Limits...
Relevance of the diversity among members of the Trypanosoma cruzi trans-sialidase family analyzed with camelids single-domain antibodies.

Ratier L, Urrutia M, Paris G, Zarebski L, Frasch AC, Goldbaum FA - PLoS ONE (2008)

Bottom Line: This result indicates that they likely derived from a unique clone.Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes.These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de General San Martín-CONICET, Buenos Aires, Argentina.

ABSTRACT
The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.

Show MeSH
(A) Natural TcTS was not inhibited by VHHSI14.                            Comparison of VHH114 inhibition activity of the TcTS present in                            supernatants from Cl-Brener T. cruzi-infected cell                            cultures versus a recombinant TcTS611/2 enzyme. The decameric form of                            VHHSI14 was used. The values represent the average of two independent                            determinations. (B) Independent recombinant TcTSs are inhibited                                to different extents by VHHSI14. A fixed mass of 0.5 ng of                            different purified TcTSs from T. cruzi were                            preincubated with increasing concentrations of VHHSI14 decameric form                            and the remaining trans-sialidase activity was analyzed by TIA. The                            values represent the average of at least three independent                            determinations.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2568053&req=5

pone-0003524-g006: (A) Natural TcTS was not inhibited by VHHSI14. Comparison of VHH114 inhibition activity of the TcTS present in supernatants from Cl-Brener T. cruzi-infected cell cultures versus a recombinant TcTS611/2 enzyme. The decameric form of VHHSI14 was used. The values represent the average of two independent determinations. (B) Independent recombinant TcTSs are inhibited to different extents by VHHSI14. A fixed mass of 0.5 ng of different purified TcTSs from T. cruzi were preincubated with increasing concentrations of VHHSI14 decameric form and the remaining trans-sialidase activity was analyzed by TIA. The values represent the average of at least three independent determinations.

Mentions: VHHSI14 was tested against supernatants of Cl-Brener T. cruzi-infected cell cultures containing TcTS released from the trypomastigote stage of the parasite. Surprisingly, VHHSI14 failed to inhibit the enzyme present in parasite supernatants while a strong inhibition activity toward the recombinant TcTS611/2 used as a control was observed (figure 6A). Supernatants from three different strains were assayed in the presence of an excess of VHHSI14 antibody, showing similar results (Table 3). The lack of inhibition by VHHSI14 antibody was not due to the presence of any compound in the medium since recombinant TcTS added in the reaction was neutralized (Table 3). Similar negative results were observed with live Cl-Brener trypomastigotes containing TcTS linked to the membrane surface of the parasites (data not shown). To analyze if the ausence of inhibition was due to the univalent nature of VHHs, we increased the avidity of this antibody fragment. To this end, we constructed a fusion protein displaying ten VHH domains per assembly, taking advantage of the decameric structure of Brucella spp. lumazine synthase [35]. This assembly allowed to increase 10 times the avidity of VHHSI14 for recombinant TcTS but did not show differences in its inhibitory capacity as compared with that of the monomeric VHHSI14 (data not shown). This construction was assayed against Cl-Brener trypomastigotes and it was still unable to inhibit the activity of TcTS present in the surface of parasites (Table 3).


Relevance of the diversity among members of the Trypanosoma cruzi trans-sialidase family analyzed with camelids single-domain antibodies.

Ratier L, Urrutia M, Paris G, Zarebski L, Frasch AC, Goldbaum FA - PLoS ONE (2008)

(A) Natural TcTS was not inhibited by VHHSI14.                            Comparison of VHH114 inhibition activity of the TcTS present in                            supernatants from Cl-Brener T. cruzi-infected cell                            cultures versus a recombinant TcTS611/2 enzyme. The decameric form of                            VHHSI14 was used. The values represent the average of two independent                            determinations. (B) Independent recombinant TcTSs are inhibited                                to different extents by VHHSI14. A fixed mass of 0.5 ng of                            different purified TcTSs from T. cruzi were                            preincubated with increasing concentrations of VHHSI14 decameric form                            and the remaining trans-sialidase activity was analyzed by TIA. The                            values represent the average of at least three independent                            determinations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2568053&req=5

pone-0003524-g006: (A) Natural TcTS was not inhibited by VHHSI14. Comparison of VHH114 inhibition activity of the TcTS present in supernatants from Cl-Brener T. cruzi-infected cell cultures versus a recombinant TcTS611/2 enzyme. The decameric form of VHHSI14 was used. The values represent the average of two independent determinations. (B) Independent recombinant TcTSs are inhibited to different extents by VHHSI14. A fixed mass of 0.5 ng of different purified TcTSs from T. cruzi were preincubated with increasing concentrations of VHHSI14 decameric form and the remaining trans-sialidase activity was analyzed by TIA. The values represent the average of at least three independent determinations.
Mentions: VHHSI14 was tested against supernatants of Cl-Brener T. cruzi-infected cell cultures containing TcTS released from the trypomastigote stage of the parasite. Surprisingly, VHHSI14 failed to inhibit the enzyme present in parasite supernatants while a strong inhibition activity toward the recombinant TcTS611/2 used as a control was observed (figure 6A). Supernatants from three different strains were assayed in the presence of an excess of VHHSI14 antibody, showing similar results (Table 3). The lack of inhibition by VHHSI14 antibody was not due to the presence of any compound in the medium since recombinant TcTS added in the reaction was neutralized (Table 3). Similar negative results were observed with live Cl-Brener trypomastigotes containing TcTS linked to the membrane surface of the parasites (data not shown). To analyze if the ausence of inhibition was due to the univalent nature of VHHs, we increased the avidity of this antibody fragment. To this end, we constructed a fusion protein displaying ten VHH domains per assembly, taking advantage of the decameric structure of Brucella spp. lumazine synthase [35]. This assembly allowed to increase 10 times the avidity of VHHSI14 for recombinant TcTS but did not show differences in its inhibitory capacity as compared with that of the monomeric VHHSI14 (data not shown). This construction was assayed against Cl-Brener trypomastigotes and it was still unable to inhibit the activity of TcTS present in the surface of parasites (Table 3).

Bottom Line: This result indicates that they likely derived from a unique clone.Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes.These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de General San Martín-CONICET, Buenos Aires, Argentina.

ABSTRACT
The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.

Show MeSH