Molecular and Biochemical Parasitology, 40 (1990) 1-12

1

Elsevier MOLBIO 01300

Plasmodium falciparum aldolase: gene structure and localization Bernhard Knapp, Erika Hundt and Hans A. Kiipper Department of Molecular Biology, BehringwerkeAG, Marburg, F.R.G. (Received 15 August 1989; accepted 26 October 1989)

A genomic clone was isolated which codes for the fructose bisphosphate aldolase of Plasmodiumfalciparum. The aldolase gene is interrupted by one intron which divides the coding region into two exons. The first one codes for one amino acid only, the initiation methionine, while the second one encodes the residual 368 amino acids of the protein. The gene, which is represented only once in the genome, is transcribed at high rates as a 2.4-kb mRNA in the P. falciparum blood stage. The aldolase gene encodes a protein of 40105 Da, which is 61-68% homologous to known eukaryotic aldolases. The protein was expressed in Escherichia coli cells in an unfused and enzymatically active form. Antisera raised against amino acids 9-96 recognize a 41-kDa protein band previously shown to protect monkeys against a P. falciparum infection. These antisera cross-react with aldolases of different species, which confirms the strong conservation of this enzyme during evolution. The aldolase could be localized in the cytoplasm of the parasite as an active and soluble form. An inactive form was found to be associated with the membrane fraction. Digestion data with phospholipase C suggest a membrane association of this polypeptide via a glycosylphosphatidylinositol anchor. Key words: Aldolase; Plasmodiumfalciparum; Gene structure

Introduction Several proteins of Plasmodium falciparum have already b e e n tested for their potential to protect m o n k e y s against a lethal malaria infection. A m o n g these antigens, a protein b a n d of 41 k D a was shown to induce protective i m m u n i t y in Saimiri m o n k e y s and was therefore considered to be a candidate for the d e v e l o p m e n t of an antim e r o z o i t e malaria vaccine [1]. T h e 4 1 - k D a polypeptide is associated with m e m b r a n e preparations of schizonts [2] and was f o u n d to be localized in the r h o p t r y organelles of the parasite [1]. T h e c o r r e s p o n d i n g coding sequence was recently isolated [3,4] and the d e d u c e d protein sequence

Correspondence address: Bernhard Knapp, Behringwerke AG, Post Box 11 40, 3550 Marburg/Lahn, F.R.G. Note: Nucieotide sequence data reported in this paper have been submitted to the GenBankT M Data base with the accession number M28881.

Abbreviations: dNTP, desoxynucleotide triphosphates; PMSF, phenylmethylsulfonyl fluoride.

revealed significant h o m o l o g y with m a m m a l i a n aldolases. Here we report the isolation of the gene for the P. falciparum aldolase and describe its structure. W e also present data about the conservation of this antigen a m o n g different species and the localization of the protein within the parasite.

Materials and Methods T h e m e t h o d s of P. falciparum cultivation, preparation of antigens and isolation of D N A and m R N A as well as analysis of this material by Southern, N o r t h e r n and W e s t e r n blot t e c h n o l o g y have b e e n described earlier [5,6]. Similarly, the expression of D N A insert fragments in the vector p E X 3 0 b has b e e n r e p o r t e d [5].

Construction and screening of a E c o R I library. 2 p,g of D N A f r o m the P. falciparum strain F C B R was incubated at 37°C overnight with 14 units of the restriction e n z y m e E c o R I in 10 m M Tris-HCl ( p H 7.5), 10 m M MgCI2, 1 m M dithiothreitol and 40% glycerol. U n d e r these conditions E c o R I

0166-6851/90/$03.50 t~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

shows star activity, resulting in DNA fragments of 50 bp to 10 kb in size. The digested DNA was fractionated on a 0.8% agarose gel and the DNA region between 500 bp and 7 kb was electroeluted and introduced into the vector hgtll by the method of Huynh et al. [7]. A genomic EcoRI* library of 5 x 105 recombinant phage clones was obtained and amplified. 105 plaques of this library were screened with nick-translated insert DNA of clone 41-7 and three phage clones hybridizing with the insert were identified. From each of these clones a D N A fragment of 3.3 kb in size could be isolated using the restriction enzymes EcoRI and SalI. The malaria specific portion of 2.3 kb was used to construct a restriction map. Based on this map, subfragments were cloned into the Bluescript vectors (Stratagene) for sequencing of double-stranded DNA using the sequenase system from USB (Cleveland, OH).

