The Membrane Glycoprotein M6a Endocytic/Recycling Pathway Involves Clathrin-Mediated Endocytosis and Affects Neuronal Synapses.

Single point mutations or variations in the expression of the gene encoding the neuronal glycoprotein M6a have been associated with psychiatric disorders such as Alzheimer's disease, depression and schizophrenia. In cultured neurons, M6a positively contributes to neurite extension, axon guidance, filopodia/spine outgrowth, and synapse formation. The endocytic processes of neuronal membrane proteins are linked to the differentiation, growth, signaling and plasticity of neurons. However, the roles of M6a and the precise mechanisms through which M6a internalizes and recycles back to the neuronal membrane are unknown. Here, by using a controlled in vitro assay, we showed that if 30-40% of M6a is endocytosed, the number of synapses in hippocampal neurons decreases. When re-establishing the levels of M6a at the cell surface, the number of synapses returned to normal values. M6a internalization involves clathrin-coated pits, probably by association between the adaptor protein 2 and the 251YEDI254 "tyrosine-based" motif located within the C-tail of M6a. Upon endocytosis, M6a is sorted to early endosome antigen 1- and Rab5-positive endosomes and then sorted back to the cell surface via Rab11-positive endosomes or to degradation via Rab7 and, finally LAMP-1-positive endosomes. Our results demonstrated that the levels of M6a at the cell surface modified the formation/maintenance of synapses, without altering the protein levels of synaptophysin or N-methyl-D-aspartate receptor type-1. This novel mechanism might be relevant during neuronal development, pruning and/or many of the neurological disorders in which the number of synapses is affected.

Improved bioavailability of inhibitors of Trypanosoma cruzi trans-sialidase: PEGylation of lactose analogs with multiarm polyethyleneglycol.

The trans-sialidase of Trypanosoma cruzi (TcTS) catalyzes the transfer of sialic acid from host glycoconjugates to terminal ß-galactopyranosides in the mucins of the parasite. During infection, the enzyme is actively shed by the parasite to the bloodstream inducing hematological alterations. Lactitol prevents cell apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. Linear polyethyleneglycol (PEG) conjugates of lactose analogs were prepared but their clearance from blood was still quite fast. With the aim of improving their circulating half-lives in vivo, we now synthesized covalent conjugates of eight-arm PEG. The star-shape of these conjugates allows an increase in the molecular weight together with the loading of the active sugar. Two approaches were used for PEGylation of disaccharide derivatives containing ß-D-Galp as the non-reducing unit. (1) Amide formation between benzyl ß-D-galactopyranosyl-(1→6)-2-amino-2-deoxy-α-D-glucopyranoside and a succinimide-activated PEG. (2) Conjugation of lactobionolactone with amino end-functionalized PEG. Two 8-arm PEG derivatives (20 and 40 kDa) were used for each sugar. Substitution of all arms was proved by (1)H nuclear magnetic resonance (NMR) spectroscopy. The bioavailability of the conjugates in mice plasma was considerably improved with respect to the 5 kDa linear PEG conjugates retaining their inhibitory properties.

Evaluation of a recombinant Trypanosoma cruzi mucin-like antigen for serodiagnosis of Chagas' disease.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and is one of the most important endemic problems in Latin America. Lately, it has also become a health concern in the United States and Europe. Currently, a diagnosis of Chagas' disease and the screening of blood supplies for antiparasite antibodies are achieved by conventional serological tests that show substantial variation in the reproducibility and reliability of their results. In addition, the specificity of these assays is curtailed by antigenic cross-reactivity with sera from patients affected by other endemic diseases, such as leishmaniasis. Here we used a highly sensitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) to evaluate a recombinant protein core of a mucin-like molecule (termed trypomastigote small surface antigen [TSSA]) for the detection of specific serum antibodies in a broad panel of human sera. The same samples were evaluated by CL-ELISA using as the antigen either a mixture of native T. cruzi trypomastigote mucins or an epimastigote extract and, for further comparison, by conventional serologic tests, such as an indirect hemagglutination assay and indirect immunofluorescence assay. TSSA showed ∼87% sensitivity among the seropositive Chagasic panel, a value which was increased up to >98% when only parasitologically positive samples were considered. More importantly, TSSA showed a significant increase in specificity (97.4%) compared to those of currently used assays, which averaged 80 to 90%. Overall, our data demonstrate that recombinant TSSA may be a useful antigen for the immunodiagnosis of Chagas' disease.

