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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2025-2032
Tandem Amino Acid Repeats From Trypanosoma cruzi Shed Antigens
Increase the Half-Life of Proteins in Blood
By
Carlos A. Buscaglia,
Julieta Alfonso,
Oscar Campetella, and
Alberto C.C. Frasch
From the Instituto de Investigaciones Biotecnológicas,
Universidad Nacional de General San Martín, Buenos Aires,
Argentina.
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ABSTRACT |
Proteins containing amino acid repeats are widespread among
protozoan parasites. It has been suggested that these repetitive structures act as immunomodulators, but other functional aspects may be
of primary importance. We have recently suggested that tandem repeats
present in Trypanosoma cruzi trans-sialidase stabilize the
catalytic activity in blood. Because the parasite releases trans-sialidase, this delayed clearance of the enzyme might
have implications in vivo. In the present work, the ability of
repetitive units from different T. cruzi molecules in
stabilizing trans-sialidase activity in blood was
evaluated. It is shown that repeats present on T. cruzi shed
proteins (antigens 13 and Shed-Acute-Phase-Antigen [SAPA])
increase trans-sialidase half-life in blood from 7 to almost 35 hours. Conversely, those repeats present in intracellular T. cruzi proteins only increase the enzyme half-life in blood up to 15 hours. Despite these results, comparative analysis of structural and
catalytic properties of both groups of chimeric enzymes show no
substantial differences. Interestingly, antigens 13 and SAPA also
increase the persistence in blood of chimeric glutathione
S-transferases, thus suggesting that this effect is inherent to these
repeats and independent of the carrier protein. Although the molecular
basis of this phenomenon is still uncertain, its biotechnological
potential can be envisaged.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PROTEINS CONTAINING tandems of amino acid
repeats are particularly widespread among several parasitic taxa like
the protozoan agents of malaria (Plasmodium spp.),
leishmaniasis (Leishmania spp.), and trypanosomiasis
(Trypanosoma cruzi and T. brucei).1-5 The
meaning of such a particular protein array in these parasites is still
a matter of debate. It has been suggested that they are involved
in binding to repetitive structures within the
parasite,5,6 binding to host-cell receptors and/or
polymerization of their associated, nonrepeated domains.7
One relevant feature concerning these repetitive motifs is that they
are readily detected by antibodies present in the sera from infected
patients, thus suggesting that they are major targets of the immune
response.1,3,8 Nevertheless, these humoral responses have
not usually been correlated with protection. These observations have
led us to speculate that repeated amino acid arrays would act as
immunomodulators in protozoan antigens.1,8,9 The precise
mechanism of this immunomodulation is not known, but it has been shown,
for instance, that repetitive and/or highly organized epitopes
could influence both antigen presentation and responsiveness of immune
cells.10-13
The trans-sialidase from T. cruzi is a developmentally
regulated protein that has been implicated in host-parasite
interactions (for review, see reference 14). The enzyme, located on the
trypanosome's surface, is responsible for transferring sialyl residues
from host's glycoconjugates to parasite molecules.15 Once
sialylated, these molecules are thought to mediate binding to, and
invasion of, mammalian cells.16,17 The
trans-sialidase displayed by the epimastigote (the parasite
form present in the reduviid vector) has a potential
trans-membrane domain and is not released, even after addition
of exogenous phospholipase.18 On the contrary, the enzyme
present in the trypomastigote (the infective form of the parasite that
circulates in the blood of the vertebrate host) is anchored by a
glycosylphosphatidylinositol (GPI) linkage to the T. cruzi
surface and is released into the environment.19 Even
though the effect of circulating trans-sialidase on T. cruzi infectivity and/or pathogeny is far from obvious,
this enzymatic activity has been detected in patient's blood during
acute human infections.20
When compared with the epimastigote trans-sialidase, the
trypomastigote enzyme contains an additional C-terminal extension made
up essentially of repetitive amino acid sequences termed SAPA (for
Shed-Acute-Phase-Antigen). SAPA-domain is not involved in the
catalytic activity of trans-sialidase, as shown by using recombinant21 and papain-digested enzymes.7 In
a previous work, we suggested that SAPA-domain stabilize the
trans-sialidase activity in blood.22 To further
test this hypothesis and to determine the sequences involved in this
stabilization, chimeras containing the trans-sialidase coding
sequence linked to several T. cruzi tandem repeats have been
generated. Some of these tandem repeats are present in
intracellularly-located proteins (antigens 1 and 36) whereas others are
in GPI-surface anchored proteins (antigen 13).23 It is
shown here that the addition of repetitive units present in T. cruzi shed proteins (antigens 13 and SAPA) significantly increase
the persistence of the chimeric trans-sialidases in circulation
when compared with the enzymes linked to repetitive units present in
intracellularly located T. cruzi proteins (antigens 1 and 36).
