cloning of the plasmodium vivax duffy receptor

Molecular and Biochemical Parasitology, 44 (1991) 125-132 125 Elsevier

MOLBIO 01458

Cloning of the Plasmodium vivax Duffy receptor

X i a n g d o n g F a n g , D a v i d C. K a s l o w , J o h n H. A d a m s a n d L o u i s H. M i l l e r

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, U.S.A.

(Received 20 June 1990; accepted 4 September 1990)

Plasmodium vivax and Plasmodium knowlesi merozoites invade only Duffy blood group-positive human erythrocytes. Soluble P. vivax and P. knowlesi merozoite proteins of 135 kDa bind specifically to Duffy blood group determinants. The gene encoding a member of the Duffy receptor gene family of P. knowlesi has been cloned. We report here the molecular cloning of the presumptive Duffy receptor gene of P. vivax, using the P. knowlesi gene as a probe. There is a single gene in P. vivax which codes for a protein of 1115 amino acids. The deduced amino acid sequence predicts a putative signal sequence at the amino-terminus and a transmembrane region followed by 45 amino acids at the carboxy-terminus. The three introns found at the 3' end of the P. knowlesi gene were conserved in P. vivax, including high homology for the sequences of the introns. Comparison of the portion of the proteins amino to the transmembrane region between P. vivax and the partial sequence of P. knowlesi indicated at least three domains. Two homologous regions were separated by a non-homologous region. The cysteines in the homologous regions were conserved in number and position, indicating that the folding is similar and suggesting that these regions may be the Duffy blood group binding domains. In both P. vivax and P. knowlesi, the non-homologous region is hydrophilic and proline-rich, although the position of the prolines is not conserved. As prolines tend to stiffen a protein, this region may act as a 'hinge region' similar to those in the immunoglobulin gene family.

Key words: Plasmodium vivax; Plasmodium knowlesi; Malaria; Duffy receptor; Merozoite invasion; Homology

Introduction

Human erythrocytes that lack Duffy b lood group antigens are refractory to invasion by Plasmod-

ium vivax, a human malaria, and by Plasmodium knowlesi, a malaria o f Old World monkeys that invades Duffy-posi t ive human erythrocytes [1,2]. Soluble proteins o f 120 and 135 kDa f rom P. knowlesi culture supernatant were identified that specifically bind to the Duffy blood group determinants [3,4]. The specificity o f their binding and immunochemica l data indicate that the soluble

Correspondence address: Louis H. Miller, Laboratory of Para- sitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, U.S.A.

Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank TM data base with the accession number M37514.

Abbreviations: PBS, phosphate-buffered saline; PCR, polymerase chain reaction.

proteins or membrane -bound forms of these proteins are the Duffy receptor. The 3' part o f c D N A encoding a member o f the Duffy receptor family was cloned by antibodies affinity purified on the 135-kDa Duffy binding protein [5]. Antibodies to multiple regions derived f rom the deduced amino acid sequence o f the P. knowlesi gene immunoprecipitated the soluble 135-kDa Duffy binding protein, indicating that the cloned gene indeed encoded a member o f the Duffy receptor family [5].

An analogous protein o f 135 kDa that binds specifically to the Duffy blood group determinants has been found in the culture supernatant o f P. vivax [6,7]. Here we report the cloning of the Duffy receptor gene o f P. vivax, using as a probe a c D N A clone f rom the P. knowlesi gene. The h o m o l o g y of the amino acid sequences and the posit ion and D N A sequence o f the three introns at the 3' end of the genes indicated that the structure of the Duffy receptor is highly conserved for receptor function and for splicing o f introns.

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

126

Materials and Methods

Genomic DNA extraction. The Salvador I strain of P. vivax [8] was grown in a chimpanzee. The genomic DNA of P. vivax was extracted as follows. Parasitized blood was collected in antico- agulant citrate phosphate dextrose (Fenwal, Deer- field, IL), filtered through a Sepacell R500A unit (Baxter, Columbia, MD) to remove leukocytes, and then passed over a column of acid-treated glass beads (Thomas Scientific, Swedesboro, NJ) to remove platelets. The parasitized cells were centrifuged at 2000 x g for 10 min at room temperature and washed twice with phosphate- buffered saline, pH 7.4 (PBS). The cells were re- suspended in an equal volume of 0.15% saponin in PBS and incubated at 37°C for 10 min. Two volumes of PBS were added, and the cells were centrifuged again and washed once with PBS. The cells were then lysed at 37°C in 10 mM Tris, pH 8.0/10 mM EDTA/10 mM NaCl/2% SDS/100 izg ml-~ proteinase K. The lysate was extracted with phenol, then chloroform, RNase treated, re- extracted with phenol, then chloroform, ethanol- precipitated and adjusted to a final concentration of 1 lag/z1-1 in 10 mM Tris, pH 8.0/1 mM EDTA.

Library construction and colony screening. P. vivax genomic DNA (5 #g) was digested with HindIII (BRL, Gaithersburg, MD) at a concentration of 1 unit izg-~ for 2 h at 37°C. The DNA fragments were fractionated by agarose gel electrophoresis, and DNA fragments of 3-5 kb were isolated from the gel on glass (GeneClean kit BIO 101, La Jolla, CA). Eluted DNA fragments (500 ng) were ligated to HindIII-digested, phosphatase- treated pUC 13 vector DNA (200 ng; Pharmacia, Piscataway, N J) and used to transform competent DH 5c~ cells (BRL). Filter lifts of 6000 colonies were screened with a 2.7-kb cDNA clone (plC1) of P. knowlesi gene [5] by hybridization in 1 M NaC1/1% SDS/50 mM Tris, pH 8.0/200 ltg ml -~ heparin at 55°C for 16 h. The filters were washed at a final stringency of 0.2 x SSC/0.1% SDS for 1 h at 55°C. Autoradiography at -70°C for 12 h was sufficient to identify a positive colony, pPvDR.