Synthesis of PEGylated lactose analogs for inhibition studies on T.cruzi trans-sialidase.

Trypanosoma cruzi, the agent of Chagas disease, expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The enzyme is shed to the medium and may affect the immune system of the host. We have previously described that lactose derivatives effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Lactitol also prevented the apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. In this paper we report covalent conjugation of polyethylene glycol (PEG) with lactose, lactobionolactone and benzyl beta-D-galactopyranosyl-(1-->6)-2-amino-2-deoxy-alpha-D-glucopyranoside (1) with the hope to improve the bioavailability, though retaining their inhibitory properties. Different conjugation methods have been used and the behavior of the PEGylated products in the TcTS reaction was studied.

Filopodial protrusions induced by glycoprotein M6a exhibit high motility and aids synapse formation.

M6a is a neuronal membrane glycoprotein whose expression diminishes during chronic stress. M6a overexpression in rat primary hippocampal neurons induces the formation of filopodial protrusions that could be spine precursors. As the filopodium and spine motility has been associated with synaptogenesis, we analysed the motility of M6a-induced protrusions by time-lapse imaging. Our data demonstrate that the motile protrusions formed by the neurons overexpressing M6a were more abundant and moved faster than those formed in control cells. When different putative M6a phosphorylation sites were mutated, the neurons transfected with a mutant lacking intracellular phosphorylation sites bore filopodia, but these protrusions did not move as fast as those formed by cells overexpressing wild-type M6a. This suggests a role for M6a phosphorylation state in filopodium motility. Furthermore, we show that M6a-induced protrusions could be stabilized upon contact with presynaptic region. The motility of filopodia contacting or not neurites overexpressing synaptophysin was analysed. We show that the protrusions that apparently contacted synaptophysin-labeled cells exhibited less motility. The behavior of filopodia from M6a-overexpressing cells and control cells was alike. Thus, M6a-induced protrusions may be spine precursors that move to reach presynaptic membrane. We suggest that M6a is a key molecule for spine formation during development.

Procyclic Trypanosoma brucei expresses separate sialidase and trans-sialidase enzymes on its surface membrane.

The procyclic stage of Trypanosoma brucei in the insect vector expresses a surface-bound trans-sialidase (TbTS) that transfers sialic acid from glycoconjugates in the environment to glycosylphosphatidylinositol-anchored proteins on its surface membrane. RNA interference against TbTS abolished trans-sialidase activity in procyclic cells but did not diminish sialidase activity, suggesting the presence of a separate sialidase enzyme for hydrolyzing sialic acid. A search of the T. brucei genome sequence revealed seven other putative genes encoding proteins with varying similarity to TbTS. RNA interference directed against one of these proteins, TbSA C, greatly decreased the sialidase activity but had no effect on trans-sialidase activity. The deduced amino acid sequence of TbSA C shares only 40% identity with TbTS but conserves most of the relevant residues required for catalysis. However, the sialidase has a tryptophan substitution for a tyrosine at position 170 that is crucial in binding the terminal galactose that accepts the transferred sialic acid. When this same tryptophan substitution in the sialidase was placed into the recombinant trans-sialidase, the mutant enzyme lost almost all of its trans-sialidase activity and increased its sialidase activity, further confirming that the gene and protein identified correspond to the parasite sialidase. Thus, in contrast to all other trypanosomes analyzed to date that express either a trans-sialidase or a sialidase but not both, T. brucei expresses these two enzymatic activities in two separate proteins. These results suggest that African trypanosomes could regulate the amount of critical sialic acid residues on their surface by modulating differential expression of each of these enzymes.

Interfering polysialyltransferase ST8SiaII/STX mRNA inhibits neurite growth during early hippocampal development.

