it plays an important role in the suppression of free radical-induced lipid peroxidation. There are eight naturally occurring homologues of vitamin E which dier in their structure and in biological activity in vivo and in vitro. Various studies have suggested that the tocopherol distribution system favours the accumulation of a-tocopherol both in the plasma and dierent tissues. Mechanisms involved in the preferential accumulation of a-tocopherol are not yet well established; however, recent data indicate that both intracellular and membrane a-tocopherol-binding proteins may be involved in these processes. A 30 kDa a-tocopherol-binding protein (TBP) in the liver cytoplasm is now known to regulate plasma vitamin E concentrations by preferentially incorporating a-tocopherol into nascent very low density (VLDL) whereas the 15 kDa TBP may be responsible for intracellular distribution of atocopherol. The 30 kDa TBP is unique to the hepatocyte whereas the 15 kDa TBP is present in all major tissues. The 15 kDa TBP speci®cally binds a-tocopherol in preference to the d- and g-tocopherol and may exclusively transport a-tocopherol to these intracellular sites. In addition, the presence of a membrane TBP (TBPpm) in tissues may regulate their a-tocopherol levels. Activity of erythrocyte TBPpm appears to be reduced in smokers, which may lead to reduced levels of a-tocopherol in these cells despite smokers have similar plasma levels of vitamin E as in non-smokers. The current status of the evidence for this directed ¯ow of a-tocopherol through interactions with these proteins (TBP and TBPpm) is discussed. # 1999 Elsevier Science Ltd. All rights reserved Keywords: a-tocopherol; vitamin E; tocopherol binding protein; tocopherol transport; rat liver; TBP; red cell membranes.
Introduction a-Tocopherol and g-tocopherol are the most common of the eight naturally occurring vitamin E homologues (a-, b-, g- and d-tocopherol and a-, b-, g- and d-tocotrienol) in the human diet. Although g-tocopherol is a more eective free radical scavenger than a-tocopherol in vitro, the reverse is true in vivo (Duthie et al., 1991). There is limited uptake of g-tocopherol into peripheral tissues and rapid clearance from plasma despite eating large amounts of g-tocopherol (Traber and Kayden, 1989). Normally, therefore, g-tocopherol, although eciently absorbed, only accounts for 10±15% of plasma tocopherol (Kayden and Traber, 1993; Traber, 1994; Traber and Kayden, 1989). This suggests that there is a selection process discriminating against the uptake or accumulation of g-tocopherol which
decreases its in vivo eectiveness. The most biologically active of the vitamin E homologues is a-tocopherol, which is present in the membranes of cells and cellular organelles, where it plays an important role in the suppression of lipid peroxidation. aTocopherol accumulates at those sites within the cell where oxygen radical production is greatest and thus where it is most required, for example, in the membranes of heavy mitochondria, light mitochondria, and endoplasmic reticulum. Therefore, protection against the peroxidation of membrane lipids by a-tocopherol is dependent on its incorporation into membranes and the extent of this protection is related to the quantity of a-tocopherol present in the membranes. However, very little is known about the transport mechanism that results in the accumulation of a-tocopherol both in the plasma as well as in cellular and intracellular membranes of tissues.
0278-6915/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00081-2
968
A. K. Dutta-Roy
As translocation of lipids through aqueous compartments is often association with speci®c proteins (Dutta-Roy et al., 1994) we have been studying whether such transport proteins for vitamin exist in tissues. These non-enzymatic, non-structural lipidbinding proteins are thought to be important both in extracellular and intracellular transport of lipids. This paper describes the role of newly identi®ed cytoplasmic and membrane tocopherol-binding proteins in the regulation of plasma level as well as of intracellular level of vitamin E. Transport of vitamin E Unlike other fat-soluble vitamins, vitamin E does not have a special carrier protein in plasma and is transported in plasma by lipoproteins (Kayden and Traber, 1993; Traber, 1994; Traber and Kayden, 1989). Dietary studies indicate that there is little or no discrimination between the vitamin E homologues in the intestine, since tocopherols are absorbed from the gut in micelles whose formation depends on bile salts and pancreatic lipase (Kayden and Traber, 1993). The vitamin E homologues are released from the enterocyte into lymph, within chylomicrons, which subsequently appear in the circulation where they are catabolized by lipoprotein lipase. Once transported into the liver there is a clear dierentiation between the vitamin E homologues with a-tocopherol being the exclusive isomer incorporated with nascent very low density lipoproteins (VLDL) for secretion into the plasma. The other isomers are mostly excreted through the biliary canaliculi (Kayden and Traber, 1993; Traber, 1994; Traber and Kayden, 1989). Mechanisms involved in the preferential