DNA sequencing. Restriction fragments ofpPvDR

were subcloned into pBluescript-SK II (Strata- gene, La Jolla, CA), and single-stranded DNA was prepared as described [9], except that pBluescript- SK II (Stratagene) was used as the plasmid and M13K07 used as the helper phage (Promega, Madison, WI). DNA was sequenced by the dideoxynucleotide terminator method (USB se- quenase version 2.0 kit, Cleveland, OH) using universal sequencing primers and oligonucleotides from known sequences. Greater than 90% of the sequence was determined from both strands. Computer-assisted sequence analysis and comparison were performed "using PCGENE (Release 6.01, Intelligenetics).

Polymerase chain reaction. The non-homologous region was amplified by polymerase chain reaction (PCR) using 10 ng pPvDR, a 5' primer (Fig. 1; nucleotide. 2129-2149 plus a BamHI site at its 5' end: ggggatccAGTGATATTGCC- GAAAGTGTA) and a 3' primer (Fig. 1; inverted and complementary sequence from pPvDR nucleotide 2729 to 2749 plus a HindIII site at its 5' end: ataagcttGGTAGAGGCCCCGTTCTTTTC). The reaction mixture contained 100 mM Tris-HC1, pH 8.3/500 mM KC1/15 mM MgC12/0.01% gelatin (Sigma, St. Louis, MO), 0.2 mM dNTPs each/600 ng of each primer/2.5 U Taq DNA polymerase (Cetus, Norwalk, CT) in a final volume of 100 p,1, overlaid with 100 #1 of mineral oil (Fisher Scien- tific, Fairlawn, N J) and was subjected to 30 cycles of amplification in a Perkin-Cetus thermal cycler. Thermal cycling conditions were 1 min at 94°C, 1 min at 42°C, and 1 rain at 72°C. The 200-bp PCR product was purified from 0.8% agarose gel, la- beled with [32p]dCTP (BRL random priming kit) to 109 cpm # g - l , and used as a probe to hybridize with P. knowlesi genomic DNA restriction digest under the conditions described below.

Southern blot analysis. P. vivax genomic DNA was digested with DraI, EcoRI and HindlII, and P. knowlesi genomic DNA with DraI and EcoRI under the conditions recommended by the manu- facturer (BRL). Restriction fragments were fractionated by agarose gel electrophoresis and transferred to GeneScreen Plus membranes (NEN, Boston, MA) as previously described [9]. The P. vivax filter was hybridized with: (1) 0.05 #g

127

AAGCTTTTAAAAATAGCAA•AAAATTT•GAAACATTGCCACAAAAATTTTATGTTTTACATATATTTAGATTCATACAATTTAG• 85 V

TGTACCCTGTTTTTTGATATATGCGCTTAAATTTTTTTTTCGCTCATATGTTTAGTTATATGTGTAGAACAAC•TGCTGAATAAATTACGTACACTTTCTGTTCTGAATAATATTAC•AC 205 v

ATACATTTAATTTTAAATACTATGAAAGGAAAAAACCGCTCTTTATTTGTTCTCCTAGTTTTATTATTGTTACACAAGGTATCATATAAGGATGATTTTTCTATCACACTAATAAATTAT 325 V III , , ,,III K D D F S I T L I N Y 3 3 V

$ i h a l p e p t i d e CATGAAGGAAAAAAATATTTAATT~TACTAAAAAGAAAATTAGAAAAAGC¥AATAATCGTGATGTTTGCAATTTTTTTCTTCATTTCTCTCAGGTAAATAATGTATTATTAGAACGAACA 445

H E G K K Y L Z I L K R K L E K A N N R O V ~ N F F L H F S Q V N N V L L E R T 7 3 ~

ATTGAAACCCTTCTAGAATGCAAAA•TG•ATATGTGAAAGGTGAAAATGGT•ATAAATTAGCTAAAGGACACCACTGTGTTGAGGAAGATAACTTAGAA•GATGGTTA•AAGGAACCAAT 560 I E T L L E ~ K N E Y V K G E N G Y K L A K G H H ~ V E E O N L E R W L Q G T N 1 1 3 ~

GAAAGAAGAAGTGAGGAAAATATAAAATATAAATATGGAGTAAcGGAACTAAAAATAAAGTATG•GCAAATGAATGGAAAAAGAAGCAGCCGCATTTTGAAGGAATCAATTTACGGGGCG 605 E R R S E E N I K Y K Y G V T E L K I K Y A Q M N G K R S S R I L K E S I Y G A 1 5 3 ~

CATAACTTTGGAGGCAACAGTTACATGGAGGGAAAAGATGGAGGAGATAAAACTGGGGAGGAAAAAGATGGAGAACATAAAACTGATAGTAAAACTGATAACGGGAAAGGTGCAAACAAT 805 H N F G G N S Y M E G K D G G D K T G E E K O G E H K T D S K T D N G K G A N N 1 0 3

T T G G T A A T G T T A G A T T A T G A G A C A T C T A G C A A T G G C C A G C C A G C G G G A A C C C T T G A T A A T G T T C T T G A A T T T G T G A C T G G G C A T G A G G G A A A T T C T C G T A A A A A T T C C T • G A A T G G T G G • g25 L V M L O Y E T S S N G Q ~ A G T L D N V L E F V T G H E G N S R K N $ S N G G 2 3 3 ~