Polysialic acid (PSA) attached to NCAM is involved in cell-cell interactions participating in structural and functional plasticity of neuronal circuits. Two polysialyltransferases, ST8SiaII/STX and ST8SiaIV/PST, polysialylate NCAM. We previously suggested that ST8SiaII/STX is the key enzyme for polysialylation in hippocampus. Here, polysialyltransferase mRNA interference experiments showed that, knock down of ST8SiaIV/PST transcripts did not affect PSA expression, but PSA was almost absent from neuronal surfaces when ST8SiaII/STX mRNA was interfered. Non-polysialylated neurons bore a similar number of neurites per cell than polysialylated neurons. However, non-polysialylated processes were shorter and a lower density of synaptophysin clusters accompanied this reduced neuritic growth. Therefore, ST8SiaII/STX expression is essential to allow a correct neuritic development at initial stages of hippocampus ontogeny.

The trans-sialidase from Trypanosoma cruzi triggers apoptosis by target cell sialylation.

The trans-sialidase, a modified sialidase that transfers sialyl residues among macromolecules, is a unique enzymatic activity expressed by some parasitic trypanosomes being essential for their survival in the mammalian host and/or in the insect vector. The enzyme from Trypanosoma cruzi, the agent of Chagas disease, is found in blood and able to act far from the infection site by inducing apoptosis in cells from the immune system. A central and still unsolved question is whether trans-sialidase-mediated addition or removal of sialic acid to/from host acceptor molecules is the event associated with the apoptosis induced by the enzyme. Here we show that lactitol, a competitive inhibitor that precluded the transference of the sialyl residue to endogenous acceptors but not the hydrolase activity of the enzyme, prevented ex vivo and in vivo the apoptosis caused by the trans-sialidase. By lectin histochemistry, the transference of sialyl residue to the cell surface was demonstrated in vivo and found associated with the apoptosis induction. The sialylation of the CD43 mucin, a key molecule involved in trans-sialidase-apoptotic process, was readily detected and also prevented by lactitol on thymocytes. Therefore, lesions induced by trans-sialidase on the immune system are due to the sialylation of endogenous acceptor molecules.

Trypanosoma cruzi surface mucins: host-dependent coat diversity.

The surface of the protozoan parasite Trypanosoma cruzi is covered in mucins, which contribute to parasite protection and to the establishment of a persistent infection. Their importance is highlighted by the fact that the approximately 850 mucin-encoding genes comprise approximately 1% of the parasite genome and approximately 6% of all predicted T. cruzi genes. The coordinate expression of a large repertoire of mucins containing variable regions in the mammal-dwelling stages of the T. cruzi life cycle suggests a possible strategy to thwart the host immune response. Here, we discuss the expression profiling of T. cruzi mucins, the mechanisms leading to the acquisition of mucin diversity and the possible consequences of a mosaic surface coat in the interplay between parasite and host.

Insights into a CRM1-mediated RNA-nuclear export pathway in Trypanosoma cruzi.

Nuclear export and import of proteins and RNAs is a regulated process that permits the control of protein expression during cell development and differentiation. In all eukaryotic organisms transport of proteins to specific cellular compartments requires specific signaling sequences. Proteins that shuttle between nucleus and cytoplasm bear nuclear localization signals (NLS) and/or nuclear export signals (NES) and some of them can carry mRNAs, as part of shuttling ribonucleoprotein complexes. In this work we describe in the protozoan parasite Trypanosoma cruzi, a CRM1/exportin1 nuclear export factor named TcCRM1. This protein contains the conserved central region (CCR) that interacts with NES sequences present within cargo molecules, and the Cys residue involved in covalent binding to the Streptomyces metabolite leptomycin B (LMB). By subcellular fractionation we show that TcCRM1, a protein of about 117 kDa, has nuclear localization. We also demonstrate that LMB inhibits the replication of T. cruzi in a dose-dependent manner. In situ hybridization experiments performed with a Texas red-coupled oligo(dT) probe revealed that LMB produced a partial short-term accumulation of a poly(A)+RNA subset in the nucleus. Some mRNAs such as HSP70, TcUBP2/1 and TcPABP1 are reduced or disappeared from the cytoplasm of LMB treated cells. In sharp contrast with metazoans, no effect was observed on two U snRNAs subcellular localization, implying that a different export route might exist for these RNAs in trypanosomes.

Gene expression analysis in the hippocampal formation of tree shrews chronically treated with cortisol.