Interestingly, persistence in blood of the gluthathione S-transferase (GST) from Schistosoma japonicum is
also increased when linked to antigen 13 or SAPA, thus suggesting that
this stabilization in blood is a general property of certain T. cruzi shed amino acid repeats.
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MATERIALS AND METHODS |
Cloning of chimeric trans-sialidases.
To express the repetitive domain of different T. cruzi
antigens3 in frame with a functional
trans-sialidase gene, a so-called "acceptor clone" has
been constructed by a polymerase chain reaction (PCR) strategy. This
acceptor clone (termed as TSac clone, Table 1) is essentially alike TS clone22 but contains a unique
EcoRI site in its 3' terminus in the frame of  gt11
phage (Pharmacia Biotech, Uppsala, Sweden) EcoRI cloning site.
Inserts recovered from EcoRI-digested gt11 clones containing
repetitive T. cruzi antigens 1, 13, and 363 were
ligated with the TSac clone previously digested with EcoRI. Chimeric trans-sialidases were further analyzed by DNA
sequencing by the dideoxynucleotide chain-termination
method,24 using the Sequenase 2.0 kit (U.S. Biochemical
Corp, Cleveland OH). Molecular properties of chimeric
trans-sialidases were analyzed with the aid of the LaserGene
software (DNASTAR Inc, Madison WI).
Purification of chimeric trans-sialidases.
Chimeric trans-sialidases expressed in Escherichia coli
strain Novablue (Novagen, Madison WI) were purified using the stretch of histidines present on the N-terminus fusion peptide (Table 1)
provided by pTrcHis vector, according to manufacturer's instructions (Invitrogen, San Diego, CA). Accurate expression of T. cruzi
repetitive sequences in frame with trans-sialidase was verified
in each case by Western blot analysis using a polyclonal antiserum
raised in mice against each repetitive domain used
(Fig 1). None of these polyclonal antisera
recognize the catalytic domain of trans-sialidase (data not
shown).
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| Fig 1.
Identification of chimeric trans-sialidases.
Detection of chimeric trans-sialidases in Western blot was
performed with antisera raised in mice against each repetitive T. cruzi antigen expressed in pGEX vector. The monoclonal antibody
against SAPA-repeats used to probe TS-3R, TS-8R, and TS-13R proteins
has been already described.25 The presence of additional
immunoreactive bands in TS-Ag 36 protein is due to partial protein
degradation. The increase in the apparent molecular weight of TS-Ag 13 protein is attributed to abnormal migration in SDS-PAGE. Molecular
weight markers are indicated in kD.
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Cloning and purification of TS-SAPA proteins containing variable
number of tandem repeats.
PCR was performed with the following oligonucleotides:
5'-GGGGCAGAATCAACGGTATCG-3' and
5'-CGGAATTCTCACCCATTGGCACTGCTGTC-3', using the
TS-SAPA clone22 as template. Because the latter
oligonucleotide primes on the repetitive unit, a collection of products
containing a randomized number of tandem repeats was obtained. The
entire mixture of PCR products was purified from agarose gels and
enzymatically digested with KpnI and EcoRI. This latter
restriction site is underlined in the repeat-priming oligonucleotide.
