A new blood stage antigen of Plasmodium falciparum highly homologous to the serine-stretch protein SERP

Molecular and Biochemical Parasitology, 44 (1991) 1-14

1

Elsevier MOLBIO 01426

A new blood stage antigen of Plasmodium falciparum highly homologous to the serine-stretch protein SERP Bernhard Knapp, U w e Nau, Erika H u n d t and Hans A. Ktipper Research Department, lmmunology/Oncology Group, Behringwerke AG, Marburg, F.R.G. (Received 29 May 1990; accepted 9 July 1990)

We have isolated the gene coding for a new protein (SERP H), highly homologous to the ll3-kDa serine-stretch protein SERP which confers protective immunity to monkeys. The gene consists of four exons interrupted by three short introns located at positions corresponding to those of the SERP gene. Both genes were shown to be linked on chromosome 2 of Plasmodium falciparum suggesting that both originate from a common ancestral gene. Both genes are transcribed in the blood-stage form as 3.8-kb mRNAs with high yield. The deduced amino acid sequence of SERP H is highly homologous to SERP, although it does not contain a serine stretch. A highly hydrophilic region specific for the protein which was shown to be identical among different P. falciparum isolates was expressed in Escherichia coli for preparation of SERP H specific antisera. A schizont polypeptide of 130 kDa within the parasitophorous vacuole was detected by Western blot analysis and immunoelectron microscopy. Like SERP, the 130-kDa protein exhibits a region homologous to cysteine proteinases, suggesting that these proteins, or their processing products, may play a role as proteinases at the time of merozoite release from the infected erythrocyte. Key words: Plasmodium falciparum; Gene structure; Serine-stretch protein; Cysteine proteinase; Gene cloning

Introduction One of the candidate antigens of Plasmodium falciparum for the development of a vaccine against the blood stage of human malaria is the serine-stretch protein SERP [1], which is also described as SERA [2], p126 [3,4,5], pl13 [6] or Pfl40 [7]. The antigen was shown to induce antibodies inhibitory for the parasite in vitro [6,8,9] and to confer protective immunity to Saimiri monkeys [7]. Recently two different groups have reported the structure of the entire gene coding for SERP [ 1,2], which consists of four exons and was shown to be localized on chromosome 2 of the P. Correspondence address: Bernhard Knapp, Behringwerke AG, P.O. Box 11 40,3550 Marburg/Lahn, F.R.G.

Abbreviations: PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SERP, serine-stretch protein; UWGCG, University of Wisconsin Genetic Computer Groups.

Note: Nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank TM and DDBJ data bases with the accession number M 37089.

falciparum genome [10]. In our hands SERP migrates as a l l3-kDa polypeptide in SDS-PAGE [1]. An N-terminal signal sequence was shown to be used for the export of SERP from the. parasite [11]. Indeed, the antigen was detected in the parasitophorous vacuole by immunoelectron microscopy [1,3,5]. A function of SERP as a proteinase has recently been suggested by alignment data [12,13]. Here we report the isolation of a gene which encodes SERP H, a protein highly homologous to SERP. We compare both antigens with reference to gene structure, gene localization, gene expression, characteristic protein regions, molecular weight and protein localization within the infected red blood cell. We further present data concerning the conservation of the antigen and discuss its possible function as a cysteine proteinase. Materials and Methods

Preparation of biological material. The cultivation of the P. falciparum strains FCBR (Colum-

0166-6851/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

bia), Palo Alto (Uganda), SGE2 (Zaire), ItG2GI (Brazil) and FVOR (Vietnam), the preparation of antigens, isolation of DNA and poly(A) + RNA as well as analysis of this material by Southern, Northern and Western blot technology have been described previously [ 1,14].

Screening of an EcoRl* library. 105 plaques of an EcoRI* library described previously [15] were screened by standard methods [16] with nicktranslated insert DNA of the ,~gtl I phage clone 41-10 which was isolated with an antiserum raised against a 41-kDa protein band of P.falciparum, as reported earlier [14]. Out of four phage clones hybridizing with the insert DNA one was choosen to isolate a malaria-specific DNA fragment of 1.25 kb in size using the restriction enzymes EcoRI and PvulI which was subcloned into the the Bluescript vector pKS (Stratagene) to obtain vector p41-10A for sequencing. Construction and screening of a cDNA library. In order to isolate additional 41-10 specific sequences, a cDNA library was prepared essentially as described by Gubler [17]. 5 #g of poly(A) + RNA was transcribed to cDNA using the MLV reverse transcriptase (BRL); ds cDNA larger than 500 bp was sized by gel electrophoresis and eluted using the GeneClean system (Bio 101 Inc., California). The ds cDNA was ligated using a 20 molar excess of an EcoRI adaptor (Pharmacia), phosphorylated at their 5' ends using polynucleotide kinase and separated from the oligonucleotides by agarose gel electrophoresis with subsequent phenol extraction. The ds cDNA carrying EcoRI sticky ends was introduced into the vector ~gtl 1 by the method of Huynh et al. [18]. A cDNA library of 2 x 105 recombinant phage clones was obtained and amplified. The library was screened with nick-translated insert DNA of vector p4110A and one phage clone hybridizing with the insert was identified. The insert DNA of 2.45 kb was subcloned into the Bluescript vector pKS (Stratagene) to yield plasmid p41-10B. Cloning of the 51 gene region using inverse polymerase chain reaction. A 260-bp XbaI-EcoRI fragment located at the 51 end of the cDNA insert was isolated from plasmid p41-10B and used

for Southern blot analysis of genomic P. falciparum DNA (strain FCBR) digested with different restriction enzymes according to procedures reported previously [1,14]. A 1.4 kb genomic SpeI-fragment hybridizing to the 260-bp probe was identified. Genomic, SpeI-restricted DNA of 1.3-1.5 kb in size was isolated, ligated and restricted with AccI. This approach resulted in an inversion of the known sequences to the ends of the genomic SpeI fragment. The oligonucleotides pl (5'-GGGGGAACTTCAAAATAAATGTTCTGAAGG-3', corresponding to bases 1365-1384 and located near a" SpeI site at its 5' end) and p2 (51-TATGACCCACAGGATCCCGTAACCT TAACCCCATT-3 I, complementary to bases 1219-1253 with a base change in position 12 resulting in a BamHI site) and 100 ng of the genomic DNA were used for the inverse polymerase chain reaction [19] which was carried out under standard conditions using the Gene-Amp TM kit of Perkin Elmer-Cetus. The 1.24 kb product was digested with SpeI and BamHI, purified by agarose gel electrophoresis and introduced into the Bluescript vector pKS to obtain plasmid p41-10C for sequencing.

