Tandem Amino Acid Repeats From Trypanosoma cruzi Shed Antigens Increase the Half-Life of Proteins in Blood

To express the repetitive domain of different T. cruziantigens3 in frame with a functionaltrans-sialidase gene, a so-called “acceptor clone” has been constructed by a polymerase chain reaction (PCR) strategy. This acceptor clone (termed as TSac clone, Table1) is essentially alike TS clone22 but contains a uniqueEcoRI site in its 3′ terminus in the frame of λgt11 phage (Pharmacia Biotech, Uppsala, Sweden) EcoRI cloning site. Inserts recovered from EcoRI-digested λgt11 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 chimerictrans-sialidases were analyzed with the aid of the LaserGene software (DNASTAR Inc, Madison WI).

Chimeric trans-sialidases expressed in Escherichia colistrain 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. cruzirepetitive 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).

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 uniqueEcoRI 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).

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 oftrans-sialidase was defined as the amount of enzyme able to transfer 10 nmoles of sialic acid to lactose in 1 minute under standard conditions.

C3H/HeN mice 60- to 90-days old (both males and females), bred in our own facilities were used.

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 

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.

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).

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 fortrans-sialidase activity as described above.

The stability in blood of the different proteins was compared with that obtained for TS-SAPA (Fig 2 and3) or GST-SAPA values (Fig 4) in every time point of the curve using the Student’s t-test. P < .05 were considered significant.

Gene fragments coding for repetitive domains present in differentT. cruzi proteins (antigens 1, 13, 36, and SAPA) were placed in cis with a functional trans-sialidase gene and expressed inE. 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).23,28As 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 theseT. 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 chimerictrans-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) (Table1). 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 thetrans-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.

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 Fig2, 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.

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 andtrans-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.

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. cruziinternal 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 chimerictrans-sialidases, the sole addition of any T. cruzirepetitive 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.

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.

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).

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.

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 ofT. cruzi) and bloodstream trypomastigotes, as shown by electron microscopy studies.23 The overall similarity betweentrans-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 Fig2, 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 trypomastigotetrans-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. cruziproteins (or even in other eukaryotic parasites) could be mediating similar stabilization effects.

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.