RNA sequencing. Polyadenylated R N A was prepared and sequenced as described previously [5] using a synthetic oligonucleotide p24 (5'GGTTTGTGTI'GATI'CATCAGCAGC-3', complementary to bases 616--639). The primer p24 annealed to the complementary mRNA region 108 bases downstream from the 3' end of the corresponding intron position. A second primer, p30 (5'-GGGGCATTCATATATTCAGTGCAATGAGCC-3', complementary to bases 507-536) annealed to the region surrounding the changed site of the spliced mRNA. This primer was used for m R N A sequencing and in addition for primer extension experiments which were performed using the protocol of Ausubel et al. [8]. Expression of P. falciparum aldolase. For expression of the P. falciparum aldolase without a fusion component, the pTRC vectors of Amann et al. [9] were used. The complete coding region was amplified by the polymerase chain reaction technique using the oligonucleotides p38 (5'-TTTATA'ITITI~CCATGGCTCATTGCACTGAATATATG-3', corresponding to bases 492-528 with base changes in positions 13, 14 and 16 resulting in a NcoI site which contains the start codon) and p39 ( 5 ' - C A ~ G G T T G G T A C C G T I T I ' T A A T A G A C A T A T T T C T F - 3 ' , complementary to bases 1597-1635 with base changes

in positions 15-17 resulting in a KpnI site downstream of the stop codon). The polymerase chain reaction was carried out using the GeneAmp T M kit of Perkin Elmer Cetus. Amplification of DNA fragments from the plasmid template was achieved by addition of 10 ng of template DNA to 50 mM KC1/10 mM Tris-C1 pH 8.3/1.5 mM MgC12/0.01% gelatin/200 IxM each of dNTP/380 ng of each primer and 2.5 units of Taq polymerase in a final volume of 100 txl. These samples were overlaid with 100 ixl of paraffin and subjected to 30 cycles of denaturation (1 min, 91°C), annealing (1 rain, 45°C), and extension (3 min, 65°C) using a DNA thermal cycler. The product was digested with KpnI, partially digested with NcoI and size-fractionated by agarose gel electrophoresis. A DNA fragment of 1.1 kb in size was isolated and ligated with the NcoI and KpnI digested pTRC 99A vector DNA. The ligated expression plasmid was used to transform Escherichia coli strain DH5ot, and several independent colonies containing inserts of the correct size were isolated and analyzed for expression as described by Amann et al. [9]. Expression products were analyzed by Western blot using an antiserum raised against a fusion protein encoded by the original 41-7 sequence. Bacterial extracts were fractionated as described previously [5].

Enzyme assay and inhibition of activity. Soluble and membrane fraction of schizonts were prepared as reported previously [6] with the exception that p-chloromercuribenzoate was omitted from the solubilization buffer. Aldolase activity assays were performed essentially as described [10]. The buffer used for solubilization of the membranes (PBS containing 1% Triton X-100, 5 mM EDTA, 2 mM PMSF and 2% antagosan) was previously shown not to influence the enzyme activity. All reagents were obtained from Boehringer, Mannheim. Inhibition of enzyme activity was tested after incubation of 400 Ixl schizont-soluble fraction from 40 ~zl pelleted schizonts (80-90% parasitemia), or 200 Ixl of E. coli soluble fraction, respectively, with 100 ~1 antiserum for 3 h at room temperature and subsequent enzyme assay of 50 ixl. In some cases, a second sample was tested after further incubation with Protein A-Sepharose (6 mg/200 ixl PBS, 1% Triton X-100) for 2 h

at room temperature and subsequent centrifugation.

a

b

Phospholipase C treatment of schizont membranes. Schizont membranes were washed twice with PBS and incubated for 30 min at 37°C with PBS and 0.3, 3 and 30 U phospholipase C (Boehringer, Mannheim) in PBS, respectively. The samples were centrifuged and supernatants were tested for enzyme activity. Furthermore, supernatants and sediments were dissolved in equal amounts of SDS sample buffer and analyzed by Western blot using an antiserum raised against the expression product of the 41-7 fragment.

