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Biochimica et Biophysica Acta, 1088 (1991) 429-432 © 1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 ADONIS 016747819100112P BBAEXP 90218

BBA Report - Short Sequence-Paper

Sequence and expression of annexin VII of Dictyostelium discoideum Michael Greenwood and Adrian Tsang Department of Biology, McGill UnicersiO', Montreal (Canada) (Received 10 December 1990)

Key words: Annexin; Calcium/phospholipid binding protein; cAMP binding protein; ()ictvostelium)

Sequence analysis reveals that a gene expressed during growth and early development of Dictyostelium discoideum encodes a polypeptide which exhibits extensive similarity with annexins, a family of caicium/phospholipid binding proteins. Comparison of the amino acid composition of the N-termini suggests that the Dictyostelium an,exJ.i ,~ a homologue of human synexin, also referred to as annexin VII.

A large number of calcium-dependent phospholipid binding proteins which have previously been implicated in unrelated functions are shown to share sequence similarity. Many names have been ascribed to these proteins. Recently, the term annexins has been proposed for this family of calcium/phospholipid binding proteins [1]. Annexins differ from the calmodulin and troponin C family of calcium binding proteins in that they lack the ' E F ' hand helix-loop-helix structure [2]. Sequence analyses show that annexins are composed of four or eight repea: units of 70-80 amint~ acid residues. Different members of annexins display 40-60% amino acid identity in this region of the protein. Based on sequence comparison, eight annexin genes have so far been characterized from mammalian species [3-6]. Recently, two annexin genes have been identified in Drosophila [7]. Annexin-like proteins have also been observed in plants [8]. Despite the progress made in the structural characterization, the functions and mode~ of action of annexins are largely unknown. The cellular slime mold Dictyostelium discoideum is an attractive system in which to study cell function and differentiation, since it is feasible to bring genetic manipulation to bear on the functions of specific proteins [9]. During our analysis of the cAMP binding

The nucleotide sequence reported here has been submitted to EMBL/Genbank and DDBJ p_ucleotide-sequence databases under accession number X56751. Correspondence: A. Tsang, Department of Biology, McGin University, 1205 Avenue Docteur Penfield, Montreal, Quebec. Canada H3A IB1.

protein CABP1, we found that the gene c~-:~U,, CABP1 hybridizes, under reduced stringent ccnditions, to several other genomic DNA fragments [10]. k ",~ ,ave isolated over 100 cDNA and genomic clones en~,.,ding the cross-hybridizing seque,ces. Sequence analysis showed that the cloned DNAs repre~e-: five new genes. The basis for the cross-hybridization appea-~ ' a restrict to a region which encodes a protei,. '/,ma:m rich in prohne, glycine and glutamine residues (unpublished data). In this report, we show by sequence comparison that one of the five cloned genes appears to encode a homologue of human synexin [5], which has been reassigned as annexin VII [1]. Human synexin has been demonstrated to possess voltage-dependent calcium channel activity in vitro [5]. For discussion purposes, we term the Dictyostelium annexin gene DdA NN7. Dd40C is the largest DdANN7 cDNA isolated from a h g t l l library. The EcoRl fragments of Dd40C were subcloned into Biuescript vectors, and their sequences were determined on both strands. To ascertain the orientation and order of the EcoRI fragments, the cDNA insert in Dd40C was amplified by polymerase chain reaction [11]. The sequence of the amplified product was determined with synthetic oligonucleotides as primers. The predicted amino acid sequence of the cDNA suggests that it contains the C-terminus but lacks the N-terminus. The sequences of two additional Dd4NN7 cDNAs were determined, and both lacked the 5' coding region. We therefore examined the sequence of a 5 kb genomic fragment which contained the 5' noncoding region and about three-quarters of the coding sequence of DdANN7. Fig. 1 shows the composite sequence of the cDNA and genomic fragments. Be-