Cloning of the 3' gene region. Starting from 1 #g of poly(A) ÷ RNA single stranded cDNA was prepared using MLV reverse transcriptase (BRL) and 1.3 #g of the oligonucleotide p3 (5'GACTCGAGTCGACGAATTC(T)I7-3') instead of oligo(dT)ls. The ss cDNA and the oligonucleotides p4 (5'-GACTCGAGTCGACGAATTC3', adaptor sequence carrying the restriction sites XhoI, Sail and EcoRI) and p5 (5'-GAGATAAAACTAGTTGTATATCTCAAATAG- 3', corresponding to bases 2745-2774 and carrying a SpeI site) were applied for PCR essentially as described by Frohman et al. [20]. A 960 bp fragment obtained by this approach was digested with Sail and SpeI for introduction into pKS to yield p4110D for sequencing. Using this approach, we did not succeed in cloning the complete 3' region of the gene. Therefore, a subfragment of p41-10D was used to screen a cDNA library prepared by hexanucleotide primers. 2 #g poly(A) ÷ RNA were converted to ds cDNA using 4 #g of random hexanucleotides (Amersham) as primers according to a protocol

of Gubler [17]. The ds cDNA was sized using a sepharose CL-4B column (Pharmacia) and DNA larger than 500 bp was pooled. The ds cDNA was ligated with an EcoRI-adaptor of Promega using the protocol of the supplier and introduced into the EcoRI site of the vector ,~gtl 1 resulting in a library of 1.5 x l0 s recombinant phages. This gene bank was screened with a labeled 270-bp fragment obtained by restriction of plasmid p41-10D with Avail (position 3431) and Sail. 13 positive phage clones were obtained. Individual phages were used for PCR using the oligonucleotides p6 (5'GGGGGATCCTGGAGCCCGTCAGTATCGGCGGAA-3', which corresponds to the 5' border of the )~gtl 1 EcoRI site and carries a BamHI site at its 5' end) or p7 (5'-GGGAAGCTTGGTAGCGACCGGCGCTCAGCTGGA-3', which corresponds to the 3' border of the/~gtl 1 EcoRI site and contains a HindlII site at its 5' end) in combination with oligo p8 (5'-AAAAGCTTATGAACCAGAAACATCTCAAGATI'TTGA-3', which corresponds to bases 3586-3615 of the 3' end of the gene and carries a HindlII site at its 5' end) according to a protocol of Giissow and Clackson [21]. Using the oligonucleotides p6 and p8, a 530-bp DNA fragment was obtained as largest product from one of the phage clones which was digested with the enzymes BamHI and HindlII and introduced into pKS to yield plasmid p41-10E for sequencing. The DNA insert of the corresponding ~gtl 1 phage clone was isolated using EcoRI and the resuiting 2.4-kb fragment was subcloned into pKS to obtain p41-1 OF.

DNA sequencing. Both strands of the insert DNAs of the plasmids p41-10 A-E were sequenced by the chain termination procedure using the sequenase system from USB (Cleveland, OH). Suitable subfragments of the insert DNAs were obtained by subcloning from convenient restriction sites into the Bluescript vectors or by using the exonuclease III/S 1-nuclease method according to Maniatis et al. [16]. Analysis of the sequencing data obtained was carried out using the UWGCG programs [22]. Pulse-field gel electrophoresis, Chromosomes from the P. falciparum isolate FCBR were prepared for electrophoresis as described by van der

Ploeg et al. [23]. Chromosomes were separated on a 1% agarose gel at 180 mA at 10°C for 27 h at a pulse frequency of 60 s-~ in both directions using the Gene Line System of Beckman. Upon completion of electrophoresis the gel was stained with ethidium bromide and DNA was transferred to a nylon membrane (Gene Screen, Dupont) by the Gene Line system according to the protocol supplied. The blot was hybridized with a labeled 800-bp EcoRI-PstI fragment of the SERP gene [1] and with a labeled 660-bp XbaI-SnaBI fragment of the plasmid p41-10B, performed as described previously for Southern blots [1,14].

Expression of partial sequences. The insert DNA of the /~gtll original clone 41-10 was subcloned into the EcoRI site of the expression vector pEX31b [24], giving rise to the plasmid pEX41-10a. The pEX31a,b,c vectors were modified by introduction of the pUC18 EcoRIHindlII polylinker into the EcoRI-HindlII sites of the vectors giving rise to the expression vectors pEX32a,b,c (B. Knapp, unpublished results). For the expression of a specific gene region which is missing in the SERP gene the oligonucleotides p9 (5'-ACCATGGCAACGCGTGGTACCCTAACATTACCTAGTGAATCACCTAGT-3', corresponding to bases 2339-2365 and carrying NcoI, MluI and KpnI sites at its 5' end) and pl0 (5'-GGGTCGACAAGCTTACAATAqq'TACTATTTTTAAAATAAGGTG-3', complementary to bases 2691-2721 and carrying Sail and HindlII sites at its 5' end) were used to amplify a 380-bp fragment by PCR. The DNA fragment was digested with KpnI and HindlII and introduced into the expression vector pEX32b, giving rise to plasmid pEX41-10b. Both plasmids were transformed into the Escherichia coli strain POP2136 which was induced to express the malaria-specific insert by a temperature shift [25]. The purification of the MS2-polymerase fusion proteins and the preparation of antisera were performed as described previously [25]. Amplification and sequencing of the gene specific fragments from different P. falciparum isolates. 0.5/~g DNA from the P. falciparum strains FCBR, Palo Alto, SGE2, ItG2G~ and FVOR were used in combination with 300 ng of 5'

and 3' oligonucleotides each to amplify genomic fragments by PCR using the Gene-Amp TM Kit of Perkin Elmer-Cetus. Before adding of DNA the probes were UV-treated for 5 min using the Stratalinker TM 1800 to avoid background amplification. The oligonucleotides p l l (5'-ACCATGGCAACGCGTGGTACCAAATGTGAAGGAAATAAAGTGACTGTG-3', corresponding to bases 736-762 and carrying NcoI, MluI and KpnI restriction sites at its 5' end) and p12 (5'GGGCTGCAGTTAAAGTTGTATTACGCTGTATTTTGT-3', corresponding to bases 1297-1320 and carrying a PstI site at its 5' end) as well as p9 and p l 0 were used to amplify 590 bp and 380 bp genomic fragments from the five parasite strains. The products were digested with KpnI and PstI and with KpnI and HindlII, respectively, and introduced into the vector pKS for sequencing.

lmmunoelectron microscopy. Immunoelectron microscopy after embedding of schizonts in LR White was performed as reported previously [1]. Results

Identification of a Agt11 clone coding for a protein homologous to SERP. By screening of a genomic Agtl 1 library from the P. falciparum strain T9.96 with an antiserum raised against a gel-purified protein band of 41 kDa, 16 phage clones were isolated [14,15]. Most of these clones code for different gene products and were identified due to serological crossreaction. Using the best-fit program of UWGCG, the malaria specific 164-bp sequence of one of the clones, 41-10, was shown to have a high grade of homology to nucleic acid positions 2142-2274 of the SERP gene isolated previously [1]. The deduced amino acid sequence of the insert DNA from clone 41-10 was found to be closely homologous to the amino acid positions 466-517 of SERP (Fig. 5). Northern blot analysis using the insert DNA of the Agtl 1 clone 41-10 and a 800-bp EcoRI-PstI fragment of the SERP gene [1] as probes reveal abundant 3.8 kb mRNAs for both genes (Fig. 1). The probes do not cross-hybridize as shown by Southern blot analysis indicating that each recognizes a different mRNA species of the same size and that both genes are indeed expressed.