Adrenal corticosteroids influence the function of the hippocampus, the brain structure in which the highest expression of glucocorticoid receptors is found. Chronic high levels of cortisol elicited by stress or through exogenous administration can cause irreversible damage and cognitive deficits. In this study, we searched for genes expressed in the hippocampal formation after chronic cortisol treatment in male tree shrews. Animals were treated orally with cortisol for 28 days. At the end of the experiments, we generated two subtractive hippocampal hybridization libraries from which we sequenced 2,246 expressed sequenced tags (ESTs) potentially regulated by cortisol. To validate this approach further, we selected some of the candidate clones to measure mRNA expression levels in hippocampus using real-time PCR. We found that 66% of the sequences tested (10 of 15) were differentially represented between cortisol-treated and control animals. The complete set of clones was subjected to a bioinformatic analysis, which allowed classification of the ESTs into four different main categories: 1) known proteins or genes (approximately 28%), 2) ESTs previously published in the database (approximately 16%), 3) novel ESTs matching only the reference human or mouse genome (approximately 5%), and 4) sequences that do not match any public database (50%). Interestingly, the last category was the most abundant. Hybridization assays revealed that several of these clones are indeed expressed in hippocampal tissue from tree shrew, human, and/or rat. Therefore, we discovered an extensive inventory of new molecular targets in the hippocampus that serves as a reference for hippocampal transcriptional responses under various conditions. Finally, a detailed analysis of the genomic localization in human and mouse genomes revealed a survey of putative novel splicing variants for several genes of the nervous system.

Lactose derivatives are inhibitors of Trypanosoma cruzi trans-sialidase activity toward conventional substrates in vitro and in vivo.

Chagas' disease, caused by Trypanosoma cruzi, affects about 18 million people in Latin America, and no effective treatment is available to date. To acquire sialic acid from the host glycoconjugates, T. cruzi expresses an unusual surface sialidase with trans-sialidase activity (TcTS) that transfers the sugar to parasite mucins. Surface sialic acid was shown to have relevant functions in protection of the parasite against the lysis by complement and in mammalian host cell invasion. The recently determined 3D structure of TcTS allowed a detailed analysis of its catalytic site and showed the presence of a lactose-binding site where the beta-linked galactose accepting the sialic acid is placed. In this article, the acceptor substrate specificity of lactose derivatives was studied by high pH anion-exchange chromatography with pulse amperometric detection. The lactose open chain derivatives lactitol and lactobionic acid, as well as other derivatives, were found to be good acceptors of sialic acid. Lactitol, which was the best of the ones tested, effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Furthermore, lactitol inhibited parasite mucins re-sialylation when incubated with live trypanosomes and TcTS. Lactitol also diminished the T. cruzi infection in cultured Vero cells by 20-27%. These results indicate that compounds directed to the lactose binding site might be good inhibitors of TcTS.

Identification of genes regulated by chronic psychosocial stress and antidepressant treatment in the hippocampus.

Analysis of differentially expressed genes in the brain is a promising tool for elucidating pathological mechanisms that lead to central nervous disorders. Stress is known to be involved in the development of psychopathologies such as depression. In the present study, we searched for differentially expressed genes in the hippocampal formation after chronic psychosocial stress and after treatment with the antidepressant clomipramine. Experiments were conducted in male tree shrews, a valid psychosocial stress model in which antidepressant drugs prevent diverse effects of stress. Because many effects of stress have been attributed to the stress-induced elevation in glucocorticoids, we screened two subtractive hippocampal cDNA libraries generated from RNA of chronic cortisol-treated animals. Using real-time PCR to measure mRNA amounts, we identified five sequences whose expression levels differed between stressed animals and controls. Transcript levels of four of them, nerve growth factor (NGF), membrane glycoprotein 6a (M6a), CDC-like kinase 1 (CLK-1) and G-protein alpha q (GNAQ) were reduced by chronic psychosocial stress. Reduced amounts of these genes, which are all related to processes of cell differentiation, is in agreement with previous findings showing a retraction of dendrites and an impairment of neurogenesis in the hippocampal formation after chronic stress. An additional expressed sequence that was also regulated by stress could not be assigned to any known gene. Treatment with the antidepressant clomipramine prevented stress effects on expression of M6a, CLK-1, GNAQ and the novel sequence, but showed no effect on NGF stress-induced down-regulation. These findings support the concept that depressive disorders are accompanied by processes of neuronal dedifferentiation, at least in the hippocampal formation, and that antidepressants prevent these processes.