430 AAJU~AC~TTTTTATTTTTTTTTTTATTTAATTTTTTATTTTATTTTTTTTTTTTTTTTTTT~ATTTTTTAATAT.4U~TTATAAA TTTTTAATTCCAAAAAAAATTAATAAAAACTTTTTTTTTTTTCTTCATTTTATTAATTTCTTACAAACAAATACCA•TAGCATC 168 A~UrGTCCTATCCA~CAAA~CAAGGgtatgtttaact~atcctcacattcacaat:acccaccaatattaatttttttttttt 252 hCTSerTyrProProAsnOtnGt

tttttt•c•a•tc•atttgtttttgttttt•tttttt•t•tttcacaccaaaaaacaaataataaaacat•aaataataataat

3~6

aataataataacaataataataataataataataaatataaatcaacatca-~qTTAT~A~CACAATCAA~TTCACCACAA~CA yTyrProProG~nS~rAsnSerFroGinPro

419

GGA~A~TATGGAGCC~CACAACj~GGTTATCCA~CACAACAAGGATACC~ACCA~AA~AAGGTTAT~A~CACAACAAGGTTAT503

GlyG•nT•rGtyA•aPr•GLnG•nG••T•rPr••r•GtnG!nGt•T•rPr•P••G•nG•nG••T•rPr•Pr•G|nG•nGt•T•r ~CACCACAACAAGGQttt~tattt~tttggattcaacttt~ttattcaactataatactcaattataaatatatatatataat

587

ProProGinGtnGt

tattaccactattcaactataatattcaatcaatcaatatttc•agttataatattcccataattatttatttttatattatta tcattttatttatttttatttatatatatatatttatttaaatatttaaaactaaactatttatttatttatttttatttatta atattttttttttttttttttttttttttttttttctttttctttttt~taaaattaattagTTATCCAC~ACAACAAGGTTAT

671

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CCACCACAACAAGGTTATCCACCACAACAAGGrTATCCACCACAACAAGGTTACCCAC•ACAACAAGGCTACCCACCA•AACAA 923 ~r~Pr~GtnG~r~[yTyrPr~Pr~G~nG~nG~yTyr~r~Pr~G~nG~nG~TyrPr~Pr~G~nG~nGlyT~rPr~Pr~GtnG[n GGTTATCCACCACAACAAGGCTACCCACCACAACAAGGTTA•C•ACCACAACAAGGTTACCCACCACAACAAGGCTACCCACCA1007 GtyTyrPr••r•G•nG•nG•yTyr•r••r•G•nGtnG•yTyrPr••r•G•nG•nG•yTyrPr••r•GtnGtnGtyTyr•r••r•

CAACAAGGTTATqCA~ACAAGGTTATCCACCA~AACAAGGTTATCCACCAGTTGGTGTACCAGTTGGTGTACCAGTTGGATTT 1091 GtnG~nG~yTvrpr~°r~G~nG~yT~r~r~pr~G~nG~nGt~T~r~r~Pr~a~GlyVat~r~atG~yva~r~alGtYPhe

Ec_onV GCACCAGGTATGGTAGTTGGATATCATCAAGGTTA•TTTGTTGGTACAA•CACTCATGATTGTAAACATGATGCTGAAGTTTTA 1175 A[aPr~G~yt~etVa~VatG[yTyrHisG~nG~yTyrPheVatCtyThr~leThrHisAspCysL~sH~sAspAtaGtuVa~Leu AGAAAAGCAATGAAAGGTATTGGTA~AAATGAATCTGATTTAATTAAAGTTTTAGC~AATAGAAATTGGGCTGAACGTGAA~AA1259 Ar~LysAta~4etLys~ty~teGtyThrAsnG~uSerAspLeu~eL~s~a|LeuA~aAsnAr~AsnTrpAtaG~uArgGtuGln AT~AAGAGAGAGTTCT~TGCC~/U~`TATAGCAAAGATTTAATTCAAGATATTAAATCTGAAACCAGTGGTAACTTTGAAAAATGT1343 •teLysArgG•uPheSerAlaLysTyrSerLysAspLeu•teG•nAsp•leLys•erGtuThr•erG•yAsnPheG•uLysCys TTAGTTGCCCTCTT•ACTGAACCAGC••ATTT•GATGTTGAACAAATTCACAGTGCTTGCGCTGGTGCAGGTACCAACGAGAAC 1427