116

-

66-

43-

m

"

29 Results

Identification of a P. falciparum gene encoding a 41-kDa protein. A protein band of 41 kDa was reported to protect Saimiri monkeys against a P.

falciparum infection [1]. Using a polyclonal antiserum raised against this protective protein band, we have isolated several clones from a hgtll expression library prepared from genomic DNA of the P. falciparum strain T9.96 [4]. Two clones, 41-2 and 41-7, reacted very strongly with the antiserum. The clone 41-2 which codes for a 29-kDa schizont protein was described previously [4]. In this paper the clone 41-7 is reported in more detail. The 266-bp insert of clone 41-7 was expressed in the vector pEX30b as a 24-kDa MS2polymerase fusion protein. Antisera raised against the purified expression product react with a 41kDa protein band as shown by Western blot analysis of polypeptides from P. falciparum schizonts (Fig. 1), which demonstrates that the clone 41-7 indeed codes for a 41-kDa protein. Two-dimensional Western blot analysis showed the same pattern for both, the serum raised against the recombinant protein and the original antiserum used for the screening of the library (results not shown). This indicates that the 41-kDa protein band contains only one major immunogenic antigen encoded by the corresponding gene of clone 41-7.

Isolation and characterization of the complete gene coding for the 41-kDa protein. A genomic hgtll library containing EcoRI* DNA fragments pre-

Fig. 1. Western blot analysis of polypeptides from P. falciparum schizonts with the antiserum raised against the 41-kDa protein band (a) and with an antiserum raised against the MS2polymerase fusion protein of the 41-7 sequence (b). Molecular weights are indicated in kDa.

pared from genomic DNA of the P. falciparum strain FCBR was screened with the insert DNA of clone 41-7. This allowed the isolation of a clone carrying a 2.38-kb EcoRI* fragment. Fig. 2 shows the nucleotide sequence and deduced amino acid sequence of this DNA fragment. It carries the entire coding region for the 41-kDa protein, which is interrupted by an intron of 452 bp. The presence of this intron was demonstrated by mRNA sequencing as described in Materials and Methods. Fig. 3 shows the mRNA sequence surrounding the splice site of the gene. The result demonstrates that the intron sequence is spliced out at the position indicated, which is further confirmed by the splice site consensus sequences GTA and T I T F A G found at the 5' and 3' boundaries of the intron. The first exon encodes only a single amino acid, the initiation methionine. Seven triplets upstream from this start codon a TGA opal stop codon is situated which confirms the predicted translation start position. The known 52 bp of the 5' non-coding region of the gene has a high A + T content of 88.5%, car-

I AA'rrri-rrrbrtCaATATi/-l-rrr I~CAgnr_aTATAA~Aa'rA'rr r ~ r / A ~ . . ~ : ; T A A P . ~ T A T A A ~ ~ ~ ~ Met

1

91 181 TA6TrA~TATATAC~TTATATA~TTCA'rrr ~ ~ C _ A T r A T I T A : i r ~ T A ~ 271 G T A T T A A C A ~ T d A C ~ ~ T r C C r T A A ~ I T A C A T A T G T A ' r r r r r rm,~TAA

"

Cr r ~ A T A A ~ ~ " TAGTAAAAi

361 C C ~ 451 TUC~`,AATAT2ATATA~rri~ri~r~t~2A~i~rri~zATA~r~ri~n%TA.rr[~iTri~GGCi~ATTGCACT~d~TATATGAA~ AlaHi sCysThrGluTyrMe tAsnAlaPr oLys

12

TT~.CC~CCGCC 'Prr~TC_,~TCA 541 A ~ T T A C ~ LysLeuP roAlaAspValAlaGluGluLeuAl aTh rTh rAl aGlnLysLeuVa IGInAI aGIyLysGIy IieLeuAlaAlaAspGluSe r

42

631

Th rGlnTh rIleLysLysArgPheAspAsnI leLysLeuGluAsnThr IleGluAsnArgAlaSe rTyrArgAspLeuLeuPheGlyThr

