Structure, Expression and Functions of Bovine Alpha ......Structure, Expression and Functions of Bovine Alpha RT-PCR and Northern blot analysis were performed using RNA ext cows to

_____________________________________________________________________________________________________ 1Department of Genetic Engineering, Faculty of Biology-Oriented Science and Technology, Kindai University, Wakayama

649-6493, Japan. *Corresponding author: E-mail: [email protected], [email protected];

Chapter 7 Print ISBN: 978-93-89246-84-1, eBook ISBN: 978-93-89246-85-8

Structure, Expression and Functions of Bovine Alpha-Tocopherol Transfer Protein (α-TTP) Yoshitomo Taguchi1* DOI:10.9734/bpi/rabr/v5

ABSTRACT

α-tocopherol transfer protein (α-TTP) of bovine consists of 282 amino acids with 98% and 83% identities to sheep and rat orthologs, respectively. Bovine α-TTP has an additional 5 amino acids (GEEVT) at the C terminus, which rat, human, mouse α-TTPs do not have. The Blast research suggested that the C-terminal sequences of α-TTP are specific for Cetartiodactyla animals. Bovine α-TTP mRNA and protein were expressed most strongly in liver, and also in lung, whereas expression of α-TTP mRNA and protein are reported to be very weak or absent in human and rodent lungs. In the lung, immunostaining suggested that α-TTP is expressed specifically in alveolar walls, which consists of alveolar cells, epithelial cells of small bronchi, and endothelial cells of pulmonary blood vessels. These results suggest that, in the lung, α-TTP is involved in supplying vitamin E to alveolar surfactant in order to protect the lung tissue from oxidative stress, and that this role may be more important in bovines than in other mammals. Keywords: Vitamin E; α-TTP; α-tocopherol; bovine; lung alveolar cells.

ABBREVIATIONS α-TTP: Alpha(α)-tocopherol transfer protein; AVED: Ataxia with vitamin E deficiency; RT-PCR: Reverse transcription polymerase chain reaction.

1. INTRODUCTION α-tocopherol transfer protein (α-TTP) is a cytosolic protein that specifically binds α-tocopherol, the most biologically active form of vitamin E. It has been shown that α-TTP is found mainly in the liver, where it regulates the amount of α-tocopherol secreted into the plasma [1-4]. By this activity of α-TTP in liver, plasma and tissues of animals are enriched in α-tocopherol, although foods of animals include many other forms of vitamin E, such as γ-tocopherol [5-11]. In human, α-TTP mRNA and protein are strongly expressed in liver [2]. In mouse α-TTP protein is strongly expressed in liver, and weakly expressed in brain and uterus [12,13]. In rat, α-TTP protein is strongly expressed in liver [1], while its mRNA is expressed strongly in liver, and very weakly in brain, spleen, lung and testis [14]. α-TTP is defective in patients with ataxia with vitamin E deficiency (AVED) [15,16]. α-TTP knockout mice have much lower plasma concentrations of α-tocopherol and are unable to maintain pregnancy because of impaired placentas [12]. They also show severe neuronal degeneration unless fed a high dose of vitamin E [13]. Studies of AVED patients and α-TTP knockout mice suggest that hepatic α-TTP governs the plasma α-tocopherol level, and that the protein also works in tissues such as brain and uterus in which α-TTP is expressed. Animals obtain vitamin E from plants, which can synthesize vitamin E on their own. Thus, plant-eating animals, such as bovines, might have quite different mechanisms for vitamin E circulation and

Structure, Expression and Functions of Bovine Alpha

accumulation than rodents and humans, and the roles of bovine of rat, mouse and human. To elucidate the role of bovine was isolated and sequenced, and the mRNA and protein expressions of bovine bovine tissues were investigated functions of bovine α-TTP are discussed.

2. STRUCTURE, EXPRESSION A full-length α-TTP cDNA was amplified by RTLC223005) [17]. The sequence of the bovine contains 846 bp encoding 282 amino acids with 88%,mouse and sheep orthologs, respectively. Tthought to form the α-TTP binding site sheep α-TTP. Bovine α-TTP has an additional 5 amino acids (GEEVT) at the C terminus, which rat, human, mouse α-TTPs do not have. Sheep (GEEVI) [19]. A protein BLAST search revealed bison, deer, dolphin, killer whale, terminal sequences, while other mammals (primates, rodents, Perissodactyla animals, such as donkeyresults suggest that the C-terminal sequences of

Fig. 1. Multiple alignment of the C

The C-terminal amino acid sequences of (sheep (Ovis aries), goat (Capra hircus(Tursiops truncatus), killer whale (Orcinus orca(Rattus norvegicus), elephant (Loxodonta africanacristata), donkey (Equus asinus), horse (were aligned. The C-terminal sequences which were suggested to be Cetartiodactyla animals are shaded.