AATCCTTACGATATTGATCATAAGAAAACGATCTCTAGTG•TATTATAAATCATGCTTTTCTTCAAAATACTGTAATGAAAAACTGTAATTATAAGAGAAAACGTCGGGAAAGAGATTGG 1045 N P Y D I D H K K T I S S A I I N H A F L Q N T V M K N ~ N Y K R K R R E R D W 2 7 5 ~

GACTGTAACACTAAGAAGGATGTTTGTATACCAGATCGAAGATAT•AATTATGTATGAAGGAACTTA•GAATTTGGTAAATAATACAGACACAAATTTT•ATAGGGATATAA•ATTTCGA 1165 D ~ N T K K D V ~ I ~ D R R Y Q L ~ M K E L T N L V N N T O T N F H N D I T F R 3 1 3 ~

AAATTATATTTGAAAAGGAAA~TTATTTATGATGCTGCAGTAGAGGGCGATTTATTACTTAAG~TGAATAA~TACAGATATAA~AAAGACTTTTG~AAGGATATAAGATGGAGTTTGGGA 1285 K L Y L K N K L I Y O A A V E G D L L L K L N N Y R Y N K O F ~ K O I R W g L G 3 5 3 ~ . . . . . . . . . K

GATTTTGGAGATATAATTATGGGAACGGATATGGAAGGCATCGGATATTCCAAAG•AGTGGAAAATAATTTGCGCAGCATCTTTGGAACTGATGAAAAGGCC•AA•AGCGTcGTAAACAG 1405 D F G D I Z M G T O M E G Z G Y S K V V E N N L R S Z F G T D E K A C Q R R K Q 3 9 3 . . . . . . . . . N . . . . . . . Q . . . . . . . O V , = * . . . . K ' D * o * K

TGGTGGAATGAATCTAAAGCACAAATTTGGACAGCAATGATGTACTCAGTTAA~AAAAGATTAAAGGGGAATTTTA~ATGGATTTGTAAATTAAATGTTGCGGTAAATATA~AACCGCAG 1525 W W N E S K A Q I W T A M M Y S V K K R L K G N F I W I B K L N V A V N Z ' E ~ Q 4 3 3 ~ . . . . . . E H * * R * * ' F * I R S * * ' E K ' V . . . . K D • T L K V * - o K

ATATATAGATGGATTCGAGAATGGGGAAGGGATTACGTGTCAGAATTGCCCACAGAAGTGCAAAAACTGAAAGAAAAATGTGATGGAAAAATCAATTAT~CTGATAAAAAAGTATGTAAG 1645

GTACCACCATGTCAAAATGCGTGTAAATCATATGATCAATGGATAACCAGAAAAAAAAATCAATGGGATGTTCTGTCAAATAAATTCATA•GTGTAAAAAACGCAGAAAAGGTTCAGACG t 7 6 3 V ~ Q N A ~ K S Y D Q W I T R K K N Q W D V L S N K F I S V K N A E K V Q T 5 1 3 ~ L ~ L * H O . . . . . . . . . " " * * K . . . . . . T * " $ K T Q " I G * K

G C A G G T A T C G T A A C T C C T T A T G A T A T A C T A A A A C A G G A G T T A G A T G A A T T T A A C • A G G T G G C T T T T G A G A A T G A A A T T A A C A A A C G T G A T G G T G C A T A T A T T G A G T T A T G C G T T T G T T C C 1885 A G I V T ~ Y O I L K Q E L O E F N E V A F E N E I N K R D G A Y I E L ~ V ~ 6 5 5 3 ~ E N ' A * A . . . . . . . . N G * K * A T . . . . . . . . . N L ' N H ' * ~ ' V K

GTTGAAGAGGCTAAAAAAAATACTCAGGAAGTTGTGACAAATGTGGACAATGCTGCTAAATCTCAGGCCACCAATTCAAATCCGATAAGTCAGCCTGTAGATAGTAGTAAAGCGGAGAAG 2005 V E E A K K N T O E V V T N V ~ N A A K S Q A T N S N ~ I S Q ~ V D S S K A E K 5 9 3 ~ . . . . R . . . . . N • K G S G V E ' K * A S * * ~ - ' T E A K " S G G K

GTTCCAGGAGATTCTACGCATGGAAATGTTAACAGTGGCCAAGATAGTTCTACCACAGGTAAAGCTGTTACGGGGGATGGTCAAAATGGAAATCAGACACCTGCAGAAAG•GATGTACAG 2125 V ~ G O S T H G N V N S G Q D S S T T G K A V T G D G Q N G N Q T ~ A E S O V Q 6 3 3 ~ • Q E ' * A ° K S ' * K * E G K * S * N E * D ~ ' $ Q $ G A ~ A S ~ S V O E K A K

CGAAGTGATATTGCCGAAAGTGTAAGTGCTAAAAATGTTGATCCGCAGAAATCTGTAAGTAAAAGAAGTGACGACACTGCAAGCGTTACAGGTATTGCCGAAGCTGGAAAGGAAAACTTA 2245

•GCGCATCAAATAGTCGACCTTCTGAGTCCACCGTTGAAGCAAATAGCC•AGGTGATGATACTGTGAACAGTGCATCTATACCTGTAGTGAGTGGTGAAAACCCATTGGTAACCCCCTAT 2365