Ligation with the TSac clone was achieved by using the unique
EcoRI and KpnI sites.22 The precise number
of repeated blocks for each clone was settled by DNA
sequencing.24 Expression and purification of three of these
recombinant trans-sialidases with a variable number of
SAPA-repeats was done as described above. Accurate expression of
TS-SAPA deletion proteins was verified by reactivity with a monoclonal anti-SAPA antibody (Fig 1).
Trans-sialidase assay.
Enzymatic activity of purified trans-sialidases was assayed by
measuring the amount of sialic acid residue transferred from sialyllactose (Sigma Chemical Co, St Louis MO) to
[14C]-lactose (Amersham, Little Chalfont,
Buckinghamshire, UK) as previously described.25 One Unit of
trans-sialidase was defined as the amount of enzyme able to
transfer 10 nmoles of sialic acid to lactose in 1 minute under standard conditions.
Animals.
C3H/HeN mice 60- to 90-days old (both males and females), bred in our
own facilities were used.
Immunization schedules and pharmacokinetics studies with chimeric
trans-sialidases.
Animals were injected by the retroorbital sinus with 100 pmoles of
chimeric trans-sialidases contained in 150 to 200 uL of sterile
saline solution (0.15 mol/L NaCl). Remnant trans-sialidase activity was assayed in blood samples (1 to 5 uL) collected from the
tail at different times after inoculation.22
Purification of S. japonicum GST chimeric proteins and
determination of GST activities.
Chimeric GST proteins containing different T. cruzi amino acid
repeats were generated by gene-fusion to S. japonicum GST
encoded by plasmid pGEX-1 (Pharmacia Biotech) using the EcoRI
cloning site, expressed in E. coli and purified by affinity on
glutathione-agarose columns as described.26 GST activities
were measured according to Habig et al.27 Briefly, serial
dilutions (0.5 ng/mL to 5 ug/mL) of GST chimeric proteins were
incubated in 2 mL of phosphate buffer 0.1 mol/L pH 6.5 containing 2.5 mmol/L glutathione (Sigma) and 0.5 mmol/L 1-Chloro-2, 4 dinitrobenzene
(Sigma). Changes in absorbance at 340 nm were recorded.
Pharmacokinetics studies with GST chimeric proteins.
Mice were intravenously injected with 1 nmol of each GST chimeric
protein extensively dialyzed against phosphate-buffered saline (PBS).
Remnant GST chimeric proteins in circulation were quantitated in blood
samples collected at different times after injection both by
determination of GST activity (see above) and by a standard
enzyme-linked immunosorbent assay (ELISA) capture technique. Briefly,
polystyrene ELISA microplates (Maxisorp; Nunc, Roskilde, Denmark) were
coated with 75 uL of an 8 ug/mL solution of protein-A purified rabbit
immunoglobulins (Igs) to S. japonicum GST in PBS. Blocking of
the plates and subsequent dilution of Ig solutions were made in
Tris-buffered saline 0.15 mol/L pH 7.6 containing 3% of extensively
dialyzed bovine serum albumin (Sigma). Appropriate dilutions of serum
samples taken at different times after injection were incubated for 1 hour at room temperature and captured GST was evaluated by the addition
of biotin-labeled anti-GST Igs raised in rabbits (2.5 ug/mL). A solution of alkaline phosphatase-conjugated avidin (Pierce,
Rockford, IL) was then added to the plate and incubated for an
additional hour. Chromogenic reaction with p-nitro phenyl phosphate
(Sigma) was allowed to proceed for 30 minutes. Plates were read at 405 nm in a microplate reader (Bio-rad Laboratories, Richmond, CA).
Sensitivity assays of chimeric proteins to blood-proteinases.