LeuVatA•aLeuLeuThrGtuPr•ALaHisPheAsp•atG•uG•n••eHis•erA•aC•sA•aGt•A•aGtyTnrAsnG•uAsn ACTATAATTGAAATTTTAGTTACCCGTAGTAATGTA•AAATGGAATACATTAAACAAATC•TCAAGAATAAACATGGTAAATCA 1511 Thr••e••eG•u•teLeu•atThrArgSerAsn•atG•r/•etG•uT•r••eL•sG•n•te•heL•sAsnLysHisG••L•sSer TTAAAAGAT•GTCTTGAATCCGAAGCAAGTGGTGACTTTAAGAAATTATTAGAGAAACTCACTGAA•CAAGAGATGAATCA•CA 1595

LeuL~sAspArgLeuGtu~erGtuAtaSerGt~AspPheLysLysLeuL~uG~ULysLeuThrGtu~r~ArgAspGtuSerpr~ EcoRl GTcATcAA~AATGCAAG~TTCAAAAGATG~TGAAGATCTCTA~AAAGcTGGTGAAGGTAAAATTGGTA~CGA~GAAAAGGAA1679 ~a~lteAsnPr~NetGtnVatSerLysAspAtaGtuAspLeuTyrLysAtaGtyGtuG~yLys~teGlyThrAspGtuLysGlu TTCATCAAAATTCT•AC•TCTCGTTCATTACCA•ATATTGCTGc•GTcGcTAGTGAATA•ATTAAAcATcACAAAAAAcACTCA •he••eLys••eLeuThr•erAr••erLeuPr•His••eA•aA•a•a•AtaSerG•uTyr••eLysHisHisLysLysHisSer Ec.__.~! CT•AT•AAAG•AATCGATT•TGAATT•TCTGGTT•AATTAAAACTGGTTTAATcGCTATCGTTACCTATGcTCTCAACCCATAT

1763

1847

Leu••eLysA•a•teAspSerG•uPheSerG•Y•er••eLysThrG••Leu••eA•a••eVaLThrT•rA•aLeuAsn•r•T•r GGTTATTTCGCTGA~ATCTTAAA~AAATCAATGAAAGGTGCTGGTAcAAATGATAACAAA~TcATTCGTACTGTTGTAACTCAA1931 G~yT~r~heA~aGtu~[eLeuAsnLysSerHetLysGlyAtaGlyThrAsnAs~:)AsnLysLeu~teArgThrVatVa~ThrGLn Ec.__~oRt ATG•A•AATATGc•A•AAATTAAAA•TG•TTATTCAA•TcTCTT•AAGAATT•ATTAGcTCATGATATT•AAG•TGATTGTAGT

E015

HetH•sAsr•4etPr•Gtn•teLysTh•A•aTyr•erThrLeuPheL•sAsnSerLeuAlaHisAsp•leG•nA•aAspCy•Ser GGTGATTTCAAAAAATTATTATTAGATATTATCTCATAAAATAATAATTTT GtyAspPheLysLysLeuLeuLeuAspltelteSer---

20(~

Fig. I. Nucleotide sequence and the deduced amino acid sequence of DdANNT. A composite of genomic and c D N A sequence+ i~ presented. The nucleotides are numbered on the right margin. The genomic mquence was determined from first nucleotide to the first EcoRt site, nucleotides 1 to 1682. The sequence of the entire Dd40C eDNA was determined+ from nucleotide 413 (marked by an asterisk) to the 3" end. The intron sequences are depicted in lower case letters. The 5' and 3' splice sites are underlined. The locations of the three internal EcoR! and EcoRV sites are also shown.