¢1

b kb

-6.1 -5.1 -4.1 -3.1

-- 2.0 -- 1.6

--1.O Fig. 1. Northern blot analysis of schizont poly(A+) RNA hybridized with 32p-labeledinsert DNA of the phage clone 41-10 (a) and with a SERP-specific800-bp EcoRl-PstI DNA fragment (b). The 1-kbladder (BRL)was used as molecularweight standard. The insert DNA of clone 41-10 was expressed in the vector pEX41-10a as a 21-kDa MS2polymerase fusion protein. Antisera raised against the purified expression product react with two protein bands of 130 kDa and 113 kDa as shown by Western blot analysis of polypeptides from P. falciparum schizonts (Fig. 2a). The latter band is due to crossreaction with SERP (Fig. 2c); because of its homology to SERP the 130-kDa polypeptide was named SERP H.

Isolation and characterization of the complete gene coding for SERP H. As reported in Materials and Methods, three genomic clones (p4110A, p41-10C, p41-10D) and three cDNA clones (p41-10B, p41-10E, p41-10F) were isolated, spanning the entire coding region (Fig. 3). The complete nucleotide sequence and the deduced amino acid sequence for SERP H are shown in Fig. 4. The 5' (nucleotides 1-300) and 3' (nucleotides 3971--4111) non coding regions are extremely A+T-rich (87% and 85.1%, respectively) as expected for non coding regions of P. falciparum DNA [26]. Taking into account intron sequences of 544 bp which are spliced out and a mRNA

abc kD 200---

116--

m

97--

3

--

Fig. 2. Western blot analysis of polypeptides from P. falciparum schizonts with an antiserum raised against the MS2polymerase fusion proteins expressed by the vectors pEX4110a (a) and pEX41-10b (b) as well as with an antiserum raised against an expression product of a SERP specific fragment (c).

of 3.8 kb in size, the non-coding regions are assumed to extend for approximately another 250 bases. The ATG codon in position 301 is assumed to be the initiation codon since further ATG trinucleotides located upstream are immediately followed by stop codons. However, the ATG codon in position 331 may also be used as initiation codon. The coding region of the SERP H gene is interrupted by three introns of 345, 92 and 107 bp (Figs. 3 and 4). The exact locations of the intervening sequences were determined by comparison of the genomic and cDNA sequencing data. All three introns of the SERP H gene start with GT and the introns two and three end with AG preceded by a polypyrimidine stretch which is in agreement with the consensus sequences described for other eukaryotic introns [27]. Only the 3' boundary of intron one shows some variation to the consensus sequence because three pyrimidine bases are changed to purines. The intron positions of the SERP H gene correspond exactly to those of the SERP gene (see Fig. 3 and ref. 1). The coding region of the SERP H gene, with an A+T content of 72.3%, is divided into four exons which encode 1041 amino acids with a calculated molecular weight of 120 152. The first exon carries an initiation codon with a stop codon 18 triplets upstream in the same reading frame and encodes a hydrophobic stretch of 21 amino acids. Together with the following five amino acids encoded by the second exon, this sequence probably forms a signal sequence. The second exon

1

3

4

rq '~I ¢1¢%~ I I ~ I

i

--

::.. =,,. . . . . . .

s~ pSI

~

pl2pl

pIQ

41 I I~"'

I0

k

I

I

B

I

C I

I I

I

D IE IF

Fig. 3. Restriction map and structure of the SERP H gene. The coding regions (boxes) are separated by three intervening sequences. The SERP H-specific regions are hatched and the protease consensus sequences are dotted. The regions corresponding to the insert DNA of phage clone 41-10 and t o t h e cloned fragments of the vectors p41-10A-F as well as positions of the oligonucleotides used are indicated.

1 aaaaaggagaaaaataataaaaataatatgatatatattacatatatatatatatatatattatattatattattaaaatgaagtaataEgaaaaatacatgaatgacaEaaaattataE i2i tctctttaa~ttatat~taca~ttt~aa&at~aaEat~tt~aatEttatt~tttEtt~a~ttaaEta~a9attt~t~taaag&ctaaaa9g~aaaatattt~t~ctttaag~ 241 gaa ta t taaiaaaaa gaaa Eaaaaa taaaaaaaaaaaaaacagaaaaagEaaaagaccaaATC~TA'iTri'r~AATFFrAAATrAAAC~ATC~TATGTCCTA rrrrri'iCClnTATATA M

I

F

F

N

F

361 ATA~gtaataataaatatataaaccataaEaaataaataaataaataaatatatatataEatatatatgtgtatct

K

L

N

R

M

I

C

P

I

F

F

L

Y

20

I

ttct tct tt tt tt tcaat ttaaat tatgtgatt tgtacaatgta

22

481 ca ta taaaaatatacacaaaaaaaaaaaaatata tatat tatatata tatatata tatatatata tatatggaaa tat tataaaata taaggggaaaaaaaaaaaat t taca t taataaa 601 atatataatatatat ttatat taaggagaatatatat tt tgcaatgtttcat tgt tatataataaataataaataaatatatatatacatatatatatttttaatgcagATGTGT~ATI'r L F

25

721 ACGCAATA i ~ i T ~ T r A A A T G T C ~ T A A A G T G A C I ~ T A T C A C A T A A T A A T C f ~ C A T A A C C ~ T A A T F 7 3 % G A T G T r A A T A A A A A % ~ G G T G T G A ~ ~ ~ T T Q Y I K E G N K V T I S H N N G H N D L D V N K N G V I

S

Q

E

N

V

F

D

65

841 ACITCAGAAA~ I T I T A A A T I T A C C I T C A A A T A A A ~ T T G G T T C C G A T C ~ T I ~ T A C ~ C A A C T A T I T C A T T C A C T G T A C C A G A T A A T ~ ~ ~ T S E S L N P S N K K V S D D L N T T T I F T V P D N

E

N

E

V

K

V

V

S

S

105

961 ~ T C C ~ T G C C A C T G T A T C T C A T A ~ T G A C A ~ T T G ~ T A C C ~ C C A A A T G T A A C C C A A T C G G T A T C T T C A T C T A C G C A T A C A C C A ~ S E S G K G A T V S H T K V T S E G L S D T Q N V T Q

S

S

T

H

T

G

S

L

145

I

N

S

185

S

V

1081 GAI~ZAACAATGTCTACAGAACAC, C A T ~ T C A C A A T C ~ C ~ f C C A A C T C g ~ T C A T C A T C ~ C ~ ~ ~ ~ ~ ~ D 8 T M S T Q H S S V S S S L P T E S S S T L N K A T V P E P I Q 1201 G G A ~ r i ~ i ~ A A A A A A T T A T A A T C ~ T E A A G G T E A C G G ~ T C A T G T G G G T C A T A 1 ~ I ~ 3 T A T A ~ C T ~ X ~ T P C C T C A C A T C T r A A T I ~ ~ ~ ~ G L L K N Y G V K V T G C G S Y F R V Y L V P H I L I Y

A

L

T

1321 G A G T C C I ~ A T ~ A A T G A T A A C C ~ T I ~ A T G ~ ~ C T I ~ T A A A T G T I ~ T A T C A T T I T A A A ~ T A T A A C ~ f - A T A A T G T G T ] ~ A A A C E S L F N D A H I D V E K G E L Q N K C S G Y H F

V

V

Y

1441

K

L

K

Y

S

V

I

Q

L

225

T

H

N

V

L

N

265

CTCAAA~CATATAAGCCTAACGAAC4~TCAAAC~gtaaaaataaataaatatatatataaatatatatatatatatatatatatataatattcactctctttttaatttcttat L