72

CAATGGTrAATITA 721 AAAGCgkTTA G A A A A T T C A ~ ' I T r ~ A ~ C A T T A T T T C A A A A G A A T G A A G C C G G T G T A C LysGI yLeuGlyLysPhe IieSe rGlyAlaI leLeuPheGluGluTh rLeuPheGlnLysAsnGluAlaGlyVal P roMe tValAsnLeu 102 GTEAACATTC C A T G C 3 % C A G A ~ T C A A C T C A A ~ T 811 T'II~C~C_~AIV,A A A A T A T A A T I ~ C ~ ~ T A A A G G T T T C , LeuHi sAsnGluAsnl le IleP roGlyI leLysValAspLysGlyLeuValAsnI leP roCysTh rAspGluGluLysSe rTh rGlnGly 132 901 T~AGATGGAT~/x~`-AC~%AAGATCK~AA/K~%GTAT~ATAAAGCTC.~3TGCAACEi~iGCTAAATGGAGAACAGTri~~~ C LeuAsl:GlyLeuAlaGluAr gCysLysGluTyrTyr LysAiaGlyAlaAr gPheAlaLysTrpAr gThrValLeuVal IleAspTh rAla 162 991 A A A G G A A A A C ~ A A ~ T G A T I T A T C A A T I V A C ~ T G G C ~ ` ~ TTC~C~AC~TATGCATCTAT~TCAACAAAATAGAT~AG2TC ~ LysGl yLgsP roThrAspLeuSe rIleHi sGluTh rAlaTrpGlyLeuAlaAr gTyrAiaSe rIieCysGlnGlnAsnAr gLeuValP ro 192 ACC.ACACI'C/~ TTGTAACTCAAAAAG'i'i'r n~TCATGTGTATITAAA 1081 A ~ C C T G A A ~ I - t ' r ~ T G C , IleValGluP roGluI leLeuAlaAspGlyP roHi sSe rIleGluValO ,sAiaValValThrGlnLysValLeuSe rCysValPheLys 222 1171

CACT A1 aLeuGlnGluAsnGiyVa iLeuLeuGluGlyAlaLeuLeuLysP roAsnMe tVa iTh rAl aGll/IX/rG i u ~ s T h r ~ a L y s ~ r~ r 252

CCACCAGCCTTAC~'I'I'iTrA~CAA 1261 ~ T G T T G ~ I - I - I t ~ i - ~ ThrGlnAspVMGIyt'heLeuThrValArgThr LeuAr gArgTnrValProPr oAiaLeuP roGlyValValPheLeuSe r G l ~ l ~ I n 1351

ITI;~A~AA~_AA~T

r IC~3GTCCACACCCAI~GGCTI~2AAC~

C

282

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Se rGluGluGluAlaSe rValAsnSeuAsnSe r IleAsnAlaLeuGlyP roHi sP roTrpAlaLeuTh rPheSe rTyrGlyAr gAlaLeu 312 1441 ~ ~ a = A " Z3"rrr~ATTA CAACIX2C GlnAlaSe rValLeuAsnTh rTrpGlnGlyLysLysGluAsnValAlaLysAlaArgGluValLeuLeuGlnArgAlaGluAlaAsnSe r 342 1531 ~ ~ TGG~ k n a ~ A T C C . A C 6 n V . C I ' I V ~ T T A T A ~ T A T G T C T A ~ C T T LeuAl aTh rTyrGlyLysTyr LysGIyGIyAI aGlyGlyGluAsnAlaGlyAl aSe rLeuTyrGluLysLysTyrValTyr** * "

369

1621 CACC.AACCAAAAATGAATAATAATAATAA~%AATAAATTACTAAATGAATGGTACTATA'I-rI - I - I ~ T ~ T A ~ T A 1711 2X3TATATATATATATATATATATACAAATATGTGAATA

TGTAATATATATCGATCAATGTATATCTACGATAT

1801 ATAAATATATATITATI'CATATCTCCci-r r rrna,GATGATATATTATAATACCTAAAATTATATATATITATTTAT2AT2ATrr zATIT~ 1891 TITAATAAri-ri-i-I-rl-rA~TGATAATAAA'rl-I-I-I-zAAACG.i-i-i-ITr CAACGiTi-zATTAAATGTGTAAATATAAATATAAAT~ 1981 T~ATATATATATATATATATATATGTA~WYATITATITATTTATTTATATATACATACATACC T G ~

~

~

~

2071 GAACACATGCTTA.i~i~riGTAT2ATATATCTEA~CITCTA~.Ti~i~i~ii~ATAAAAAATGTCAAAGCAGGAAATAAAAA~AATT2AAC~ 2161 AAAAAAATATAATTAATGATGTACACITATAGATATTGATACAAGAAAAACATTATATATG.rl-i-rr i-i-rr rC.i-l.iC.FPi-l-i-f-l-l-l-iTi-i-i, 2251 t"ri"ri"rAATTATAACAAAAAATATITATTATAATATATAA'rI-i-igAATGAATGA~TI~AATGAGCCA, i-ITi~TTTATAi-I-I-zAA~ 1341 T/~ FrATAATAATAACGTACATATATAAAATCK3TGATTGAATT Fig. 2. Nucleotide sequence of the P. falciparum aldolase gene and deduced amino acid sequence of the coding region.

