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

13 Nov 2008

Marcia Triunfol

Citation: Ratier L, Urrutia M, Paris G, Zarebski L, Frasch AC, Goldbaum FA (2008). Relevance of the diversity among members of the Trypanosoma cruzi trans-sialidase family analyzed with camelids single-domain antibodies. PLoS ONE. 3(10): e3524.

To be able to invade and survive once inside the cells of its host, T. cruzi has developed at least two main essential mechanisms. These mechanisms rely on the existence of molecules required to invade the host cells and on others required to evade the host immune system.

To evade the host immune system, T. cruzi has a mucin membrane coat comprised of a diverse repertoire of proteins. The mucin coat contains sialic acid, which is responsible for many aspects of the parasite-host interaction. However, T. cruzi itself is unable to make sialic acid. To obtain the necessary component the parasite has an enzyme known as T. cruzi trans-sialidase (TcTS) that ‘borrows’ sugar molecules from the host’s glycoconjugates and transfers them to its own alfa-galactoses enzymes present in the parasite coat.

TcTS is therefore essential for parasite survival, making it a great target for drug development. However, due to some structural and folding particularities of the enzyme, finding an antibody capable of tightly binding to the 12-aminoacid antigenic repeat motif of TcTS has been a major challenge for molecular engineers. One of the problems is that the active site of TcTS is located in a deep cleft of the protein for which access by conventional antibodies (antibodies comprised by two heavy-chains and two light-chains) has been shown to be very difficult.

To overcome the problem, the authors of this recent study published in PLoS ONE tried to engineer an antibody against TcTS using heavy-chain antibodies derived from llamas. Members of the camelidae family (the family of camels and llamas) have a unique type of antibody that is constituted by heavy-chains but no light-chains. The variable domain of this type of antibody, known as VHH or nanobody, is the smallest domain able to bind an antigen, and is therefore used when access to antigen molecules is limited.

In this study, the researchers used a modified version of a technique called antibody phage display in which proteins (among which are potential antibodies) are fused to a surface protein of a phage. Once fused, these proteins can be displayed on the surface of the phage and then screened according to whether they inhibit the expression of the target of interest (in this case the TcTS antigene). To generate potential camelidae antibodies that would inhibit TcTS action, the researchers immunized two llamas with a recombinant version of TcTS. The use of a recombinant version of TcTS increases the chances of raising antibodies that would recognize the active site of TcTS. The proteins from the two immunized llamas were then fused with a surface phage protein and used as described above. The whole process takes place inside bacteria.

The study successfully found four antibodies that showed strong inhibition against a recombinant version of TcTS (the same used to immunize the llamas) and which most likely bund to the active site of the protein or to a close location. The fact that these antibodies share the same consensus sequence indicates that there is just one available solution for inhibiting TcTS, (at least in the phage library used in the study). Nevertheless, these antibodies failed to inhibit TcTS present in trypomastigote extracts. For this disparity between the effects on recombinant TcTS and on TcTS from parasite extracts the authors offered no definitive explanation. It might be that some other TcTS-like enzymes exist in the natural parasite environment, which could promote different levels of interaction between antibodies, which ultimately would lower the whole concentration of the antibodies necessary to inhibit TcTS activity. However, addition of a recombinant TcTS enzyme to the natural extract caused its complete inhibition.

Additional findings suggested by the different extents that each individual recombinant TcTS was inhibited by a series of antibodies indicate that a diverse family of TcTS enzymes exists in T. cruzi. It is possible that a great number of TcTSs that vary in one or two amino acid positions are produced by T. cruzi. This would indeed explain why one single antibody may not be able to inhibit the TcTS activity, as the activity is probably the product of many TcTs enzymes working together, which may be the key mechanism used by T. cruzi to ‘distract’ the host immune system.

2008 Ratier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Source

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

Authors:
Ratier L, Urrutia M, Paris G, Zarebski L, Frasch AC, Goldbaum FA

References:
PLoS ONE 2008: Vol. 3 no. 10, pp. e3524

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.

PMID: 18949046
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