Isolation and sequence analysis of a plasmodium Falciparum gene encoding glucosamine fructose -6- Phosphate aminotransferase from parasite line Nf54

Abstract

The purpose of this study was to Isolate and analyse the DNA and amino acid sequence of the enzyme glucosamine fructose-6- phosphate aminotransferase (GFAT) fromPlasmodium falciparum parasite line NF54. GFAT is the rate limiting step enzyme in the reaction that catalyses the interconversion of D-fructose-6- phosphate and glucosamine-6-phosphate. GFAT plays a role in the pathway that leads to the biosynthesis of Uridine Diphosphate N-acetyl glucosamine, an important precursor in the synthesis glycophosphatidylinositol (GPI) anchors. GPI anchors are crucial for the survival of the intraerythrocytic stage Plasmodium falciparum because of their role in membrane anchoring of merozoite surface proteins involved in parasite invasion of erythrocytes.GPI also function as dominant parasite toxin in the context of infection. Glucosamine fructose-6- phosphate aminotransferase gene was isolated from in vitro cultivated Plasmodium falciparum NF54 using PCR based methods. The PCR product was generated using specific .primers designed flanking the complete open reading frame of 3D7 parasite line using either genomic DNA or double stranded complementary DNA (cDNA) isolated from parasite line NF54 as template. Primers for sequencing were also designed based on the sequence available in the database of P. falciparum strain 3D7. TheNF 54 (GFAT) gene of2.5kb purified from the agarose gel was sequenced A ' partial nucleotide sequence of approximately 550bp of the amplified gene was sequenced. A blast search performed on the translated nucleotide sequence confirmed that the partial nucleotide sequence encoded homologue of GFAT (from 3D7) as anticipated. The highest probability score was for P. falciparum (e value of -66) and a low probability score (e- value 0.007) for the homosapiens glucosamine fructose-6- phosphate aminotransferase (accession number 05421.2). The P. falciparum NF54 GFAT sequenced shares up to 95 % identity with the database nucleotide sequence from line 3D7. The translated amino acid sequence ofNF54 gene (GFAT) was aligned to that of 3D7 line to determine whether or not the variation existed within the gene. Alignment of the deduced amino acids of the two. sequences showed that the partial gene sequenced is perfectly conserved between lines 3D7 and NF54. It also showed the relatedness of3D7 and NF54 P. falciparum parasite lines. As per the result of BLAST search, it is localized in chromosome 10 section 5 of 7. Comparisons of the database sequence of P. falciparum putative glucosamine fructose-6- phosphate aminotransferase with glucosamine fructose-o-phosphate aminotransferase of other species (bacteria and yeast) indicated that the residues are also substituted in these organisms, probably, an indication of its important role. The significance of this study is that, even though only a partial sequence of this gene was obtained, this is the :Q.rstime to show that Plasmodium NF54 has the OFAT gene. The identity between the NF54 GFAT and the putative 3D7 GFAT combined with the low overall similarity between the homosapien and P. falciparum homologues (0.15%) is encouraging for the development of compounds that specifically bind the parasites GFAT. This study also provides evidence for the existence of a classical aminosugar biosynthetic pathway in P. falciparum. This is an important contribution since this gene can be investigated as a novel drug target.