Conservation of key amino acids among the cysteine proteinases of multiple malarial species

MOLEIUIR

i?ikEMlcAL PARAsI?oLoGy

ELSEVIER

Molecular and Biochemical Parasitology 75 (1996) 255-260

Short communication

Conservation

of key amino acids among the cysteine proteinases of multiple malarial species1 Philip J. Rosenthal*

Department of Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94143, USA

Received 8 August 1995; accepted 6 October 1995

Keywords:

Plasmodium;

Malaria;

Cysteine

proteinase;

Cysteine

Intraerythrocytic malaria parasites degrade hemoglobin as a major source of amino acids for parasite protein synthesis [l]. To degrade hemoglobin, parasites transport erythrocyte cytosol to an acidic food vacuole. Hemoglobin is then separated into heme, which is processed to hemozoin, and globin, which is hydrolized to free amino acids [l]. Both cysteine and aspartic malarial proteinases have been identified, localized to the food vacuole, and shown to cleave denatured and native human hemoglobin [2-61. Cysteine proteinase inhibitors blocked hemoglobin degradation [2], killed cultured parasites [7], and cured murine malaria infections [8]. Malarial hemoglobin-degrading proteinases are thus promising new targets for antimalarial chemotherapy.

* Correspondence address: Box 08 11, University of California, San Francisco, CA 94143. Tel: + 1 415 2068845; Fax: + 1 415 2066015; e-mail: [email protected]. ’ Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBankTM, and DDJB data bases under the accession numbers U33420-U33426.

We have previously characterized the genes encoding papain-family cysteine proteinases of three malarial species, the human parasites Plasmodium falciparum [9] and Plasmodium vivax [lo], and thk murine parasite Plasmodium vinckei [l 11. Coniparisons of the predicted malarial proteinase sequences with those of other papain-family proteinases identified a number of characteristics that were unique to the malarial enzymes included unusually long proform sequences, a 28-33amino acid insert located near the C-terminus of the proteins, and the conservation of a number of amino acids predicted by molecular modeling to be located near the active site [lo]. We hypothesized that amino acids located near the active site may have been conserved to maintain optimal proteolytic specificity in the hydrolysis of hemoglobin by malaria parasites. As the identification of biochemical differences between parasite and host cysteine proteinases may be critical in the development of drugs directed against the malarial enzymes, we have further tested our hypothesis by characterizing the cysteine proteinase sequences of additional malarial species.

0166-6851/96/$15.00 Q 1996 Elsevier Science B.V. All rights reserved SSDI 0166-6851(95)02517-O

protease

P.J. Rosenthal / Molecular and Biochemical Parasitology 75 (1996) 255-260

256

PRE

PRO I

1w

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MATURE

I 2330 ’

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Fig. 1. (A) Map of PCR strategy and results. Predicted pre-, pro- and mature regions and amino acid numbers, based on the P. of the four oligonucleotides used for PCR and the extent of the proteinase genes cloned or amplified for each species are shown. Key for the Plasmodium species (with reference, strain, or source noted): FA, falciparum [9]; VX, oioax [IO]; VI, uinckei [ll]; RE, reichenowi (Biomedical Primate Research Centre, Rijswijk, The Netherlands); CN, cynomolgi (Vietnam strain); FR, fragile (CDC); BE, berghei (ANKA); OV, ouale (CDC); GA, gallinaceum (8A); MA, malariae (WR314). Oligonucleotide sequences (I = inosine): PCl, AAAGGATCCAA(AG)TA(CT)GCI(AT)(CG)IAA(AG)TT(CT)T-I; PC2, AAAGAATTCAAIACITCI(ACGT)(ACGT)ICCIA(CT)ICC(AG)CA; PC3, TTTGAAT-TCCCAI(CG)(AT)(AG)TTI(CT)(GT)IA(CT)IATCCA(AG)TA; PC4, AAAGGATCCTG(CT)GGI(AT)(CG)ITG(CT)TGGGCITT. (B) Deduced amino acid sequences of malarial cysteine proteinases. Numbering of amino acids at the left margin is based on the P. falciparum proteinase [9]; italicized numbering above individual amino acids is based on the standard papain numbering system [15] and corresponds to that in Table 2. Amino acid positions encoded by oligonucleotides used for PCR ( x ), identities (I), conservative substitutions (:), and the locations of the predicted proform cleavage site (O), highly conserved papain-family amino acids (*), and amino acids conserved among the plasmodia ( + ) are labelled.

