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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.

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Inhibition of TcTS activity by llama 7006 serum.Serial dilutions of pre-immune and post-recombinant                            TcTSΔ1443-SAPA immunization sera, were analyzed by TIA as                            indicated under Materials and                            Methods.
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pone-0003524-g001: Inhibition of TcTS activity by llama 7006 serum.Serial dilutions of pre-immune and post-recombinant TcTSΔ1443-SAPA immunization sera, were analyzed by TIA as indicated under Materials and Methods.

Mentions: Two llamas were immunized using different recombinant TcTS constructions. Llama 7006, was immunized with pTcTSΔ1443 (lacking the 1443 epitope and retaining the SAPA repeats). This recombinant protein was used since deletion of the internal epitope between amino acids 433 and 447, called epitope 1443, increases the production of neutralizing antibodies in mouse models of infection [29], [30]. The second camelid, named llama 9210, was immunized with protein from the clone pTrcTS611/2 (entire globular core of TcTS without SAPA repeats) [31]. Llama 9210 showed a late TcTS inhibitory response and at lower level than llama 7006 (data not shown). Due to the high polyclonal inhibitory response detected in serum from llama 7006 after the fourth immunization, we engaged in the construction of a VHH library from the RNA of lymphocytes isolated from this animal fifteen days after the last immunization (Figure 1). The absence of 1443 epitope and/or the presence of SAPA repeats that increase the half-life in blood could be responsible for the difference in the inhibitory response between both llamas.


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)

Inhibition of TcTS activity by llama 7006 serum.Serial dilutions of pre-immune and post-recombinant                            TcTSΔ1443-SAPA immunization sera, were analyzed by TIA as                            indicated under Materials and                            Methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003524-g001: Inhibition of TcTS activity by llama 7006 serum.Serial dilutions of pre-immune and post-recombinant TcTSΔ1443-SAPA immunization sera, were analyzed by TIA as indicated under Materials and Methods.
Mentions: Two llamas were immunized using different recombinant TcTS constructions. Llama 7006, was immunized with pTcTSΔ1443 (lacking the 1443 epitope and retaining the SAPA repeats). This recombinant protein was used since deletion of the internal epitope between amino acids 433 and 447, called epitope 1443, increases the production of neutralizing antibodies in mouse models of infection [29], [30]. The second camelid, named llama 9210, was immunized with protein from the clone pTrcTS611/2 (entire globular core of TcTS without SAPA repeats) [31]. Llama 9210 showed a late TcTS inhibitory response and at lower level than llama 7006 (data not shown). Due to the high polyclonal inhibitory response detected in serum from llama 7006 after the fourth immunization, we engaged in the construction of a VHH library from the RNA of lymphocytes isolated from this animal fifteen days after the last immunization (Figure 1). The absence of 1443 epitope and/or the presence of SAPA repeats that increase the half-life in blood could be responsible for the difference in the inhibitory response between both llamas.

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