K

W

K

T

Y

P

N

E

E

S

K

1561 a t t t t c t t t £ a gGTC,A A C ~ T T C G G A T G T A A C ~ T A ~ T C C C A A A A T I ~ ' A C G T C C A T I ' T A C T ~ C A T A C A A G T I ' ~ A ~ ~ ~ ~ ~ E D S D V R K Y R I P K L E R P F T S I Q V Y

T

A

N

S

K

A

G

V

I

E

T

K

N

1681 ATr-ATAATATAAGGACCC~`TATPCCTGgtttgtcaagaaattaaaaataacaaaatgaaataaattacatatatatatatatatatatatatatatgtgtatatattcatttatcatata Y N R T D P D 1801 t t t c t t t a t t t t agATACI?GTGATGCAATI~CACTGACTG'ITI"r I " I G A A T G G T A A C G T T A A T A ~ ~ % ~ ' m _ A C C T I ~ T I ~ T A A A T C A C A T G A T C D A A T D C F L N G N V N I E K C F Q C T L L V

Q

1921 ATGTFI~AAG TAC G T A T C A A G T C ~ T G A ~ C ~ A T G A A C G A A A T A A ~ C A ~ A T C , C F K Y V S S E M K K M N E I V

C E

2041 T A A A A T A T A T A A A A A ~ C A A A T A A A C L-L"~"r ~G A A A T ~ T I T A A T T A A T T T ~ C , K I Y K A N K P E I S K D L N

A K L

D

D

2 T F N

I P

~ T C C G ~ N E Y K L

A

Q

E

AT~TrATCAA~TGAATTATrAC, D L D Y Q F K N E

2161 T C , A T A C T A G T G G A A C A T T A C , A A G A A T A C ~ T r A C ~ T C ~ T A T A T A T A A T A A ~ C T A C ~ T T ~ K T - A ~ C G A A A A T A T T G T A A D T S T L E E Y E L G N A E D Y N N L T R L L K S 2281 ~ T A C A C , R N T

279

S

CTA~TAA~TGTI~ATGAATGGATATrAAATA~~CAT~AC A I C K N V D E W I L N

R

G

L

T

L

~ T C A C P S E S

H

S

L D

L E

T I

K

K

D

K

2521 A G ~ T G A T G A G A A T A A T A T A A A T A A ~ T A C T A A T r A T G T I T A ~ G A ~ T G A T G A T G A T r A T G A T A A C A A T A G T ~ A ~ T A ~ b ~ E A T C , A A A G C C D D E N N I N N G D T N Y V Y D D D D D Y D N N S E K D

D

324

S

H

E

359

_ ~ ~ ~ ~ S I D N I

L

S

399

AATATTGTAAAT~ATrAAA~ E Y C K L L K K

V

439

N

.CT L

479

T

519

I

V

T

L

Q

G

K

CT~/3TGAATCATC C A G T A A A ~ ~ C S E S S S K D S Y L N

2401 TTTTAATC~ACAAGGATAAAAACC~TAA~.~TC.ATATC, A G T A A A A A T T C G A ~ T I ~ A T C . A ~ ~ C F N D K D K N E D D D M S K N S K E E F K N D D K N S D

315

~ Q

N

~ N

~ D

N

599 639

S

N

K

559

Y

E

S

CCATAAAC, C,A A A A ~ P I K E N

2641 G A A T G G T G T E A T ~ u A T I T A C , A A A A A T A T G G T A A C C A A A T A A A A T T G A A A T C A C C I T A T I ? T A A A A A T A G ~ 3 % A A T A ~ T T A T C ~ A T A T I ~ ~ ~ N G V D L E K Y G N Q I K L K P Y F K N S K Y C Y E Y C N

R

W

R

D

K

T

S

H

TCGA~A~TATTAGATGTA~TGGTCA~TIT.AC~ F E T R C M R G Y G H

F

R

S

S

A

679

E

ITIT~C~TATEAC, A~GAAAA~-ITI-I-~C~~ F L R L D E K K F L P L

E

S

N

Y

719

C r A A A T r A C C A A A T A ~ C A A A T T T A ~ T A C A A A A ~ ~ ~ ~ P K L P N W T N L W G D T K L F N K K V H R Y I

G

N

2761 TATA ~ I S

T Q

~ E

V

E

E

Q

G

~TrA~TATI~T~n%AGTTACA~f N C G L C W I F A

2881 A T T A T A T ~ T T G T T C T A ~ G A A A A C C T A T A ~ T A G A T G T G A ~ T C ~ T C C A T ~ _ ~ L Y V A N C S K R P I D R C E 3001 TCCGTATTCATATACAAGTGCAC, G r A A T T ~ P Y S Y T S A G N C

G

S

S N

3121 AGGATITATATCACATC~AACATCTTATTI?AAAAATAATATGGATTTATTTATAGATA" G F I H E T S Y K N N M D L I D M 3241 TATAGGT~ATC, A'I"IT ~ % A T C ~ T A A A ~ I G Y D F N G K G

V

3361 T E A ~ T G G T T A A ~ , ~ ~ T A T I ~ T G A A G C , Y W L I R N S W S Y 3481 A C T A G A T I ' 5 ~ C ~ T A C A ~ T A C C T A A A ~ L D L G T I H V P K 3601 A T C I ~ S Q

K P

V

L L

K

" R E

V

Q

K

G

CATAGTA.I%3.1~,.IL~TAGAACACCTC~CCATC~TATTA~TC~TTATATTAA H S M C G D T P D H A A N I I G Y G Y K

W K

S

D W

E K

V

K

N

Y

I

N I

C N

V

3721 Af-AAATACTC CATATATr A A A A C ~ / Z A T r A A A G A T T C A C A A A T A A A C ~ T ~ A T A T G A T A A ~ A T A A A T V _ 4 ~ A ~ C A A A A G A ~ X I ~ A ~ ~ ~ ~ Q I L H I L K H I K D S Q K R G L V K Y D N I N E T K D E

H

T

C

T I ~ T D L Y

~ K

F

E

S

E

3841 T C ~ T A T C ~ T G T A A A A A A T I ~ I ~ r I - ~ % A C C A A G T C ~ T C ~ T A A A C ~ T C A T r A T T C A C A E K Y E E C K" K F C L T K W N E C K 3961

K

K

G

T

V

E

K

V

1-IT1-I-~%A F F K 879

T~TI'r CI~TAATACTGATTITATGTATAG~TATrATAATAAT~ATC,~ACCAC~ N V Y F L R H N T D M Y S L Y Y N N Y E

C D

H

Y

S

P

A G

C Y

~

T E

~ L

G G

799

TC

AAATFITAC,~%TA%~C~TTG~CAA.~-z-~-~TACATACTGTTGTA G N F R V D M L G P K N C L Y N F

uTrI-i~3TACATGC~CA~.GCC~TC.~ATCAGA"I~GAAA ~ T A F V H G Q 8 D E S D E T N

759

TrATATATATAAAGACTCAAGATGT I I Y I K T Q D V

T K

D

TC,A ~ A T C ~ T C , N D Y D N

G

S

M

P

R

E

839

T

CACAA~fCTGTC, G A C ~ T H N S V E K K R ~

V ~

E

919 959

N

S

Q

D

999

C

N

F

C

1039

~ D

T~ATGTAtaagaggatatataaaggaacaatataaaaataaattaatatatatatatatatatatatatatatatatataataaattacttttggatggattacgatttttttttttttt Y

V

1041

4081 tttttttt tttttcttattaataatggcac

Fig. 4. Nucleotide sequence of the SERP H gene of the FCBR strain of P. falciparum and the deduced amino acid sequence of the coding regions• The nucleotide sequence of the coding regions is shown in capital letters while the sequence of the non-coding regions is indicated in lower case. The potential N-glycosylation sites are denoted by asterisks.

contains no repetitive segments and encodes 257 amino acids of which 23.3% are serine (14.4%) or threonine (8.9%) residues. The third exon codes for only 45 amino acids and the fourth exon has an open reading frame of 2154 bp encoding 718 amino acids. With the exception of a dimer of the tetrapeptide PSES located in position 502-509 the protein sequence reveals no repetitive elements. Seven potential N-glycosylation sites for asparagine residues were found at positions 84, 130, 459, 554, 583, 684 and 984.

tophan residues, are identical. The alignment reveals no significant homology for the N-terminal 240 amino acids of the two proteins as well as for a second segment encoded within exon 4. The N-terminal region of SERP H lacks the serine stretch which is characteristic for SERP; however, its high content of serine and threonine is similar to that of the corresponding SERP region. A 121-amino-acid region between residue positions 503 and 624 is highly specific for SERP H, while SERP contains a different fragment of 52 amino acids. By sequence analysis using the peptide structure program of UWGCG this SERP H specific protein region was found to be highly hydrophilic which is in agreement with the number of charged amino acid residues (32 acidic and 18 basic) found in this region.

Amino acid sequence comparison of SERP H and SERP. Fig. 5 shows an alignment of the amino acid sequences of SERP H and SERP. Both sequences show an identity of 38% indicating a significant homology of both antigens. Considering only the amino acids from position 243-503 and 624-1041, the identity between both proteins increases to 48.8%. In this regions 25 of 26 cysteine residues, 7 of which are found within the 50 C-terminal amino acids, as well as 9 of 11 tryp-

Localization of the genes encoding SERP and SERP H. Using the Gene Line system for pulse field gel electrophoresis we were able to separate chromosomes one to five of the P.falciparum iso-

1 MIFi%r~XIM~ICPIFFLYIINVI,I ' I X } Y F I K C I I e a V I ~ S ~ S Q I ~ F - Z I A % P S N K I C V ~ D D I / V I T T I S ~ ' T V P ~ i

....

, .....

.

.

.

.

.

.

.

.

.

.

.

.

i00 .

.

.

.

I01 KVVSSSESGKGATVSHTKVTSEGLSD'I~PN...~SSTHTPC~LDSTMSTEQ{-ISS'~oQSS~SSETLNKA'IVPEIPIQINSGLLKNYNGVKVT 198 G .................. SCGSYFRVYLVPHILIYALTKYSVIQLESL. F N I A N A R I ~ G ~ C S I D G Y I - I F K L V V Y I ~ .

197

KPNEES 277

17s

274

278 KSEDSDVRKYRIPKLERPFTSIQV..YTANSKAGVIETRNYNIRTDIPDTCDAIATDC~IEKC~'q)C'I~uL'VQKKDKSHECFKYVSSI~I

375

376 KVKAQ[X)FNI:~..EYKLIESIDNILSKIYKKANKPFEISKDLINLEDLDYQFKNELLEY . . . . . . . . . . . . . .

IYI~TRLLKSHSD~IIV 473

474 T ~ G K L E N ' r ~ e ~ L m ~ , ' I ~ . ~ S ~ S P S E S S S K S D S Y U ~ r F ~ K D m ~ E D K D D M S K ~ S K E E F ~ S ~ ~ ~ 57a . . . . . . . I I " " 574 Y D F D { X ) D Y I ~ S Y E K D M Y E S P I ~ D L E K Y ( ~ Q I K L K S P Y F K N S K Y ( 3 w ' Y E Y ~ K T S C I S Q I ~ F A S K L H F E T I R C M R G Y G ::

:

:

:

:

::

::I

:

:

:

:

673 :

I:

674 HFRSSALYVANCsKRKPID~LEFLRILDEKKFLPLESNYPY~YTSAGNSCPKLPNSWTNI~FNKKVIn~YI(i~KGFIs~~ 773 702

774 D L F I ~ [ C ~ I Y I ~ G I ~ H q C _ ~ G V ~ R T P ~ I I ~ I N K K G E K R S Y W L I R N S W S ~ K N C L Y N F I I ~ I

873

874 ~ F K L D L G T Y : H V P ~ F M Y S L ~ E ......T S Q D F E S E I ~ D ~ ....... ~DESDE'INKEGKNVI-~SVEKKi 959 803 S ~ ~ ] ~ ' I - I ~ d ~ E S I ~ I Y D ~ ' ~ P E ~ L ~ K K N L F S ~ i I F C ~ ~ ~ 902 960 QILHILKHIKDSQIKRGLVKYDNIN~..-~'KuEI-ITCSRVN~KKFCL~SI~-"YCLT6LYKC, EI:X2NFCYV 1041

Fig. 5. Comparison of the amino acid sequence of SERP H with the amino acid sequence of SERP [1] using the gap program of UWGCG. Identity is denoted by lines and conserved amino acid substitutions are indicated by colons.

late FCBR as described in Materials and Methods (results not shown). The chromosomes were hybridized with probes specific for the genes encoding SERP and SERP H using conditions which do not result in crossreaction between the homologous genes. Both genes were found to be localized on chromosome 2. Localization of the SERP gene on chromosome 2 has also been described for the P. falciparum Brazilian clone ItG2F6 [10]. Recently, we found that the nucleotide sequence upstream from the Xbal site in position 1077 of the SERP H sequence is identical to the nucleotide sequence from the 3' XbaI restriction site of the 5.6-kb XbaI genomic clone carrying the SERP gene [1] suggesting that the SERP gene is immediately followed by the SERP H gene. The TAA stop codon of the SERP gene is located about 1 kb upstream from the ATG start codon of the SERP H gene. This 1-kb segment should contain the 3' non-coding region of SERP, the 5' non-coding region of SERP H and the promoter region for the SERP H gene (B. Knapp, in preparation).

Conservation of the SERP H gene. The nucleotide sequence from position 2236-2399 of the SERP H gene from the P.falciparum strain FCBR is found to be identical to the insert DNA of clone 41-10 obtained from genomic DNA of clone T9.96. Furthermore, DNA fragments corresponding to nucleotide position 2339-2721, coding for the SERP H specific fragment from amino acid position 499-626, were amplified and sequenced for the P. falciparum strains FCBR, Palo Alto, SGE2, ItG2G~ and FVOR. This 383-bp fragment of the SERP H gene was found to be identical in its sequence for all the parasite strains analysed. In addition we have investigated the variability within the N-terminal part of SERP H, as the corresponding sequence of SERP shows some variability within different strains [1,2,4]. We have isolated a DNA fragment of five different P. falciparum strains by PCR corresponding to bases 736-1320 which encodes the N-terminal amino acids 31-225 of SERP H. The fragments amplified from genomic DNA of the strains Palo Alto, SGE2 and ItGaGl were identical in their nucleotide sequence to the FCBR sequence. Only the fragment originating from the FVOR strain shows two base pair transitions: the A in position 808 is

exchanged by a G resulting in a N ~ D substitution and the T in position 820 is replaced by a C resulting in a S ~ P exchange on the amino acid level. These data show that the SERP H specific protein regions are highly conserved among different P. falciparum strains and allow us to speculate that the conservation is extended to regions of SERP H outside these sequences.

