Acta Tropica 111 (2009) 255262

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Molecular analysis of surface glycoprotein multigene family TrGP expressed on

the plasma membrane of Trypanosoma rangeli epimastigotes forms
a , N. Lander b , E. Rodrguez b , G. Crisante c , N. Anez
c , J.L. Ramrez b , M.A. Chiurillo a,
C.P. Pena
a
b
c

Laboratorio de Gentica Molecular Dr. Yunis-Turbay, Decanato de Ciencias de la Salud, Universidad Centroccidental Lisandro Alvarado, Barquisimeto, Venezuela
Centro de Biotecnologa, Fundacin Instituto de Estudios Avanzados, Caracas, Venezuela
Centro de Investigaciones Parasitolgicas J.F. Torrealba, Universidad de los Andes, Mrida, Venezuela

a r t i c l e

i n f o

Article history:
Received 5 February 2009
Received in revised form 12 April 2009
Accepted 5 May 2009
Available online 9 May 2009
Keywords:
Trypanosoma rangeli
gp85/trans-sialidase
Multigenic family
TrGP

a b s t r a c t
Trypanosoma rangeli, a non-pathogenic hemoagelate that in Central and South America infects humans,
shares with Trypanosoma cruzi reservoirs and triatomine vectors, as well as geographical distribution.
Recently, we have described in T. rangeli a truncated gene copy belonging to the group II of the transsialidase superfamily (TrGP). This superfamily, collectively known in T. cruzi as gp85/TS, includes members
that are involved in host cell invasion and infectivity. To conrm the presence of this superfamily in the
genome of T. rangeli and obtain a better knowledge of its characteristics, we designed a PCR and RT-PCR
cloning strategy to allow sequence analysis of both genomic and transcribed copies. We identied two
full-length copies of TrGP, some pseudogenes, and N- and C-terminal sequences of several genes. We also
analyzed the expression and cellular localization of these proteins in epimastigote forms of a Venezuelan
T. rangeli isolate using polyclonal antibodies made against a recombinant peptide from the N-terminal
region of a TrGP member. We conrmed that TrGP is a multigenic family that shares many features with
T. cruzi gp85/TS, including the telomeric location of some of its members, and by immunouorescence
analysis that its location is at the surface of the parasite.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Trypanosoma rangeli despite being non-pathogenic to humans
shares a wide range of vertebrate hosts and triatomine vectors
with Trypanosoma cruzi, the etiological agent of Chagass disease,
and produces serological cross-reactivity with this parasite (Grisard
et al., 1999). Furthermore, the morphological similarity of these
two parasites, the lack of an appropriate specic diagnostic procedure, and the absence of clinical manifestations, contribute to the
underestimation of infections caused by T. rangeli (Guhl and Vallejo,
2003).
During the T. rangeli life cycle, the triatomine vectors become
infected after feeding with the blood of infected animals. The parasite subsequently replicates within the insects gut, and at some
point, the epimastigote forms cross the midgut epithelium to reach
the haemocoel. Once in the haemolymph, epimastigotes either
invade and multiply within hemocytes, or divide as free parasites in the haemolymph. Finally parasites invade and multiply
within the salivary glands transforming into infective metacyclic

Note: Nucleotide sequence data reported in this paper are available in the
GenBankTM database under the accession numbers FJ404790FJ404809.
Corresponding author. Tel.: +58 251 2591985; fax: +58 251 2591886.
E-mail address: mchiurillo@ucla.edu.ve (M.A. Chiurillo).
0001-706X/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2009.05.003

