Enzymically inactive members of the trans-sialidase family from Trypanosoma cruzi display beta-galactose binding activity.

trans-sialidase is a unique sialidase in that, instead of hydrolizing sialic acid, it preferentially transfers the monosaccharide to a terminal beta-galactose in glycoproteins and glycolipids. This enzyme, originally identified in Trypanosoma cruzi, belongs to a large family of proteins. Some members of the family lack the enzymatic activity. No function has been yet assigned to them. In this work, the gene copy number and the possible function of inactive members of the trans -sialidase family was studied. It is shown that genes encoding inactive members are not a few, but rather, are present in the same copy number (60-80 per haploid genome) as those encoding active trans -sialidases. Recombinant inactive proteins were purified and assayed for sialic acid and galactose binding activity in agglutination tests. The enzymatically inactive trans -sialidases were found to agglutinate de-sialylated erythrocytes but not untreated red blood cells. Assays made with mouse and rabbit red blood cells suggest that inactive trans -sialidases bind to beta, rather than alpha, terminal galactoses, the same specificity required by active trans -sialidases. A recombinant molecule that was made enzymatically inactive through a mutation in a single amino acid also retained the galactose binding activity. The binding was competed by lactose and was dependent on conservation of the protein native conformation. Therefore, at least some molecules in the trans -sialidase family that have lost their enzymatic function still retain their Gal-binding properties and might have a function as lectins in the parasite-host interaction.     

Enzymatically inactive trans-sialidase from Trypanosoma cruzi binds sialyl and beta-galactopyranosyl residues in a sequential ordered mechanism

Host/parasite interaction mediated by carbohydrate/lectin recognition results in the attachment to and invasion of host cells and immunoregulation, enabling parasite replication and establishment of infection. Trypanosoma cruzi, the protozoan responsible for Chagas disease, expresses on its surface a family of enzymatically active and inactive trans-sialidases. The parasite uses the active trans-sialidase for glycoprotein sialylation in an unusual trans-glycosylation reaction. Inactive trans-sialidase is a sialic acid-binding lectin that costimulates host T cells through leucosialin (CD43) engagement. The co-mitogenic effect of trans-sialidase can be selectively abrogated by N-acetyllactosamine, suggesting the presence of an additional carbohydrate binding domain for galactosides, in addition to that for sialic acid. Here we investigated the interaction of inactive trans-sialidase in the presence of beta-galactosides. By using NMR spectroscopy, we demonstrate that inactive trans-sialidase has a beta-galactoside recognition site formed following a conformational switch induced by sialoside binding. Thus prior positioning of a sialyl residue is required for the beta-galactoside interaction. When an appropriate sialic acid-containing molecule is available, both sialoside and beta-galactoside are simultaneously accommodated in the inactive trans-sialidase binding pocket. This is the first report of a lectin recognizing two distinct ligands by a sequential ordered mechanism. This uncommon binding behavior may play an important role in several biological aspects of T. cruzi/host cell interaction and could shed more light into the catalytic mechanism of the sialic acid transfer reaction of enzymatically active trans-sialidase.     

Directed mutagenesis of the Trypanosoma cruzi trans-sialidase enzyme identifies two domains involved in its sialyltransferase activity

Of the increasing number of sialidases found to be made by microorganisms,the trypanosome trans-sialidase is unique in its added abilityto efficiently carry out a sialyltransferase reaction usingpreformed glymonjugates. The enzyme is predicted to have a multidomainstructure, with one domain containing sequence and expectedstructural features found in bacterial sialidases. The trans-sialidaseis very similar in overall sequence to another trypanosome enzymethat has only sialidase activity. Hybrid expression constructscontaining pieces of these trypanosome transsialidase and sialidasegenes were used to determine which regions of trans-sialidaseare required for sialyltransferase activity. Two domains werefound to influence the enzymatic activity: the N-terminal catalyticdomain, and a downstream domain that resembles an Fn3-like module. mutagenesis enzyme sialyltransferase trypanosome     

NMR spectroscopic and molecular modeling investigations of the trans-sialidase from Trypanosoma cruzi

Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the transfer of sialic acid from a range of sialic acid donor compounds to acceptor molecules, catalyzed by Trypanosoma cruzi trans-sialidase (TcTS). We demonstrate here that NMR spectroscopy is a powerful tool to monitor the trans-sialidase enzyme reaction for a variety of donor and acceptor molecules. The hydrolysis or transfer reactions that are catalyzed by TcTS were also investigated using a range of N-acetylneuraminosyl-based donor substrates and asialo acceptor molecules. These studies showed that the synthetic N-acetylneuraminosyl donor 4-methylumbelliferyl alpha-d-N-acetylneuraminide (MUN) is hydrolyzed by the enzyme approximately 3-5 times faster than either the disaccharide Neu5Acalpha(2,3)Galbeta1Me or the trisaccharide Neu5Acalpha(2,3)Lacbeta1Me. In the transfer reaction, we show that Neu5Acalpha(2,3)Lacbeta1Me is the most favorable substrate for TcTS and is a better substrate than the naturally-occurring N-acetylneuraminosyl donor alpha1-acid glycoprotein. In the case of MUN as the donor molecule, the transfer of Neu5Ac to different acceptors is significantly slower than when other N-acetylneuraminosyl donors are used. We hypothesize that when MUN is bound by the enzyme, the orientation and steric bulk of the umbelliferyl aglycon moiety may restrict the access for the correct positioning of an acceptor molecule. AutoDock studies support our hypothesis and show that the umbelliferyl aglycon moiety undergoes a strong pi-stacking interaction with Trp-312. The binding properties of TcTS towards acceptor (lactose) and donor substrate (Neu5Ac) molecules have also been investigated using saturation transfer difference (STD) NMR experiments. These experiments, taken together with other published data, have clearly demonstrated that lactose in the absence of other coligands does not bind to the TcTS active site or other binding domains. However, in the presence of the sialic acid donor, lactose (an asialo acceptor) was observed by NMR spectroscopy to interact with the enzyme's active site. The association of the asialo acceptor with the active site is an absolute requirement for the transfer reaction to proceed.     

Crystal structure of an enzymatically inactive trans-sialidase-like lectin from Trypanosoma cruzi: the carbohydrate binding mechanism involves residual sialidase activity

Trans-sialidases are surface-located proteins in Trypanosoma cruzi that participate in key parasite-host interactions and parasite virulence. These proteins are encoded by a large multigenic family, with tandem-repeated and individual genes dispersed throughout the genome. While a large number of genes encode for catalytically active enzyme isoforms, many others display mutations that involve catalytic residues. The latter ultimately code for catalytically inactive proteins with very high similarity to their active paralogs. These inactive members have been shown to be lectins, able to bind sialic acid and galactose in vitro, although their cellular functions are yet to be fully established. We now report structural and biochemical evidence extending the current molecular understanding of these lectins. We have solved the crystal structure of one such catalytically inactive trans-sialidase-like protein, after soaking with a specific carbohydrate ligand, sialyl-α2,3-lactose. Instead of the expected trisaccharide, the binding pocket was observed occupied by α-lactose, strongly suggesting that the protein retains residual hydrolytic activity. This hypothesis was validated by enzyme kinetics assays, in comparison to fully active wild-type trans-sialidase. Surface plasmon resonance also confirmed that these trans-sialidase-like lectins are not only able to bind small oligosaccharides, but also sialylated glycoproteins, which is relevant in the physiologic scenario of parasite infection. Inactive trans-sialidase proteins appear thus to be β-methyl-galactosyl-specific lectins, evolved within an exo-sialidase scaffold, thus explaining why their lectin activity is triggered by the presence of terminal sialic acid.     

Trypanosoma rangeli sialidase: cloning, expression and similarity to T.cruzi trans-sialidase

Sialidases are hydrolytic enzymes present from virus to highereukaryotes, catalyzing the removal of sialic acid from glycoconjugates.Some protozoa Trypanosomatidae secrete high levels of sialidaseinto the medium. We have now purified the secreted sialidasefrom Trypanosoma rangeli Its N-terminal sequence reveals 100%identity with the corresponding region of the trans-sialidasefrom T.cruzi Trans-sialidase, although homologous to viral andbacterial sialidases, displays a novel sialyltransferase activityand is involved in host cell invasion. Several homologous trans-sialidase-likegenes were cloned from genomic DNA of T.rangeli, and groupedin three subfamilies. Active siali-dase-encoding genes werefound in one of them. The re-combinant sialidase shows similarproperties to those of the native enzyme, including undetectabletrans-sialidase activity. Nevertheless, it has an overall identityof 68.9% with the catalytic domain of T.cruzi trans-sialidase,increasing to 86.7% admitting conservative substitutions. Onlythree other eukaryotic sialidases have been previously cloned,none of them showing significant homology to trans-sialidase.The isolation of a highly similar sialidase is relevant to furtheridentify the molecular determinants allowing trans-sialidaseactivity. As a first approach, chimeric constructs between sialidaseand trans-sialidase were generated, one of them rendering asialidase with three times lower K_m than the natural enzyme. eukaryotic sialidase gene family glycosidase parasite sialic acid     

Recombinant human acid alpha-glucosidase generated in bacteria: antigenic, but enzymatically inactive.

