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.
Citation
Masters of Science, in biochemistrySponsorhip
University of NairobiPublisher
Department of biochemistry University of Nairobi