Close-up of the TS/antibody interface.

<p>Highlight of the spatial distribution of the epitope residues (in stick representation, colored yellow). TS is shown in orange, light Fab chain in magenta and heavy chain in cyan. For clarity not all the epitope residues are shown nor labeled (see text for full analysis). On top of the cartoon secondary structure representation, residues are represented in lines for the three chains. As a reference for TS positions within the reaction center, the catalytic amino acids Y₃₄₂ (on the floor of the pocket) and D₅₉ are highlighted as green sticks; Y₁₁₉ (colored red) forms part of the epitope, normally flexible in free TS. Note how the mAb light chain precludes free mobility of Y₁₁₉, which plays a key function in <i>trans</i>-glycosylation.</p

Reactivity of mAb 13G9 with <i>T. cruzi</i> parasites.

<p><b>A</b>) <i>T. cruzi</i> surface labeling by the 13G9 mAb. Epifluorescence microscopy of <i>T. cruzi</i> trypomastigotes, seeded onto poly-L-lysine-treated coverglasses, and immunolabeled with 13G9 mAb followed by a secondary FITC-labeled antibody. <b>B</b>) Inhibition of parasite sialylation. Trypomastigotes obtained from cell cultures made in ‘low sialyl-donors’ conditions, were sialylated with TS and sialyllactose, in the presence of mAb 13G9. Total sialic acid was quantified by the thiobarbituric acid method and referred to re-sialylated parasites in the absence of mAb as 100% (approximately 1.2 pmoles of sialic acid/10⁶ parasites). <b>C</b>) Effect of mAb 13G9 in infection assays on mammalian cells. Parasites were preincubated for 1 h with 13G9 antibody (100 µg/ml) before infection. After 24 hrs, infected cultures were fixed and stained with Hoescht 33342. At least 300 cells were counted.</p

Residues that display a significant change in solvent-accessible surface, comparing the separate and complexed structures of TS and mAb 13G9.

<p>*also establishes hydrogen bonds.</p>†<p>also establishes salt bridge.</p>§<p>no direct contact is observed.</p

Data processing and refinement statistics.

a<p>Values in parentheses apply to the high-resolution shell.</p>b<p>; N<i>_h</i>, multiplicity for each reflection; I<i>_i</i>, the intensity of the <i>i</i>^th observation of reflection <i>h</i>; <i>, the mean of the intensity of all observations of reflection <i>h</i>, with ; is taken over all reflections; is taken over all observations of each reflection.</i></p><i>c<p>; ; R_cryst and R_free were calculated using the working and test hkl reflection sets, respectively.</p>d<p>Total refined protein residues equal 3172, from which 28 terminal amino acids (the <i>N-</i> and <i>C-</i>termini on the 9 chains; plus residues: TS#399, TS#409 (in chains A, B & C), Fab#27, Fab#29 (in chain H), Fab#137, Fab#139 (in chain I), all flanking unmodeled gaps) were not included in the Ramachandran analysis (as implemented in Coot v 0.6.2-pre-1).</p></i

Biochemical characterization of the TS-13G9 mAb interaction.

<p><b>A</b>) Surface plasmon resonance analysis of TS-mAb 13G9 interaction kinetics. mAb 13G9 was immobilized onto a CM5 sensor chip and the indicated concentrations of TS were injected in the mobile phase. <b>B, C</b>) mAb 13G9 inhibition of TS activity. TS (2 ng) was mixed with increasing amounts of purified Fab (<b>B</b>), or whole 13G9 IgG (<b>C</b>) and remnant TS activity was assayed. <b>D</b>) Competition assay for mAb 13G9-TS binding. TS activity was assayed (2 ng, 30 min) in the presence of the neutralizing mAb (3 and 6 µl of hybridoma culture supernatant diluted 1/20) and increasing amounts (from 0 to 8 ng) of the inactive TS (iTS) were added. Student's <i>t</i> test was used. * <i>p</i><0.05; ** <i>p</i><0.005 comparing against TS activity without mAb addition <b>E</b>) Specificity of the mAb 13G9. Trypomastigotes were biotinylated, washed and mAb 13G9 added to pull-down reacting proteins. Western blots were developed with anti-SAPA (left) and streptavidin (right).</p

Sialoconjugate substrates modeled in the TS reaction center, in the context of the immunocomplex structure.