Genetic deficiency of acid alpha-glucosidase (GAA) results in glycogen storage disease type II. To investigate whether we could generate a functional recombinant human GAA protein for future enzyme replacement therapy, we subcloned the GAA cDNA into the bacterial expression plasmid pMaI and analyzed the recombinant protein produced. This nonglycosylated recombinant human GAA was found to be antigenic by reacting with polyclonal rabbit antibody to human placental GAA using ELISA and Western techniques. However, the protein was not enzymatically active, suggesting that glycosylation may play a role in enzymatic function.     

Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis

The trans-sialidase from Trypanosoma cruzi catalyzes the transfer of a sialic acid moiety from sialylated donor substrates to the terminal galactose moiety of lactose and lactoside acceptors to yield alpha-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl alpha-sialoside substrates and the acceptor substrate lactose were independently varied, we have demonstrated that this enzyme follows a ping-pong bi-bi kinetic mechanism. This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed. Mass spectrometric analysis of the protein directly demonstrates the formation of a covalent intermediate, while the observation of release of a full equivalent of p-nitrophenol by the mutant in a pre-steady state burst provides further support. The active site nucleophile is confirmed to be Tyr342 by trapping of the sialyl-enzyme intermediate using the D59A mutant and sequencing of the purified peptic peptide. The role of D59 as the acid/base catalyst is confirmed by chemical rescue studies in which activity is restored to the D59A mutant by azide and a sialyl azide product is formed.     

Enzymatic activity, protein expression, and gene sequence of cruzipain in virulent and attenuated Trypanosoma cruzi strains.

Protein expression, characterized in Western blots and gelatinolytic activity, of cruzipain (Cr), the major Trypanosoma cruzi cysteine proteinase, was compared among 3 attenuated T. cruzi strains (TUL 0, TCC, and Y null) and their virulent counterparts (TUL 2, Tulahuen, and Y). All attenuated strains displayed a weaker gelatinolytic activity as compared with their virulent counterparts. The electrophoretic mobility and immunological reactivity revealed quantitative and qualitative differences, with the attenuated parasites showing bands of less density in all strains and lower mobility in 2 of them, as compared with the virulent strains. Sequence analysis of 1 Cr gene in the Tulahuen and TCC strains indicated 37/1404 base pair substitutions, corresponding to 20 amino acid changes in the attenuated strain. A similar comparative analysis of 1 Cr gene between Y and Y null strains showed 13/1404 base pair substitutions, corresponding to 8 amino acid changes in the attenuated strain. Although enough variability exists in the Cr gene to allow for less- or nonfunctional isoforms of the protein, further clones should be analyzed to establish whether attenuation is regularly associated with specific sequence changes of this enzyme.     

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.     

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.     

Trypanosoma cruzi: isolation and characterization of two clones derived from neonatal mice

The Tulahuen strain of Trypanosoma cruzi was cloned in 15 C3H/Anf neonatal mice. Ten of these 15 neonates became parasitemic before the 12th day and died before the 19th day after the inoculation of a single bloodstream trypomastigote. Two clones were selected and maintained, while the other isolates which did not grow in a liquid metacyclic stage culture (LMC) medium were eventually discarded. The kinetics of in vitro growth and transformation from epimastigote to metacyclic trypomastigote of these two clones were characterized in LMC medium at 27 degrees C. Infectivities for vertebrate cells in vitro were retained by these two clones during the period of cultivation. The tropism for brain, heart, lungs, esophagus, stomach, large intestine, liver, pancreas, spleen, lymph nodes, kidneys, bladder, and skeletal muscles was also examined in the mice. The communication describes the establishment and characterization of T. cruzi clones. The utilization of these cloned parasites should produce some advantages in generating reproducible data in investigations.     