431 tween the Dd40C eDNA and the genomic sequences there is an overlap of 967 bp. The coding region of the genomic fragment was deduced on the basis of the following observations: (1) noncoding sequences of Dictyostelium genes are typically rich in AT [12]; (2) it is the only open reading frame in this region of the genomic fragment; (3) the size of the DdANN7 transcript (see Fig. 3) suggests that the coding region is about 1300 nucleotides in length, which is the size of the open reading frame estimated from the genomic and eDNA sequences. The genomic fragment harbours two sequences, downztream of the translation initiation codon, which have the characteristics of introns. These two sequences are rich in AT, and are flanked by consensus splice sequences [13,14]. Moreover, the second intron is spanned by the e D N A Dd40C. Thus, we assign these two sequences as introns in deducing the protein translation region. The predicted polypeptide encoded by DdANN7 consists of 462 amino acids with a calculated molecular mass of 51 171 Da. Based on amino acid composition the polypeptide can be divided into two regions. The amino acid composition of the 165 residues in the N-terminus is very limited, with Pro, Gin, Gly and Tyr constituting 83%. This region contains 17 perfect and 2 imperfect copies of Gln-Gly-Tyr-Pro-Pro-Gln. Eighteen of these repeats are arranged in tandem. Similar repeats are found in a number of proteins including human synexin [5] and CABP1 [13]. These repeats have been postulated to form unusual helical structures [15]. The predicted amino acid sequence was used to search for similar sequences in the GenBank and EMBL data bases. Fhe C-terminus exhibits 35-41% sequence identity with all known mammalian annexins. The high degree of similarity between DdANN7 and mammalian annexins suggests that DdANN7 encodes an annexin protein. The strongest homology is to human synexin or annexin VII [5], 41% amino acid identity and 81% when conserved substitutions are included. However, the homology of this region to other annexins are comparable. Thus, on the basis of similarity in the C-termini alone we are unsure if DdANN7 is a new member of the annexin family or the homologue of human synexin. The N-termini of annexins are highly divergent. Most annexins have short N-termini of less than 50 residues. H u m a n synexin is unique in that its N-terminus consists of 167 armno acids. Besides sharing an N-terminus of similar size, the polypeptide encoded by DdANN7 has in common with human synexin in its amino acid composition. Pro, Gly, Tyr and G i n make up 61% for human synexin and 83% for its Dictyostelium counterpart. Charged amino acids accoant for less than 2% in both cases and they are confined to the ends of the N-termini. Taken together, these results suggest that the polypeptide encoded by Dd4~IN7 is a homologue of human synexin, and we tentatively name it as annexin

D: MSYPPNQGYPPQSN S PQPGQYGAPQQGYpPQQGypPQQGYp pQQGy ppQQGyppQQCyp p QQGYP PQQGYPPQQGY P POQGYP PQQGYP PQQGYP PQQGyPPQQGyp FQQGyp pQQGy p p QGYPPQQ~YPP~/GVPVCV PVG FAPGh"VVGYHQGy FVGT ITHDCKH H: HSYPGYPPTGYPPFPGYPPAGQES S FP PSGQYPYPSCFPPMGGGAYPQVpS SGYPGACGY PA PGGYP&pGGYPGAPQPGGAP SYPGVP PGQG rcvP PGGAG FS GypQ ppsQSYGGG PAQV P LPGG F PGGQMPSQYPGGQ PTYP SQ PATVTQVTQGT IR PAANFDA IR D: DAEVLRICMqKG I GTNES ULI KVLANRNNAEREQ I KREFSAKYSKDLIQDI KS ET SGNFEK H:

DAE I LRKABKGFGTDEQAI VDVX~'ABRS NDQRQK I KAAFKTSYGKDL 1KDLKS ELSGNME E

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Fig. 2. Comparison of human and Dictyostelium annexin VIIs. The deduced amino acid sequencesof human s.~aexin(H) and-Dd.ANN7 (D) are shown. The amino termini of the two sequencesare displayed independently.The carboxylportions of the two sequencesare aligned with the TFASTA programme[16]. Identicalresiduesare indicated by double dots C), while singledots (.) representconservedsubstitutiors. Gaps (-) are inserted to achievemaximumalignment. VII according to the scheme proposed by Crumpton and D e d m a n [1]. A comparison of the amino acid sequences of h u m a n synexin and Dictyostelium annexin VII is shown in Fig. 2. We have examined by R N A blot hybridization the accumulatior~ of DdANN7 m R N A during growth and development. DddNN7 D N A hybridizes to a single band of 1.5 kb on an RNA blot during growth and the early stages of development. The transcript levels decline after aggregation (Fig. 3). Since the average size of the poly(A) tail and untranslated regions of Dic-

0 2 4 6 8 1012141618 Kb

1.5-

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Fig. 3. Northern blot analysis of Dcl,4NN7. Cells of strains NC4 were grown on lawns of bacteria and allowed to develop on filters. At 2 h intervals during development,cell samples were harvested and RNA was extracted. 5 /~g of total cellular RNA from each sample were separated by electrophoresis and transferred to a Nytran membrane (Schleicher and Schuell). The blot was probed with a radiolabelled 565 bp EcoRV-EcoRI fragment according to the manufacturers suggestions.The numbers indicate the time, in h, after starvation when the samples were obtained, o, RNA extracted from vegetative cells.