tripomastigote forms (Grisard et al., 1999). Recent data based on

kDNA and spliced leader (SL) gene markers, indicate a complex
parasitevector relationship, and suggest a co-evolution of the vector with T. rangeli isolates, such that each triatomine species would
select the sub-population that can be transmitted to the vertebrate
host (Vallejo et al., 2003; Maia Da Silva et al., 2007).
Among the most prominent genes shared by T. rangeli and T.
cruzi are those encoding for a large GPI-anchored glycoproteins
family named trans-sialidase (TS) superfamily, that according to
sequence identity, molecular weight, and function are classied
into a variable number of groups by different authors (Colli, 1993;
Cross and Takle, 1993; Frasch, 2000), although they can be gathered into two main groups (Frasch, 2000): Group I includes genes
encoding proteins with trans-sialidase and sialidase activity in T.
cruzi and T. rangeli, respectively. The sialidases expressed in T.
rangeli epimastigotes forms (TrSial) are strict hydrolytic enzymes
that release sialic acid residues from the host cell surface glycoconjugates (Pontes de Carvalho et al., 1993; Buschiazzo et al., 1997).
Group II molecules are devoid of enzymatic activity and include
the gp85 family or gp85/TS (8090 KDa), FL-160 (160 kDa) and Tc13
subgroups of proteins (Frasch, 2000).
The gp85/TS family includes proteins with variable degrees of
identity, characterized by the presence of two conserved neuraminidase motifs: ASP box (SxDxGxTW), and the VTV motif
(VTVxNVfLYNR), but lacking critical residues in the FRIP motif (Phe-

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Arg-Ile-Pro) that are important in determining catalytic activity

(Colli, 1993; Cross and Takle, 1993; Frasch, 2000; Buschiazzo et al.,
2000). In T. cruzi, gp85/TS members are expressed in infective trypomastigotes forms and intracellular amastigotes stages, and by
their capacity to adhere to the host cell surface and the extracellular matrix, some of these members are implicated in cell invasion
(Magdesian et al., 2001; Yoshida, 2006; Alves and Colli, 2007).
Recently, we have described that T. rangeli contains and

expresses genes of gp85/TS superfamily (Anez-Rojas

et al., 2005).
The rst ORF of gp85/TS described in T. rangeli (TrGP-1) was a telomeric truncated copy that conserves the motifs that characterize the
family, but lacks the GPI anchor site, and the C-terminal hydropho
bic tail (Chiurillo et al., 2002; Anez-Rojas
et al., 2005). In this work
we have further studied T. rangelis gp85/TS members and we have
conrmed that they represent a multigenic family. Additionally, we
evaluated the expression and cellular localization of these proteins
in T. rangeli epimastigotes using immunodetection methods.
2. Materials and methods
2.1. Parasites
T. rangeli isolates, provided by Drs. Palmira Guevara from
Universidad Central de Venezuela (Caracas, Venezuela), and Ns
tor Anez
from Universidad de los Andes (Mrida, Venezuela)
were cultured in biphasic bloodagar/NNN media at 25 C. We
used T. rangeli Venezuelan strains: M/CAN/VE/82/DOG82 and
MHOM/Ve/99/CH-99 for DNA isolation; MHOM/Ve/99/CH-99 for
RNA isolation from experimental infections and parasitic cultures;
and IRHO/Ve/98/Triat-1 for protein expression experiments.
Fourth instar nymphs of Rhodnius prolixus used in this study
were obtained from the Centro de Investigaciones Parasitolgicas J.F. Torrealba, Universidad de los Andes (Mrida, Venezuela).
Triatomines were articially fed with cultures containing exponentially grown T. rangeli. After the infective meal, triatomines were
kept at 27 C, at 70% humidity. Triatomines haemolymph was collected by sectioning a leg from the bugs. The presence of agellates
in the collected haemolymph was observed under light microscopy
in fresh and Giemsa stained preparations.
2.2. Nucleic acid isolation
DNA from T. rangeli culture epimastigote forms was isolated
using Wizard Genomic DNA Purication Kit (Promega). Total RNA
was puried from epimastigote parasites obtained from culture
or infected triatomines using TRIzol reagent (Invitrogen) following
manufacturers instructions.
2.3. Cloning of TrGP genes by PCR and reverse transcriptase-PCR
Genomic DNA or total RNA, from M/CAN/VE/82/DOG82 and
MHOM/Ve/99/CH-99 T. rangeli strains, respectively, were used as
template in reactions with Platinum Taq DNA Polymerase High
Fidelity (Invitrogen), or one-step AccessQuickTM RT-PCR System
(Promega), respectively. A set of forward (atgF: 5 -CACGTGCCCAACATGTCCCGGCAT-3 ; atgF2: 5 -ATGGCCTTTGGCAGTACGGC-3 ; slF:
5 -CTAACGCTATTATTGATACAGTTTCTG-3 ), and reverse primers
(nR1: 5 -GATGATACCCTCGGCAAGTG-3 ; nR2: 5 -TTTGTTGCCCTTTGCAATTG-3 ; nR3: 5 -GGCCTGCATCACAAATAC-3 ; nR4: 5 TCATGGAGACAAGCCCTTTTC-3 ) were used to clone sequences
encoding N-terminal TrGP regions by PCR and RT-PCR reactions. While vtvF: 5 -GTCTTTTTGTACAACCGCCC-3 and oligo-dT:
5 -CCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTT-3 )
primers
were
employed to clone C-terminal sequences by RT-PCR. We designed
atgF, atgF2, nR1, nR2, nR3, nR4 and vtvF primers based on the
nucleotide sequence of the TrGP-1 gene (GenBank accession no.