AATGGTTTGAGG•ATT•GAAAGACAATAGTGATAGCGATGGACCTG•GGAATCAATGGCGAATCCTGATTCAAATAGTAAAGGTGAGACGGGAAAG•GGCAAGATAATGATATGGCGAAG 2485 N G L R H S K D N S O S D G ~ A E S M A N ~ O S N S K G E T G K G Q O N O M A K 7 5 3 ~ T S A S ' A L A G E N G E V ~ N G T O T E ~ K E D G E K A D ~ Q K D Z E V K G * K

G C T A C T A A A G A T A G T A G T A A T A G T T C A G A T G G T A C C A G C T C T G C T A C G G G T G A T A C T A C T G A T G C A G T T G A T A G G G A A A T T A A T A A A G G T G T T C C T G A G G A T A G G G A T A A A A C T G T A G G A 2605 A T K O S S N S S D G T S S A T G D T T D A V D R E I N K G V ~ E O R O K T V G 793 Q O T • D R S Q G S L G ~ H T D E R A - L G E T H M E K D T E ~ A G G S T L T ~ K

A G T A A A G A T G G A G G G G G G G A A G A T A A C T C T G C A A A T A A G G A T G C A G C G A C T G T A G T T G G T G A G G A T A G A A T T C G T G A G A A C A G C G C T G G T G G T A G C A C T A A T G A T A G A T C A A A A A A T G A C 2725 S K D G G G E D N S A N K D A A T V V G E D R I R E N S A G G S T N O R S K N D 8 3 3 ~ E Q N V S V A S D N G V ~ G S G N . . . . . . . . . . . . . . . . . . . . . . K

ACGGAAAAGAACGGGG~T~TA~C~CTGACAGTAAACAAAGTGAGGATG~AACTGCGCTAAGTAAAA~GAAAGTTTAGAATCAA~AGAAAGTGGAGATAGAA~TACTAATGATACAACT 2845 T E K N G A S T ~ O S K Q S E D A T A L S K T E S L E S T E S G D R T T N D T T 8 7 3 ~ . . . . . . . . ~ - - - - * * N ' G . . . . . G A • ' ' K * N ' ' V H K * ~ D N ' * K

A A C A G T T T A G A A A A T A A A A A T G G A G G A A A A G A A A A G G A T T T A C A A A A G C A T G A T T T T A A A A G T A A T G A T A C G C C G A A T G A A G A A C C A A A T T C T G A T C A A A C T A C A G A T G C A G A A G G A C A T 2965 N S L E N K N G G K E K D L Q K H D F K S N D T ~ N E E ~ N S D Q T T D A E G H 9 1 3 ~ H G . . . . . . . N • ' ' F . . . . . M N ' * * I ~ S D O ~ S . . . . I * T * ' ' K

MLNOOASSOHTSSDQTSSO~TSSDQTSSDHTSSDHTSSDQ K

GACAGGGATAGCATCAAAAATGATAAAGCAGAAAGGAGAAAGCATATGAATAAAGATACTTTTACGAAAAATA~AAATAGTCACCATTTAAATAGTAATAATAATTTGAGTAATGGAAAADR~S~KN~KAER~KHMNK~TFTKNTN~HHLN~NNNL~NGK ~̀533085 H , * N V R , ~ E Z K S S E D * S . G D * M R - S * , N E • Y , H . * ' N ' R * K

TTAGATATAAAAGAATACAAATACA~AGATGTCAAAGCAACAAGGGAAGATAT~ATATTAATGTCTTCAGTACGCAAGTGCAACAATAATATTTCTTTAGAGTACTG~AACTCTGTAGAG 3205 L D I K E Y K Y R D V K A T R E O I I L M S S V R K ~ N N N I S L E Y ~ N S V E 9 9 3 ~ " N R O Q * E H . . . . . . . . K . . . . . E * N . . . . R A ' V K ' * ' T ~ * K

GACAAAATATCATCGAATACTTGTTCTAGAGAGAAAAGTAAAAATTTATGTTGCTCAATATCGGATTTTTGTTTGAACTATTTTGACGTGTATTCTTATGAGTATCTTAGCTGCATGAAA 3325 D K I S S N T B S R E K S K N L ~ S I $ O F ~ L N Y F D V Y S Y E Y L S B M K 1 0 3 3 ~ " R M L ' $ . . . . . . R R . . . . . . . . . . . . . . . E L . . . . F Y N ' * " K

AAGGAATTTGAAGATCCATCCTACAAGTGCTTTACGAAAGGGGGCTTTAAAGGTATGCAGAAAAAGATGCTGAATAGAGAAAGGTGTTGAGTAAATTAAAAAGGAATTAATTTTAGGAAT 3445 . . . . . ~ $ Y K ~ F T K G G F K . . . . . . . . . . . . . . . . . . . • * * * • ~ * * E S S T * . . . . K