accumulation of a-tocopherol are not yet fully understood; however, recent studies indicate that both intracellular- and membrane a-tocopherol-binding proteins may be involved in these processes (Dutta-Roy et al., 1994; Traber, 1994). Cytoplasmic a-tocopherol-binding proteins Studies on the absorption and transport of various forms of vitamin E indicate that plasma atocopherol concentration is regulated speci®cally by the preferential incorporation into nascent VLDL Traber, 1994; Traber and Kayden, 1989). The 30 kDa hepatic a-tocopherol-binding protein (TBP) is thought to be involved in this regulation and is found only in the liver cytosol in various species (Traber, 1994). A cDNA clone for TBP from rat liver has been isolated (Arita et al., 1995; Sato et al., 1993). Polyclonal antibodies against rat liver TBP were used for immunological screening of a lgt11 rat liver cDNA library. The amino acid sequence from the clone contains all sequences determined by direct analysis of the puri®ed protein. Comparison
of the primary structure of rat TBP with proteins revealed a notable homology with bovine and human cellular retinaldehyde-binding protein (Sato et al., 1993). There are two isoforms of rat liver TBP structurally related with respect to amino acid sequence, amino acid composition, molecular weight and substrate speci®city. It is suggested that both isoforms can be translated from a single mRNA followed by dierential post-translantional modi®cation in the liver cells. The isolated cDNA clone has a relatively large 3'-untranslated region which has a role in RNA translation, transport or stability. The region of the isolated cDNA has four AUUUA motifs which are generally involved in the selective degradation of the messenger causing marked reductions in mRNA stability (Arita et al., 1995). Like rat liver TBP, human liver TBP also shows remarkable similarity with cellular retinaldehyde binding protein, and also with yeast SEC14p (SEC14 gene product). SEC14 gene product is required for protein secretion through the Golgi complex in yeast (Bankitis et al., 1990). Human liver TBP gene is localized in chromosome 8. Fluorescence in situ hybridization also revealed a single TBP gene corresponding to the 8q13.1±13.3 region of chromosome 8 (Arita et al., 1995), which is identical to the locus of a clinical disorder, Friedreich's ataxia phenotype, with selective vitamin E de®ciency (Hamida et al., 1993a,b; Ouachi et al., 1995). Increasing evidence suggests that the hepatic 30 kDa TBP appears to be responsible for discrimination between the homologues of vitamin E by incorporating a-tocopherol from lysosomes to the endoplasmic reticulum for packaging in VLDL (Traber 1994). This could explain why the g-tocopherol, although eciently absorbed, only accounts for 10±15% of plasma tocopherol. How the hepatic TBP channels a-tocopherol into nascent VLDL awaits further study. a-Tocopherol is widely distributed in organelles of various tissues but the 30 kDa TBP resides only in hepatocytes, thus its role as a general intracellular carrier of a-tocopherol in dierent tissues seems unlikely (Dutta-Roy et al., 1994). However, translocation of lipophilic compounds through aqueous compartments are often mediated by a speci®c carrier protein (Dutta-Roy et al., 1994). The recently identi®ed low molecular weight (approx. 15 kDa) TBP in the cytosol of various tissues including liver, may be involved in the intracellular distribution and metabolism of a-tocopherol in tissues (DuttaRoy 1997; Dutta-Roy et al., 1993a,b, 1984; Gordon et al., 1995). Unlike the 30 kDa TBP, the 15 kDa TBP is present in all major tissues. The 15 kDa TBP speci®cally binds a-tocopherol in preference to the d- and g-tocopherol and may exclusively transport a-tocopherol to intracellular sites. Although the mechanism of intracellular transport by the 15 kDa protein is still unknown, the path of a small
Tocopherol-binding proteins
hydrophobic molecule through the aqueous cytoplasm is not linear. Partition may favour membrane binding by several orders of magnitude; thus signi®cant membrane association will markedly decrease the cytosolic transport rate. The presence of high concentrations of soluble binding protein for hydrophobic a-tocopherol would compete with membrane association and thereby increase transport rate. We have examined the mechanism of transfer of a-tocopherol from puri®ed 15 kDa TBP to mitochondria and phospholipids vesicles. Preliminary studies indicated that a-tocopherol is transferred from TBP by collisional interaction of the protein with an acceptor membrane. The rate of transfer increased markedly when membranes contain anionic phospholipids; this suggests that positively charged residues on the surface charges of the 15 kDa TBP may interact with the membranes. Neutralization of surface lysine residues of 15 kDa TBP decreased the atocopherol transfer rate; however, which domains of the 15 kDa TBP are critical for interaction with anionic acceptor membranes is not known. Thus, 15 kDa TBP may function in intracellular transport of a-tocopherol to decrease their membrane association, as well as to target a-tocopherol to speci®c subcellular sites of utilization.