GST or GST-SAPA protein solutions (30 ug/mL) in normal mouse serum or
normal mouse blood diluted (1:1 vol/vol) in Alsever's solution (sodium
azide 0.02% wt/vol was added in both cases) were incubated at 37°C
with occasional agitation. At different times, aliquots (10 uL) were
subjected to Western blot analysis using a polyclonal anti-GST
antiserum developed in rabbits. Reaction was visualized by the addition
of peroxidase-conjugated goat anti-rabbit Igs (DAKO, Ejby, Denmark)
followed by the chromogenic substrate 4-Chloro-1-naphtol (Bio-rad).
TSac or TS-SAPA protein diluted to 5 ug/mL in normal mouse serum
were incubated at 37°C with occasional agitation. At
different times, aliquots (2 uL) were assayed for
trans-sialidase activity as described above.
Statistical analysis.
The stability in blood of the different proteins was compared with that
obtained for TS-SAPA (Fig 2 and
3) or GST-SAPA values (Fig 4) in every time point of the curve
using the Student's t-test. P < .05 were considered
significant.
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| Fig 2.
Pharmacokinetics studies of chimeric
trans-sialidases. Mice intravenously inoculated with 100 pmoles
(about 10 ug) of the indicated protein, were bled at the indicated days
after injection and measured for remnant trans-sialidase
activity in serum samples. Data are expressed as the mean ± standard
deviation (SD) (n = 3 animals). Values recorded 30 minutes after
injection are indicated as day 0 values and were taken as 100%
trans-sialidase activity. One out of two experiments with
similar results is shown. Asterisks (*) denote significant differences
(P < .05) to TS-SAPA protein.
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| Fig 3.
Pharmacokinetics studies of TS-SAPA deletion proteins.
Mice intravenously inoculated with 100 pmoles of the indicated protein,
were bled at the indicated days after injection and quantitated for
remnant trans-sialidase activity in blood as indicated in the
legend to Fig 2. Data are expressed as the mean ± SD (n = 3 animals). TS-3R, TS-8R, and TS-13R proteins contain 3, 8, or 13 SAPA-repetitive units, respectively. One out of two experiments with
similar results is shown. Asterisks (*) indicate significant
differences (P < .05) to TS-SAPA protein.
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| Fig 4.
Pharmacokinetics studies of GST chimeric proteins. Mice
intravenously inoculated with 1 nmol (about 50 ug) of the indicated
protein were bled at the indicated days after injection and quantitated
for remnant GST in blood using a GST capture assay. Sera from
PBS-inoculated mice were used as negative controls. Values recorded 30 minutes after injection are indicated as day 0 values and were taken as
100% circulating GST. Data are expressed as the mean ± SD (n = 4 animals). One out of two experiments with similar results is shown.
Asterisks (*) denote significant differences (P < .05) to
GST-SAPA protein.
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RESULTS |
Chimeric trans-sialidases containing different T. cruzi
repetitive units are enzymatically active and display similar specific
activities.
Gene fragments coding for repetitive domains present in different
T. cruzi proteins (antigens 1, 13, 36, and SAPA) were placed in
cis with a functional trans-sialidase gene and expressed in E. coli (Fig 1). These T. cruzi repeats have been
previously shown to be associated to membrane anchored and possibly
shed into the milieu proteins (antigens 13 and SAPA) or to
intracellularly located proteins (antigens 1 and 36).23,28
As shown in Table 1, chimeric trans-sialidases thus obtained
have similar structure, containing (from N- to C-terminus)
the pTrcHis fusion peptide, the trans-sialidase catalytic
domain, and, except for the TSac protein, the different repetitive
units. In addition, TS-Ag 13 and TS-SAPA proteins contain a nonrepeated
stretch of amino acids in their C-termini that, in the latter case, is
compatible with a GPI-anchoring signal.19 Even though these
T. cruzi repeated units are variable in size and amino acid
sequence, they all display hydrophilic characteristics and confer a
negative net charge at pH 7 to the resultant chimeric trans-sialidase (Table 1).