falciparum proteinase, are labelled. The locations and orientations

Plasmodial DNAs were obtained through the generosity of a number of collaborators and amplified with proteinase-specific oligonucleotide primers. PCR reactions with each DNA utilized two oligonucleotides that encode consensus papain-family cysteine proteinase sequences (PC3, PC4) and two oligonucleotides that encode sequences conserved among the three previously characterized malarial cysteine proteinases, but not among other papain-family proteinases (PC 1, PC2) (Fig. 1A). PCR conditions were optimized for each reaction; all reactions utilized Taq polymerase, annealing at 40-45°C and 1.5-3.0 mM MgCl,. Some oligonucleotide combinations did not provide successful amplifications with certain DNAs, and so different portions of the proteinase genes were amplified for different species (Fig. 1A). For Plasmodium malariae, only a nested PCR approach utilizing first the oligonucleotides PC1 and PC2, and then the internal oligonucleotides PC3 and PC4 led to successful amplification. PCR products were cloned into a plasmid vector and sequenced by standard methods. The deduced plasmodial cysteine proteinase sequences were aligned and compared (Fig. 1B). For the eight sequences extending into the predicted proteinase proform, considerably greater similarity in sequence was seen than is generally

the case among papain-family proteinases. Within the predicted mature form of the proteinase, the malarial sequences were highly conserved. All strongly conserved papain-family amino acids [12] were conserved among the malarial proteinases. The malarial proteinases also all contained an insert located near the C-terminus of the proteins (amino acids 499-527 in P. falciparum) between highly conserved sequences. A large insert at this location has been seen in only one other papainfamily enzyme, a proteinase of Dictyostelium discoideum [12]. Interestingly, the size and sequence of the insert was not well conserved among the malarial species. The percentage identities of the sequences within a 167-amino acid stretch available for all of the malarial species were determined. This region included both highly conserved papain-family sequences, regions that are quite poorly conserved, and the unique insert region discussed above. All of the malarial cysteine proteinase sequences were at least 45% conserved within the region evaluated (Table 1). This is considerably greater conservation of sequence than that with the cysteine proteinases of other species. For example, sequence identities between the sequence of the P. falciparum proteinase and those of human cathepsin L and papain in this region were

P.J. Rosenthal / Molecular and Biochemical Parasitology 75 (1996) 255-260

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28.6 and 23.6%, respectively [9]. Certain malarial species were very highly conserved, suggesting strong evolutionary relatedness. In partitular, the proteinase sequence of the chimpanzee parasite Plasmodium reichenowi was

to that of P. falciparum from the primate malarias Plasmodium cynomolgi and Plasmodium fragile were about 900/o identical to that of P. nearly identical and sequences

vivax.

P.J. Rosenthal / Molecular and Biochemical Parasitology 75 (1996) 255-260

258

Table 1 Percentage amino acid identity among malarial cysteine proteinases”

FA vx ov MA

FA

Vx

ov

MA

RE

CN

FR

VI

BE

GA

_ 63.4 62.1 58.4

63.4 74.0 72.6

62.1 74.0 74.6

58.4 72.6 74.6

99.4 63.4 62.1 58.4

63.4 92.6 13.4 73.1

63.8 81.4 71.8 71.8

52.3 49.1 51.4 45.4

56.1 52.6 54.2 52.0

61.3 60.0 55.4 56.6

“Identities within the region corresponding to P. falciparum amino acids 361-527 were determined based on the alignment shown in 1, except within a poorly conserved stretch (from amino acids 499 to 527) where the alignment providing maximum identity without added gaps was arranged for each species combination. Abbreviations for the malarial species are as in Fig. 1.

Beyond the strong similarities noted, a comparison of the sequences of functional genes is probably of limited value in determining evolutionary relatedness, as functional constraints may have driven sequence divergences. In the case of the cysteine proteinases, an important functional constraint would appear to be the structure of the

hemoglobin substrate for the proteinases. Primate hemoglobins are all very similar in sequence, while those of rodents and birds differ from primate hemoglobins by 15-30% (Table 2). It would be expected that proteinases directed toward similar substrates would maintain similarities of sequence. This appears to be the case, as, despite

Table 2 Conservation of key amino acids among malarial species Species”

FA Vx ov MA RE CN FR VI BE GA Enzyme” Papain [ 151 Cathepsin L [16] Cathepsin B [17] Cathepsin H [18] Theileria parva [19] Trypanosoma brucei [20] Trypanosoma cruzi [21] Schistosoma mansoni cathepsin B [22] Schistosoma mansoni cathepsin L [23]

Host

Human Human Human Human Chimpanzee Rhesus Rhesus Mouse Mouse Chicken

Hb Identityb

Residue”

(%tx /%p )

18 21 64 67 69

loo/loo loo/loo loo/loo loo/loo loo/loo 97.9194.5 97.9194.5 85.8/80.1 85.8/80.1 70.9169.2

DLDHFN DLDHFN DADHFN _ _ DHFN DLDHFNL DLDHFN DLDHFNL DNDNFA DMDHFN - _ DHFCVNAG-

L L L L

NSNYWV NQNLDA DSNYAAGGANE NAQLSAVNAGCS DNQLEY VQNLDA DQSLNA DREI GS NMNLSGL

V D AGS MDGGAS

133 157 158 160 178 205 207

L L L

N N N N NS N N N N

S S S S S S S S

S S -d

S S S G S

E E

F F

-

-

D

-

F

-

F V

L L L G

N D DGT G D

A G

G S

A N AGV

T A E E

M S S I T

“Abbreviations for the malarial species are as in Fig. l.bPercent identity between host and human doand a globin chains.‘Papain numbering system for residues located near the active site in the modeled structure of the P. falciparum proteinase [lo]. Standard one-letter abbreviations for amino acids are used.dSequence not available.‘References for the papain-family proteinase sequences listed are in parentheses.

P.J. Rosenthal / Molecular and Biochemical Parasitology 75 (1996) 255-260

the fact that P. falciparum appears to be more related to avian than other primate malarias [13], the P. falciparum proteinase sequence was generally more similar to those of human and other primate malarias than it was to those of rodent or bird malarias (Table 1). The principal reason for our sequence comparison was to expand on initial studies showing that a number of amino acids predicted to be located near the proteinase active site were highly conserved among malarial species, but not other papain-family cysteine proteinases [lo]. Our present results. now encompassing ten malarial species infecting humans, other primates, rodents, and birds, show that the conservation of sequence at 12 amino acid positions near the active site is strong among the malarial species, and strongest among the species that infect humans and other primates (Table 2). Conservation between the malarial proteinases and other papain-family enzymes at these sites is poor. Our results suggest two important conclusions regarding the potential of the malarial cysteine proteinases as targets for antimalarial drug development. First, the identification of Plasmodium-specific active site sequences should expedite, with the use of molecular modeling techniques [14], the design of inhibitors that can distinguish between fairly similar host and parasite proteinases. Second, it is likely that an inhibitor of the P. falciparum cysteine proteinase will be effective against the cysteine proteinases of all human malaria parasites. Therefore, cysteine proteinase inhibitors have the potential to be both broadly effective and highly specific antimalarial drugs.

Acknowledgements I thank A. Waters (P. reichenowi, P. cynomolgi, P. ,fiagile, P. ovale), A. Thomas (P, reichenowij, D. Kaslow (P. gallinaceum), W. Collins (P. malariae), E. Davidson and G. Kurzban (P. bevghei) for their generous gifts of malarial DNA

and G. Lee and A. Li for their expert technical assistance. This work was supported by the National Institutes of Health, the UNDP/World

259

Bank/WHO Special Program for Research and Training in Tropical Diseases, and the American Heart Association. P.J.R. is an Established Investigator with the American Heart Association.

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