Cloning and characterisation of a fish aldolase B gene

BiochirnicaL et Biophysika Acta

ELSEVIER

Biochimica

et Biophysics

Acta 1263 (1995) 75-78

Short Sequence-Paper

Cloning and characterisation of a fish aldolase B gene * Lynda Llewellyn

a*b,Vimi P. Ramsurn a,b,Glen E. Sweeney a,*, Trevor Wigham b, Cecilia R.A Santos ‘, Deborah M. Power ’

a School of Molecular and Medical Biosciences, Uniuersiry of Wales, P.O. Bon 911, Cardis CFI 3US. UK b School of Pure and Applied Biology, University of Wales, P.O. Box 915, Cardifi CFI 3TL, UK ’ Unidade de Ciencias e Tecnologias Agrarias, Unuersidade do Algarue, Campus de Gambelas, Faro 8000, Portugal Received 28 December

1994; revised 18 April 1995; accepted 21 April 1995

Abstract A full length cDNA clone representing an aldolase mRNA was isolated from a sea bream (Sparus aurutu) liver cDNA library. Sequencing of this clone revealed it to encode a 364 amino acid protein with 74% amino acid identity to human aldolase B and slightly

lower similarity to human aldolase A and C. In view of the sequence data and of Northern blot analysis showing strong expression of a 1.6 kb transcript in liver it was concluded that the cloned gene represents aldolase B. This clone represents the first aldolase gene to be sequenced

from any fish species thus providing

Keywords: Aldolase;

new data on the evolution of the vertebrate aldolase gene family.

Fish; Sea bream; (Spar-us aurata)

Fructose-1,6-bisphosphate aldolase (E.C. 4.1.2.13) catalyses the reversible cleavage of fructose-l ,6-bisphosphate in the glycolytic pathway, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Many invertebrates possess a single qpe of aldolase whereas in most vertebrates in which aldolases have been studied three related isoforms, each encoded by a separate gene, are present [I]. These are aldolase A, the major form which is found mostly in muscle and is associated primarily with glycolysis, aldolase B pres’ent in liver and involved in the metabolism of fructose, and aldolase C found in brain and neural tissue. The role of aldolase B in fructose metabolism is illustrated by the association of mutations in the human aldolase B gene with hereditary fructose intolerance [2]. Correct regulation of aldolase gene expression may be required for normal cell development since the expression of aldolase genes is altered in certain disease states [3]. The evolution of the aldolase gene family has been the subject of extensive research [4-61. However, these studies have been hampered by a lack of comparative sequence

* The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number X82278. * Corresponding author. E-mail: [email protected]. Fax: + 44 222 874116. 0167-478 l/95/$09.50 0 1995 El sevier Science B.V. All rights reserved SSDI 0167.4781(95)00096-8

data since, with the exception of the recently described aldolase C of Xenopus Zuevis [7], all vertebrate aldolase genes cloned to date have come from mammals or birds. Aldolase B genes have been isolated and sequenced from chicken [8], rat [9], sheep [lo] and human Ill]. Here we report the cloning and sequencing of a full-length cDNA for aldolase B from a teleost fish, the sea bream (Sparus aurutu). This is the first aldolase sequence to be reported from any fish species. A cDNA library in the vector AzapII containing approx. 300000 recombinants was constructed from poly(A)+ RNA isolated from sea bream liver. To evaluate the quality of the library, ten clones were picked at random and, following automatic excision into pBluescript SK-, the 5’ and 3’ end of each clone was sequenced using a Pharmacia ALF automated sequencer. Database searches revealed one of the clones sequenced to be similar to vertebrate aldolase B. The complete sequence of this clone was then determined following subcloning of appropriate restriction fragments into pUC18. The sequence obtained (Fig. 1) spans 1342 bases and contains an open reading frame encoding a protein of 364 amino acids (predicted molecular mass 39.6 kDa). A polyadenylation signal (AATAAA) is present 10 bases before the poly(A) tail. Fig. 2 shows a comparison of the deduced protein sequence with chicken and human al-

L. Llewellyn et al./Biochimica

16

et Biophysics Acta 1263 (1995) 75-78

from the sea bream sequence. Instead the amino acids at these positions are identical to those seen in mammalian aldolase A and C and also in Drosophila aldolase and so may indicate conservation of amino acids present in the ancestral aldolase from which all the vertebrate isoforms are derived. In view of the higher sequence similarity to aldolase B, the strong expression in liver and the computer analyses discussed below we conclude that the cloned gene represents sea bream aldolase B. A phylogenetic tree obtained using the protpars program from the Phylip package [ 151 is shown in Fig. 4. This supports the identification of the cloned gene as aldolase B. Similar trees (data not shown) were generated when the sequences were analysed using the clustal V program 1161 and by the method of Hein [17]. As illustrated in Fig. 4, it is believed that the gene duplication giving rise to aldolase

dolase B [8,11], human aldolase A [12], human aldolase C [ 131 and the p isoform of aldolase from Drosophila [ 141. The sea bream aldolase B shows 74%, 73% and 72% amino acid identity with human aldolases B, C and A respectively and 65% amino acid identity with p aldolase from Drosophila. Northern blot analysis of RNA from sea bream liver and kidney revealed strong expression of a 1.6 kb transcript in liver but no detectable expression in kidney (Fig. 3). Although the sea bream aldolase is only slightly more similar to human aldolase B than to human aldolases A and C, a number of residues seen only in aldolase B isoforms, such as Lys-242 and Tyr-362, are present in the sea bream sequence. On the other hand, certain residues that had previously been stated to be characteristic of aldolase B [lo], such as Ala-150 and Asn-2 18, are absent

1 1

M T H Q F T CTG TTC CCT Cl'2Tn: CCG TCG TTG TTT GAC AAA ACT CTC AAG ATG ACT CAC CAG TTC

6PSLSPEQKKELSDIA 59 CCA TCC CTG AGT CCA GAG CAG AAG AAG GAG CTC TCT GAC ATT GCT C:G A&

Ai& Gik G:T

26 119

P G K G I L A A D E S T G T M G K R CCA GGA AAG GGA ATC Cl-GGCT GCG GAT GAA TCA ACA GGA ACC ATG GGA AAG CGT l&

C:G

46 179

N I N V E N AAC ATC AAC GTG GAG MC

T&

I E E N R R C F R D I L An: GAG GAG AAC CGT CGC l-GCl-l-C CGT GAC ATC CTC T&

Gk

G

I

2:;