Recent Advances in Biological Structure, Expression and Functions of Bovine Alpha-Tocopherol Transfer Protein (

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accumulation than rodents and humans, and the roles of bovine α-TTP might be different from those t, mouse and human. To elucidate the role of bovine α-TTP, a full-length cDNA of bovine

was isolated and sequenced, and the mRNA and protein expressions of bovine α-TTP in different bovine tissues were investigated [17]. In this article, the features of structure, expression and

TTP are discussed.

XPRESSION AND FUNCTIONS of BOVINE α-TTP

TTP cDNA was amplified by RT-PCR from tissues of bovine liver (GenBank accession: . The sequence of the bovine α-TTP cDNA indicated that the open reading frame

contains 846 bp encoding 282 amino acids with 88%, 83%, 83% and 98% identity with the human, rat, mouse and sheep orthologs, respectively. The domains containing the α-helix and β-sheet, which are

TTP binding site [18], are well conserved among bovine, rat, human, mouse and TTP has an additional 5 amino acids (GEEVT) at the C terminus, which rat,

TTPs do not have. Sheep α-TTP has similar 5 amino acid C-terminal sequence . A protein BLAST search revealed that many Cetartiodactyla animals, such as goat, dolphin, killer whale, water buffalo, antelope, yak, camel and whale, have similar C

terminal sequences, while other mammals (primates, rodents, elephant, bat, mole, leopard animals, such as donkey, horse and rhinoceros) do not have them (Fig.

terminal sequences of α-TTP are specific for Cetartiodactyla animals.

of the C-terminal amino acid sequences (7 to 12 amino acids) ofα-TTP orthologs

terminal amino acid sequences of α-TTP orthologs of bovine (Bos taurus), and other animals

Capra hircus), bison (Bison bison), deer (Odocoileus virginianusOrcinus orca), human (Homo sapiens), mouse (Mus musculus

Loxodonta africana), bat (Rhinolophus sinicus), mole (), horse (Equus caballus) and rhinoceros (Ceratotherium simum

terminal sequences which were suggested to be specific for orthologs of animals are shaded.

Recent Advances in Biological Research Vol. 5 Tocopherol Transfer Protein (α-TTP)

TTP might be different from those length cDNA of bovine α-TTP

TTP in different . In this article, the features of structure, expression and

PCR from tissues of bovine liver (GenBank accession: TTP cDNA indicated that the open reading frame

83% and 98% identity with the human, rat, sheet, which are

, are well conserved among bovine, rat, human, mouse and TTP has an additional 5 amino acids (GEEVT) at the C terminus, which rat,

terminal sequence animals, such as goat,

, have similar C-mole, leopard and

and rhinoceros) do not have them (Fig. 1). These animals.

terminal amino acid sequences (7 to 12 amino acids) of

), and other animals Odocoileus virginianus), dolphin

Mus musculus), rat ), mole (Condylura

Ceratotherium simum)) specific for orthologs of


RT-PCR and Northern blot analysis were performed using RNA extcows to examine α-TTP expressionsecond-strongest signal was observed in lung. Immunoblot analysis showed that the protein was also strongly expressed in liver and less strongly expressed in lung mRNA and protein is very weak or absent in human and rodent lungs TTP plays a more important role in bovine lung. bovine lung than in human and rodent lungs is unclear. Further studies of the expression of the lungs of other ruminants might shed some light on this issue.immunostaining with anti-α-TTP anticells, epithelial cells of small bronchi, and endothelial cells of pulmonary blood vessels (Fig. 2) The surfaces of lung alveoli are covered with a lipid monolayer called alveolar surfactant. The surfactant is produced in alveolar cells (type II), accumulated in the lamellar body in the secreted to the lung alveolar lumen. The main constituents of the surfactant are phospholipids and cholesterol, but α-tocopherol is also present as an antialveolar cells of α-TTP thus suggest that, alveolar surfactant to protect the lung tissue from oxidative stress (Fig.proteins which can transport lipids such as cholesterol and phospholipids, can also transport tocopherol and is involved in hepatic type II cells express ABCA3, an ABC protein that can transport lipids, and that is involved in supplying phospholipids to the surfactant [23raise the possibility that in the lung, αalveolar surfactant (Fig. 3).

Fig. 2. Immunostaining of lung tissue from the cow with anti Immunostaining was performed as described previously appear dark brown from oxidized diaminobenzidine, while nuclei appear blue from counterstaining with hematoxylin. Scale bars, 100 µm.