Membrane a-tocopherol binding protein (TBPpm) Vitamin E is transported in plasma by lipoproteins (Kayden and Traber, 1993; Traber, 1994; Traber and Kayden, 1989). Therefore, LDL receptors may be involved but do not play an obligatory role in the uptake of a-tocopherol bound to LDL because LDL receptor de®ciency does not impair atocopherol uptake. In addition, it has been shown that deposition of a-tocopherol to tissues are dierent indicating that the existence of a tissue speci®c a-tocopherol uptake system (TBPpm) by cell membranes. For example, despite high plasma levels of vitamin E, red blood cell membranes contain relatively low levels of the vitamin compared with other peripheral tissues (Bellizzi et al., 1997b). This suggests the existence of a selective vitamin E uptake and/or regeneration system in human red blood cell membranes. Thus, a-tocopherol binding sites on human red blood cells are thought to be involved in the uptake of a-tocopherol from the plasma (Kitabchi and Wimalasena, 1982). We have recently characterized a plasma membrane a-tocopherol-binding protein (TBPpm) in human erythrocytes and liver and suggested that this may regulate a-tocopherol levels in these cells (Bellizzi et al., 1997a,b; Dutta-Roy et al., 1994). Scatchard analysis indicates two binding sites for a-tocopherol: one with a high anity and low capacity and the other with a low anity and high capacity. Two independent classes of a-tocopherol binding sites were evi-
969
dent on human erythrocytes with widely dierent dissociation constant values (Kd) and capacities (n) for the two classes of binding sites. The high-anity binding sites had a dissociation constant (Kd1) of 90 2 11 nM with a binding capacity (n1) of 9002 145 sites per cell. The low anity binding sites had a dissociation constant (Kd2) of 5.2 2 0.45 mM, with a binding capacity (n2) 105,400 2 12,400 sites per cell. The normal concentration of vitamin E in plasma is about 22 mM; it is likely that both of these sites in erythrocytes may have an important role in the eective uptake of plasma a-tocopherol. a-Tocopherol binding sites are also present in the membranes of dierent cell types such as cultured aortic endothelial cells (Kunisaki et al., 1980) and rat adrenal cells (Kitabchi et al., 1980). Competition for the binding of a-tocopherol to human erythrocytes was eective with other homologues of a-tocopherol (b-tocopherol, g-tocopherol and d-tocopherol) and their potency was almost equal to a-tocopherol itself (Bellizzi et al., 1997a). This is in contrast with the speci®city of intracellular a-tocopherol-binding proteins (both 30 kDa and15 kDa) which are highly speci®c to a-tocopherol (Dutta-Roy et al., 1993a, 1994; Gordon et al., 1995). The 30 kDa protein in liver is involved in regulating plasma levels of a-tocopherol through the preferential incorporation of a-tocopherol into nascent VLDL (Dutta-Roy et al., 1994; Traber, 1994), whereas the 15 kDa protein is responsible for intracellular transport and distribution of a-tocopherol in cells (Dutta-Roy et al., 1994). The rigorous speci®city of these intracellular proteins (both 30 kDa and 15 kDa proteins) for a-tocopherol is probably required to maintain the physiological levels of a-tocopherol (a-tocopherol, 90±95%, and other isomers mainly, g-tocopherol, 5±10%) in the plasma as well as in the cellular and intracellular membranes. In contrast, human erythrocytes have very little a-tocopherol, and therefore require a constant supply of a-tocopherol from the plasma lipoproteins to prevent membrane lipid peroxidation and thereby maintain membrane integrity. However, appropriate plasma levels of a-tocopherol may not always be maintained for several reasons, such as the dietary presence of various isomers of vitamin E, the pattern of distribution of tocopherols within the circulation following ingestion and, their residence time in each carrier species (lipoproteins and chylomicrons). Therefore, TBPpm, due to lack of stringent binding speci®city for a-tocopherol, may enable erythrocytes to use all these vitamin E homologues to prevent oxidative damage, since all these vitamin E isomers have antihaemolytic activity in human erythrocytes but to dierent degrees. It is also important to note that chylomicrons carry all the dietary tocopherols and may be used by circulating erythrocytes by virtue of having their non-speci®c tocopherol binding sites. The eects of glucose metabolism in erythrocytes seem
970
A. K. Dutta-Roy
to have no eect on the a-tocopherol binding sites in contrast to inhibition of ATPase activities of human erythrocytes (Bellizzi et al., 1997a), and atocopherol binding activity of cultured aortic endothelial cells (Kunisaki et al., 1993). To understand the role of the uptake system, we have compared the a-tocopherol content and binding activity of red blood cells from smokers and non-smokers. The speci®c binding of [3H]a-tocopherol to red blood cells from smokers was lower compared with that of non-smokers (30.6 2 3.2 fmol per 3 108 red blood cells vs. 41.7 2 3.7 fmol per 3 108 red blood cells, P = 0.05). Red blood cells from smokers contained less (1.82 0.4 mg/g Hb) a-tocopherol than non-smokers (2.8 2 0.3 mg/g Hb), (P < 0.05), despite plasma levels of a-tocopherol being similar: 12.9 2 0.8 mM in non-smokers vs. 12.7 2 0.5 mM in smokers. The reduced a-tocopherol levels in red blood cells from smokers may be due to impairment of a-tocopherol uptake activity. The reduced levels of a-tocopherol in smokers red blood cells was not associated with any changes in cell membrane ¯uidity. At present it is not known whether supplementation of smokers with vitamin E would normalise the a-tocopherol uptake activity of red blood cells. Conclusions It appears from various studies that a-tocopherol is the most biologically active among all the vitamin E isomers. This is now strongly supported by the discovery of various tocopherol-binding proteins in tissues and their binding preference for a-tocopherol. It appears also that for eective in vivo function of vitamin E the participation of these proteins at dierent levels are required. The activity of both hepatic 30 kDa TBP and ubiquitous 15 kDa TBP may be critical for the regulation of a-tocopherol levels in plasma, and cell membranes respectively, whereas TBPpm may be involved in sequestration of tocopherols from the plasma by the peripheral tissues. However further studies are required to understand the directed ¯ow of dietary a-tocopherol to tissues through interactions with membrane and cytoplasmic proteins (TBPpm and TBPs).
AcknowledgementsÐThis work was supported by the Scottish Oce Agriculture, Environment and Fisheries Department and Henkel Corporation, USA.
