Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax

Abstract

Background: Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites.

Results: We describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs) sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098) after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot. The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa) in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7), suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from P. vivax, despite an amino acid identity of 44% between the proteins.

Conclusion: We identify a novel DHS protein in the more benign malaria parasite,P. vivax, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, P. yoelii, and human P. falciparum strains.

Figure 1

Amino acid alignment between a putative DHS protein from P. vivax and two different P. falciparum strains, the rodent malaria parasite P. yoelii, human and the mosquito Anopheles gambiae. Numbering refers to DHS in the two P. falciparum species. Lane 1: P. falciparum strain Dd2 (accession number AF290977); lane 2: P. falciparum strain 3D7 (accession number NC_004317); lane 3: P. yoelii (XM-724232); lane 4: P. vivax (AJ549098) ; lane 5: human (U26266), lane 6: Anopheles gambiae (XM-316567). Gaps (-) were introduced to obtain maximum alignment. Arterisks label amino acid identities, colons (:) and dots (.) label amino acid similarities. The spermidine binding site (243–329 refering to human DHS numbering) is marked by bold amino acids. The NAD binding site from serine105 to aspartic acid 342 is marked in bold letters. The active center of the DHS protein from glutamine 324 to lysine 329 is bolded black.

Figure 2

A: Expression and purification of recombinant putative six histidine tagged deoxyhypusine synthase from P. vivax by Nickel-chelate chromatography. 12% SDS PAGE protein gel: 1) protein marker: 10 kDa ladder; 2) non induced bacterial cell lysate 3) induced bacterial cell lysate with 100 mM IPTG after 4 hours of induction; 4 and 5) wash fractions obtained after purification; 5 and 6) eluate fractions of purified putative deoxyhypusine synthase. B: Western Blot analysis of the expressed, purified P. vivax DHS protein. lane 0) standard molecular size marker (Roth); lane 1) protein extract of purified DHS protein from P. vivax by Nickel-chelate chromatography. lane 2) protein extract of purified human DHS protein. The blot was probed with a 1: 800 diluted polyclonal antiserum against human DHS.