Trypanosoma cruzi: protection of mice with epimastigote antigens from immunologically different parasite strains

Antigens, prepared by the aid of pressure, from epimastigotes of strains of T. cruzi belonging to the different immunological groups described, conferred equal protection in mice against lethal infections of T. cruzi trypomastigotes of the T strain, which belongs to one of those immunological groups. Humoral antibodies were detected by the direct agglutination and immune fluorescent tests in all the immunized groups. The B and T strains produced earlier antibody responses than G and L strains. The weakest antibody response was induced by antigens obtained from the L strain. All the immunized mice sacrificed 21 days after challenge infection showed prominent inflammatory reactions at the tissue level, as well as free amastigote-like bodies. Four months after challenge injection, myocardium, liver, and spleen appeared histologically normal when compared to uninfected control mice. However, histological alterations were detected usually in striated muscle. The latter tissue seemed to be the best to check residual infections.     

The high resolution structures of free and inhibitor-bound Trypanosoma rangeli sialidase and its comparison with T. cruzi trans-sialidase

The structure of the recombinant Trypanosoma rangeli sialidase (TrSA) has been determined at 1.6A resolution, and the structures of its complexes with the transition state analog inhibitor 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (DANA), Neu-5-Ac-thio-alpha(2,3)-galactoside (NATG) and N-acetylneuraminic acid (NANA) have been determined at 1.64A, 2.1A and 2.85A, respectively. The 3D structure of TrSA is essentially identical to that of the natural enzyme, except for the absence of covalently attached sugar at five distinct N-glycosylation sites. The protein exhibits a topologically rigid active site architecture that is unaffected by ligand binding. The overall binding of DANA to the active site cleft is similar to that observed for other viral and bacterial sialidases, dominated by the interactions of the inhibitor carboxylate with the conserved arginine triad. However, the interactions of the other pyranoside ring substituents (hydroxyl, N-acetyl and glycerol moieties) differ between trypanosomal, bacterial and viral sialidases, providing a structural basis for specific inhibitor design. Sialic acid is found to bind the enzyme with the sugar ring in a distorted (half-chair or boat) conformation and the 2-OH hydroxyl group at hydrogen bonding distance of the carboxylate of Asp60, substantiating a direct catalytic role for this residue. A detailed comparison of TrSA with the closely related structure of T.cruzi trans-sialidase (TcTS) reveals a highly conserved catalytic center, where subtle structural differences account for strikingly different enzymatic activities and inhibition properties. The structure of TrSA in complex with NATG shows the active site cleft occupied by a smaller compound which could be identified as DANA, probably the product of a hydrolytic side reaction. Indeed, TrSA (but not TcTS) was found to cleave O and S-linked sialylated substrates, further stressing the functional differences between trypanosomal sialidases and trans-sialidases.     

Resialylation of sialidase-treated sheep and human erythrocytes by Trypanosoma cruzi trans-sialidase: restoration of complement resistance of desialylated sheep erythrocytes.

Trypanosoma cruzi trans-sialidase (TS) is a recently described enzyme which transfers alpha(2-3)-linked sialic acid from host-derived sialylated glycoconjugates to parasite surface molecules [Schenkman et al. (1991) Cell, 65, 1117]. We report here on the ability of TS to transfer sialic acid from donor sialyl-alpha(2-3)lactose to sialidase-treated sheep and human erythrocytes. Up to approximately 50% resialylation of both desialylated red cells could be attained. Resialylation of desialylated sheep erythrocytes restores their resistance to lysis by human complement. This ascribes a possible biological role for T. cruzi TS and demonstrates directly that sialic acid is solely responsible for preventing alternative pathway activation of human complement by sheep erythrocytes.     

In vivo infection by Trypanosoma cruzi: the conserved FLY domain of the gp85/trans-sialidase family potentiates host infection

Trypanosoma cruzi is a protozoan parasite that infects vertebrates, causing in humans a pathological condition known as Chagas' disease. The infection of host cells by T. cruzi involves a vast collection of molecules, including a family of 85 kDa GPI-anchored glycoproteins belonging to the gp85/trans-sialidase superfamily, which contains a conserved cell-binding sequence (VTVXNVFLYNR) known as FLY, for short. Herein, it is shown that BALB/c mice administered with a single dose (1 μg/animal, intraperitoneally) of FLY-synthetic peptide are more susceptible to infection by T. cruzi, with increased systemic parasitaemia (2-fold) and mortality. Higher tissue parasitism was observed in bladder (7·6-fold), heart (3-fold) and small intestine (3·6-fold). Moreover, an intense inflammatory response and increment of CD4+ T cells (1·7-fold) were detected in the heart of FLY-primed and infected animals, with a 5-fold relative increase of CD4+CD25+FoxP3+ T (Treg) cells. Mice treated with anti-CD25 antibodies prior to infection, showed a decrease in parasitaemia in the FLY model employed. In conclusion, the results suggest that FLY facilitates in vivo infection by T. cruzi and concurs with other factors to improve parasite survival to such an extent that might influence the progression of pathology in Chagas' disease.     