REFERENCES
Arita M., Sato Y., Miyata A., Tanabe T., Takjahashi E., Kayden H. J., Arai H. and Inoue K. (1995) Human alpha tocopherol transfer protein cDNA cloning, expression and chromosomal localization. Biochemical Journal 306, 437±443.
Bankaitis V. A., Aitken J. R., Cleves A. E. and Dowhan W. (1990) An essential role for a phospholipid transfer protein in yeats Golgi function. Nature 347, 561±562. Bellizzi M., Dutta-Roy A. K., Duthie G. G. and James W. P. T. (1997a) Vitamin E binding activity of red blood cells in smokers. Free Radical Research 27, 105± 112. Bellizzi M. C., Dutta-Roy A. K. and James W. P. T. (1997b) High D-glucose does not aect binding of a-tocopherol to human erythrocytes. Molecular and Cellular Biochemistry 170, 187±193. Duthie G. G., Gonzalez B. M., Morrice P. C. and Arthur J. R. (1991) Inhibitory eects of isomers of tocopherol on lipid peroxidation of microsomes from vitamin E de®cient rats. Free Radical Research Communications 15, 35±40. Dutta-Roy A. K. (1997) a-Tocopherol-binding proteins: Puri®cation and characterization. Methods in Enzymology 282, 278±297. Dutta-Roy A. K., Gordon M. J., Campbell F. M., Duthie G. G. and James W. P. T. (1994) Vitamin E requirements, transport and metabolism: Role of a-tocopherolbinding proteins. Journal of Nutritional Biochemistry 5, 562±570. Dutta-Roy A. K., Gordon M. J., Leishman D. J., Paterson B. J., Duthie G. G. and James W. P. T. (1993b) Puri®cation and partial characterization of an a-tocopherol-binding protein from rabbit heart cytosol. Molecular and Cellular Biochemistry 123, 139±144. Dutta-Roy A. K., Leishman D. J., Gordon M. J., Campbell F. M. and Duthie G. G. (1993a) Identi®cation of a low molecular mass (14.2 kDa) atocopherol-binding protein in the cytosol of rat liver and heart. Biochemical and Biophysical Research Communications 196, 1108±1112. Gordon M. J., Campbell F. M., Duthie G. G. and DuttaRoy A. K. (1995) Characterization of a novel a-tocopherol-binding protein from bovine heart cytosol. Archives of Biochemistry and Biophysics 318, 140±?. Hamida B. M., Belal S., Sirugo G., Hamida C. B., Panayides K., Ionannou P., Beckmann J., Mandel J. L., Hentati F., Koenig M. and Middleton L. (1993a) Friedreich's ataxia phenotype not linked to chromosome 9 and associated with selective autosomal vitamin E de®ciency in two inbred Tunisian families. Neurology 43, 2179±2183. Hamida C. B., Doer¯inger N., Belal S., Linder C., Reutenauer I., Dib C., Gyapay G., Vignal A., Lepaslier D., Cohen D., Pandolfo M., Mokini V., Novelli G., Hentati F., Hamida B. M., Mendel J. L. and Koenig M. (1993b) Localization of Friedreich's ataxia phenotype with selective vitamin E de®ciency to chromosome 8 by homozygosity mapping. Nature Genetics 5, 195± 200. Kayden H. J. and Traber M. G. (1993) Absorption, lipoprotein transport, and regulation of plasma concentrations of vitamin E in humans. Journal of Lipid Research 34, 343±358. Kitabchi A. E. and Wimalasena J. (1982) Speci®c binding sites for d-a tocopherol on human erythrocytes. Biochimica et Biophysica Acta 684, 200±206. Kitabchi A. E., Wimalasena J. and Berker J. (1980) Speci®c receptor sites for a-tocopherol in puri®ed isolated adrenocortical cell membrane. Biochemical and Biophysical Research Communications 96, 1739±1746. Kunisaki M., Umeda F., Yamauchi T., Masakado M. and Nawata H. (1993) High glucose reduces speci®c binding for d-alpha tocopherol in cultured aortic endothelial cells. Diabetes 42, 1138±1146. Ouachi K., Arita M., Kayden H. J., Hentati F., Benhamida M. M., Sokol R., Arai H., Inoue K., Mandel J. L. and Koening M. (1995) Ataxia with isolated vitamin E de®eciency is caused by mutation in the
Tocopherol-binding proteins a-tocopherol transfer protein. Nature Genetics 9, 141± 145. Sato Y., Arai A., Miyata S., Tokita K., Yamamoto K., Tanabe T. and Inoue K. (1993) Primary structure of atocopherol transfer protein from rat liver. Homology with cellular retinaldehyde-binding protein. Journal of Biological Chemistry 268, 17705±17710.
971
Traber M. G. (1994) Determinants of plasma vitamin E concentrations. Free Radical Biology and Medicine 16, 229±239. Traber M. G. and Kayden H. G. (1989) a-tocopherol as compared g-tocopherol is preferentially secreted in human lipoproteins. Annals of New York Academy of Sciences 570, 95±108.
www.elsevier.com/locate/foodchemtox
Molecular Mechanism of Cellular Uptake and Intracellular Translocation of a-Tocopherol: Role of Tocopherol-binding Proteins A. K. DUTTA-ROY Rowett Research Institute, Aberdeen, Scotland, UK SummaryÐVitamin E (a-tocopherol) is a lipid-soluble antioxidant which is present in cellular membranes where it plays an important role in the suppression of free radical-induced lipid peroxidation. There are eight naturally occurring homologues of vitamin E which dier in their structure and in biological activity in vivo and in vitro. Various studies have suggested that the tocopherol distribution system favours the accumulation of a-tocopherol both in the plasma and dierent tissues. Mechanisms involved in the preferential accumulation of a-tocopherol are not yet well established; however, recent data indicate that both intracellular and membrane a-tocopherol-binding proteins may be involved in these processes. A 30 kDa a-tocopherol-binding protein (TBP) in the liver cytoplasm is now known to regulate plasma vitamin E concentrations by preferentially incorporating a-tocopherol into nascent very low density (VLDL) whereas the 15 kDa TBP may be responsible for intracellular distribution of atocopherol. The 30 kDa TBP is unique to the hepatocyte whereas the 15 kDa TBP is present in all major tissues. The 15 kDa TBP speci®cally binds a-tocopherol in preference to the d- and g-tocopherol and may exclusively transport a-tocopherol to these intracellular sites. In addition, the presence of a membrane TBP (TBPpm) in tissues may regulate their a-tocopherol levels. Activity of erythrocyte TBPpm appears to be reduced in smokers, which may lead to reduced levels of a-tocopherol in these cells despite smokers have similar plasma levels of vitamin E as in non-smokers. The current status of the evidence for this directed ¯ow of a-tocopherol through interactions with these proteins (TBP and TBPpm) is discussed. # 1999 Elsevier Science Ltd. All rights reserved Keywords: a-tocopherol; vitamin E; tocopherol binding protein; tocopherol transport; rat liver; TBP; red cell membranes.
Introduction a-Tocopherol and g-tocopherol are the most common of the eight naturally occurring vitamin E homologues (a-, b-, g- and d-tocopherol and a-, b-, g- and d-tocotrienol) in the human diet. Although g-tocopherol is a more eective free radical scavenger than a-tocopherol in vitro, the reverse is true in vivo (Duthie et al., 1991). There is limited uptake of g-tocopherol into peripheral tissues and rapid clearance from plasma despite eating large amounts of g-tocopherol (Traber and Kayden, 1989). Normally, therefore, g-tocopherol, although eciently absorbed, only accounts for 10±15% of plasma tocopherol (Kayden and Traber, 1993; Traber, 1994; Traber and Kayden, 1989). This suggests that there is a selection process discriminating against the uptake or accumulation of g-tocopherol which
decreases its in vivo eectiveness. The most biologically active of the vitamin E homologues is a-tocopherol, which is present in the membranes of cells and cellular organelles, where it plays an important role in the suppression of lipid peroxidation. aTocopherol accumulates at those sites within the cell where oxygen radical production is greatest and thus where it is most required, for example, in the membranes of heavy mitochondria, light mitochondria, and endoplasmic reticulum. Therefore, protection against the peroxidation of membrane lipids by a-tocopherol is dependent on its incorporation into membranes and the extent of this protection is related to the quantity of a-tocopherol present in the membranes. However, very little is known about the transport mechanism that results in the accumulation of a-tocopherol both in the plasma as well as in cellular and intracellular membranes of tissues.
0278-6915/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00081-2
968
A. K. Dutta-Roy
As translocation of lipids through aqueous compartments is often association with speci®c proteins (Dutta-Roy et al., 1994) we have been studying whether such transport proteins for vitamin exist in tissues. These non-enzymatic, non-structural lipidbinding proteins are thought to be important both in extracellular and intracellular transport of lipids. This paper describes the role of newly identi®ed cytoplasmic and membrane tocopherol-binding proteins in the regulation of plasma level as well as of intracellular level of vitamin E. Transport of vitamin E Unlike other fat-soluble vitamins, vitamin E does not have a special carrier protein in plasma and is transported in plasma by lipoproteins (Kayden and Traber, 1993; Traber, 1994; Traber and Kayden, 1989). Dietary studies indicate that there is little or no discrimination between the vitamin E homologues in the intestine, since tocopherols are absorbed from the gut in micelles whose formation depends on bile salts and pancreatic lipase (Kayden and Traber, 1993). The vitamin E homologues are released from the enterocyte into lymph, within chylomicrons, which subsequently appear in the circulation where they are catabolized by lipoprotein lipase. Once transported into the liver there is a clear dierentiation between the vitamin E homologues