<p>(<b>A</b>) α(2,3)sialyllactose (SL, carbons in yellow) and MU-NANA (carbons in purple) are shown in stick representation, colored according to atom elements (oxygen in red, nitrogen in blue). The carbohydrates are grafted from PDB models 1S0I (for SL) and 1S0J (MU-NANA), after structural superposition of the TS molecules onto the immunocomplex, resulting in their specific positions within the TS catalytic pocket. TS and Fab molecules are shown in ribbons (TS in green, Fab light chain in magenta and heavy chain in blue), with their corresponding solvent-accessible surfaces on top. The surface has been cut to highlight the inner architecture of the TS catalytic pocket: this orientation does not allow appreciating that the site is open from above and beneath the plane of the paper. TS Y₁₁₉ (green sticks) is seen directly obstructing the sialic acid position, and its normal mobility is hindered by antibody's light chain S₃₀ (magenta sticks). (<b>B</b>) A similar representation as in panel (A), in a rotated orientation scene, to highlight the ‘roof’ formed by residues S₆₆–G₆₇ of the Fab light chain (in magenta sticks to the top left of the panel) in direct contact with TS residues R₃₁₁–W₃₁₂ (in pale green sticks, to the right of the figure). Note the expected clash of the glucosyl residue in sialyllactose against loop 66–67, and the better fit of the smaller MU-NANA substrate, still quite restricted in free torsional movements. Y₁₁₉ is again shown (strong green sticks), precluding entrance of the sialic acid moiety of both modeled sugar compounds.</p

Trypanosoma cruzi trans-sialidase in complex with a neutralizing antibody: Structure/function studies towards the rational design of inhibitors

Trans-sialidase (TS), a virulence factor from Trypanosoma cruzi, is an enzyme playing key roles in the biology of this protozoan parasite. Absent from the mammalian host, it constitutes a potential target for the development of novel chemotherapeutic drugs, an urgent need to combat Chagas' disease. TS is involved in host cell invasion and parasite survival in the bloodstream. However, TS is also actively shed by the parasite to the bloodstream, inducing systemic effects readily detected during the acute phase of the disease, in particular, hematological alterations and triggering of immune cells apoptosis, until specific neutralizing antibodies are elicited. These antibodies constitute the only known submicromolar inhibitor of TS's catalytic activity. We now report the identification and detailed characterization of a neutralizing mouse monoclonal antibody (mAb 13G9), recognizing T. cruzi TS with high specificity and subnanomolar affinity. This mAb displays undetectable association with the T. cruzi superfamily of TS-like proteins or yet with the TS-related enzymes from Trypanosoma brucei or Trypanosoma rangeli. In immunofluorescence assays, mAb 13G9 labeled 100% of the parasites from the infective trypomastigote stage. This mAb also reduces parasite invasion of cultured cells and strongly inhibits parasite surface sialylation. The crystal structure of the mAb 13G9 antigen-binding fragment in complex with the globular region of T. cruzi TS was determined, revealing detailed molecular insights of the inhibition mechanism. Not occluding the enzyme's catalytic site, the antibody performs a subtle action by inhibiting the movement of an assisting tyrosine (Y119), whose mobility is known to play a key role in the trans-glycosidase mechanism. As an example of enzymatic inhibition involving non-catalytic residues that occupy sites distal from the substrate-binding pocket, this first near atomic characterization of a high affinity inhibitory molecule for TS provides a rational framework for novel strategies in the design of chemotherapeutic compounds.Fil: Buschiazzo, Alejandro. Instituto Pasteur de Montevideo; UruguayFil: Muia, Romina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Larrieux, Nicole. Instituto Pasteur de Montevideo; UruguayFil: Pitcovsky, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Mucci, Juan Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Campetella, Oscar Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin