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
The epitope recognized by inhibitory llama antibodies maps to the                            TcTS catalytic site.View of the TcTS active site shown as surface representation using the                            program Chimera. (A) Free TcTS, (B) DANA-TcTS (shows the conformational                            change upon binding of DANA to TcTS) and (C) shows a 90°                            rotation of TcTS-DANA structure highlighting the arginine 311 residue                            protruding from the active site. PDBs used are 1MS4 and 1MS8. Residues                            involved in the catalytic site are colored as follows: mutated residues                            that were analyzed in this work (Trp312 and Tyr119) in red, other                            catalytic amino acids (Arg35, Asp59, Asp96, Met98, Arg314, Arg245,                            Glu230 and Tyr342) in yellow, space-fill model of DANA in green and                            Arg311 in blue.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2568053&req=5

pone-0003524-g005: The epitope recognized by inhibitory llama antibodies maps to the TcTS catalytic site.View of the TcTS active site shown as surface representation using the program Chimera. (A) Free TcTS, (B) DANA-TcTS (shows the conformational change upon binding of DANA to TcTS) and (C) shows a 90° rotation of TcTS-DANA structure highlighting the arginine 311 residue protruding from the active site. PDBs used are 1MS4 and 1MS8. Residues involved in the catalytic site are colored as follows: mutated residues that were analyzed in this work (Trp312 and Tyr119) in red, other catalytic amino acids (Arg35, Asp59, Asp96, Met98, Arg314, Arg245, Glu230 and Tyr342) in yellow, space-fill model of DANA in green and Arg311 in blue.

Mentions: Epitope mapping analysis of the binding of the SI VHHs to recombinant TcTS is shown in Figure 5. Comparing free TcTS and DANA-TcTS structures (Figure 5A and 5B), it can be seen that Tyr119 suffers a conformational change upon binding to DANA [8], [33].


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)

The epitope recognized by inhibitory llama antibodies maps to the                            TcTS catalytic site.View of the TcTS active site shown as surface representation using the                            program Chimera. (A) Free TcTS, (B) DANA-TcTS (shows the conformational                            change upon binding of DANA to TcTS) and (C) shows a 90°                            rotation of TcTS-DANA structure highlighting the arginine 311 residue                            protruding from the active site. PDBs used are 1MS4 and 1MS8. Residues                            involved in the catalytic site are colored as follows: mutated residues                            that were analyzed in this work (Trp312 and Tyr119) in red, other                            catalytic amino acids (Arg35, Asp59, Asp96, Met98, Arg314, Arg245,                            Glu230 and Tyr342) in yellow, space-fill model of DANA in green and                            Arg311 in blue.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003524-g005: The epitope recognized by inhibitory llama antibodies maps to the TcTS catalytic site.View of the TcTS active site shown as surface representation using the program Chimera. (A) Free TcTS, (B) DANA-TcTS (shows the conformational change upon binding of DANA to TcTS) and (C) shows a 90° rotation of TcTS-DANA structure highlighting the arginine 311 residue protruding from the active site. PDBs used are 1MS4 and 1MS8. Residues involved in the catalytic site are colored as follows: mutated residues that were analyzed in this work (Trp312 and Tyr119) in red, other catalytic amino acids (Arg35, Asp59, Asp96, Met98, Arg314, Arg245, Glu230 and Tyr342) in yellow, space-fill model of DANA in green and Arg311 in blue.
Mentions: Epitope mapping analysis of the binding of the SI VHHs to recombinant TcTS is shown in Figure 5. Comparing free TcTS and DANA-TcTS structures (Figure 5A and 5B), it can be seen that Tyr119 suffers a conformational change upon binding to DANA [8], [33].

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