with a-tocopherol being the exclusive isomer incorporated with nascent very low density lipoproteins (VLDL) for secretion into the plasma. The other isomers are mostly excreted through the biliary canaliculi (Kayden and Traber, 1993; Traber, 1994; Traber and Kayden, 1989). Mechanisms involved in the preferential accumulation of a-tocopherol are not yet fully understood; however, recent studies indicate that both intracellular- and membrane a-tocopherol-binding proteins may be involved in these processes (Dutta-Roy et al., 1994; Traber, 1994). Cytoplasmic a-tocopherol-binding proteins Studies on the absorption and transport of various forms of vitamin E indicate that plasma atocopherol concentration is regulated speci®cally by the preferential incorporation into nascent VLDL Traber, 1994; Traber and Kayden, 1989). The 30 kDa hepatic a-tocopherol-binding protein (TBP) is thought to be involved in this regulation and is found only in the liver cytosol in various species (Traber, 1994). A cDNA clone for TBP from rat liver has been isolated (Arita et al., 1995; Sato et al., 1993). Polyclonal antibodies against rat liver TBP were used for immunological screening of a lgt11 rat liver cDNA library. The amino acid sequence from the clone contains all sequences determined by direct analysis of the puri®ed protein. Comparison
of the primary structure of rat TBP with proteins revealed a notable homology with bovine and human cellular retinaldehyde-binding protein (Sato et al., 1993). There are two isoforms of rat liver TBP structurally related with respect to amino acid sequence, amino acid composition, molecular weight and substrate speci®city. It is suggested that both isoforms can be translated from a single mRNA followed by dierential post-translantional modi®cation in the liver cells. The isolated cDNA clone has a relatively large 3'-untranslated region which has a role in RNA translation, transport or stability. The region of the isolated cDNA has four AUUUA motifs which are generally involved in the selective degradation of the messenger causing marked reductions in mRNA stability (Arita et al., 1995). Like rat liver TBP, human liver TBP also shows remarkable similarity with cellular retinaldehyde binding protein, and also with yeast SEC14p (SEC14 gene product). SEC14 gene product is required for protein secretion through the Golgi complex in yeast (Bankitis et al., 1990). Human liver TBP gene is localized in chromosome 8. Fluorescence in situ hybridization also revealed a single TBP gene corresponding to the 8q13.1±13.3 region of chromosome 8 (Arita et al., 1995), which is identical to the locus of a clinical disorder, Friedreich's ataxia phenotype, with selective vitamin E de®ciency (Hamida et al., 1993a,b; Ouachi et al., 1995). Increasing evidence suggests that the hepatic 30 kDa TBP appears to be responsible for discrimination between the homologues of vitamin E by incorporating a-tocopherol from lysosomes to the endoplasmic reticulum for packaging in VLDL (Traber 1994). This could explain why the g-tocopherol, although eciently absorbed, only accounts for 10±15% of plasma tocopherol. How the hepatic TBP channels a-tocopherol into nascent VLDL awaits further study. a-Tocopherol is widely distributed in organelles of various tissues but the 30 kDa TBP resides only in hepatocytes, thus its role as a general intracellular carrier of a-tocopherol in dierent tissues seems unlikely (Dutta-Roy et al., 1994). However, translocation of lipophilic compounds through aqueous compartments are often mediated by a speci®c carrier protein (Dutta-Roy et al., 1994). The recently identi®ed low molecular weight (approx. 15 kDa) TBP in the cytosol of various tissues including liver, may be involved in the intracellular distribution and metabolism of a-tocopherol in tissues (DuttaRoy 1997; Dutta-Roy et al., 1993a,b, 1984; Gordon et al., 1995). Unlike the 30 kDa TBP, the 15 kDa TBP is present in all major tissues. The 15 kDa TBP speci®cally binds a-tocopherol in preference to the d- and g-tocopherol and may exclusively transport a-tocopherol to intracellular sites. Although the mechanism of intracellular transport by the 15 kDa protein is still unknown, the path of a small
Tocopherol-binding proteins
hydrophobic molecule through the aqueous cytoplasm is not linear. Partition may favour membrane binding by several orders of magnitude; thus signi®cant membrane association will markedly decrease the cytosolic transport rate. The presence of high concentrations of soluble binding protein for hydrophobic a-tocopherol would compete with membrane association and thereby increase transport rate. We have examined the mechanism of transfer of a-tocopherol from puri®ed 15 kDa TBP to mitochondria and phospholipids vesicles. Preliminary studies indicated that a-tocopherol is transferred from TBP by collisional interaction of the protein with an acceptor membrane. The rate of transfer increased markedly when membranes contain anionic phospholipids; this suggests that positively charged residues on the surface charges of the 15 kDa TBP may interact with the membranes. Neutralization of surface lysine residues of 15 kDa TBP decreased the atocopherol transfer rate; however, which domains of the 15 kDa TBP are critical for interaction with anionic acceptor membranes is not known. Thus, 15 kDa TBP may function in intracellular transport of a-tocopherol to decrease their membrane association, as well as to target a-tocopherol to speci®c subcellular sites of utilization.