AF426022). slF and oligo-dT primers were designed based on T.

rangeli SL sequence (GenBank accession no. M62864) and poly-A
tail, respectively. The information of the 3 region of TrGP acquired
from the cDNA recombinants obtained by using oligo-dT primer
allowed us to design a cR primer (5 -TCCACTGTGCCCCACTCA-3 ),
which was used with atgF as forward primer and genomic DNA
as template to amplify full-length TrGP gene sequences. The PCR
products were then cloned into pGEM-T Easy vector (Promega),
and transformed into Escherichia coli strain TOP10F. According
to the portion of TrGP included in the recombinants they were
classied in three groups: (1) containing the N-terminal (trgpN);
(2) containing the C-terminal (trgpC); and (3) a full-length gene
copy. From each group we sequenced and analyzed several
clones.
2.4. DNA sequence analysis
Nucleotide sequences of TrGP recombinants were obtained using
BigDye Terminator v3.1 Cycle Sequencing Kit in an ABI PRISM 310
Genetic Analyzer (Applied Biosystems). Nucleotide and protein
sequence alignments were performed using DNAMAN v. 5.2.2
software (Lynon BioSoft). BLAST algorithms were used to search
for homologous nucleic acid or protein TrGP sequences in GenBank
and T. cruzi GeneDB databases, at http://www.ncbi.nlm.nih.gov
and http://www.genedb.org. Genomic or cDNA sequences were
annotated and submitted to the GenBank (accession nos.:
FJ404790FJ404809). Motif scanning for predicted protein
sequences was performed using the ExPASy proteomic server
(http://www.expasy.org).
2.5. Assessing the presence of TrGP copies in the telomere
To determine whether the telomeric location was a common feature in TrGP copies, we designed a simple PCR assay
based on the conserved T. rangeli subtelomeric sequences (SubTr)
described by Chiurillo et al. (2002), who using Balb-31 digestion
and hybridization experiments showed its exclusive subtelomeric location. We used vtvF as forward primer, which anneals at
3 end of TrGP copies (VTV motif), and as reverse primer TrF3:
5 -CCCCATACAAAACACCCTT-3 that anneals at SubTr. Sequences
were amplied in a 50 l nal volume, using 0.4 mM each primer,
0.2 mM dNTP, 1.5 mM MgCl2 , 1.25 U of Taq Platinum DNA polymerase (Invitrogen) and the following cycling conditions: 94 C for
3 min, followed by 35 cycles of 94 C for 1 min, 57 C for 30 s, 72 C
for 2 min, and a nal elongation at 72 C for 10 min. Amplied products were separated in a 0.8% agarose gel, and visualized with UV
light after stain with ethidium bromide. DNA of TrTel-4 recombinant (GenBank accession no. AF426022) containing a conrmed
TrGP telomeric copy was used as positive control (Chiurillo et al.,
2002). Some PCR fragments were puried from agarose gel using
Wizard SV Gel, and PCR Clean-Up System (Promega) following the
manufactures recommendations. Then, the fragments were cloned
into pGEM-T Easy vector (Promega), and transformed into E. coli
strain TOP10F. The nucleotide sequence of these recombinants was
obtained by automatic sequencing.
2.6. Expression and purication of recombinant TrGP
For indirect immunouorescence microscopy assays we generate antibodies against TrGP. To avoid any cross-reaction with
TrSial, the anti-TrGP antibody was prepared from the expression of a 706 bp fragment of the TrGP-1 gene encoding the
N-terminal domain (235 aa) of the putative protein. In a PCR
reaction using TrTel-4 as template (Chiurillo et al., 2002), we
used a forward 5 -TAGGATCCATGGCCTTTGGCAGTACGGC-3 and
reverse 5 -TCATGGACTCGAGCCCTTTTCCTCTCC-3 primers, contain-

