Genomic Cloning and Sequence Analysis of Trypanosoma brucei rhodesiense Gene Encoding Putative N-glycosylation Enzyme

Ano: 2010 | Volume: 38 | Número: 3

Autores: Waren Navarra Baticados, Noboru Inoue, Chihiro Sugimoto, Hideyuki Nagasawa, Abigail Morales Baticados

Palavras-chave: trypanosoma brucei rhodesiense, putative oligosaccharyl transferase, n-glyc

Resumo Inglês:

Background: Trypanosoma brucei rhodesiense is a haemoflagellate parasite of zoonotic significance. Aside from its public
health importance, this parasite subspecies gained notoriety because of their effective system to circumvent the immune response
of vertebrate host. The parasite cell surface is covered with millions of VSG dimers, which serve as an almost infinite repertoire
of biomolecules needed for evasion of host immune system. Around two decades ago, it was resolved that all trypanosome
VSG is associated with one or more N-linked oligosaccharides, with a range of structures including high mannose and complex
types. This complex process of protein modification known as N-linked glycosylation is catalyzed by oligosaccharyl transferase
(OST). In general, the incorporation of glycan structures can alter protein’s antigenic properties and recently it was established
that glycan molecules associated with VSG were found to be important in several aspects of trypanosome-host interaction,
especially during parasite evasion of the host defense mechanisms. Therefore, our major interest is to clone and characterize the
trypanosome OST.
Materials, Methods & Results: The template genomic DNA for PCR amplification was extracted as described previously. In
an attempt to clone Trypanosoma brucei rhodesiense putative oligosaccharyl transferase, an amplicon of ~2000 bp was obtained
having an open reading frame of 2057 bp and deduced primary structure composed of 685 amino acid residues (TbrOST II).
Comparison of TbrOST II ORF with annotated putative oligosaccharyl transferase in the genome of other organisms revealed
sequence identity to other kinetoplastid. TbrOST II had high nucleotide (Ns) and amino acid (As) sequence similarity with the
genomes of T. brucei gambiense (Ns:99%; As:78%) and T. brucei (Ns:95-98%; As:77%-98%). There was also significant
nucleotide and amino acid sequence identity in the genomes of T. cruzi (Ns:74%; As:63%), Leishmania infantum (Ns:70-83%;
As:46-57%), L. braziliensis (Ns:69-81%; As:46-55%) and L. major (Ns:69-80%; As:46-57%). Sequence similarity (71-77%)
from other origins was also exhibited. The nucleotide sequence alignments and analysis were performed using the Oxford
University Mac Vector 6.5 sequence analysis software and CLC Workbench 5.6 software.
Discussion: The nucleotide BLAST results indicate that sequence identity is higher between species of the same genus rather
than of the same family. It is known that T. brucei, T. gambiense and T. rhodesiense are members of the Brucei-complex or
Brucei group. Although T. brucei brucei has more similarities with T. brucei rhodesiense than T. brucei gambiense, these
parasites are morphologically indistinguishable. This is the probable reason why high sequence identity was displayed by other
subspecies of the Brucei group. In addition, the high percent identity possessed by TbrOST II with other trypansomatids agrees
with the evolutionarily conserved characteristics of the established OST. The DNA sequence data of TbrOST II showing
similar sequences in the genome of other organisms further corroborate the previous reports regarding the ubiquitous nature of
OST in other life forms. Based on the size of the amplicon and significant percentage of nucleotide and amino acid sequence
identity to homologues within the genome of related species and various organisms, the results strongly indicate that TbrOST
II is a trypanosome oligosaccharyl transferase gene candidate that should be fully characterized and subjected to functional
genomic studies. The study reports the molecular cloning and sequencing of a potential oligosaccharyl transferase gene in T.
brucei rhodesiense (TbrOST II). The sequence data has been deposited in the GenBank with accession number GU475126.