G T T A T A A A C A T T T T T G T A C C C A A A A T T C T T T T T G C A G A C A A G A C T T A C T T T G C C G C G G C G G G A G C G T T G C T G A T A C T G C T G T T G T T A A T T G C T T C A A G G A A G A T G A T C A A A A A T G A G T A A 3565 V ~ n t f o n I e n d s ~ l b K ~ ~ i ~ i ~ ! i i i ~ i i ~ ~ R K M Z K N 0 1 ~ t 0 7 7 V

t r a n s m e m b f a n e ~e i o n c t o I | $ m 1 ¢ t = i l CCAGAAAATAAAATAAAATAACATAAAATAAAA~AAAAA~TAGAATAACAATT~AAATAAAA~AAAATGAGAAATGCCTGTTAA~GCACAGTTAATTCTAA~GA~TCCATTTGTGAAGTT 3055 V i n t ¢ o n II s t a r t s TTAAAGAGAGCACAAATGCATAGTCATTATGTCCATGCATATATACACATATATGTACGTATATATAATAAACGCACACTTT•TTGTT•GTACAGTTCTGAAGAAG•TACATTTAATGAG 3805

i n t r o n Z I e n d t ~ l S E E A T F N E 1085 Y E . . . . " D * K

TTTGAAGAATACTGTGATAATATT~ACAGAATCCCTCTGATGCCTAACAGTAATTCAAATTTC~AGAGCAAAATTCCATTTAAAAAGAAATGTTACATCATTTT~CGTTTTTCTTTTTTT 3925 V F E E Y D N I H R I ~ L M ~ N I ~ i n t ~ o n I I I s a { t s 1102 V • V , * ~ * D - - * T - * - * * ' o - * K

CTTTTTT~TTTCTTTTTTA~ATAT~GAACACATG~AGCCATCAACCCCCCTGGATTATTCATGATGCTAC~GGTAAGTAAAAG~AATT~TGATTGTA~TGCTGATGTAATTTTAGTCA 4045 V i n t ( o n I I I e n d s ~ l N I E H M Q ~ S T ~ L 0 Y S 1115 V

o . . . . . Q F . . . . . . K

T T T T G C T T G C T G C A A T A A A C G A G A A A A T A T A T C A A G C T T 4 0 8 5 V

Fig. 1. Nucleotide sequence and deduced amino acid sequence of P. vivax Duffy receptor and a comparison of its predicted protein sequence with that of P. knowlesi. The P. vivax Duffy receptor nucleotide sequence is shown on the upper line, amino acid sequence on the middle line, and the amino acid sequence of P. knowlesi on the bottom line with asterisks indicating identity at the amino acid level. Spaces are inserted in P. knowlesi protein sequence for an optimal alignment. The repeated sequence (SSDHTSSDQT) of P. knowlesi is separated to another line for an optimal alignment. Cysteine residues are highlighted by reverse print, and proline residues in a dashed box. The predicted signal peptide sequences of P. vivax is shaded, and the transmembrane-spanning hydrophobic sequence of P. vivax and P. knowlesi gene are lightly shaded. The beginning and end of the three introns are indicated below the

nucleotide sequence. V, nucleotide or amino acid sequence of P. vivax Duffy receptor; K, amino acid sequence of P. knowlesi.

128

[32p]plC1 DNA; (2) 0.05 #g [32p]pPvDR DNA; and (3) 0.05 #g [32p]HindIII/EcoRI fragments (2.7 and 1.4 kb) of pPvDR DNA, and the P. knowlesi filter with 0.05 #g [32p]PCR product of P. vivax for 16 h at 55°C. Unbound label was removed by two washes in 2 x SSC, 0.5% SDS at room temperature for 15 min, followed by one wash in 0.2 x SSC, 0.1% SDS at 55°C for 60 min. Hy- bridizations were visualized by autoradiography at -70°C.

Results

Cloning of P. vivax Duffy receptor gene. A 2.7- kb cDNA clone of the P. knowlesi Duffy receptor gene family, plC1, was used as a hybridization probe in Southern blot analysis of P. vivax genomic DNA (Fig. 2A). p lC1 hybridized to a HindIII fragment (4.1 kb), two EcoRI fragments (3.8 and 2.3 kb), and DraI fragments of 2.7 kb and 150-300 bp. A size-selected (3-5 kb) HindIII genomic DNA library of P. vivax was constructed in pUC 13 and screened with p 1C 1. A 4.1-kb HindIII fragment was cloned and named pPvDR.

Characterization of the P. vivax Duffy receptor gene and sequence comparison with P. knowlesi. The P. vivax sequence was determined by using the dideoxynucleotide terminator method. Trans- lation most likely begins at the ATG at nucleotide

- - e r " - - m r - - e r - -

2 3 . 1 ~ I ~

4.4 O w i 2 . 3 _ _ 2.0

0 .31 - -

A B C D

Fig. 2. Southern blot analysis of P. vivax genomic DNA. P. vivax genomic DNA digests of DraI, EcoRI, and HindIII were fractionated by agarose gel electrophoresis and then transferred to nylon membrane and hybridized with (A) p lCl ; (B) pPvDR; (C) 2.7-kb HindIII/EcoRI fragment of pPvDR; and (D) 1.4-kb HindIII/EcoRI fragment of pPvDR at 55°C for 16 h. The filter was washed at a final stringency of 0.2 x SSC/0.1% SDS, at 55°C for 60 min. The blot was stripped with 0.2 M NaOH

between hybridizations.

position 227-230, because this ATG is followed by a typical eukaryotic signal sequence (Fig. 1). The sequence consists of positively-charged amino acids, a hydrophobic region of 12 amino acids, and a signal between amino acid 22 and 23 that fits the eukaryotic consensus for cleavage. The cleavage site has a value of 6.74, as determined by using PSIGNAL program in PCGENE with 6.0 as the cutoff value.