When assayed for trans-sialidase activity, these chimeric
proteins render similar specific activities (Sp. Act.) ranging between 0.48 (for TS-Ag 1 protein) and 1.09 U/nmol (for TS-SAPA protein) (Table
1). Furthermore, Sp. Act. of chimeric trans-sialidases are
similar to that of the recombinant enzyme expressed by the TSac clone
(0.56 U/nmol, Table 1). Thus, as previously shown for the
trans-sialidase molecule containing
SAPA-domain,7,21 the presence of any of these T. cruzi repeats in cis does not seem to significantly interfere with
the catalytic activity.
Repetitive domains present in T. cruzi shed antigens but not
in T. cruzi internal antigens stabilize trans-sialidase
activity in blood.
To determine the degree of stabilization of trans-sialidase
activity in blood mediated by different T. cruzi repetitive
units, 100 pmoles of either TS-Ag 1, TS-Ag 13, or TS-Ag 36 protein were intravenously administered in mice and remnant enzymatic activity monitored in serum samples obtained at different times after
inoculation. Equivalent amounts of TSac (lacking any C-terminal
extension) and TS-SAPA proteins were used as controls. As shown in Fig
2, TS-Ag 13 protein has an estimated half-life in blood of 32 to 34 hours and can be detected in circulation up to 3 days after injection.
This persistence in blood is similar to that of TS-SAPA protein
(estimated half-life in blood of 37 to 38 hours, Fig 2) and represents
a fivefold increase as compared with the half-life in blood of TSac
protein (7 hours, Fig 2). On the other hand, trans-sialidase
activity of both TS-Ag 1 and TS-Ag 36 proteins is rapidly cleared from
mice blood, with estimated half-lives of 16 to 18 hours and 15 to 19 hours, respectively (Fig 2). Thus, it can be concluded that repeated
motifs present in T. cruzi shed antigens SAPA and 13 are more
efficient in stabilizing trans-sialidase activity in blood than
those present in T. cruzi internal antigens 1 and 36.
The repetitive units in the SAPA-domain are required to enhance the
half-life in blood of trans-sialidase.
To further characterize the sequences involved in the improved
pharmacokinetics of trans-sialidase activity of TS-SAPA
protein, a set of deletion clones derived from TS-SAPA gene containing different number of SAPA-repeats was generated (Fig 1). All of them
lack the GPI-anchoring signal present in the SAPA-domain (Table 1).
Proteins expressed and purified from three of these clones (TS-3R,
TS-8R, and TS-13R containing 3, 8, and 13 SAPA-repetitive units,
respectively) display similar Sp. Act. (about 0.65 U/nmol) to that
reported for the entire TS-SAPA protein (Table 1).
These proteins were intravenously injected in mice and
trans-sialidase activity monitored in blood samples throughout
several days. As shown in Fig 3, persistence in blood of TS-8R and
TS-13R proteins (both with estimated half-lives in blood of 37 to 39 hours) is almost indistinguishable from that of the entire
TS-SAPA molecule, which contains a hydrophobic C-terminal
44-amino acid extension in addition to 13 repetitive units (Table 1).
Thus, it can be concluded that the repetitive units present in antigen SAPA, and not its GPI-anchoring signal, are involved in the
stabilization of trans-sialidase activity in blood.
On the other hand, TS-3R protein results in a trans-sialidase
with shorter half-life in blood (about 12 to 13 hours; Fig 3). This
experiment shows that a minimal number of repetitive units is required
to stabilize the circulating trans-sialidase activity.
Repetitive shed antigens from T. cruzi could also enhance the
stability in blood of a protein unrelated to trans-sialidase.
We next asked if 13- and SAPA-repeats were also able to stabilize in
blood a protein unrelated to trans-sialidase. We chose the 28 kD-GST domain from S. japonicum encoded in pGEX vector as a
model. GST proteins bearing amino acid repeats from T. cruzi internal antigens (1, 30, and 36) and from proteins spontaneously released by the parasite (antigens 13 and SAPA) on their C-termini were
expressed, purified, and analyzed.26
The enzymatic activity of the GST domain present in each chimeric
protein was determined as the change in optical density values recorded at 340 nm. These values are about 0.01 Absorbance Units.minute -1 .ug GST-1 in proteins
containing or lacking repetitive motifs (data not shown). These results
suggest that, as is the case of trans-sialidase, the folding
and/or functional activity of GST is not severely modified by
the addition in cis of any of these T. cruzi repeats.
Each of these GST chimeric proteins was intravenously administered in
mice in equimolar amounts (1 nmol/mouse) and their rate of clearance
from blood followed by determination of remnant GST activity in blood
samples taken at different times after injection. Values recorded 30 minutes after injection were taken as 100% GST activity in each case.
Except for GST-Ag 1, GST-Ag 13, and GST-SAPA-injected mice that retain
8%, 25%, and 21% of their initial GST activities, respectively, the
rest of the animals show undetectable GST activity in blood as short as
24-hours after injection (data not shown). Due to the low sensitivity
of the enzymatic assay, we decided to try a GST capture procedure to
determine the persistence of GST chimeras in blood (see Materials and Methods).
Three groups of GST chimeric proteins can be differentiated according
to their persistence in blood using this GST capture assay (Fig 4). The
first group includes GST-SAPA and GST-Ag 13 proteins, both with an
estimated half-life in blood of 22 to 24 hours. The second group of
proteins is characterized by a significant lower half-life in blood
(about 10 to 13 hours) and includes GST chimeras containing repeats
from internal T. cruzi antigens 1, 30, and 36. The third group
is composed just by GST protein lacking repeats (estimated half-life in
blood of 3 hours), that is not detected in circulation as short as
24-hours after injection (Fig 4). As is the case with chimeric
trans-sialidases, the sole addition of any T. cruzi
repetitive domain to the carrier protein seems enough to exert a
moderate effect on its stability in blood; but this effect is much more
evident when the repetitive motifs added are the ones present in T. cruzi shed antigens 13 or SAPA (Fig 2 and 4).
As shown in Fig 5, a bias in the detection
of different GST chimeras can be disregarded. The GST capture assay
employed herein is able to detect changes in protein concentration
between 0.2 to 50 ng/well for every chimeric GST (Fig 5), thus
suggesting that its sensitivity is unaffected by the presence in cis of
different repetitive T. cruzi domains.
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| Fig 5.
Standardization of GST capture assay. GST chimeric
proteins diluted in normal mouse serum were tested in the GST capture
assay described under Materials and Methods. Mean optical
density values are shown (SD values did not exceed 10% of
mean values in any case; not shown). TS-SAPA and cruzipain protein
diluted in normal mouse serum were used as negative controls. Samples
were tested in triplicate. One out of three experiments with similar
results is shown.
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Pharmacokinetics data shown in Fig 4 were further confirmed by Western
blots of serum samples shown by a polyclonal anti-GST antiserum (data
not shown). Taken all together, these results show that the GST domain
of S. japonicum circulates in mouse bloodstream for longer
periods and in intact form when associated to T. cruzi repeats
SAPA and 13.
Evaluation of possible mechanisms involved in the stabilization of
carrier proteins mediated by SAPA amino acid repeats.
There are several possible mechanisms through which chimeric proteins
containing SAPA-repeats might increase their half-life in blood.
Experiments were performed to analyze two likely alternatives: first,
whether these T. cruzi tandem repeats prevent or inhibit degradation by blood-proteinases and second, whether SAPA-domain interacts with other proteins in the bloodstream of the vertebrate host.
GST or trans-sialidase molecules containing or not the
repetitive extension were incubated at 37°C either with blood or
plasma recovered from normal mice. At different times,
aliquots were taken and analyzed for proteolytic degradation. As shown
in Fig 6, the presence of SAPA-repeats does
not significantly affect the course of enzyme inactivation (in the case
of trans-sialidase molecules, Fig 6A) or protein degradation
(in the case of GST molecules, Fig 6B).
