Cloning, characterisation and expression of the apolipoprotein A-I gene1 in the sea bream (Sparus aurata)

Biochimica et Biophysica Acta 1442 (1998) 399^404

Short sequence-paper

Cloning, characterisation and expression of the apolipoprotein A-I gene1 in the sea bream (Sparus aurata) Lynda Llewellyn

a; b

, Vimi P. Ramsurn a; b , Trevor Wigham b , Glen E. Sweeney Deborah M. Power c

a;

*,

a

c

School of Molecular and Medical Biosciences, University of Wales, P.O. Box 911, Cardi¡ CF1 3US, UK b School of Pure and Applied Biology, University of Wales, P.O. Box 915, Cardi¡ CF1 3TL, UK Unidade de Ciencias e Tecnologias Agrarias, Universidade do Algarve, Campus de Gambelas, Faro 8000, Portugal Received 31 July 1998; accepted 12 August 1998

Abstract A full length cDNA clone representing apolipoprotein A-I was isolated from a sea bream (Sparus aurata) liver library. The clone encodes a 261 amino acid protein which shows highest amino acid identity (38%) with salmon apolipoprotein A-I. Northern blot analysis showed strong expression of a 1.4 kb transcript in liver with lower expression in intestine. Expression of apolipoprotein A-I in intestine was markedly reduced by treatment with triiodothyronine (T3 ). ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Apolipoprotein A-I; Teleost ¢sh; Triiodothyronine (T3 ); (Sea bream); (Sparus aurata)

Apolipoprotein A-I (apoA-I) is the major protein in plasma high density lipoprotein (HDL) and is a potent activator of the enzyme lecithin:cholesterol acyl transferase (LCAT), which facilitates the removal of free cholesterol to the liver for excretion [1]. Reduced levels of HDL and apoA-I are inversely related to a risk of coronary heart disease [2]. Apolipoprotein A-I is a member of a family which also includes apolipoproteins A-II, A-IV, B, C-I, C-II, CIII and E [3,4]. ApoA-I genes have been isolated from a variety of mammalian species including human [5], rat [6] and rabbit [7] and from non-mamma* Corresponding author. Fax: +44 (1222) 874116; E-mail: sweeneyge@cardi¡.ac.uk 1 The sequence data reported in this paper have been submitted to EMBL/GenBank data libraries under accession No. AF013120.

lian species, including chicken [8], salmon [9] and trout [10]. Phylogenetic analysis of salmon apoA-I with other apolipoproteins suggests that teleosts diverged from tetrapods prior to the emergence of the longer apolipoproteins apoA-IV and apoE, which contain additional internal repeats, and therefore it may be that teleosts do not possess apoA-IV and apoE [9], although two apolipoprotein genes have been cloned from lamprey, which show highest similarity with rat apoA-IV [11]. It has been suggested that the apolipoproteins have evolved from a common ancestral gene which is very similar to apoC-I [3]. In mammals, apoA-I is synthesised predominantly in the liver and intestine, although it is also found in the muscle and brain of certain ¢sh and birds. It is synthesised as a preprotein, proapoA-I, and cleaved in the plasma to form the mature protein [12]. Apolipoproteins share common structural fea-

0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 1 7 1 - 7

BBAEXP 91181 20-10-98

400

L. Llewellyn et al. / Biochimica et Biophysica Acta 1442 (1998) 399^404

Fig. 1. A: Base sequence of the sea bream apolipoprotein A-I cDNA clone and the putative amino acid sequence of the encoded protein. B: Translation in vitro of the sea bream apolipoprotein A-I gene. Rabbit reticulocyte lysate, containing added 35 S-methionine, was incubated in the absence (lane 1) or presence (lane 2) of sea bream apolipoprotein A-I mRNA made by transcription of the cloned gene in vitro with T3 RNA polymerase. Labelled proteins were fractionated on an SDS-PAGE gel and visualised by autoradiography. Translated apolipoprotein A-I is indicated with an arrow, the sizes of molecular weight markers are shown in kDa.