Comparative rates of sialylation by recombinant trans-sialidase and inhibitor properties of synthetic oligosaccharides from Trypanosoma cruzi mucins-containing galactofuranose and galactopyranose

The mucin-like glycoproteins of Trypanosoma cruzi have novel O-linked oligosaccharides that are acceptors of sialic acid in the trans-sialidase (TcTS) reaction. The transference of sialic acid from host glycoconjugates to the mucins is involved in infection and pathogenesis. The O-linked chains may contain galactofuranose in addition to the acceptor galactopyranose units. Thus far, the galactofuranose form was found in the mucins of strains belonging to the less infective lineage. The acceptor properties of the chemically synthesized oligosaccharides were now studied in order to correlate their structure with the ability to act as substrates. Recombinant TcTS and sialyllactose as donor were used. The reactions were followed by HPAEC-PAD. The K(m) values were calculated for the free sugars, the sugar alditols and the benzyl glycosides. All the compounds showed to be good acceptors of sialic acid. Thus, the introduction of galactofuranose in the mucins of the strains of lineage 1 would not be responsible for the diminished virulence of the strains. The oligosaccharides and derivatives inhibited the transfer of sialic acid to the substrate N-acetyllactosamine with IC(50) values between 0.6 and 4 mM.     

Trypanosoma cruzi: conformational preferences of antigenic peptides bearing the immunodominant epitope of the B13 antigen.

The Trypanosoma cruzi recombinant protein B13 contains tandemly repeated domains and shows high sensitivity in the serological diagnosis of Chagas' disease. It has been shown that the immunodominant epitope of B13 is contained in the GDKPSLFGQAAAGDKPSLF-NH(2) sequence and that the hexapeptide AAAGDK seems to be the "core" of that epitope. Three peptides containing that "core" sequence, one corresponding to the entire repeat motif GDKPSLFGQAAAGDKPSLF-NH(2), pB13, and two smaller fragments, FGQAAAGDK-NH(2), S4, and QAAAGDKPS-NH(2), S5, have been tested in competitive ELISA with recombinant protein B13 in the solid phase against 40 chagasic sera from Brazilian patients. The median percentage inhibition for pB13, S4, and S5 were, respectively, 91, 86, and 68%. The possibility that the distinct antigenic activity of those peptides correlates with the existence of preferential conformational properties has been investigated by CD and NMR spectroscopy. Results indicate their propensity to adopt a helical configuration, centered in the AAAGDK sequence, and whose extent and stability directly correlates with the peptides' antigenicity. The data are discussed in the light of the existence of conformational preferences involving immunodominant epitopes in tandemly repeated antigens.     

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop

Abstract A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.     

The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains

The EAL domain (also known as domain of unknown function 2 or DUF2) is a ubiquitous signal transduction protein domain in the Bacteria. Its involvement in hydrolysis of the novel second messenger cyclic dimeric GMP (c-di-GMP) was demonstrated in vivo but not in vitro. The EAL domain-containing protein Dos from Escherichia coli was reported to hydrolyze cyclic AMP (cAMP), implying that EAL domains have different substrate specificities. To investigate the biochemical activity of EAL, the E. coli EAL domain-containing protein YahA and its individual EAL domain were overexpressed, purified, and characterized in vitro. Both full-length YahA and the EAL domain hydrolyzed c-di-GMP into linear dimeric GMP, providing the first biochemical evidence that the EAL domain is sufficient for phosphodiesterase activity. This activity was c-di-GMP specific, optimal at alkaline pH, dependent on Mg(2+) or Mn(2+), strongly inhibited by Ca(2+), and independent of protein oligomerization. Linear dimeric GMP was shown to be 5'pGpG. The EAL domain from Dos was overexpressed, purified, and found to function as a c-di-GMP-specific phosphodiesterase, not as a cAMP-specific phosphodiesterase, in contrast to previous reports. The EAL domains can hydrolyze 5'pGpG into GMP, however, very slowly, thus implying that this activity is irrelevant in vivo. Therefore, c-di-GMP is the exclusive substrate of EAL. Multiple-sequence alignment revealed two groups of EAL domains hypothesized to correspond to enzymatically active and inactive domains. The domains in the latter group have mutations in residues conserved in the active domains. The enzymatic inactivity of EAL domains may explain their coexistence with GGDEF domains in proteins possessing c-di-GMP synthase (diguanulate cyclase) activity.