Preparation of SERP H specific antisera. The SERP H specific fragment ranging from amino acid position 499 to 625 was expressed using the plasmid pEX41-10b which was prepared as described in Materials and Methods. The MS2polymerase fusion protein shows a molecular weight of 36 000, which is 9 000 higher than the size expected (results not shown). Using antisera raised against the expression product only one malarial antigen of 130 kDa was detected by Western blot analysis of schizont proteins (Fig. :2b), indicating that the antisera are highly specific for SERP H. Localization of SERP H. The SERP H specific antisera were used to localize the corresponding antigen within the parasitized red blood cell. The SERP H antigen was found in the soluble as well as in the membrane fraction of schizonts, but was not detected in isolated merozoites by Western blot analysis (results not shown). Immunoelectron microscopy using gold-labeled second antibody showed the antigen to be localized mainly within the parasitophorous vacuole (Fig. 6) as was also reported for SERP [1,3,5]. Homology to cysteine proteinases. Recently it has been suggested that SERP or a processing product thereof may function as cysteine proteinase since homology to known cysteine proteinases was found in two regions forming the active site, region one containing amino acid residues 573-595 and region two carrying residues 745 to 787 of SERP [12]. Fig. 7 shows an alignment of both regions forming the active site of cysteine proteinases from different species (barley, carica, papaya, rat, slime mold) with homologous regions of the P. falciparum proteins SERP and SERP H. The protein regions of SERP H containing amino acids 644-666 and 816--858 were

trast to SERP, SERP H carries a cysteine residue at this position, the only cysteine residue which was found to be changed in SERP in comparison to the homologous SERP H regions (Fig. 5). These data suggest that SERP H or a proteolytical cleavage product of this antigen, may function as a cysteine proteinase. Discussion We have determined the complete nucleotide sequence and the structure of a gene coding for a 130-kDa P. falciparum protein, an antigen which is highly homologous to the 113-kDa polypeptide SERP and which was therefore called SERP H. Table I shows a comparison of the characteristics of SERP and SERP H. Both genes are expressed in high yield as shown by Northern blot analysis. The genes encoding SERP and SERP H are localized on the same P. falciparum chromosome and the coding regions are arranged in tandem, with a distance of only 1 kb containing the non-coding regions of the genes and the promoter region for the SERP H gene (B. Knapp, in preparation). The SERP gene linked to SERP H corresponds to allele 2 [2]. Also, allele 1 is located on chromosome 2, as only this chromosome is detected with SERP-specific probes. However, the exact localization of allele 1 in relation to allele 2 remains to be elucidated. For SERP H we found no evidence for the presence of multiple alleles by Southern blot analysis and PCR of different gene fragments. The genes for SERP and SERP H exhibit similar structures: four exons are interrupted by three introns located at exactly corresponding positions in both genes. Considering localization and structure of both genes, we suggest that they originate from a common ancestral gene by a dupli-

Fig. 6. Immunoelectron micrograph of a P.falciparum schizont section reacted with an antiserum raised against the fusion protein expressed by pEX41-10b, x 18 000.

found to be homologous to the corresponding regions of cysteine proteinases. Six residues in region one and 11 residues in region two were found to be identical for all species compared. The alignment of SERP H and SERP (Fig. 5) reveals an identity of 70% and 68% for regions one and two, respectively, exceeding the average value of identity. Outside these two active site regions, the similarity between the different proteinases is lower and is also reduced between SERP and SERP H. The active site cysteine, histidine and asparagine residues are highlighted in Fig. 7. Cysteine forms an ion pair with the imidazole group of histidine and with asparagine completing the essential 'catalytic triad' of the cysteine proteinases [28,29]. These residues are conserved in all the proteins shown, with the exception that SERP has changed the cysteine to a serine residue. Therefore, SERP has been suggested to be an intermediate between a cysteine and a serine proteinase [13]. In con# B a r l e y aleurain

.157aa

Papain ~Carica~apaya) P~tca~epsin H

.137aa .128aa .127aa .131aa .573aa

~

.643aa

~LCWIFASKLHFETI • _ ,_ ,_ _, ,_

Rat c a t ~ p s i n

L

slime mould cathepsin PIa~i~SERP PIa~i~SERP H

~

S

#

~

.. 123aa.. ~ V L A V ~ Y C

..ll8aa.. ..125aa.. ..122aa.. ..128aa.. ..150aa..

~ •

# V-~GVFYWLI K

N

~

NKVDHAVAAVGYC PNYILI[e~IR DKV~4AVLAVSYG-----E~NGLLYWIV~ KDLD[~&'VLVVGYGYEGTD-SNKDKYWL IK NSUX4GI L IVGYSAI@~I ~ V I ~ S W C , K[II~__ IY IYEADHAVNI~ECdKKKSYWI~

..150aa.. ~ I I G Y G N Y I N K K G E K R S ~ W L I I % N S W S ~ _,_ _**_ **__***_

**

,__

Fig. 7. Amino acid sequence alignment of the two regions comprising the active sites of five different cysteine proteinases (sequence data are taken from ref. 12) with homologous regions of SERP and SERP H. The three active site residues are indicated by #; residues identical across all sequences are indicated by asterisks and positions with only one conservative amino acid replacement are indicated by dashes.

10 TABLE ! Comparison of the characteristics of SERP and SERP H Characteristic

SERP

SERP H

mRNA size Gene localization Gene structure Amino acids Molecular weight Signal peptide Repetitive regions Conservation in different strains Non-homologous protein regions

3.8 kb chromosome 2 4 exons 985 113 kDa + + high aa 1-238 (two repeats) aa 503-549 parasitophorous vacuole serine/cysteine proteinase

3.8 kb chromosome 2 4 exons 1041 130 kDa + very high aa 1-242 (no repeats) "aa 504--623 (hydrophilic) parasitophorous vacuole cysteine proteinase

Antigen localization Possible function

cation or an amplification event. This multiplication process should have occurred earlier in the evolution of the P. falciparum genome than the duplication process resulting in the two different alleles of the SERP gene which are very similar in sequence. Therefore, the SERP gene seems to be subjected to an evolutionary pressure for duplication. SERP H migrates as a 130-kDa band in SDSPAGE, in contrast to the 120 kDa calculated from the deduced amino acid sequence. The difference in size is obviously the result of an unusual SDS binding behaviour of the highly hydrophilic SERP H specific protein region, as fusion proteins containing this region migrate at a higher molecular weight than expected from the sequence expressed whereas fusion proteins of other SERP H regions migrate quite normally in SDS gels (results not shown). An unusual migration behavior has also been described for other P. falciparum antigens [25]. Both proteins, SERP and SERP H, carry very hydrophobic regions at their N-termini typical for signal sequences. Recently, an N-terminal fragment of the SERP gene was expressed in a cell-free translation/translocation system and was found to be translocated cotranslationally across canine pancreatic microsomes in the presence of the signal recognition particle, suggesting that SERP is exported from the parasite cell via the endoplasmic reticulum [11]. Indeed, SERP was found by immunoelectron microscopy to be localized in the parasitophorous vacuole. A similar

transport way is assumed for SERP H because it carries a hydrophobic stretch at its N-terminus and shows the same localization within the parasitized erythrocyte as SERP. The SERP H gene encodes an extended hydrophobic stretch of 21 amino acid residues at its N-terminus. However, the translation may also be initiated at the methionine residue in position 11 which would form a hydrophobic sequence long enough to function as a signal peptide. Indeed, it has been shown that initiation of translation at methionine residue 11 forms a protein translocated across canine pancreatic microsomes (K. Lingelbach, unpublished results). The alignment of SERP and SERP H reveals two heterogenous protein regions which however were found to be highly conserved among different P. falciparum strains for the SERP H gene: the N-terminal 240 amino acids and a highly hydrophilic protein segment encoded within exon 4 of the SERP H gene. The N-terminal variations were found mainly in the fragment encoded by exon 2; it is not caused by a shuffling of exon 2 during evolution because the 3 ~ region of this exon codes for 36 amino acid residues found to be highly conserved between both genes. Most striking is the deletion of two repeat regions within SERP H in comparison to SERP, one coding for six octapeptides rich in glycine residues and another coding for 37 consecutive serine residues. However, the high serine and threonine content is common to the N-terminal parts of both proteins, suggesting that these may be important for the