ries stop codons in all three reading frames and contains no A T G sequences. This sequence pattern is in agreement with non-coding regions of P. falciparum genes. The calculated molecular weight of 40 105 daltons is in good agreement with the molecular weight of 41 kDa, estimated from Western blot analysis, and gives further evidence

that the start codon assumed is correct. Northern blot analysis using the insert D N A of clone 41-7 as a probe revealed a 2.4-kb m R N A which was found to be very abundant in schizonts and trophozoites (results not shown). This result demonstrates that the gene is strongly expressed during different blood stages of P. falciparum.

ment with the aldolase sequence of the P. falciparum strain K1 described by Certa et al. [3]. Only one difference is found at amino acid position 171, where an asparagine residue is replaced by an isoleucine residue in strain FCBR, caused by an A ---> T transversion in nucleotide position 1016. The corresponding sequence of the K1 isolate begins at position 184 and ends at position 1765. The comparison of sequence data of the intron and the 3' non-coding region between both strains revealed minor variations caused by deletions or insertions of single nucleotides or dinucleotides. The strong conservation of the nucleotide sequence of the aldolase gene among different P. falciparum strains is further confirmed by the 266-bp insert sequence of the clone 41-7 originating from strain T9.96, which is found to be identical to nucleotide positions 527-792 in Fig. 2 The identity of the encoded protein with aldolase is also supported by the fact that antisera raised against the 41-kDa protein band or against the expression product of the 41-7 fragment both specifically inhibit the enzyme activity of P. falciparum aldolase (Table I).

Fig. 3. Determinationof the intronpositionby directmRNA sequencing. The splice site between exon 1 and exon 2 is indicated by an arrow. Primer extension analysis data using the p30 oligonucleotide revealed an extension DNA fragment of 354 bp (results not shown) suggesting that the 5' non-coding region of the corresponding mRNA extends to at least 322 bp. The 3' noncoding region of 772 bp determined so far is extremely A + T rich (84.2% A + T ) as expected for non-coding regions of P. falciparum DNA. Taking into account a mRNA of 2.4 kb, the 3' noncoding region is assumed to extend for approximately another 200 bases. The coding region of this gene with an A + T content of 64.4% comprises 369 amino acids. It does not code for a signal sequence or for any repetitive segments. The gene shown in Fig. 2 codes for the P. falciparum aldolase of strain FCBR, and is in good agree-

Conservation of the antigen between different species. Fig. 4 shows a Western blot analysis of total proteins from P. falciparum, Plasmodium chabaudi, Plasmodium vinckei and Plasmodium berghei as well as of different mammalian cell lines with an antiserum raised against the expression product of the insert DNA of clone 41-7 which carries the N-terminal amino acids from residue 9 to residue 96. A protein band of 41 kDa was detected not only for different Plasrnodium species but also for the different mammalian cell lines. These data suggest a very strong conservation of the aldolase enzyme during evolution. Comparison of the P. falciparum aldolase sequence to each of the other aldolase sequences reveals 61-68% homology. The alignment of the aldolase sequences of five different species resuits in a consensus sequence including 36% of the amino acid residues (Fig. 5). This consensus sequence shows distinct regions of highly conserved residues suggesting that these are involved in enzyme function. The P. falciparurn aldolase has 6 additional amino acids at the N terminus com-

6 TABLE I Inhibition of P. falciparum aldolase activity by specific antisera Serum

Specificity

NRS

Enzyme activity (%)

-

A

B

100

100

808

Anti 41 kDa

32

12

909

Anti 41-7

36

12

83

63

910 770 771

Anti 31-1 lrd [5]

102 106

92 89

774 775

Anti HRP II [5]

93 93

102 97

See Materials and Methods for details. Schizont soluble fraction was incubated with normal rabbit serum (NRS) and sera raised against the 41-kDa band, the 41-7 antigen and two control antigens, respectively, and subsequently tested for aldolase activity before (A) and after depletion of the sample with protein A-Sepharose (B). pared to the m a m m a l i a n aldolases. Strong homology starts at residue 21 (Fig. 5).