Although the open reading frame from genomic DNA ends at nucleotide 3414, the mature mRNA is likely to be formed by the removal of introns and the splicing of exons. The reasons for predict- ing the presence of introns in the P. vivax gene were as follows. The P. knowlesi gene has three introns at the 3' end of the gene [5]. There were open reading frames 3' to the stop codon of P. vivax that are homologous to the three exons at the 3' end in the P. knowlesi gene. We therefore defined the introns of P. vivax by comparison of the amino acid sequence of exons of P. knowlesi to homologous regions in P. vivax and by the consensus splice sequences for malaria and other eukaryotic genes (GTA at the 5' end and YAG at the 3' end) [11,12]. The three intron sequences so de- lineated are highly homologous to the P. knowlesi introns (Fig. 3). Thus, the evidence for the ex- istence of three introns in P. vivax is based on homology of the 3' exons and homology of the three introns between P. vivax and P. knowlesi. Because mRNA of P. vivax was not available, we were not able to confirm the presence of introns in P. vivax at this time.

The complete DNA sequence and structure of the P. vivax Duffy receptor gene are shown in Figs. 1 and 4. The deduced amino acid sequence encoded by P. vivax Duffy receptor gene predicts a polypeptide of 1115 amino acids that contains a 22-amino acid putative signal sequence at the amino-terminus, an 18-amino acid transmembrane region followed by 45 amino acids at the carboxy- terminus. There was no significant similarity to any proteins in the Swiss-Prot 13 database (Intel- ligenetics).

Comparison of the predicted amino acid sequences between P. vivax and P. knowlesi re- veals striking conservation of several major fea- tures (Fig. 4). In the sequence amino to the transmembrane region, two areas of high homology

INTRON I exon I i n t r o n I exo¢l I I

V "--AAG~G~A~cA~AAAAAGATGCTGAATAGA~AAA~GTGTTGA~TAAATTAAAAAGGAATTAAT~TTAGGAATGTTATAAA~ATTTTTGTACCCAAAATT~TTTTT-G~AG~ACA~--

K ---cAG~GTATACA~AAAAGATGATcAATAGAcAAAGATGTTACGTA~AT~GAAAATAAATTCATTTTAGGAATGTTATAAATTTTTTTGTAATCAATATT~TTTTTTG~AG~GCA---

129

INTRON II exon I I intron 11

V ...T~GTAACCAGAAAATAAAATAAAAT . . . . . . . . . . A A ~ A ~ U ~ A A ~ A A A A ~ A A A A A ~ A ~ A A T A A C A A ~ T A A A A ~ A A A A ~ A - A A A ~ A ~ A A A ~ c C ~ T A A T ~ c A ~ A G ~ A A T T ~ A A c G A ~ c C A T ~ T G A A ~ T ~ T A A A ~

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

K -~-~GA/~TAACCAAAAAA~AATA~A~AAAT~AGAATAAGAA~AAGAA~TAGAGTGGAAGCTAGA~TAA~AATTAAAATAAAAAATAAAATAGAAAATGcTGTTAATGCACAATTAATTCTATATATT~CATGTGTGCAATTTTAAGG

e x o n 111

V AGAGCACAAATGCATAGTCATTATGTCCATGCA . . . . . . . . TATATACACATATATGTACGTATATATAATAAACGCACACTTTCTTGTTCGTACAG/TIC...

K AGAG•AAAAATG•GAAA•CA•TA•A•GCATGcA•GTATAcATATA•AGACATATATGTACCAATA•ATAATAAT-GCACA•TTCCTTGT•CGTACAG/••A--•

INTRON I I I

exon I I I i n t r o n 111 exon IV V ~ACA/GTAATTcAAAT~TCAAGAGCAAAATTCCATTTAAAAAGAAATGT~ACATCATTT~GCG~T~TTT~TT~CTITTTTT~TTCTTTTTTAG~AIA~-~

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

K ...ACG/GTAATTCAAATTTCAAAAGCAAAATTTCATTTATGAAGAAATATTACACCATTCTGCATTATTCCTTTTA . . . . . . . . . TTTCTTCTTTAG/ATA...

Fig. 3. Comparison of the introns between the P. vivax and the P. knowlesi genes. The beginning and end of the three introns are indicated, and the exon sequences are in bold type. Spaces are inserted in the sequences for a better alignment.

H i n d III A T G

k-- iii',iiiiiiiiiJiiiiiiiiiii' iiiiiiiiiiiiiiiiiiiljiiiiii' iiiiiiiiiiiii',i;.!

i

/ i I I I

I I I I

R e p e a t

11 12 13 H i n d III

t T r a n s m e m b r a n e

r e g i o n

• m

I T A A

P. v ivax

P. knowlesi

t.....l

100 bp

Fig. 4. The structure of P. vivax Duffy receptor gene and its comparison with that of P. knowlesi. Exons are shown as boxes, introns as solid lines, and 5' and 3' noncoding sequences as dashed lines. The homologous regions between P. vivax and P, knowlesi Duffy receptor gene are shown in black, and the non-homologous region is shown as an open box. The amino terminal part in P. vivax is shown as stippled box because of unknown homology between the P. vivax and P. knowlesi genes in this region. Three introns (11, 12, and I3) and the transmembrane region are indicated. The start codon of the P. vivax gene, the stop

codon, and the repeat region of P. know/esi gene are marked on the figure.

are separated by a middle, non-homologous region (275 amino acids in P. vivax, and 242 amino acids in P. knowlesi). 65.4% of the amino acids in P. vivax are identical in P. knowlesi gene in the amino homologous region and 61.0% in the carboxy homologous region. A repeat pentamer sequence in P. knowlesi, not present in P. vivax, separates the carboxy homologous region. Both

proteins are cysteine-rich in these two homologous regions (3.5% in the amino and 5.9% in the carboxyl homologous region, respectively). All of the cysteines are positionally conserved (Fig. 1).