A& GETGtCT& A& Gk A!C l& G&

299

CAG AAA l-CAAGC AAC GGC AAA CTC TIC CCC CAG GTC GTT AAG GAG AAG GGC ATT GTr OTC

106 359

GIKVDKGTAPLMGTDKETTT GGC Al-CAAG GTT GAC AAA GGC ACA GCT CCT m

126 419

Q G L D G L S E R C A Q Y K K D G C D F CAA GGT CTT GAT GGC Cl-2TCT GAG CGG TGC GCC CAG TAC AAG AAG GAC GGT 'TGTGAT ?TC

146 479

AKWRCVLKISDGCPFALA GCC AAG 'IGGAGG TGT GTC CTC AAG ATC TCG GAC GGC 'IGC CCG TN3 GCT CTC GCC A&

166 539

ENANVLARYASICQMNGLVP GAG AAC GCC AAT GTC CTT GCC AGA TAC GCC AGC ATC TGC CAG ATG AAC GGC CTG On; CCT

186 599

I V E P E I L P D G D H D L Q R C Al-?GTT GAG CCA GAG Al-C Cl% CCT GAC GGA GAC CAT GAC Cl-ZCAG CGC %C

206 659

T E K V L A A V Y K A L S D H H V ACA GAA AAG GTT Cn: GCG GCT GTA TAC AAG GCT CTG TCC GAC CAC CAC GTT Tic Ck

226 719

G T L L K P N M V T P G Ii S C P K K F GGT ACT Cl-2 Cn: AAG CCC AAC An: Gl-CACT CCT GGA CAC TCC TGC CCT AAG AAG TTC A:,

246 779

P Q E V A M A T V T A L R R T V P A S V CCT CAG GAG GTT GCC ATG GCC ACA GTG ACC GCT TII:AGG CCC ACT GTC CCT GCC TCT GTG

266 839

P G I C F L S G G Q S E E E A S I H CCT GGC ATC TGC TTC CT'ZTCC GGA GGC CAG AGT GAG GAG GAG GCC TCC ATC CAC Ck

286 899

A I N Q V P L H R P W K L T F S Y G GCC ATC AAC CAG GTG CCC CTC CAT CGC CCC TGG AAG CT0 ACC l-l-C TtT TAC GGC C:T $A

306 959

L Q A S A L CTG CAG GCC TCT GCT CW

CO.2 AT-2 AiT

TtCTk C:C GiGA$GCk

T:C

ATG GGC ACA GAT AAA GAG ACC ACC ACA

A A W Q G X D A N K GCA GCC TGG CAG GGC AAA GAT GCC AAC AAG $A

GtT

C:G T:T G& Gk

$C

G?T A:, C:G

326 1019

V CiA G'IT 'I& G&

346 1079

S G S A D Q A S Q Q S L Y T A S Y V Y TCG GGC TCA GCT GAC CAG GCC TCC CAG CAG TCC CTG TAC ACT GCC AGC TAC GTC TAT TAA

TRAKINGLASKGEY ACC AGG GCC AAG ATT AAT GGC CTG GCT TCC AAA GGC GAG TAC :G

C&

1139

ATA C'TGTAA ATA CAT CAA CAT TZA AGG GGC GAA AAA AAG TTA AAA AAA AGT ACA n;c ACC

1199

AAA 'IGTGAC TAC GAC CAA AAC CCT CCG Cm

1259

AGG GGG AGA CAT GTT CAA TGT TAA ACT ACT GTT 'TGTCTA CTC AGT GAA ATG ACT GGA AAT

1319

AAA TTA AGT GAC CAA AAA i&A AAA

GAT Tl'CGCT CTT CCC TAG ACT ACC AAC AGC

Fig. 1. Base sequence of the sea bream aldolase B cDNA clone and the putative amino acid sequence of encoded protein.

L. Llewellyn et al. / Biochimica et Biophysics Acta 1263 (1995) 75-78

B occurred before that giving rise to aldolases A and C. However, it is unclear whether this latter duplication occurred before or after the evolutionary separation of teleost fish and other vertebrates. It is interesting to note that the human aldolase A and C proteins are more closely related (81% amino acid identity) than fish and human aldolase B (74% amino acid identity). Electrophoretic analysis of aldolase proteins from fish [l] has indicated the presence of three isoforms in shark and perch. Assuming the different protein bands observed reflect the products of different genes and not post-translational modifications then at least some fish species must have three aldolase genes. In contrast, four aldolase isoforms can be detected in salmonids, which given their tetraploid genomes suggests the presence of only two aldolase genes prior to genome duplication. In order to further clarify the phylogenetic relationships between vertebrate aldolases it will be important to clone muscle and brain isoforms from teleosts such

77

A

B

Fig. 3. Expression of aldolase B in liver and kidney. (A) 5 pg of sea bream liver (L) and kidney (K) total RNA were fractionated on a formaldehyde agarose gel and stained with ethidium bromide prior to transfer to hybond N nylon membrane. (B) The membrane was hybridised under conditions of high stringency (final washes at 65°C in 0.1 X SSC, 0.1% SDS) to a 32P-labelled PslI fragment comprising residues l-646 of the aldolase cDNA. After washing the membrane was exposed to XARS film for 3 days at - 70°C with intensifying screens. SB-AIB Ch-AIB Hum-AI0 Hum-AIA Hum-Al C Or-AI SB-AIB Ch-AIS Hum-AIB Hum-AIA Hum-AIC Dr-A

I

;;:;I; I8

Hum-A Hum-AIA Hum-AIC Dr-AI

SB-AIB Ch-AI8 Hum-AI6 p$::;: Dr-AI SB-AIB Ch-AI0 Hum-Al B Hun-AIA Hun-AIC Dr-AI

%

SB-AIS Ch-AIB Hum-Al B Hum-A I A Hum-AIC Or-Al SB-AIS Ch-AIB Hun-AI0 Hum-AIA Hum-A/C Or-Al SB-AIB Ch-AIB Hum-Al 0 Hum-AIA Hum-AIC Dr-AI SB-AIB Ch-AIB Hum-Al0 Hum-A I A Hum-AIC Dr-AI SB-A IS Ch-AIB Hum-Al B Hum-A IA Hum-AIC Dr-AI

Fig. 2. Alignment of the amino acid sequences of sea bream aldolase B (SB-AIB) with chicken aldolase B (Ch-AIB), human aldolase B (Hum-AIB), human aldolase A (Hum-AM), human aldolase C (Hum-AIC) and the p isoform of Drosophila aldolase @r-AI). The boxed residues are identical to those in the sea bream sequence.

L. Llewellyn et al. /Biochimica et Biophysics Acta 1263 (1995) 75-78

78

References

100%

HUMANALDOLASEA 58.5 %

HUMANALDOLASEC

I

DROSOPlULAALDOLASE

Fig. 4. Phylogenetic tree derived using the Phylip package showing the relationship between the aldolase sequences illustrated in Fig. 2. Bootstrap values, calculated for 1000 replicates, are shown for each branch.

as sea bream and salmonids. It will also be of great interest to analyse aldolase genes in primitive fish such as Lampreys and in cephalochordates such as Amphioxus since it is possible that these organisms may only have a single aldolase gene representing the ancestor of all the isoforms currently seen in vertebrates. We thank Dr. SM. Hinsull for operating the ALF automated sequencer. This work was funded by a grant from the E.C. (contract No. AIR2-CT93-1483).

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