Genomic organization of the aldolase gene. The mammalian genome contains three different genes coding for the aldolase subunits A, B and C forming the tetrameric enzyme, which are expressed to different extents in various tissues [11]. Trypanosoma brucei has four copies of one aldolase gene arranged as two copies of a t a n d e m a

b

c

d

e

f

g

h

i

repeat [12]. In contrast, our data suggest that only one single copy of the aldolase encoding sequence exists per genome of P. falciparum. The m a p of the aldolase gene represented by the genomic 2.38-kb EcoRI* fragment is shown in Fig. 6a. The genomic D N A of the P. falciparum strain F C B R was digested with different restriction enzymes and probed with the two HindlII fragments of the 2.38-kb fragment. The Southern blot (Fig 6b) shows the pattern expected for a single aldolase gene represented by the m a p of Fig. 6a.

k

m

41 k D -

Fig. 4. Western blot analysis of polypeptides from P. falciparum (a) P. chabaudi (b), P. vinckei (c), P. berghei (d), as well as cell lines obtained from human lung (e), human larynx carcinoma (f), monkey lung (g), fetal calf lung (h), fetal horse lung (i) and fetal pig lung (k) with an antiserum raised against the fusion protein originating from the 41-7 sequence.

Expression of the P. falciparum aldolase in bacterial cells. The coding sequence of the P. falciparum aldolase gene was amplified by the polymerase chain reaction and introduced into the expression vector p T R C 9 9 as described in Materials and Methods in order to express the complete enzyme in E. coli without a fusion component. A dominant protein band of 41 k D a was found to be expressed in bacteria carrying the recombinant plasmid (Fig. 7). Part of the 41-kDa protein could be localized in the soluble E. coli fraction without any appearance of degradation products; the second band at 22.5 k D a is also found on Western blots of bacteria harboring the parent plasmid (data not shown) and is caused by crossreaction with a bacterial protein. The molecular weight of the E. coli expressed P. falci-

A B

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tlvaeELattA q K i v ~ - Ui ~ ~L----A

GKG-

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A

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301

2O0

cpssla_Iqen ~ .c0ssial_qen ~

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Y

Fig. 5. Amino acid sequence alignment of five aidolases from different species. (A) T. brucei [12]; (B) human liver [31]; (C) rat liver [32]; (D) rabbit muscle [33]; (E) P. falciparurn (this study); (F) consensus sequence resulting from this alignment•

parum aldolase was shown to be identical to the 41-kDa band of schizonts, indicating that the insert DNA represents the complete coding region.

A [kbl

2.0

Aldolase activity of the soluble fraction of E. coli cells expressing the P. falciparum aldolase was seven-fold more than that of control cells carry-

jRsal _

Drat Dral Rsal

--

Hinc II

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a

b

m

c

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Fig. 6. Genomic organization of the aldolase gene. (A) Restriction map and structure of the P. falciparum aldolase gene. The coding region is boxed. (B) Genomic Southern blot analysis of P. falciparum DNA. DNA was digested with the restriction enzymes NstI (a), HindlII (b), RsaI (c), HindlI (d), DraI (e), XbaI (f), EcoRI (g) and HpaI (h), gel-fraetionated, blotted and hybridized with a combination of nick-translated 560-bp and 270-bp HindIII fragments originating from the coding region of the aldolase gene. 3' end-labeled kDNA digested with EcoRI and HindIII was used as a molecular size marker (M).

a

b

c

d

e

a

b

c

d

e

kD

-66-

-36-29 -

-24Q

--20

-14-

A

ilililill .....

schizonts, and was also found in isolated merozoites by Western blot analysis using antisera against the 41-7 fragment (Fig. 8a); the identical pattern is obtained using the antiserum against the 41-kDa band (results not shown). By immunoelectron microscopy, the antigen was found to be evenly distributed in the parasite cytoplasm (results not shown). Aldolase activity, however, was found to be associated only with the soluble fraction and not with the m e m b r a n e bound component. T r e a t m e n t with phospholipase C rendered the membrane bound component soluble, but still inactive. Incubation with 1% Triton X-100 also solubilized the antigen, whereas treatment with 1 M NaCI released only part of the m e m b r a n e bound fraction (Fig. 8b). In all cases aldolase activity could not be demonstrated for the released antigen.