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| Fig 6.
Sensitivity of chimeric proteins to blood-proteinases.
(A) TSac and TS-SAPA proteins were incubated with normal mouse serum
and assayed for trans-sialidase activity in samples taken at
different times. Data are expressed as the mean ± SD. (B) GST or
GST-SAPA protein incubated either with mouse serum or mouse blood for
the indicated times, were assayed for proteolytic degradation as
indicated in Materials and Methods. Controls are indicated as  (mouse serum alone) and + (GST or GST-SAPA-purified protein in PBS).
(MK) Molecular weight markers (in kD) are indicated at right.
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Another suitable hypothesis would be that proteins linked to
SAPA-repeats increase their persistence in circulation due to the
interaction of the repetitive domain with blood protein/s. It has been
described, for instance, that certain surface proteins in pathogenic
Gram+ cocci allow evasion of immune defense mechanisms by
acting as receptors of blood proteins that eventually mask the
bacteria.29,30 This seems not to be the case with T. cruzi SAPA-repeats. Immunoprecipitation experiments performed with
GST or GST-SAPA protein (in solution or coupled to Sepharose) incubated
with normal mouse serum show no additional band/s in SDS-PAGE analyses
(data not shown). Altogether, these results suggest that the increase
in the circulating half-life of SAPA-repeats carrier proteins could not
be attributed to a modification of their sensitivity to
blood-proteinases or to the binding to endogenous circulating
protein/s.
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DISCUSSION |
The ability of different T. cruzi repetitive antigens in
stabilizing associated polypeptides in blood has been analyzed in two
experimental models: T. cruzi trans-sialidase and S. japonicum GST fusion proteins. Three of these antigens (denoted as
1, 30, and 36) have been shown to code for the repetitive units of
proteins intracellularly-located in the parasite.6,23 Thus,
it might be expected that these repeated structures accomplish other
function in vivo (if any at all) than protection against clearance from bloodstream of their nonrepeated carrier domains. This is not the case
with antigens SAPA and 13 that are spontaneously released into the
environment by the parasite. The latter codes essentially for the
repetitive extension of a protein located on the surface and on cell
boundaries of both amastigotes (the replicative, intracellular stage of
T. cruzi) and bloodstream trypomastigotes, as shown by electron
microscopy studies.23 The overall similarity between trans-sialidase and the natural N-domain of antigen 13 is
enough to show that they are indeed members of the same superfamily of molecules28; even though, the repetitive unit present in
antigen 13 shows no evident similarity to the 12 amino acid-long unit of SAPA-repeats (Table 1).
Despite this lack of amino acid sequence similarity, both antigen SAPA
and 13 produce the same effect on trans-sialidase activity when
placed in cis: enhancement of its half-life in blood. As shown in Fig
2, both TS-SAPA and TS-Ag 13 proteins remain in circulation up to 3 days or longer whereas TS-Ag 1 and TS-Ag 36 proteins are rapidly
cleared from the bloodstream after intravenous injection in mice. These
results cannot be attributed to thermal stability differences between
both groups of chimeric enzymes because, as previously shown,
trans-sialidases containing or lacking repetitive extensions
show negligible changes in their thermal inactivation when assayed at
37°C.7,22 Furthermore, improved half-life in blood of TS-SAPA and TS-Ag 13 proteins can hardly be attributed to
physical modifications introduced by the presence of these repeats. As
shown in Table 1, all of chimeric trans-sialidases display
similar molecular properties characterized by the prevalence of
negatively charged residues and a highly hydrophilic nature. Furthermore, these proteins show almost unaltered catalytic properties (Table 1), probably reflecting the lack of interaction between the
enzymatic domain and the repetitive extension. Thus, it might be
concluded that the stabilization of chimeric trans-sialidase activity in blood mediated by T. cruzi antigens SAPA and 13 is dependent on the specific amino acid sequence of their repetitive units.