The middle, non-homologous region is proline- rich and cysteine-free in both P. vivax and P. knowlesi (Fig. 5A and B); however, the positions of the prolines are not conserved in the non-

130

o 3- E

2 -

"5 1-

P. v i v a x

Non homologous

380 480 580 680 780 880 980 1080

E z

100 200 300 400 500 600 700

i

P. k n o w l e s i

P. v i v a x

'"1 . . . . . . . . . i . . . . . . . . . i . . . . . . . . . I . . . . . . . . . ~ . . . . . . . . . i . . . . . . . . . i . . . . . . . . . ~ , "

380 480 580 680 780 880 980 1080 1 oo 2oo 300 400 500 600 700

P. k n o w l e s i

P. v i v a x

380 480 580 680 780 880 980 1080 1 100 200 300 400 500 600 700

P. k n o w l e s i

A. Cys te ine B. Pro l ine C. Aromatic residues

Fig. 5, Comparison of the distributions of structurally important amino acid residues between P. vivax and P. knowlesi Duffy receptor gene. (A) Cysteine residues; (B) Proline residues; (C) Aromatic residues (phenylalanine, tryptophan, and tyrosine). The plots of the amino acid residues were computed at intervals of five amino acids by using PRESIDUE program in PCGENE. The number of amino acids is shown on the X axis, and the number of residues per interval of five amino acids is shown on the Y

axis. The regions of homology and non-homology are indicated on each figure (see Fig. 4).

homologous region. This non-homologous region is relatively poor in aromatic residues when com- pared to the homologous regions (Fig. 5C), which partially explains the hydrophilicity of this region.

Since there are 2 or 3 homologous genes in the Duffy receptor family of P. knowlesi, it was important to determine whether the non-homologous region of P. vivax would hybridize to any one of the other two possible P. knowlesi genes. The non- homologous region of P. vivax which was synthe- sized by PCR was used as a probe to hybridize to P. knowlesi genomic DNA. None of the three EcoRl fragments of 4 kb, 6 kb and 10 kb hybridized to the non-homologous region of P. vivax (data not shown), indicating that the 10 kb and 4 kb genomic EcoRI fragments of P. knowlesi are also non-homologous in the middle region to the P. vivax gene.

Analysis of pPvDR in the genome. It was previously shown that there are two other regions in the P. knowlesi genome which are homologous to the cloned P. knowlesi gene [5]. To determine if there is also a family of genes in P. vivax, pPvDR was hybridized with restriction digests of genomic P. vivax DNA (Fig. 2B). Two bands were observed on HindIII digestion: a major band at 4.1 kb and

a faint, diffuse band at >20 kb. The upper one was in the area of the gel where the restricted DNA began to run and may have been incom- pletely digested DNA. The DraI digest had a single band of strong intensity at 2.7 kb, a diffuse band of weak intensity at around 4.4 kb (which is probably nonspecific hybridization), and a se- ries of bands at around 150-300 bp. Except for the weak 4.4-kb band, these sizes were consistent with the predicted DraI restriction sites within pPvDR (2683 bp, 152 bp, 199 bp, 211 bp, 314 bp, 319 bp). A PCR fragment from the last 3 t DraI site to the 3' HindIII site of pPvDR hybridized to a small fragment at around 300 bp, but not to the weak 4.4 kb band of P. vivax (data not shown), suggesting that the 4.4 kb fragment may result from nonspecific binding. The EcoRI digest gave two fragments. As there was an EcoRI site within pPvDR, we probed separately with the 2.7 and 1.4 kb HindIII/EcoRI fragments from pPvDR (Figs. 2C and 2D). The 2.7-kb fragment hybridized with the 3.8-kb EcoR! fragment and not the 2.3-kb fragment of P. vivax genomic DNA. The 1.4-kb fragment only hybridized with the 2.3-kb genomic fragment. The data of the EcoRI digest were consistent with a single copy in P. vivax, whereas in P. knowlesi there was hybridization with three

chromosomes and expression of at least two Duffy receptor family genes [5].

Discussion

We have cloned the presumptive gene encoding the Duffy receptor for P. vivax, using as a probe a homologous gene from P. knowlesi [5]. Whereas the gene from P. knowlesi hybridizes with three regions in the P. knowlesi genome, the data in P. vivax are consistent with a single locus in the P. vivax genome. The evidence that we have indeed cloned the Duffy receptor is based on the following. First, antibodies to different regions of the sequence derived from the P. knowlesi gene immunoprecipitated the 120 and 135 kDa Duffy blood group-binding proteins of P. knowlesi. Sec- ond, the similar position and sequence of three introns between P. vivax and P. knowlesi indicate that the P. knowlesi gene and the P. vivax gene are homologous. Third, the high degree of amino acid homology, including the conserved position of cysteines, also indicates that the P. vivax and P. knowlesi genes are members of a homologous gene family.

A comparison of the sequences between P. vivax and P. knowlesi identifies potential functional domains. We assumed that there is sequence homology between the regions of P. vivax and P. knowlesi that bind to the Duffy blood group determinants. From the large size of the soluble Duffy binding protein (135 kDa), it is likely that the large portion of the protein amino to the transmembrane region is extracellular and the short 45 amino acid carboxyl-terminus is cytoplasmic. A comparison of the genomic DNA sequence of P. vivax to the partial cDNA sequence of P. knowlesi suggests at least three domains in the extracellular portion of the protein. Two homologous regions are separated by a non-homologous region. The two homologous regions are rich in cysteines; the non-homologous region is devoid of cysteines. The positions of the cysteines in the homologous regions are conserved, indicating a similar folding between the two proteins. We would predict that these regions will be involved in binding to the Duffy blood group determinants, but proof must await expression in mammalian cells and the study of the fine specificity of erythrocyte binding be-

131

tween P. knowlesi and P. vivax. The homology of the three introns in the 3 ~

region of the gene indicates conservation during evolution for an important function (e.g., protein recognition sites for splicing). Waters et al. ana- lyzed the phylogenetic relationship of eight different Plasmodium strains by comparing the DNA sequences of small subunit rRNA genes (Waters et al., personal communication). It was found that P. vivax is more closely related in evolution to the simian malarias, P. knowlesi and Plasmod- ium fragile, than to Plasmodium falciparum, ro- dent malarias, and avian malarias.