BBAEXP 91181 20-10-98

L. Llewellyn et al. / Biochimica et Biophysica Acta 1442 (1998) 399^404

401

Fig. 1. (continued)

tures, multiple internal repeats of 22 amino acids (22mers) which are each a tandem array of two 11-mers [13]. The level of apoA-I mRNA is controlled by hormonal and nutritional factors [14,15]. An increase in thyroxine (T4 ) is associated with elevated levels of apoA-I [16]; increases in hepatic and intestinal apoA-I were observed in hyperthyroid rats [17] and Soyal et al. [18] reported an increase in apoA-I expression in rat liver following chronic administration of triiodothyronine (T3 ). In this paper we report the cloning and characterisation of apolipoprotein A-I from a teleost ¢sh, the sea bream (Sparus aurata), analyse its expression in various tissues and investigate whether the mRNA levels are in£uenced by thyroid hormones. A cDNA library in the vector VzapII containing approx. 300 000 recombinants was constructed from poly(A)‡ RNA isolated from sea bream liver [19]. In order to clone genes representing abundant liver mRNAs such as that encoding apolipoprotein A-I, liver poly(A)‡ RNA (0.1 Wg) was reverse transcribed and the total cDNA radiolabelled with 32 P and used to probe the cDNA library. Approx. 500 clones were screened and 10% showed positive hybridisation. These clones will be derived from highly abundant mRNAs. Ten of the selected clones were excised from the VzapII vector into pBluescript SK and restriction mapped and of these, the ¢ve clones with the largest inserts were partially sequenced using a Pharmacia ALF automated sequencer. Four of these clones were derived from the same transcript, indicating that the mRNA they represent constitutes at

least 4% of total liver mRNA. Database searches revealed the cloned gene to have high similarity with salmon apoA-I, which also encodes a highly abundant transcript, representing 7% of total salmon liver mRNA [8]. The complete sequence of one of the putative apoA-I clones was determined following subcloning of appropriate restriction fragments into pUC 18. The sequence encodes a putative polypeptide of 261 amino acids with a predicted molecular mass of 29.6 kDa (Fig. 1A). To con¢rm that the cloned gene contained a functional open reading frame it was linearised, transcribed in vitro with T3 RNA polymerase, and translated in vitro using the rabbit reticulocyte lysate system (Promega) in the presence of 35 S-methionine. A product of the expected size was produced (Fig. 1B). The alignment of the putative sea bream apoA-I with salmon, trout, chicken and mouse apoA-I is shown in Fig. 2. The cloned protein shows 38% amino acid identity with salmon apoA-I, 34% identity with trout and 25 and 22% identity with chicken and human apoA-I respectively. The amino acid sequence of apoA-I is divided between a prepeptide and a mature peptide, which is made up of internal repeats. Such internal repeats can be identi¢ed in sea bream apoA-I which also retains proline as the ¢rst amino acid of seven of the eight 22-mers. Helical wheel analysis (not shown) showed that these internal repeats can form amphipathic helices, typical of the lipid binding modules of apolipoproteins [3]. To analyse tissue expression of the sea bream

BBAEXP 91181 20-10-98

402

L. Llewellyn et al. / Biochimica et Biophysica Acta 1442 (1998) 399^404

Fig. 2. Alignment of the amino acid sequence of sea bream apolipoprotein A-I with salmon, trout, chicken and mouse apolipoprotein A-I. Identical residues are indicated by (*) and conservative replacements by (.).

apoA-I gene, juvenile sea bream (average starting weight 85 þ 9.4 g) were divided between two tanks (500 l) supplied with aerated sea water at 18 þ 1³C. After acclimatisation to the experimental conditions for 1 week one group was injected i.p. with T3 (1 Wg/g body weight) and the other with an equivalent volume of physiological saline. Fish were sacri¢ced 6 h after treatment and tissues were taken for subsequent extraction of RNA. Samples of liver from untreated adult ¢sh (224 þ 13.4 g) were also taken

for RNA extraction. Northern blot analysis of RNA from sea bream tissues showed strong expression of a 1.4 kb transcript in adult and juvenile sea bream liver, weaker expression of the transcript in juvenile sea bream intestine, and no detectable expression in kidney, gill, muscle or brain (Fig. 3). The expression pattern in sea bream is therefore the same as that in mammals and the level of expression of apoA-I does not alter in the liver during the transition from juvenile to adult. There was no ex-