74

PCR and Northern blot analysis were performed using RNA extracted from various tissues of TTP expression [17]. α-TTP mRNA expression was strongest in liver, while the

strongest signal was observed in lung. Immunoblot analysis showed that the protein was also strongly expressed in liver and less strongly expressed in lung [17]. In contrast, expression of mRNA and protein is very weak or absent in human and rodent lungs [1,2,12-14], suggesting that TTP plays a more important role in bovine lung. The reason why α-TTP is expressed more strongly in bovine lung than in human and rodent lungs is unclear. Further studies of the expression of

other ruminants might shed some light on this issue. In lung tissue from the cow, TTP antibody was observed in alveolar walls, which consist of alveolar

cells, epithelial cells of small bronchi, and endothelial cells of pulmonary blood vessels (Fig. 2) The surfaces of lung alveoli are covered with a lipid monolayer called alveolar surfactant. The surfactant is produced in alveolar cells (type II), accumulated in the lamellar body in the secreted to the lung alveolar lumen. The main constituents of the surfactant are phospholipids and

tocopherol is also present as an anti-oxidant agent [20]. The expression in lung TTP thus suggest that, in the bovine lung, α-TTP serves to recruit α

alveolar surfactant to protect the lung tissue from oxidative stress (Fig. 3). ABCA1, one of the ABC proteins which can transport lipids such as cholesterol and phospholipids, can also transport tocopherol and is involved in hepatic α-TTP-mediated secretion [21,22]. On the other hand, alveolar type II cells express ABCA3, an ABC protein that can transport lipids, and that is involved in supplying

23,24]. The expression of bovine α-TTP in lung alveolar cells thus e the possibility that in the lung, α-TTP and ABCA3 function together to transport α

. Immunostaining of lung tissue from the cow with anti-bovine α-TTP polyclonal antibody

was performed as described previously [17]. Immunohistochemically stained regions appear dark brown from oxidized diaminobenzidine, while nuclei appear blue from counterstaining

Scale bars, 100 µm.


racted from various tissues of mRNA expression was strongest in liver, while the

strongest signal was observed in lung. Immunoblot analysis showed that the protein was also . In contrast, expression of α-TTP

, suggesting that α-pressed more strongly in

bovine lung than in human and rodent lungs is unclear. Further studies of the expression of α-TTP in In lung tissue from the cow,

body was observed in alveolar walls, which consist of alveolar cells, epithelial cells of small bronchi, and endothelial cells of pulmonary blood vessels (Fig. 2) [17]. The surfaces of lung alveoli are covered with a lipid monolayer called alveolar surfactant. The surfactant is produced in alveolar cells (type II), accumulated in the lamellar body in the cell, and secreted to the lung alveolar lumen. The main constituents of the surfactant are phospholipids and

. The expression in lung TTP serves to recruit α-tocopherol to

3). ABCA1, one of the ABC proteins which can transport lipids such as cholesterol and phospholipids, can also transport α-

. On the other hand, alveolar type II cells express ABCA3, an ABC protein that can transport lipids, and that is involved in supplying

TTP in lung alveolar cells thus TTP and ABCA3 function together to transport α-tocopherol to

TTP polyclonal antibody

stained regions appear dark brown from oxidized diaminobenzidine, while nuclei appear blue from counterstaining


Fig. 3. Hypothetical fu The surfaces of lung alveoli are covered with a lipid monolayer called alveolar surfactant. surfactant is produced in alveolar cells (type II), accumulated in the lamellar body in the cell, and secreted to the lung alveolar lumen.cholesterol, but α-tocopherol is also present as an antiexpressed in lung alveoar cells of bovine, suggesting that the tocopherol to alveolar surfactant. In alveolar type II cells, ABCA3, one of the ABC proteins which can transport lipids, is expressed, and involved in supplying phospholipids to the surfactant other hand, ABCA1, one of the ABC proteins which can transport lipids such as cholesterol and phospholipids, can also transport α[21,22]. The expression of bovine αTTP and ABCA3 could function together to transp

COMPETING INTERESTS Author has declared that no competing interests exist.

REFERENCES

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4. Traber MG, Arai H. Molecular mechanisms of vitamin E transport. Annu Rev Nutr. 1999;1934355.