11

function of both proteins. The second heterogenous region of 121 amino acid residues of SERP H is opposed to a 47-amino-acid segment in SERP. This region was found to be highly hydrophilic for SERP H (42% of the residues are charged) leading to an increase of the apparent molecular weight of the protein and may originate from insertion and deletion events during evolution. However, this protein region was found to be highly conserved among different parasite isolates suggesting that it is essential for the specific function of the protein. To avoid cross-reaction with SERP, antisera were prepared against the highly hydrophilic SERP H specific protein region in order to localize the antigen within the parasitized erythrocyte. Similarly to SERP, SERP H was found to be localized within the parasitophorous vacuole by immunoelectron microscopy. Attachment of SERP H to the parasite membrane and/or the membrane surrounding the parasitophorous vacuole cannot be excluded, as part of the protein was detected within the membrane fraction by Western blot analysis. An important question is that of the function of both proteins localized within the parasitophorous vacuole. SERP has been suggested to be a serine proteinase with a conformation of a cysteine proteinase because the essential cysteine is changed to a serine residue within the active site of the protein [13]. Our data show that P. falciparum expresses a second protein highly homologous to SERP and likewise found to be localized within the parasitophorous vacuole. In contrast to SERP, SERP H carries the essential cysteine residue within the proposed active site of the protein and therefore indeed may be a cysteine proteinase. Proteinases are known to be of major importance in the life cycle of Plasmodium. The hemoglobin hydrolysis is catalyzed by a combination of cathepsin D-like and acid proteases [31,32], neutral or alkaline endopeptidases [33,34] and aminopeptidases [35,36]. In addition, in vitro experiments with protease inhibitors suggested that crucial events such as merozoite release from mature schizont infected red blood cells, processing of different parasite proteins as well as erythrocyte invasion by merozoites could be mediated by specific proteases [37-43]. A 76-kDa antigen of P. falciparum was reported to be a serine pro-

tease which is activated by phospholipase C and may be related to merozoite invasion [44]. SERP and SERP H may function as proteinases within the parasitophorous vacuole, where they may be involved in the merozoite release from mature schizont-infected erythrocytes or/and in the processing of parasite proteins like the 195-kDa precursor protein of merozoite surface antigens. This is supported by the finding that SERP was identified in immune complexes formed upon schizont rupture in the presence of immune serum, which inhibits merozoite release [6]. The relatively high molecular weights of both SERP and SERP H suggest that they may be precursors of proteinases, which are activated by proteolytic cleavage. Indeed SERP was found to be proteolytically processed to polypeptides of 50, 47 and 18 kDa during merozoite release [3,45]. The 50-kDa subunit results from a 56-kDa intermediate product by cleavage with a trypsinlike enzyme. By inhibition of this proteinase with leupeptin, the 56-kDa intermediate protein was found and the release of merozoites was inhibited [46,47]. The authors conclude that the 56 to 50 kDa proteolysis may be required for the release of merozoites. The 50-kDa end product could be the activated form of a proteinase which then supports the release of merozoites. The SERP H proteinase may be activated by a similar process. Investigations on the proteinase activity of SERP and SERP H will be necessary to reveal the biological role of both proteins.

Acknowledgements We are indebted to K.-J. Abel for preparation of the oligonucleotides used in this study, E. Spangenberg-Beyer for electron microscopy and to K. Diehl, M. Gleisner, A. Greese, H.-H. Hahn and V. Isenberg for skillful technical assistance. This work was supported by the Bundesministerium fur Forschung und Technologie.

References 1 Knapp, B., Hundt, E., Nau, U. and Kuepper, H.A. (1989) Molecular cloning, genomic structure and localization of a blood stage antigen of Plasmodiumfalciparum characterized by a serine stretch. Mol. Biochem. Parasitol. 32, 73-84.

12 2 Li, W.-B., Bzik, D.J., Horii, T. and Inselburg, J. (1989) Structure and expression of the Plasmodium falciparum SERA gene. Mol. Biochem. Parasitol. 33, 13-26. 3 Delplace, P., Fortier, B., Tronchin, G., Dubremetz, J.F. and Vetoes, A. (1987) Localization, biosynthesis, processing and isolation of a major 126-kDa antigen of the parasitophorous vacuole of Plasmodium falciparum. Mol. Biochem. Parasitol. 23, 193-201. 4 Weber, J.L., Lyon, J.A. and Camus, D. (1987) Blood stage antigen genes of Plasmodium falciparum. In: Molecular Strategies of Parasitic Invasion (Agabian, N., Goodman, H. and Noguiera, N., eds.), pp. 37%388, Alan R. Liss., New York. 5 Coppel, R.L., Crewther, P.E., Culvenor, J.G., Perrin, L.H., Brown, G.V., Kemp, D.J. and Anders, R.F. (1988) Variation in p126, a parasitophorous vacuole antigen of Plasmodiumfalciparum. Mol. Biol. Med, 5, 155-166. 6 Chulay, J.D., Lyon, J.A., Haynes, J,D., Meierovics, A.I., Atkinson, C.T. and Aikawa, M. (1987) Monoclonal antibody characterization of Plasmodium falciparum antigens in immune complexes formed when schizonts rupture in the presence of immune serum. J. Immunol. 139, 2768-2774. 7 Perrin, L.H., Merkli, B., Loche, M., Chizzolini, C., Smart, J. and Richle, R. (1984) Antimalarial immunity in saimiri monkeys. J. Exp. Med. 160, 441--451. 8 Banyal, H.S. and Inselburg, J. (1985) Isolation and characterization of parasite-inhibitory Plasmodium falciparum monoclonal antibodies. Am. J. Trop. Med. Hyg. 34, 1055-1064. 9 Lyon, J.A., Thomas, A.W., Hall, T. and Chulay, J.D. (1989) Specificities of antibodies that inhibit merozoite dispersal from malaria-infected erythrocytes. Mol. Biochem. Parasitol. 36, 77-88. 10 Biggs, B.A., Kemp, D.J. and Brown, G.V. (1989) Subtelomeric chromosome deletions in field isolates of Plasmodium falciparum and their relationship to loss of cytoadherence in vitro. Proc. Nail. Acad. Sci. USA 86, 2428-2432. 11 Ragge, K., Arnold, H.-H., Tuemmler, M., Knapp, B., Hundt, E. and Lingelbach, K. (1990) In vitro biosynthesis and membrane translocation of the serine-rich protein of Plasmodium falciparum. Mol. Biochem. Parasitol. 42, 93-100 12 Higgins, D.G., McConnell, D.J. and Sharp, P.M. (1989) Malarial proteinase? Nature 340, 604. 13 Eakin, A.E., Higaki, J.N., McKerrow, J.H., Craik, C.S., Mottram, J.C., Coombs, G.H. and North, M.J. (1989) Cysteine or serine proteinase? Nature 342, 132. 14 Knapp, B., Hundt, E. and Kuepper, H.A. (1989) A new blood stage antigen of Plasmodium falciparum transported to the erythrocyte surface. Mol. Biochem. Parasitol. 37, 47-56. 15 Knapp, B., Hun&, E. and Kuepper, H.A. (1990) Plasmodium falciparum aldolase: gene structure and localization. Mol. Biochem. Parasitol. 40, 1-14. 16 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 17 Gubler, U. (1988) A one tube reaction for the synthesis of blunt ended double-stranded DNA. NucleicAcids Res. 16, 2726. 18 Huynh, T.V., Young, R.A. and Davis, R.W. (1985) Construction and screening cDNA libraries in Agtl0 and Agtl 1. In: DNA Cloning. A Practical Approach (Glover, D.M., ed.), pp. 49-78. IRL Press, Oxford.