BBAEXP 91181 20-10-98

L. Llewellyn et al. / Biochimica et Biophysica Acta 1442 (1998) 399^404

Fig. 3. Northern blot analysis of apolipoprotein A-I in sea bream liver and intestine. Total RNA (2 Wg) extracted from adult sea bream liver (AL) and from liver (L), intestine (I), kidney (K), gill (G), muscle (M) and brain (B) of juvenile sea bream was fractionated on a 1% formaldehyde agarose gel and stained with ethidium bromide (A) before transfer to a Hybond Nfp nylon membrane. Membranes were hybridised under high stringency conditions at 65³C and probed with the complete cDNA for apoA-I labelled with [K-32 P]dCTP using the random priming technique (Amersham). The hybridisation solution contained 6USSC, 5UDenhardt's solution, 100 Wg/ml tRNA and 0.1% (w/v) SDS. Following hybridisation for 24 h, the membranes were washed twice for 30 min at 42³C and twice at 65³C in 1USSC and 0.1% (w/v) SDS. After washing the membrane was exposed to XAR5 ¢lm for 8 h at 370³C with intensifying screens (B).

pression of apoA-I in sea bream muscle although it has been detected in salmon muscle [9]. The level of expression of apoA-I in the intestine following treatment of juvenile sea bream with T3 was investigated by dot blot analysis. L-Actin, a gene which is expressed at the same levels in all immature tissues and whose expression is not altered by hormone levels, was used as a control for the experiment. Treatment with T3 signi¢cantly decreased the expression of apoA-I in sea bream intestine, whereas actin levels remained constant following treatment with T3 (Fig. 4). Hence the e¡ect of thyroid hormones on apoA-I expression in sea bream is the opposite to that observed in mammals where increased T3 levels resulted in an increase in apoA-I in the intestine [17]. This could re£ect transcriptional regulation of the apoA-I gene or else an e¡ect on the stability of the apoA-I mRNA. Unfortunately knowledge of thyroid hormone metabolism in sea bream is too limited to allow informed speculation as to the physiological signi¢cance of the e¡ects of T3 on intestinal apoA-I

403

Fig. 4. Dot blot analysis of apolipoprotein A-I in sea bream intestine (I). Total RNA (0.5, 1 and 2 Wg) from untreated ¢sh (Icon) or T3 injected ¢sh (I+T3 ) was denatured and applied in 2 Wl aliquots to duplicate Hybond Nfp membranes. The membrane was hybridised under high stringency conditions at 65³C and probed with the complete cDNA for either apoA-I or L-actin labelled with [K-32 P]dCTP using the mega-prime technique (Amersham). The hybridisation solution contained 6USSC, 5UDenhardt's solution, 100 Wg/ml tRNA and 0.1% (w/v) SDS. Following hybridisation for 24 h, the membranes were washed twice for 30 min at 42³C and twice at 65³C in 1USSC and 0.1% (w/v) SDS. After washing the membrane was exposed to XAR5 ¢lm for 8 h at 370³C and with intensifying screens. The level of radioactivity was quanti¢ed by cutting discs (two for each sample) of the membrane where radioactivity was detected and counting the signal by scintillation counting.

expression in this species. It will be interesting to determine the e¡ect of T3 on apoA-I regulation in the sea bream liver and whether the level of apoA-I is controlled by the same regulatory system in both the intestine and the liver. This work was supported by the European Union AIR programme (Contract No. AIR2-CT93-1483).

References [1] C.J. Fielding, V.G. Shore, P.E. Fielding, A protein cofactor of lecithin:cholesterol acyltransferase, Biochem. Biophys. Res. Commun. 46 (1972) 1493^1498. [2] G.J. Miller, N.E. Miller, Plasma high-density lipoprotein concentration and development of ischaemic heart disease, Lancet i (1975) 16^19. [3] W.-H. Li, M. Tanimura, C.-C. Luo, S. Datta, L. Chan, The apolipoprotein multigene family: biosynthesis, structure, structure-function relationships and evolution, J. Lipid Res. 29 (1988) 245^271. [4] C.-C. Luo, W.-H. Li, M.N. Moore, L. Chan, Structure and