Results obtained in the S. japonicum GST model were in close
agreement with those depicted for the trans-sialidase model, suggesting that the persistence in bloodstream is an inherent property
to certain T. cruzi amino acid repeats and not dependent on the
linked polypeptide. Chimeric GST proteins containing different T. cruzi tandem arrays, though not modified in their enzymatic activities, clearly differ in their half-lives in blood (Fig 4).
Even though the role of T. cruzi circulating molecules is still
uncertain, it could be postulated that this novel mechanism might be
operating in vivo as part of the strategy displayed by the parasite to
establish and/or maintain the infection. In this context, it is
noteworthy that the trans-sialidase expressed by the
epimastigote (the parasite form present in the midgut of the reduviid
bug host) shares many features with trypomastigote
trans-sialidase but lacks SAPA-repeats.18
Whether the mechanism mediated by antigens SAPA and 13 to stabilize
carrier proteins in blood depends on preventing the catabolism of
carrier protein in certain tissues and/or other alternative/s is currently under investigation. In the case of the 28 kD-GST protein,
its rapid clearance might be explained in part by kidney filtration
(Fig 4). However, this mechanism seems rather unlikely to explain the
rapid loss of the 76 kD TSac protein from circulation (Fig 2) and to
justify the differences in half-lives in blood recorded for proteins
almost identical in their molecular size (about 60 kD) like GST-Ag 30, GST-Ag 36, and GST-SAPA (Fig 4).26 On the other hand, the
possibility of an increased protection against blood-proteinases or
binding to endogenous circulating protein/s mediated by T. cruzi SAPA-repeats might be excluded (Fig 6 and data not shown).
The potential implications of these findings could be readily
envisaged. Considerable efforts are currently under way to increase the
circulating half-life of certain therapeutic proteins. Among the
several strategies that are being analyzed, randomized
mutagenesis,31 gene fusions to endogenous
proteins,32-34 and conjugation to inert polymers like
polyethylene glycol35,36 can be mentioned. In the case of
intravenously administered hormones, these potentially long-acting
analogs seem a promising alternative to daily injections for treatment.
The work described herein provides a reasonable alternative for the
improvement of protein half-life in blood: the exploitation of natural
occurring sequences. As shown herein, both T. cruzi repeats
SAPA and 13 promote the persistence of nondegraded, enzymatically
active, proteins in blood when linked in cis (Fig 2 and 4 and
data not shown). Thus, it seems reasonable to predict that other
repetitive sequences present in blood-released T. cruzi proteins (or even in other eukaryotic parasites) could be mediating similar stabilization effects.
| |
ACKNOWLEDGMENT |
We thank Dr J.J. Cazzulo for critical reading of the manuscript, F. Fraga for excellent care and maintenance of the animals, E. Spinedi
(from CITECA, INTI) for the ELISA determinations, and C. Labriola for
kindly providing the purified cruzipain.
| |
FOOTNOTES |
Submitted June 5, 1998; accepted November 7, 1998.
Supported by the United Nations Development
Programme/World Bank/World Health Organization (WHO)
Special Program for Research and Training in Tropical Diseases (TDR),
the Department for Research Cooperation (SAREC) from the Swedish
International Development Cooperation Agency (SIDA), the Agencia
Nacional de Promoción Científica y Tecnológica and
the Consejo Nacional de Investigaciones Científicas y
Técnicas (CONICET), Argentina. The research of A.C.C.F. was
supported in part by an International Research Scholars grant from the
Howard Hughes Medical Institute, Washington, DC. C.A.B. is
a fellow and O.C. and A.C.C.F. are researchers from the CONICET. J.A.
is a student fellow from the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Argentina.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to C.A. Buscaglia, Instituto de Investigaciones
Biotecnológicas, UNSAM, Predio INTI, edificio 24, Av. General Paz
y Albarellos, 1650 San Martín, Buenos Aires, Argentina; e-mail:
cbusca{at}inti.gov.ar.
| |
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