Membrane a-tocopherol binding protein (TBPpm) Vitamin E is transported in plasma by lipoproteins (Kayden and Traber, 1993; Traber, 1994; Traber and Kayden, 1989). Therefore, LDL receptors may be involved but do not play an obligatory role in the uptake of a-tocopherol bound to LDL because LDL receptor de®ciency does not impair atocopherol uptake. In addition, it has been shown that deposition of a-tocopherol to tissues are dierent indicating that the existence of a tissue speci®c a-tocopherol uptake system (TBPpm) by cell membranes. For example, despite high plasma levels of vitamin E, red blood cell membranes contain relatively low levels of the vitamin compared with other peripheral tissues (Bellizzi et al., 1997b). This suggests the existence of a selective vitamin E uptake and/or regeneration system in human red blood cell membranes. Thus, a-tocopherol binding sites on human red blood cells are thought to be involved in the uptake of a-tocopherol from the plasma (Kitabchi and Wimalasena, 1982). We have recently characterized a plasma membrane a-tocopherol-binding protein (TBPpm) in human erythrocytes and liver and suggested that this may regulate a-tocopherol levels in these cells (Bellizzi et al., 1997a,b; Dutta-Roy et al., 1994). Scatchard analysis indicates two binding sites for a-tocopherol: one with a high anity and low capacity and the other with a low anity and high capacity. Two independent classes of a-tocopherol binding sites were evi-
969
dent on human erythrocytes with widely dierent dissociation constant values (Kd) and capacities (n) for the two classes of binding sites. The high-anity binding sites had a dissociation constant (Kd1) of 90 2 11 nM with a binding capacity (n1) of 9002 145 sites per cell. The low anity binding sites had a dissociation constant (Kd2) of 5.2 2 0.45 mM, with a binding capacity (n2) 105,400 2 12,400 sites per cell. The normal concentration of vitamin E in plasma is about 22 mM; it is likely that both of these sites in erythrocytes may have an important role in the eective uptake of plasma a-tocopherol. a-Tocopherol binding sites are also present in the membranes of dierent cell types such as cultured aortic endothelial cells (Kunisaki et al., 1980) and rat adrenal cells (Kitabchi et al., 1980). Competition for the binding of a-tocopherol to human erythrocytes was eective with other homologues of a-tocopherol (b-tocopherol, g-tocopherol and d-tocopherol) and their potency was almost equal to a-tocopherol itself (Bellizzi et al., 1997a). This is in contrast with the speci®city of intracellular a-tocopherol-binding proteins (both 30 kDa and15 kDa) which are highly speci®c to a-tocopherol (Dutta-Roy et al., 1993a, 1994; Gordon et al., 1995). The 30 kDa protein in liver is involved in regulating plasma levels of a-tocopherol through the preferential incorporation of a-tocopherol into nascent VLDL (Dutta-Roy et al., 1994; Traber, 1994), whereas the 15 kDa protein is responsible for intracellular transport and distribution of a-tocopherol in cells (Dutta-Roy et al., 1994). The rigorous speci®city of these intracellular proteins (both 30 kDa and 15 kDa proteins) for a-tocopherol is probably required to maintain the physiological levels of a-tocopherol (a-tocopherol, 90±95%, and other isomers mainly, g-tocopherol, 5±10%) in the plasma as well as in the cellular and intracellular membranes. In contrast, human erythrocytes have very little a-tocopherol, and therefore require a constant supply of a-tocopherol from the plasma lipoproteins to prevent membrane lipid peroxidation and thereby maintain membrane integrity. However, appropriate plasma levels of a-tocopherol may not always be maintained for several reasons, such as the dietary presence of various isomers of vitamin E, the pattern of distribution of tocopherols within the circulation following ingestion and, their residence time in each carrier species (lipoproteins and chylomicrons). Therefore, TBPpm, due to lack of stringent binding speci®city for a-tocopherol, may enable erythrocytes to use all these vitamin E homologues to prevent oxidative damage, since all these vitamin E isomers have antihaemolytic activity in human erythrocytes but to dierent degrees. It is also important to note that chylomicrons carry all the dietary tocopherols and may be used by circulating erythrocytes by virtue of having their non-speci®c tocopherol binding sites. The eects of glucose metabolism in erythrocytes seem
970
A. K. Dutta-Roy
to have no eect on the a-tocopherol binding sites in contrast to inhibition of ATPase activities of human erythrocytes (Bellizzi et al., 1997a), and atocopherol binding activity of cultured aortic endothelial cells (Kunisaki et al., 1993). To understand the role of the uptake system, we have compared the a-tocopherol content and binding activity of red blood cells from smokers and non-smokers. The speci®c binding of [3H]a-tocopherol to red blood cells from smokers was lower compared with that of non-smokers (30.6 2 3.2 fmol per 3 108 red blood cells vs. 41.7 2 3.7 fmol per 3 108 red blood cells, P = 0.05). Red blood cells from smokers contained less (1.82 0.4 mg/g Hb) a-tocopherol than non-smokers (2.8 2 0.3 mg/g Hb), (P < 0.05), despite plasma levels of a-tocopherol being similar: 12.9 2 0.8 mM in non-smokers vs. 12.7 2 0.5 mM in smokers. The reduced a-tocopherol levels in red blood cells from smokers may be due to impairment of a-tocopherol uptake activity. The reduced levels of a-tocopherol in smokers red blood cells was not associated with any changes in cell membrane ¯uidity. At present it is not known whether supplementation of smokers with vitamin E would normalise the a-tocopherol uptake activity of red blood cells. Conclusions It appears from various studies that a-tocopherol is the most biologically active among all the vitamin E isomers. This is now strongly supported by the discovery of various tocopherol-binding proteins in tissues and their binding preference for a-tocopherol. It appears also that for eective in vivo function of vitamin E the participation of these proteins at dierent levels are required. The activity of both hepatic 30 kDa TBP and ubiquitous 15 kDa TBP may be critical for the regulation of a-tocopherol levels in plasma, and cell membranes respectively, whereas TBPpm may be involved in sequestration of tocopherols from the plasma by the peripheral tissues. However further studies are required to understand the directed ¯ow of dietary a-tocopherol to tissues through interactions with membrane and cytoplasmic proteins (TBPpm and TBPs).