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Hypothetical functions of α-TTP in bovine lung

The surfaces of lung alveoli are covered with a lipid monolayer called alveolar surfactant. surfactant is produced in alveolar cells (type II), accumulated in the lamellar body in the cell, and secreted to the lung alveolar lumen. The main constituents of the surfactant are phospholipids and

tocopherol is also present as an anti-oxidant agent [20]. It was shownof bovine, suggesting that the α-TTP in lung might serve to recruit

In alveolar type II cells, ABCA3, one of the ABC proteins which can transport lipids, is expressed, and involved in supplying phospholipids to the surfactant [

ABC proteins which can transport lipids such as cholesterol and phospholipids, can also transport α-tocopherol and is involved in hepatic α-TTP-mediated secretion

. The expression of bovine α-TTP in lung alveolar cells thus raise the possibility that in lung TTP and ABCA3 could function together to transport α-tocopherol to alveolar surfactant.

declared that no competing interests exist.

Sato Y, Arai H, Miyata A, Tokita S, Yamamoto K, Tanabe T, et al. Primary structure of alphatocopherol transfer protein from rat liver. Homology with cellular retinaldehyde-binding protein. J Biol Chem. 1993;268(24):17705-10.

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The surfaces of lung alveoli are covered with a lipid monolayer called alveolar surfactant. The surfactant is produced in alveolar cells (type II), accumulated in the lamellar body in the cell, and

of the surfactant are phospholipids and It was shown that α-TTP is

TTP in lung might serve to recruit α-In alveolar type II cells, ABCA3, one of the ABC proteins which can

[23,24]. On the ABC proteins which can transport lipids such as cholesterol and

mediated secretion TTP in lung alveolar cells thus raise the possibility that in lung α-

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Biography of author(s)

Dr. Yoshitomo Taguchi

Department of Genetic Engineering, Faculty of Biology649-6493, Japan.

He is presently working as an associate professor at Department of Genetic Engineering, Faculty of Biologyand Technology, Kindai University, Japan. He received his Ph. D in Agricultural ChemistrPh. D program, he analyzed the functions of antiof which cause multi-drug resistance of cancer cells by transporting many kinds of drugs out of thhe has taught a variety of courses in chemistry and biology field such as biochemistry, microbiology, and protein engineeringHe has been investigating the functions of ABC (specifically with α-tocopherol. He has also engaged in research on transgenic animals and embryology of animals.

_________________________________© Copyright 2019 The Author(s), Licensee terms of the Creative Commons Attribution License (use, distribution, and reproduction in any medium, provided the original work is properly cited. DISCLAIMER This chapter is an extended version of the article published by the same authorAnnual Research & Review in Biology, 12(5): 1-7, 2017 Reviewers’ Information (1) Zhong Zheng, School of Dentistry, University of California, Los Angeles, USA.(2) Anuj Kumar Gupta, Yashraj Biotechnology Ltd., India.(3) Alicia Kohli Bordino, Italian University Institute of Rosario, Argentina.


77

Department of Genetic Engineering, Faculty of Biology-Oriented Science and Technology, Kindai University, Wakayama

He is presently working as an associate professor at Department of Genetic Engineering, Faculty of Biologyand Technology, Kindai University, Japan. He received his Ph. D in Agricultural Chemistry at Kyoto University, Japan. During Ph. D program, he analyzed the functions of anti-cancer drug transporters, P-glycoprotein (ABCB1) and MRP1 (ABCC1), both

drug resistance of cancer cells by transporting many kinds of drugs out of the cells. At Kindai University, he has taught a variety of courses in chemistry and biology field such as biochemistry, microbiology, and protein engineeringHe has been investigating the functions of ABC (ATP Binding Cassette) transporters such as MRP1, and α

. He has also engaged in research on transgenic animals and embryology of animals.

________________________________________________________ Book Publisher International, This is an Open Access article distributed under the

terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

le published by the same author in the following journal with CC BY license. 7, 2017.

(1) Zhong Zheng, School of Dentistry, University of California, Los Angeles, USA. Biotechnology Ltd., India.

(3) Alicia Kohli Bordino, Italian University Institute of Rosario, Argentina.


University, Wakayama

He is presently working as an associate professor at Department of Genetic Engineering, Faculty of Biology-Oriented Science y at Kyoto University, Japan. During

glycoprotein (ABCB1) and MRP1 (ABCC1), both e cells. At Kindai University,

he has taught a variety of courses in chemistry and biology field such as biochemistry, microbiology, and protein engineering. α-TTP which binds

. He has also engaged in research on transgenic animals and embryology of animals.

________________________________________________ , This is an Open Access article distributed under the

), which permits unrestricted

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Structure, Expression and Functions of Bovine Alpha ......Structure, Expression and Functions of Bovine Alpha RT-PCR and Northern blot analysis were performed using RNA ext cows to