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

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. Material, Methods and 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.


INTRODUCTION
Trypanosoma brucei rhodesiense is a haemoflagellate parasite of zoonotic significance.A wide range of mammalian fauna, especially domestic livestock and wild bovids, serves as reservoir host [23,26].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.Unique VSG are alternately produced by sequential expression of about a thousand trypanosome VSG gene reservoir per parasite; a phenomenon described as antigenic variation [5,6].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 [7,19,21,24].This complex process of protein modification is generally known as glycosylations, one of which is N-linked glycosylation.
The central event in the N-linked glycosylation process is catalyzed by oligosaccharyl transferase (OST) [11].It catalyzes the co-translational addition of preassembled oligosaccharide complexes (Dol-PP-GlcNAc 2 Man 9 Glc 3 ) to an asparagine residue in an Asn-Xaa-Ser/Thr consensus sequon (Xaa can be any amino acid excluding proline) of the growing nascent polypeptide chain being translocated into the endoplasmic reticulum through a structure called translocon [4].
In general, the incorporation of glycan structures to different protein moieties is precedent towards proper protein folding and stability, intracellular targeting, intercellular recognition, hormone synthesis, anti-apoptotic response, control of salt/osmotic stress, and cell surface expression of some glycoproteins [8,9,15,17].Glycans 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 [16,20,21].Therefore our major interest is to clone and characterize the trypanosome OST.

Laboratory animals
Female 8-week-old BALB/c mice 1 were used in the study.The animal room was maintained at 22 ± 3 0 C with a 12:12 h of light-dark cycle.All experiments were conducted according to the guidelines for the care and use of laboratory animals, Obihiro University of Agriculture and Veterinary Medicine, Japan.

DNA extraction
The template genomic DNA for PCR amplification was extracted as described previously [2].Briefly, T. brucei rhodesiense IL2343 genomic DNA was extracted by adding 9 volumes of extraction buffer (0.2 M NaCl 8 , 10 mM Tris-HCl 3,8 pH 8.0, 10 mM EDTA 3 pH 8.0 and 1% SDS), proteinase K 6 to a final concentration of 100 µg/mL and followed by 6 h incubation at 55°C with gentle agitation.Overnight incubation was performed after additional proteinase K was placed.Genomic DNAs were phenol-chloroform-isoamyl alcohol 3 extracted, ethanol precipitated, and resuspended in Tris-EDTA buffer, pH 8.0 or deionized water.The concentration of the sample DNA was determined by spectrophotometry.

Polymerase chain reaction amplification of putative OST gene
The primers were designed from the nucleotide sequences of T. brucei genomic clones as guided by EMBL-EBI Parasite Genomes WU-Blast 2 database search (www.ebi.ac.uk/blast/para-sites.html) for African trypanosomes with L. major putative OST STT3 subunit sequence as the query [EMBL Q9U5N8 (AJ251127.1)].PCR amplication was performed using a forward primer 9  (5'-TGG TAC GAC TAC ATG AGC TGG TAC CCG CT-3') and a reverse primer 9 (5'-TGG ATC TCC TTC GCT GGC GGG TAC TG-3').Distilled water was used as template for negative control reaction.The samples were programmed to a temperature-step cycle of 94°C at 10 min, 94°C at 30 s, 60°C at 30 s for a total of 30 cycles followed by 4 min extension at 72°C.The PCR products were analyzed by electrophoresis on 1% TAE (Tris-acetate-EDTA) agarose gel.The PCR product was then processed for cloning after agarose gel extraction using a commercial kit 10 according to the manufacturer's instructions.

Cloning and sequencing of PCR products
The PCR product was ligated into EcoR V site of pT7 bluescript plasmid vector 11 using Takara solution I ligation kit 12 .Ligation reaction was transformed into DH5 α competent E.coli cells and plated on Luria Britani'sampicillin (LB-amp) agar dishes.The presence of insert was confirmed by restriction digest against Hind III & Xba I site from the cloning site of the plasmid vectors flanking the PCR product.
Prior to sequencing, twenty-five cycles of Bigdye PCR was carried out in a total volume of 5 µL and were performed using the following standard condition: 96 0 C at 2 min, 96 0 C at 10 s, 50 0 C at 5 s and 60 0 C at 4 min.Sequencing was started by the single strand dideoxynucleotide-chain-termination method using a cycle sequencing kit 13 , DNA sequence analyzer 13 and T7 promoter primer and pUC/M13 reverse primers 9 .The second set 9 [forward (5'-GAC ATA CAG CGT CAG TTT GC-3'); reverse (5'-GAT GAA TGT GAG TGA AGA GAG C-3')] and the third set 9 [forward (5'-CGT TCG GAT TCT TCA AAC CTA CAG-3') and reverse (5'-AAT ACG GGC ATC TTC AGG CG-3')] primers were used to obtain the partial nucleotide sequence.The nucleotide sequence alignments and analysis were performed using sequence analysis softwares 14,15 .