B

Fig. 7. Expression of P. falciparum aldolase in E. coli. Bacteria before (a) and after induction (b); induced bacteria after mechanical disruption (c); aliquots of supernatant (d) and sediment (e); P. falciparurn schizonts (f). (A) Coomassie Blue stain. (B) Western blot analysis of the same samples using an antiserum against the 41-7 sequence, previously adsorbed on E. coli cells. ing the p T R C plasmid, and was specifically inhibited by an antiserum against the 41-7 sequence (Table II). Localization of the P. falciparum aldolase. The P. falciparum aldolase was localized in the m e m brane fraction as well as in the soluble fraction of

Discussion

We have determined the complete nucleotide sequence and the structure of a gene encoding a 41-kDa protein which was reported to protect Saimiri monkeys against a P. falciparum infection. The 41-kDa protein was previously identified as the P. falciparum aldolase [3]. Its amino acid sequence was deduced f r o m an isolated genomic D N A fragment. The authors concluded from their data that the methionine residue in position 8 (Fig. 2) corresponds to the initiation codon. As, however, the expression of this putative coding region in bacteria and yeast resulted in the synthesis of a smaller protein c o m p a r e d to the par-

TABLE II Expression of P. falciparum aldolase in E. coli and inhibition of activity by a specific antiserum Vector

Serum

pTRC 99

NRS anti 41-7

pTRC 99/P. falciparurn aldolase

-

Aldolase activity (mU (rag bacterial prorein)-1) 7.0 4.9 4.9 51.7

NRS 48.1 anti 41-7 14.9 Soluble fractions of bacteria expressing P. falciparum aldolase or carrying the parent plasmid, respectively, were tested for aldolase activity before and after incubation with normal rabbit serum (NRS) or an antiserum against the 41-7 fragment.

a

b

c

-

A



d

f

g

h

I

k

I

m

-41 kD-

B

Fig. 8. Western blot analysis using an antiserum against the fusion protein expressed from the 41-7 sequence. (A) Total schizonts (a), purified merozoites (b), schizont soluble (c) and membrane fraction (d). (B) Membrane fraction after treatment with 1 M NaC1 (e,f), 1% Triton X-100 (g,h), 0,3 U phospholipase C (i,k) and PBS (l,m). (e,g,i,1), sediments; (f,h,k,m), supernatants.

asite aldolase, Certa and Ghersa [34] claimed an extension of the coding region at the N-terminus. They postulated an unusual translation initiation with a TAG sequence corresponding to nucleotide position 505-507 in Fig. 2. In contrast to these data we have demonstrated by direct mRNA sequencing using two different primers that this TAG sequence is part of a 5' boundary consensus sequence of an intron. We therefore propose that a 452 bp intron is spliced out resulting in an aldolase mRNA, the translation of which is initiated at an ATG start codon instead of a TAG codon at the same position proposed by Certa and Ghersa. Upstream from the ATG codon the nucleotide sequence was confirmed on the mRNA level, suggesting that there is no further intron in the sequence determined so far. This short fragment of 52 bp carries stop codons in all three reading frames and therefore excludes any extention of the coding region to the 5' end. The high A + T content of 88.5% of this region is typical for non-coding regions of P. falciparum genes. Further support that the translation initiation assumed is correct is given by the fact that the third base preceding the start methionine is an A, which is typical for the initiation of translation in eukaryotes [13]. Furthermore, the calculated molecular weight of 40105 Da and the size of the P. falciparum aldolase expressed in E. coli in a soluble and active form are in good agreement with the molecular weight of the parasite enzyme estimated from Western blot analysis. These data

reveal an unusual gene structure: from two exons separated by an intervening sequence, the first encodes only one amino acid, the initiation methionine. An intervening sequence following immediately the start codon was also described for the LC3 gene of the myosin light chain [14]. Our study has shown that the aldolase enzyme is conserved during evolution. Among different P. falciparum strains only minor, if any, differences in the primary protein structure could be detected. The aldolase is also conserved among different species. Antibodies raised against the expression product of the 41-7 sequence crossreact with the aldolases of different mammalian species. Comparing aldolase sequences from five different species, 36% of the amino acid residues are found to be identical (Fig. 5). These include residues which are essential for the enzymatic function: such as the substrate-binding residues Lys-152 and Arg-154 considered as binding sites for C-1 phosphate [15,16] and Lys-ll3 as binding site for C-6 phosphate [17], as well as the additional active-site residues Asp-40, Ile-83, Phe-150, Ile-193, Glu-195, Glu-197, Lys-237, Leu-278 and Ser-307 [18]. The most divergent region of the P. falciparum aldolase is its N terminus (Fig. 5). The amino acid and nucleotide sequence homology between the P. falciparum aldolase and the enzymes of other species starts at a position corresponding to residue 21. In comparison to mammalian aldolases the P. falciparurn aldolase has a short N-terminal