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257

Fig. 1. (A) Comparison of nucleotide sequences of the 5 UTR region in trgpN recombinants, obtained by RT-PCR using slF primer. Conserved nucleotides are shaded in black
(100%), and light gray (>66% identity). The ATG initiation codons are shown in italics. As an example, the nucleotide sequence of the 5 -terminal coding region of a T. cruzi
gp85/TS family member (GenBank accession no. XM 807627) is also aligned. In this last case, conserved nucleotide with trgpN are indicated with (*) (100%), and (:) (5075%)
symbols. Alignments were done with DNAMAN v. 5.2.2 (Lynnon Biosoft) software, and then manually corrected to include T. cruzi sequences. (B) Alignments of deduced
signal peptide sequences from members of TrGP gene family and the N-terminal of a T. cruzi gp85/TSA representative. The signal peptide predicted using SignalP 3.0 program
(Bendtsen et al., 2004) in TrGP proteins are enclosed in a light gray box. The potential initiator methionines are indicated by .

ing BamHI and XhoI restriction sites, respectively. The PCR product
was digested with BamHI and XhoI restriction enzymes and for
the expression of a recombinant peptide fused to Schistosoma
japonicum glutathione S-transferase (GST), the digested fragment
was cloned into pGEX-5X-2 vector (Amersham Biosciences). Following electroporation with the TrGP construct, the recombinant
protein, named TrGPNLast , was expressed in E. coli BL21 (DE3)
pLysS (Invitrogen). After growing the recombinant bacteria in LB
medium, protein expression was induced by adding isopropyl-d-thiogalactopyranoside to a nal concentration of 1 mM, and
incubating for 6 h at 37 C. Cells were collected by centrifugation
and the pellet was resuspended in lysis buffer (25 mM TrisHCl, pH
7.8; 2 mM MgSO4 ; 50 mM NaCl, 0.1% Triton X-100, 10 mM lysozyme)
plus Set VII protease inhibitors (Calbiochem) and 1 U/ml DNase I
(Calbiochem), and then incubated for 30 min at 4 C. The lysate was
centrifuged at 10,000 g for 10 min at 4 C. The recombinant protein was recovered from the pellet and its expression conrmed
by SDS-PAGE. Finally, the protein was puried by passive dialysis
from acrylamide strips with 50 mM NaHCO3 , 0.1% SDS under constant shaking for 24 h at room temperature. Puried fractions were
reanalyzed by SDS-PAGE (MW 50-KDa). The same procedure was
performed for GST purication.

2.7. Production of anti-TrGP antibodies

Anti-TrGPNLast polyclonal antibodies were obtained by immunizing New Zealand rabbits with four doses (15 days each) of the
puried protein as antigen. Each rabbit received a rst dose consisting of 200 g of the antigen with Freunds complete adjuvant (1:1).
The following doses were 100 g/rabbit with Freunds incomplete
adjuvant.