BBAEXP 91181 20-10-98

404

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

L. Llewellyn et al. / Biochimica et Biophysica Acta 1442 (1998) 399^404 evolution of the apolipoprotein multigene family, J. Mol. Biol. 187 (1986) 325^340. S.W. Law, B. Brewer Jr., Nucleotide sequence and the encoded amino acids of the human apolipoprotein A-I gene, Proc. Natl. Acad. Sci. USA 81 (1984) 66^70. M.S. Boguski, N. Elshourbagy, J.M. Taylor, J.I. Gordon, Comparative analysis of repeated sequences in rat apolipoprotein A-I, A-IV and E, Proc. Natl. Acad. Sci. USA 82 (1985) 992^996. J. Pan, Q. Hao, T.T. Yamin, P.H. Dai, B. Chen, S. Chen, P.A. Kroon, Y. Chao, Rabbit apolipoprotein A-I mRNA and gene, Eur. J. Biochem. 170 (1987) 99^104. L. Byrnes, C.-C. Luo, W.-H. Li, C. Yang, L. Chan, Chicken apolipoprotein A-I: cDNA sequence, tissue expression and evolution, Biochem. Biophys. Res. Commun. 148 (1987) 484^492. R. Powell, D.G. Higgins, J. Wol¡, L. Byrnes, M. Stack, P.M. Sharp, F. Gannon, The salmon gene encoding apolipoprotein A-I: cDNA sequence, tissue expression and evolution, Gene 104 (1991) 155^161. G.P. Decluve, J. Min Sun, J.R. Davie, Expression of rainbow trout apolipoprotein A-I genes in liver and hepatocellular carcinoma, J. Lipid Res. 33 (1992) 251^262. M. Pontes, X. Xu, D. Graham, M. Riley, R.F. Doolittle, cDNA sequences of two apolipoproteins from lamprey, Biochemistry 26 (1987) 1611^1617. J.I. Gordon, H.F. Sims, S.R. Lentz, C. Edelstein, A.M. Scanu, A.W. Strauss, Proteolytic processing of human preproapolipoprotein A-I, J. Biol. Chem. 258 (1983) 4037^4044.

[13] W.M. Fitch, Phylogenetics constrained by the crossover process as illustrated by human hemoglobins and a thirteen cycle, eleven amino acid repeat in human apolipoprotein A-I, Genetics 86 (1977) 623^644. [14] W. Patsch, K. Kim, W. Wiest, G. Schon¢eld, E¡ects of sex hormones on rat lipoproteins, Endocrinology 107 (1980) 1074^1085. [15] A. Ribeiro, M. Mangeney, P. Cardot, C. Loriette, Y. Rayssiguier, J. Chambez, G. Bereziat, E¡ect of dietary ¢sh oil and corn oil on lipid metabolism and apolipoprotein gene expression by rat liver, Eur. J. Biochem. 196 (1991) 499^ 507. [16] Y. Vandenbrouck, B. Janvier, C. Loriette, G. Bereziat, M. Mangeney-Andreani, Thyroid hormone modulates apolipoprotein A-I gene expression at the post-transcriptional level in Hep G2 cells, Eur. J. Biochem. 231 (1995) 126^ 132. [17] N.O. Davidson, R.C. Carlos, M.J. Drewek, T.G. Parmer, Apolipoprotein gene expression in the rat is regulated in a tissue-speci¢c manner by thyroid hormone, J. Lipid Res. 29 (1988) 1511^1522. [18] S.M. Soyal, C. Seelos, Y.C. Lin-Lee, S. Sanders, A.M. Gotto, D.L. Hachey, W. Patsch, Thyroid hormone in£uences the maturation of apolipoprotein A-I messenger RNA in rat liver, J. Biol. Chem. 270 (1995) 3996^4004. [19] L. Llewellyn, V.P. Ramsurn, G.E. Sweeney, T. Wigham, C.R.A. Santos, D.M. Power, Cloning and characterisation of a ¢sh aldolase B gene, Biochim. Biophys. Acta 1263 (1995) 75^78.

BBAEXP 91181 20-10-98