10 extension of 6 residues. A similar N-terminal overhang was reported for the T. brucei aldolase [12], which was discussed to function in aggregation or glycosome assembly. The short N-terminal sequence of the P. falciparum aldolase might similarly lead to aggregation with other molecules. Two-dimensional Western blot analysis on P. falciparum showed the same pattern for both, the antiserum raised against the 41-kDa band used for initial screening and the antiserum against the cloned aldolase fragment, indicating that this enzyme is the main if not the only constituent of this protective band. A 41-kDa protein was successfully used to protect Saimiri monkeys from experimental P. falciparum infection [1]. The question arises to the identity of this antigen and to the mechanism of immune attack involved in the protective effect. The protein used for vaccination was obtained by immunoadsorption on monoclonal antibodies inhibiting the in vitro growth of P. falciparum asexual blood stages and reacting with the rhoptries by immunofluorescence [1]. Monoclonal antibody defined complexes of about 80 and 40 kDa located in the rhoptries have been described by several groups [2,19-23]. The 80-kDa component is now believed to be identical with the 76 kDa serine protease [2,22,24], whereas the identity of the 40 kDa component(s) is still a matter of discussion [22,35]. A rhoptry location, however, is unlikely for a glycolytic enzyme. We show the P. falciparum aldolase to be located in the soluble fraction of schizonts in an enzymatically active form. By immunoelectron microscopy, the antigen was found in the parasite cytoplasm, but apparently not associated with the rhoptries or with any other membranous structures of the parasite. However, a membrane bound component was observed in Western blot analysis which did not show any enzymatic activity. Comparison by twodimensional Western blot analysis using an antiserum raised against the 41-7 purified sequence shows a similar pattern for both the soluble and the membrane bound component (results not shown). In the presence of phospholipase C, which specifically cleaves glycosylphosphatidylinositol anchors, the P. falciparum aldolase was

released from the membrane into the soluble fraction, ruling out the possibility that the insolubility is due to association with the cytoskeleton as reported by Pagliaro et al. [25]. In contrast to the 76-kDa protease which was shown to be activated upon release by phospholipase C [24], the insoluble component of aldolase remains inactive after this treatment. One possible explanation of these data is that the aldolase varies its conformation and structure by binding to the membrane so that the enzyme activity is lost. This would also explain the fact that the membranebound form is not detected by immunoelectron microscopy. The existence of an inactive membrane-associated aldolase is in accordance with the data of Howard et al. [35] who found a rhoptry-specific protein complex of 82, 70, 67, 39 and 37 kDa lacking aldolase activity and therefore concluded that none of the smaller components can be related to aldolase. The phospholipase C digestion experiment suggests that the aldolase of the membrane fraction is not anchored to the membrane by a hydrophobic sequence, but via a glycosylphosphatidylinositol anchor which has been shown for several P. falciparum proteins [24,26-29]. However, the aldolase sequence lacks the C-terminal hydrophobic stretch required for glycosylphosphatidylinositol anchor attachment [30]. Alternatively the protein may be bound to a second polypeptide anchored to the membrane which is released by phopholipase C. The latter suggestion is supported by the fact that incubation with high salt solubilizes part of the membrane bound form of aldolase. This binding protein could indeed be the 76-kDa serine protease of P. falciparum, which was shown to be anchored to the rhoptry membrane [24]. The biological significance of this association remains to be elucidated. The 41-kDa protein band was reported to carry protective properties against a malaria infection. Because aldolase as a glycolytic enzyme is localized in the cytoplasm of the parasite an immune attack against this soluble active form is unlikely, but protective antibodies might act via the inactive membrane bound form. Further investigations are needed to exactly localize the mem-

11

brane bound form of the aldolase. A vaccine development based on this antigen will however be hampered by the high degree of homology between human and parasite aldolase. Therefore it is necessary to identify immunogenic regions unique to the parasite enzyme.

Acknowledgements We thank K.J. Abel for preparation of the oligonucleotide probes and D. Assmann, V. Isenberg and U. Nau for skilful technical assistance. This work was supported by the Bundesministerium fiir Forschung and Technologie.

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Plasmodium falciparum aldolase: gene structure and localization.

A genomic clone was isolated which codes for the fructose bisphosphate aldolase of Plasmodium falciparum. The aldolase gene is interrupted by one intr...
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