2.8. GPI-anchored proteins analysis

T. rangeli GPI-anchored membrane proteins were isolated using
the partition Triton X-114 method previously described by Ko
and Thompson (1995). Rabbit immunization and production of
polyclonal serum against GPI-anchored proteins were carried out

according to Anez-Rojas
et al. (2006).

2.9. Western blot analysis

Proteins were resolved by SDS-PAGE and electro-transferred to
HybondTM ECL nitrocellulose membranes (Amersham Biosciences).
Blots were blocked with a solution of 5% non-fat milk in TBST
(50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween-20) and incubated
with primary antibody (1:5000) for 1 h at room temperature. Then,
blots were washed three times for 15 min with TBST and incubated with goat anti-rabbit HRP (Calbiochem) (dilution 1:10000)
for 1 h. Finally, blots were washed three times again with TBST and
immunodetections were developed using 3,3 diaminobenzidine as
substrate.

2.10. Indirect immunouorescence

The immunouorescence assays were carried out following the
methodology described by Figarella et al. (2007) with some modications. Briey, for each preparation 1 106 parasites were xed
with 4% paraformaldehyde solution and 0.1% glutaraldehyde in PBS
overnight at 4 C. The next day parasites were washed with PBS
and resuspended in 0.5 ml of blocking solution (100 mM Na2 HPO4 ,
100 mM glycine, pH 7.2) for 15 min at room temperature. For permeabilization, parasites were incubated in 0.5 ml of 0.2% Triton
X-100 in PBS during 5 min. Immediately, parasites were washed
with 1% BSA in PBS, and they were incubated overnight at 4 C with
200 l of rabbit anti-TrGPNLast (1:1000). Parasites were washed
twice again with 1% BSA and incubated for 1 h at 4 C in darkness
with a secondary antibody (Alexa Fluor 488 goat anti-rabbit, Invitrogen) diluted 1:1000. Then, 4 ,6-diamidino-2-fenilindol (DAPI,
Santa Cruz Biotechnologies) was added to a nal concentration
of 0.25 g/ml and the preparation was incubated for 20 min at
room temperature. Finally, cells were washed once with 1% BSA
in PBS and twice with dH2 O. Cells were resuspended in 15 l
of dH2 O, and 5 l of the suspension was placed onto a clean
coverslip and left to air-dry. Preparations were mounted with
2 l of Fluoromount-GTM (SouthernBiotech) and examined with
a confocal microscope D-Eclipse C1 (Nikon). Preparations without primary antibody were used as a negative control. Images
were processed with software EZ-C1 FreeViewer Silver Version 3.00
(Nikon).

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Fig. 2. (A) Multiple amino acid sequence alignment of three TrGP genes. Alignments were done by Clustal W. Conserved residues are shaded in black (100% conservation)
and gray (67% conservation). Continuous line boxes enclose the conserved motifs that characterize proteins of the gp85/TS superfamily. Discontinuous line boxes include
the amino acids sequence of TrGPNLast recombinant protein used to produce anti-TrGP serum. The dotted line marks the C-terminal hydrophilic domain of TrGP-4 deduced
protein. (B) Homology tree representing Clustal W multiple alignment of deduced amino acid sequences from members of group I and II of T. cruzi and T. rangeli TS gene
superfamily. The length of the pathway connecting each pair of nodes roughly indicates the level of dissimilarity between sequences. A rule of homology level is placed on top
of the graph. Sequences are: T. rangeli: TrGP-4 (FJ404803), TrGP-1 (AF426022), TrGP-3 (FJ4044802), TrSial 1 (L14943) and TrSial 2 (U83180); T. cruzi: Tcasp (U77951), TcASP-2
(AY186573), TcTS/gp85 (XP820450), TcTS 1 (X57235) and TcTS 2 (L26499). Nucleotide sequences were analyzed using the DNAMAN version 5.2.2 software. In parenthesis
GenBank accession numbers.

3. Results
3.1. Cloning and sequence analysis of TrGP
Considering that N-terminal region of TrGP-1 has a lower identity with T. cruzi gp85/TS (50%), and T. rangeli sialidase (2530%)
than the full-length translated gene, we decided to characterize several fragments of this region. All trgpN recombinants corresponded
to non-interrupted ORFs (between 450 and 748 bp), and 12 out the
14 recombinants showed sequence variations. A GenBank BLASTN
search with trgpN sequences revealed identities between 85 and

88% with TrGP-1, and of 5055% with T. cruzi gp85/TS members. On

the other hand, when an analysis by BLASTX was conducted, they
showed 7080% of identity with TrGP-1, and of 4045% (5564%
considering similarities) with T. cruzi gp85/TS.
The alignment of amino acid sequences deduced from the group
of trgpN cDNA clones obtained from parasites recovered from the
triatomine haemolymph resulted in an identity of 90% among
them. However, when sequences from cDNA recombinants derived
from culture epimastigotes were included in the analysis, the overall percentage of identity dropped to 67%. The same trend was
observed at the nucleotide level.

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259

Fig. 3. PCR detection of TrGP telomeric copies. (A) Schematic representation of T. rangeli telomere organization. The sense of primers used to amplify TrGP telomeric copies
is shown. Discontinuous blocks mean variable sequence length. (B) Agarose gel electrophoresis of amplied products with vtvF and TrF3 primers. Lanes: 1, 1 Kb DNA ladder
(Promega); 2, TrTel-4 recombinant; 3, T. rangeli M/CAN/VE/82/DOG82; 4, T. rangeli MHOM/Ve/99/CH-99; 5, T. cruzi YBM (M/HOM/VE/92/YBM).

The analysis of trgpN sequences obtained by RT-PCR using SL

derived primer allowed the detection of the 5 UTR of TrGP genes
(average length: 153 bp). A BLASTN analysis with TrGPs 5 UTRs
revealed a high percentage of identity (80%) with the 5 region
demarked by the putative rst and second methionine codons
present in most of T. cruzi gp85/TS genes (Fig. 1A). Although in T. cruzi
gp85/TS, it is not possible to assure which one of these two methionines is the real initiation codon, the predicted rst methionine in
TrGP corresponds to the second one in T. cruzi gp85/TS (Fig. 1B). Different from T. cruzi gp85/TS proteins, where no N-terminal signal
peptide was predicted, the in silico analysis of the N-terminal region
of T. rangeli TrGP protein family showed this feature (Kozak, 1989).
Three full-length TrGP clones, namely TrGP-2 (2069 bp), TrGP3 (2088 bp) and TrGP-4 (2292 bp) were sequenced. The alignment
of these three sequences with TRGP-1 showed 72% identity at
nucleotide level, which reached 80% when only TrGP-1, 3 and 4
were aligned. The in silico translation of TrGP-2 and one of the
trgpC-cDNA recombinants revealed that they were interrupted by
many stop codons in all its possible frames, and therefore they
should be regarded as a TrGP pseudogenes. A BLASTX search with
TrGP-3 and TrGP-4 nucleotide sequences resulted in a sequence
identity/similarity of 4348%/5763% to T. cruzi gp85/TS members,
being the highest percentage of identity with the T. cruzi amastigote surface protein-2 (ASP-2) subfamily (GenBank accession nos.
AY186573 and U77951).
As shown in Fig. 2A, the deduced amino acid sequences of TrGP1, 3 and 4 shared many features with all members of gp85/TS