19 Triglia, T., Peterson, M.G. and Kemp, D.J. (1988) A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 16, 8186. 20 Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998-9002. 21 Giissow, D. and Clackson, T. (1989) Direct clone characterisation from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res. 17, 4000. 22 Devereaux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395. 23 Van der Ploeg, L.H.T., Smits, M., Ponnudarai, T., Vermeulen, A., Meuwissen, J.H.E.T. and Langsley, G. (1985) Chromosome-sized DNA molecules of Plasmodium falci-parum. Science 229, 658-661. 24 Strebel, K., Beck, E., Strohmaier, K. and Schaller, H. (1986) Characterization of foot and mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. J. Virol. 57, 983-991. 25 Knapp, B., Shaw, A., Hundt, E., Enders, B. and Kuepper, H.A. (1988) A histidine-alanine-rich recombinant antigen protects Aotus monkeys from P. falciparum infection. Behring Res. Commun. 82, 349-359. 26 Weber, J.L. (1987) Analysis of sequences from the extremely A+T-rich genome of Plasmodiumfalciparum. Gene 52, 103-109. 27 Mount, S.M. (1982) A catalogue of splice junction sequences. Nucleic Acids Res. 10, 459-472. 28 Husain, S.S. and Lowe, G. (1968) Evidence for histidine in the active site of papain. Biochem. J. 108, 855-859. 29 Drenth, J., Jansonius, J.N., Koekoek, R. and Wolthers, B.G. (1971) The structure of papain. Adv. Protein Chem. 25, 79-115. 30 Horii, T., Bzik, D.J. and Inselburg, J. (1988) Characterization of antigen-expressing Plasmodium falciparum cDNA clones that are reactive with parasite inhibitory antibodies. Mol. Biochem. Parasitol. 30, 9-18. 31 Sherman, I.W. and Tanigoshi, L. (1983) Purification of Plasmodium lophurae cathepsin D and its effects on erythrocyte membrane proteins. Mol. Biochem. Parasitol. 8, 207-226. 32 Levy, M.R. and Chou, S.C. (1974) Some properties and susceptibility to inhibitors of partially purified acid protease from Plasmodium berghei and from ghosts of mouse red cells. Biochim. Biophys. Acta 334, 423-430. 33 Cook, L., Grant, P.T. and Kermack, W.O. (1961) Proteolytic enzymes of the erythrocytic forms of rodent and simian species of malarial plasmodia. Exp. Parasitol. I l, 372-379. 34 Gyang, F.N., Poole, B. and Trager, W. (1982) Peptidases from Plasmodiumfalciparum cultured in vitro. Mol. Biochem. Parasitol. 5, 263-273. 35 Charet, P., Aissi, E., Maurois, P., Bouquelet, S. and Biguet, J. (1980) Aminopeptidase in rodent Plasmodium. Comp. Biochem. Physiol. 65B, 519-524. 36 Vander Jagt, D.L., Baack, B.R. and Hunsaker, L.A. (1984) Purification and characterization of an aminopeptidase from Plasmodiumfalciparum. Mol. Biochem. Parasitol. 10, 45-54. 37 Holder, A.A. and Freeman, R.R. (1984) The three major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high-molecular-weight pre-

13 cursor. J. Exp. Med. 160, 624-629. 38 David, P.H., Hadley, T.J., Aikawa, M. and Miller, L.H. (1984) Processing of a major parasite surface glycoprotein during the ultimate stages of differentiation in Plasmodium knowlesi. Mol. Biochem. Parasitol. 11,267-282. 39 Hall, R., Osland, A, Hyde, J.E., Simmons, D.L., Hope, I.A. and Scaife, J.G. (1984) Processing, polymorphism and biological significance of P190, a major surface antigen of the erythrocytic forms of Plasmodiumfalciparum. Mol. Biochem. Parasitol. 11, 61-80. 40 Lyon, J.A. and Haynes, J.D. (1986) Plasmodiumfalciparum antigens synthesized by schizonts and stabilized at the merozoite surface when schizonts mature in the presence of protease inhibitors. J. Immunol. 136, 2245-2251. 41 Banyal, H.S., Misra G.C., Gupta, C.M. and Dutta, G.P. (1980) Involvement of malarial proteases in the interaction between the parasite and the host erythrocyte in Plasmodium knowlesi infections. J. Parasitol. 67, 623-626. 42 Dejkriengkraikhul, P. and Wilairat, P. (1983) Requirement of malarial protease in the invasion of human red cells by merozoites of Plasmodium falciparum. Z. Parasitenkd. 69, 313-317.

43 Hadley, T.J., Aikawa, M. and Miller, L.H. (1983) Plasmodium knowlesi: studies on invasion of rhesus erythrocytes by merozoites in the presence of protease inhibitors. Exp. Parasitol. 55, 306-311. 44 Braun-Breton, C., Rosenberry, T.L. and Pereira da Silva, L. (1988) Induction of the proteolytic activity of a membrane protein in Plasmodiumfalciparum by phosphatidyl inositolspecific phospholipase C. Nature 332, 457459. 45 Bhatai, A., Delplace, P., Fortier, B., Dubremetz, J.F. and Vernes, A. (1987) Immunochemical analysis of a major antigen of Plasmodiumfalciparum (P126) among ten geographic isolates. Am. J. Trop. Med. Hyg. 36, 15-19. 46 Debrabant, A. and Delplace, P. (1989) Leupeptin alters the proteolytic processing of P126, the major parasitophorous vacuole antigen of Plasmodiumfalciparum. Mol. Biochem. Parasitol. 33, 151-158. 47 Delplace, P., Bhatai, A., Cagnard, M., Camus, D., Colombet, G., Debrabant, A., Dubremetz, J.-F., Dubreuil, N., Prensier, G., Fortier, B., Haq, A., Weber J. and Vernes, A. (1988) Protein p126: a parasitophorous vacuole antigen associated with the release of Plasmodium falciparum merozoites. Biol. Cell 64, 215-221.