The characteristics of the non-homologous regions are similar. The lengths of the region in P. vivax and P. knowlesi are similar (275 and 242 amino acids, respectively). Both are proline rich, although the position of the proline residues are not conserved. P. vivax contains no aromatic amino acids in this region, and P. knowlesi contains only one. The absence of aromatic amino acids is one of the reasons for the greater hydrophilicity of the region. This region may be similar to the hinge region in immunoglobulins, which is also proline-rich and hydrophilic [ 13,14]. Prolines tend to stiffen a protein and may produce a stalk that keeps the amino terminal homologous domain further from the membrane where it can interact with the Duffy blood group determinant on erythrocytes.

This non-homologous region in P. vivax is also non-homologous to the other members of the Duffy receptor gene family of P. knowlesi but it did hybridize to Plasmodium cynomolgi genomic DNA (data not shown). As P. vivax and P. cynomolgi are biologically similar (e.g., both cause relapses), the divergence of this region probably occurred after the separation in evolution of P. knowlesi from P. vivax and P. cynomolgi.

It has been presumed that the parasite evolved during the evolution of its primate hosts. The conservation of the Duffy blood group system in primates may be the reason for the homology in the Duffy receptor family between P. vivax and P. cynomolgi.

Acknowledgements

We thank Dr. William Collins (Center for Dis-

132

ease Control , Atlanta, GA) who kindly supplied the P. vivax; Dr. Jeannine Gocayne for technical assistance in s ingle-strand D N A extraction; and Drs. Thomas Wellems and David Davies for help-

ful discussion.

References

1 Miller, L.H., Mason, S.J., Clyde, D.F. and McGinniss, M.H. (1976) The resistance factor to Plasmodium vivax in blacks: the Duffy blood group genotype FyFy. New Engl. J. Med. 295, 302-304.

2 Miller, L.H., Mason, S.J., Dvorak, J,A., McGinniss, M.H. and Rothman, I.K. (1975) Erythrocyte receptors for (Plas- modium knowlesi) malaria: Duffy blood group determinants. Science 189, 561-563.

3 Haynes, J.D., Dalton, J.P., Klotz, F.W., McGinniss, M.H., Hadley, T.J., Hudson, D.E. and Miller, L.H. (1988) Receptor-like specificity of a Plasmodium knowlesi malaria protein that binds to Duffy antigen ligands on erythrocytes. J. Exp. Med. 167, 1873-1881.

4 Miller, L.H., Hudson, D. and Haynes, J.D. (1988) Identi- fication of Plasmodium knowlesi erythrocyte binding proteins. Mol. Biochem. Parasitol. 31,217-222.

5 Adams, J.H., Hudson, D.E., Torri, M., Ward, G.E., Wellems, T.E,, Aikawa, M. and Miller, L.H. (1990) The Duffy receptor family of Plasrnodium knowlesi is located within the micronemes of invasive malaria merozoites. Cell 63;

141-153. 6 Wertheimer, S.P. and Barnwell, J.W. (1989) Plasmodium

vivax interaction with the human Duffy blood group gly- coprotein: identification of parasite receptor-like protein. Exp. Parasitol. 69, 340-350.

7 Bamwell, J.W., Nichols, M.E. and Rubinstein, P. (1989) In vitro evaluation of the role of the Duffy blood group in erythrocyte invasion by Plasmodium vivax. J. Exp. Med. 169, 1795-1802.

8 Collins, W.E., Contacos, P.G., Stanfill, P.S. and Richard- son, B.B. (1973) Studies on human malaria in Aotus monkeys, I. Sporozoite transmission of Plasmodium vivax from El Salvador. J. Parasitol. 59, 606--608.

9 Dente, D., Cesareni, G. and Cortese, R. (1983) pEMBL: a new family of single-stranded plasmids. Nucleic Acids Res. 11, 1645-1655.

10 Kaslow, D.C., Syin, C., MtzCutchan, T.M. and Miller, L.H. (1989) Comparison of the primary structure of the 25 kDa ookinete surface antigens of Plasmodium falciparum and Plasmodium gallinaceum reveal six conserved regions. Mol. Biochem. Parasitol. 33, 283-288.

11 Weber, J.L. (1988) A review. Molecular biology of malaria parasites. Exp. Parasitol. 66, 143-170.

12 Mount, S.M. (1981) A catalogue of splice junction sequences. Nucleic Acids Res. 10, 459-472.

13 William, R.C. (1986) The Experimental Foundation of Modem Immunology, 3rd ed., Wiley, New York.

14 Kabat, E.A., Wu, T.T., Reid-Miller, M., Perry, H.M. and Gottesman, K.S. (1987) Sequences of Proteins of Immuno- logical Interest, 4th ed., U.S. Department of Health, Edu- cation and Welfare, Washington, DC.

cloning of the plasmodium vivax duffy receptor

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