family: the putative N-terminal signal peptide, two highly conserved copies of the sialidase motif SxDxGxTW, a complete copy
of the subterminal element VTVxNVfLYNR, the hydrophobic tail,
the potential GPI anchor signal sequence, and the absence of many
critical residues for catalytic activity. Within the C-terminal region
of TrGP-4 deduced protein there is an amino acid tandem repeat
(TR) composed by seven partially conserved copies of eleven highly
hydrophilic residues (Fig. 2A). Using the translated amino acid
sequences of TrGP-3 and TrGP-4, and several members of group
I and II of TS superfamily from T. rangeli and T. cruzi, we did a Clustal
W alignment to construct the homology tree shown in Fig. 2B. This
tree shows that although TrGP sequences share the branch with T.
cruzi gp85/TS members, they make their own cluster. Sequences of
the group I of the TS superfamily, both TrSial as TcTS, are grouped
at a second branch.
3.2. Presence of TrGP in telomere
PCR reactions combining primers based on the conserved structures of T. rangeli subtelomeric sequences (Chiurillo et al., 2002)
and VTV motif of TrGP amplied many fragments (Fig. 3A). These
amplicons formed a smear from 1 to >10 Kb in two T. rangeli
isolates, with some discrete fragments ranging between 1 and
2 Kb (Fig. 3B, lanes 3 and 4). This result indicates that TrGP
copies are abundant at T. rangelis telomeres, and the different
size bands can represent the characteristic length polymorphism
of the telomeric regions. The PCR fragment obtained with the

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Fig. 4. (A) Detection of TrGPNLast recombinat protein using anti-GPI-anchored proteins of T. rangeli. Lanes: 1, GST; 2, TrGPNLast . (B) Immunouorescence microscopy analysis
of T. rangeli permeabilized epimastigotes. (a) DAPI stain of nucleus (n) and kinetoplast (k) is shown in blue. (b) Immunodetection of TrGP (green) performed with polyclonal
anti-TrGPNLast antibodies. Fluorescense is observed at the surface membrane and the agellar pocket of T. rangeli epimastigotes. (c) Overlap of a and b images. Scale bar: 5 m.
T. rangeli strain: IRHO/Ve/98/Triat-1. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

positive control (lane 2, 960 bp) and the failure of amplication

when T. cruzi DNA was used as template (lane 5) indicate the high
specicity of the reaction. When cloned and sequenced, some of
the most abundant PCR products from TrGP genes displayed the
SubTr telomeric sequence associated with them, conrming their
telomeric/subtelomeric location. Different to TrGP-1, one of these
fragments showed a telomeric TrGP that includes a TGA stop codon
which coincides with the 3 end of full-length gp85/TS genes (not
shown).
3.3. TrGP expression and cellular localization
Since the DNA sequence used to generate the recombinant
peptide TrGPNLast has only <20% of identity with TrSial gene, antiTrGPNLast antibodies should be specic for TrGPs. Similar to the

report of Anez-Rojas
et al. (2005) with a different antibody generated from the N-terminal region of TrGP-1, when the anti-TrGPNLast
antibodies were used in Western blot assays against T. rangeli epimastigotes extracts they detected a 73-kDa protein band (not
shown). The size differences between the native TrGP and the
deduced protein from the DNA sequences (7583 kDa) could be
explained by post-translational modications, and the removal of
the signal peptide and the hydrophobic tail. When the fused protein TrGPNLast was incubated with an antibody generated against
the GPI-anchored proteins fraction of T. rangeli, the fused peptide,
but not a recombinant GST fragment, was recognized by these antibodies (Fig. 4A).
The cellular localization of TrGP proteins in T. rangeli epimastigotes cells was assessed by immunouorescense confocal
microscopy using anti-TrGPNLast antibodies, and in permeabilized
parasites we found that they reacted exclusively with cell surface
components (Fig. 4B). The immunouorescent label was evenly
distributed over the entire cellular surface of the parasite with a
granular appearance, including the agellar pocket and the agel-