432 O'ostelium genes is a b o a t 200 bp, the coding region o f D d A N N 7 is estimated to be 1.3 kb. In addition, gen o m i c analysis by D N A blot hybridization suggests that annexin VII is e n c o d e d by a single gene in Dict vc.~telium (data n o t shown). The sequence r e p o r t e d here represents the first annexin gene characterized from a lower eukaryote. This raises the possibility of using genetic tools to e x a m i n e the function of this interesting family o f proteins. Also, we show tl'~a: ~he N - t e r m i n i o f a n n e x i n s m a y be conserved, hence providing f u r t h e r s u p p o r t to the idea that the N-termini of a n n e x i n s are functionally i m p o r t a n t . This work was s u p p o r t e d b y a g r a n t f r o m the N a tional C a n c e r Institute o f C a n a d a . M.G. was a recipient o f a N S E R C p o s t g r a d u a t e scholarship.

References 1 Crumpton, M.J. and Dedman, J.R, (1990) Nature 345, 212. 2 Kretsinger, R.H. and Creutz, C.E. (1986) Nature 320, 573. 3 Si~dhof, T.C., Slaughter, C.A., Leznicki, I., Barjon, P. and Reynolds, G.A. (!988) Proc. Natl. Acad. Sci. USA 85, 664-668. 4 Hauptmann, R., Maurer-Fogy, 1., Krystek, E., Bodo, G., Andree. and Reutelingsperger, C.P,M. (1989) Eur. J. Biochem. 185, 63-71.

5 Bums, A L.. Magendzo, K.. Shirvan, A., Srivastava, M., Rojas, E., Alijani, M.R. and Pollard, H.B. (1989) Prec. Natl, Acad. SCi. USA 86. 3798-3802. 6 Peplinsky, R.B., Tizard, R., Mattaliano, R.J,, Sinclair, LK., Miller, G.T.. Browning, J,L, Chow, E.P., Burne, C., Huang, K,-S., Pratt, D., Wachter, L,, Hession, C., Frey, A.Z. and Wallner, B.P. (1988) J. Biol. Chem. 263. 10799-10811. 7 Johnston, P.A., Perin, M.S., Reynolds, G.A.. Wasserman, S.A. and Siidhof, T.C. (1990)J. Biol. Chem. 265, 11382-11388. 8 Smallwood, M., Keen, J.N. and Bowles, D.J. (1990) Biochem. J, 270, 157-161. 9 Devreotes, P, (1989) Science 245, 1054-1058. 10 Tsang, A.. Grant, C., Kay, C., Bain, G., Greenwood, M., Nece, T. and Tasaka, T. (1988) Dev. Gen. 9, 237-245. 11 Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis. K.B, and Erlich, H.A, (1988) Science 239, 487-491. 12 Kimmel, A.R. and Firtel, R.A, (1982) in The Development of Dictyostelium discoideum (Loomis, W.F., ed.), pp. 234-324, Academic Press, New York. 13 Grant, C.E, Bain, G. and Tsang, A. (1990) Nucleic Acids Res. 18, 5457-5463. 14 Csank, C., Taylor, F.M. and Martindale, D.W. (1990) Nucleic Acids Res. 18, 5133-5144. 15 Matsushima, N., Creutz, C.E. and Kretsinger, R.H. C990) Prot. Struct. Funct. Genet. 7, 125-155. 16 Pearson, W.F. and Lipman. D.J. (1988) Prec. Natl. Acad. Sci. USA 85. 2444-2448.

Sequence and expression of annexin VII of Dictyostelium discoideum.

Sequence analysis reveals that a gene expressed during growth and early development of Dictyostelium discoideum encodes a polypeptide which exhibits e...
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