RESULTS
A putative trypanosome oligosaccharyl transferase gene was successfully amplified using T. brucei rhodesiense crude DNA as the template.Genomic clone of approximately 2000 bp was obtained after PCR amplification (Figure 1).Nucleotide sequencing revealed that the amplified band was composed of 2057 bp partial nucleotide sequence (Figure 2).The deduced partial primary structure of the T. brucei rhodesiense putative oligosaccharyl transferase clone II (TbrOST II) was composed of 685 amino acid sequence (Figure 2).Determination of the sequence homology in other organisms was carried out using NCBI Basic Local Alignment Search Tool (BLAST) (http:// blast.ncbi.nlm.nih.gov/Blast.cgi)[1].
Sequence analysis showed that TbrOST II had significant nucleotide (Ns) and amino acid sequence (As) percent identity to putative oligosaccharyl transferase subunit coming from other kinetoplastid genome in the NCBI public database.
When compared with the related trypanosome species, the DNA size of T. brucei rhodesiense putative oligosaccharyl transferase was 200 bp than T. brucei gambiense (1842 bp, FN554968.1).In addition, the sequence homologues from T. brucei (2466 bp, XM_839672.1)(2466 bp, XM_839671.1)(2406 bp, AC159432.1)(2406bp, XM_839670.1),exceeded TbrOST II clone by >400 bp.The Latin American trypanosome species, T. cruzi, with percent identity to TbrOST II on the other hand had a molecular weight of 2397 bp.(XM_803446.1).Furthermore, the data showed that the annotated DNA size of the homologues under genus Leishmania was greater than that of genus Trypanosoma with a disparity ranging from

DISCUSSION
The surface coat of trypanosome species were previously reported to have marked diversities in Nglycosylation [20].This prompted us to hypothesize that trypanosomes also possess N-glycosylation enzyme.During the attempt to clone T. brucei rhodesiense putative oligosaccharyl transferase, a genomic clone of ~2000 bp was acquired.
Notably, L. major putative OST STT3 subunit sequence was used as the query during the primer design for PCR amplification of TbrOST II.The efficient amplification of the gene in genus T. brucei rhodesiense using primers designed from L. major stt3 gene as the query strongly indicate that TbrOST II seems to be conserved within the family Trypanosomatidae.The nucleotide BLAST results indicates 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 [18,23].This is the probable reason why high sequence identity was displayed by other subspecies of the Brucei group.This also indicates that the functional unit of this putative oligosaccharyl transferase is conserved within the Brucei complex trypanosomes.In addition, the high percent identity possessed by TbrOST II with other trypansomatids agrees with the evolutionarily conserved characteristics of the established OST [25,27].Consequently, DNA identity searches within the public databases obtained homologues in the genome of mammalian, nematode, arthropod and algae species.Selected organisms include Mus musculus (Ns:71%, NM_ 024222.2;As:29%, NP_077184.2),Schistosoma mansoni (Ns:73%, XM_002577919.1;As:29%, XP_002577965.1),Drosophila pseudoobscura pseudoobscura (Ns:77%, XM_002134519.1;As:31%, XP_002134555.1),Thalassiosira pseudonana (Ns:77%, XM_002288187.1;As:46%, XP_002288223.1)and Phaeodactylum tricornutum (Ns:73%, XM_002185331.1;As:46%, XP_ 002185367.1).Moreover, obtained amino acid sequence identity in the genome of fly, nematode, mouse and algae homologues also exhibited lower sequence similarity (29-46%) than the previously documented percent identity (~50%) of reported oligosaccharyl transferase in other eukaryotic species [25].

CONCLUSIONS
Finally, even currently regarded as putative, 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 trans-ferase 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 of GU475126.