lum tip. The same result was observed using non-permeabilized

epimastigotes (data not shown). Preimmune serum did not react
with these parasites.
4. Discussion
Since the T. rangeli genome sequence has not yet been completed, the analysis by a low throughput sequencing strategies can
generate valuable information about multigene families encoding
surface antigens. Herein we proved that TrGP constitutes a multigenic family, and showed for the rst time the existence of complete
copies of the gp85/TS gene family in T. rangelis genome. Moreover, the information obtained from 5 and 3 UTR regions of TrGP
genes may help to the optimization of T. rangeli gene expression
vectors.
Using a new PCR strategy based on subtelomeric sequences,
we also conrmed the presence of more TrGP copies at T. rangelis
telomeres. In protozoan parasites, telomeres are usually enriched
in contingency genes such as surface antigenic determinants, and
in the case of T. cruzi, gp85/TS or related sequences, together with
retrotransposon elements, are a structural part of its telomeres
(Chiurillo et al., 1999; Kim et al., 2005). Like many trypanosomatidss surface protein gene families, the TrGP family contains
pseudogenes, which through recombination could contribute to the
TrGP repertoire variability, a hypothesis that has been proposed to
explain the existence of numerous pseudogenes of gp85/TS in the
genome of T. cruzi (El-Sayed et al., 2005; Azuaje et al., 2007). On
the other hand, herein we demonstrated that TrGP pseudogenes
could be transcribed to mRNA. This fact can be explained by the
polycistronic nature of trypanosomatids transcription (Worthey et
al., 2003), however, some evidences indicating a role for pseudogenes in the control of gene expression in kinetoplastid parasites
have been reported for other genes (Taylor and Rudenko, 2006;
Durand-Dubief et al., 2007).

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Trypanosomatid parasites have a high prevalence of genes coding for proteins containing TR domains such as TS, mucins, and
mucin-associated surface proteins (MASP), and although TR do not
have identical sequences, their hydrophilic properties have been
retained (Goto et al., 2008). Many of the T. cruzi proteins that
have been conrmed as serological antigens have TR domains (Da
Silveira et al., 2001), and some of them have been implicated in
evasion of the host immune response, thus contributing to parasite
survival (Buscaglia et al., 1999; Goto et al., 2008). Considering that
the in silico analysis of TR domain described in TrGP-4 revealed
immunogenic properties and no signicant amino acid sequence
identity with those reported in T. cruzi available databases, we could
speculate that they could be exploited to develop more accurate
diagnostic methods to distinguish mixed infection by T. cruzi and T.
rangeli.
Another interesting nding was the high percentage of identity among cDNA clones from T. rangeli recovered from triatomine
haemolymph. Although several forms and stages of the parasite

coexist in this compartment (Anez,

1983; Palu et al., 2001), our
results suggest a uniform expression of TrGP proteins. However, we
cannot rule out a bias caused by the primers used to amplify these
sequences.
The high degree of conservation of T. cruzi gp85/TS genes and T.
rangeli TrGPs suggests that these proteins play an important role in
the parasites life cycle. The key question is why T. rangeli needs to
express gp85/TS proteins when this parasite does not seem to be
very effective in invading or multiplying within mammalian cells. In
T. rangeli vector infections pathological effects are mainly observed
during its multiplication in the haemolymph and hemocytes, and
the invasion of the salivary glands of the triatomine bug (Guhl and
Vallejo, 2003). We can speculate that TrGP proteins may be necessary during the migration and multiplication of the parasite through
and in different triatomine tissues and compartments. In the case
of TrSial, it has been proposed that its enzymatic activity may constitute a mechanism to regulate the attachment of T. rangeli to the
vectors salivary glands (Basseri et al., 2002). Nevertheless, since
its natural host is unknown, we cannot discard that TrGP expression may also be implicated in the survival of the parasite in the
vertebrate host.
Acknowledgements
This work was supported by FONACIT grant N S1-2002000542
and CDCHT-UCLA 007-ME-2007. To Mrs. M. E. Camargo for technical
assistance. M.G. Rojas and M. Sayegh for performing DNA automatic
sequencing. Mrs. Sharon Sumpter for revising the English of the MS.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.actatropica.2009.05.003.
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