AcknowledgementsÐThis work was supported by the Scottish Oce Agriculture, Environment and Fisheries Department and Henkel Corporation, USA.
REFERENCES
Arita M., Sato Y., Miyata A., Tanabe T., Takjahashi E., Kayden H. J., Arai H. and Inoue K. (1995) Human alpha tocopherol transfer protein cDNA cloning, expression and chromosomal localization. Biochemical Journal 306, 437±443.
Bankaitis V. A., Aitken J. R., Cleves A. E. and Dowhan W. (1990) An essential role for a phospholipid transfer protein in yeats Golgi function. Nature 347, 561±562. Bellizzi M., Dutta-Roy A. K., Duthie G. G. and James W. P. T. (1997a) Vitamin E binding activity of red blood cells in smokers. Free Radical Research 27, 105± 112. Bellizzi M. C., Dutta-Roy A. K. and James W. P. T. (1997b) High D-glucose does not aect binding of a-tocopherol to human erythrocytes. Molecular and Cellular Biochemistry 170, 187±193. Duthie G. G., Gonzalez B. M., Morrice P. C. and Arthur J. R. (1991) Inhibitory eects of isomers of tocopherol on lipid peroxidation of microsomes from vitamin E de®cient rats. Free Radical Research Communications 15, 35±40. Dutta-Roy A. K. (1997) a-Tocopherol-binding proteins: Puri®cation and characterization. Methods in Enzymology 282, 278±297. Dutta-Roy A. K., Gordon M. J., Campbell F. M., Duthie G. G. and James W. P. T. (1994) Vitamin E requirements, transport and metabolism: Role of a-tocopherolbinding proteins. Journal of Nutritional Biochemistry 5, 562±570. Dutta-Roy A. K., Gordon M. J., Leishman D. J., Paterson B. J., Duthie G. G. and James W. P. T. (1993b) Puri®cation and partial characterization of an a-tocopherol-binding protein from rabbit heart cytosol. Molecular and Cellular Biochemistry 123, 139±144. Dutta-Roy A. K., Leishman D. J., Gordon M. J., Campbell F. M. and Duthie G. G. (1993a) Identi®cation of a low molecular mass (14.2 kDa) atocopherol-binding protein in the cytosol of rat liver and heart. Biochemical and Biophysical Research Communications 196, 1108±1112. Gordon M. J., Campbell F. M., Duthie G. G. and DuttaRoy A. K. (1995) Characterization of a novel a-tocopherol-binding protein from bovine heart cytosol. Archives of Biochemistry and Biophysics 318, 140±?. Hamida B. M., Belal S., Sirugo G., Hamida C. B., Panayides K., Ionannou P., Beckmann J., Mandel J. L., Hentati F., Koenig M. and Middleton L. (1993a) Friedreich's ataxia phenotype not linked to chromosome 9 and associated with selective autosomal vitamin E de®ciency in two inbred Tunisian families. Neurology 43, 2179±2183. Hamida C. B., Doer¯inger N., Belal S., Linder C., Reutenauer I., Dib C., Gyapay G., Vignal A., Lepaslier D., Cohen D., Pandolfo M., Mokini V., Novelli G., Hentati F., Hamida B. M., Mendel J. L. and Koenig M. (1993b) Localization of Friedreich's ataxia phenotype with selective vitamin E de®ciency to chromosome 8 by homozygosity mapping. Nature Genetics 5, 195± 200. Kayden H. J. and Traber M. G. (1993) Absorption, lipoprotein transport, and regulation of plasma concentrations of vitamin E in humans. Journal of Lipid Research 34, 343±358. Kitabchi A. E. and Wimalasena J. (1982) Speci®c binding sites for d-a tocopherol on human erythrocytes. Biochimica et Biophysica Acta 684, 200±206. Kitabchi A. E., Wimalasena J. and Berker J. (1980) Speci®c receptor sites for a-tocopherol in puri®ed isolated adrenocortical cell membrane. Biochemical and Biophysical Research Communications 96, 1739±1746. Kunisaki M., Umeda F., Yamauchi T., Masakado M. and Nawata H. (1993) High glucose reduces speci®c binding for d-alpha tocopherol in cultured aortic endothelial cells. Diabetes 42, 1138±1146. Ouachi K., Arita M., Kayden H. J., Hentati F., Benhamida M. M., Sokol R., Arai H., Inoue K., Mandel J. L. and Koening M. (1995) Ataxia with isolated vitamin E de®eciency is caused by mutation in the
Tocopherol-binding proteins a-tocopherol transfer protein. Nature Genetics 9, 141± 145. Sato Y., Arai A., Miyata S., Tokita K., Yamamoto K., Tanabe T. and Inoue K. (1993) Primary structure of atocopherol transfer protein from rat liver. Homology with cellular retinaldehyde-binding protein. Journal of Biological Chemistry 268, 17705±17710.
971
Traber M. G. (1994) Determinants of plasma vitamin E concentrations. Free Radical Biology and Medicine 16, 229±239. Traber M. G. and Kayden H. G. (1989) a-tocopherol as compared g-tocopherol is preferentially secreted in human lipoproteins. Annals of New York Academy of Sciences 570, 95±108.