The surface coat of the mammal-dwelling infective trypomastigote stage of Trypanosoma cruzi is formed by highly diverse immunogenic mucins.

A thick coat of mucin-like glycoproteins covers the surface of Trypanosoma cruzi and plays a crucial role in parasite protection and infectivity and host immunomodulation. The appealing candidate genes coding for the mucins of the mammal-dwelling stages define a heterogeneous family termed TcMUC, which comprises up to 700 members, thus precluding a genetic approach to address the protein core identity. Here, we demonstrate by multiple approaches that the TcMUC II genes code for the majority of trypomastigote mucins. These molecules display a variable, non-repetitive, highly O-glycosylated central domain, followed by a short conserved C terminus and a glycosylphosphatidylinositol anchor. A simultaneous expression of multiple TcMUC II gene products was observed. Moreover, the C terminus of TcMUC II mucins, but not their central domain, elicited strong antibody responses in patients with Chagas' disease and T. crusi infected animals. This highly diverse coat of mucins may represent a refined parasite strategy to elude the mammalian host immune system.

Differential accumulation of mutations localized in particular domains of the mucin genes expressed in the vertebrate host stage of Trypanosoma cruzi.

The surface of Trypanosoma cruzi is covered by mucin-type glycoproteins involved in parasite protection, attachment and immunoevasion. The gene family coding for the mucins expressed by the parasite in the vertebrate host, named TcMUC, is composed of several hundred members and presents high variability. The genes encoding mucins expressed in the insect-dwelling parasite stages are part of a much more homogeneous family, named TcSMUG. Here, we addressed the organization and evolution of physically linked T. cruzi mucin genes by sequencing large chromosomal fragments containing these genes. Specific accumulation of mutations was restricted to particular domains of TcMUC genes, showing that these regions have, or have had, an accelerated evolution rate. Sequence analysis of several TcMUC genes allowed for the identification of members sharing features of TcMUC I and II, thus evidencing that one group of genes was generated from the other. The highly conserved intergenic regions of both TcMUC and TcSMUG families contained TG-rich microsatellites that were not present in unrelated genes in the cosmids, suggesting a role for homologous recombination in shuffling and/or amplification of T. cruzi mucin genes. The comparison of putative homologous TcMUC II genes from different strains of T. cruzi showed that their central variable domains are conserved. This conservation was always higher at the DNA level suggesting positive selection in these particular regions of TcMUC II genes.

Differential regulation of polysialyltransferase expression during hippocampus development: Implications for neuronal survival.

Polysialyltransferases ST8SiaII/STX and ST8SiaIV/PST add polysialic acid (PSA) to the neural cell adhesion molecule (NCAM). Surface-located PSA is involved in cell-cell interactions participating in structural and functional plasticity of neuronal circuits. This study was undertaken to investigate the polysialyltransferase regulation pattern during hippocampal development. Polysialyltransferase expression levels analyzed by real-time RT-PCR indicated that ST8SiaII/STX mRNA is markedly down-regulated in vivo, decreasing abruptly at about the first week of postnatal development. ST8SiaII/STX mRNA is also down-regulated in hippocampal cells in culture, accompanying the morphological differentiation of neuronal interconnectivity. In contrast, ST8SiaIV/PST levels remain comparatively low during hippocampus ontogeny. Immunolabeling of primary hippocampal culture assays demonstrated that PSA expression parallels ST8SiaII/STX mRNA levels. In comparison, polysialyltransferase mRNA levels are not regulated in neuroblastoma cells during their proliferation. Sequence analysis of the 3'-untranslated region of ST8SiaII/STX cDNA indicated putative regulatory motifs. This information and the observed changes in mRNA half-life during development suggest that ST8SiaII/STX might be also regulated at the posttranscriptional level. To understand the reasons for the tight control of ST8SiaII/STX expression during development, we overexpressed the enzyme in hippocampal primary cultures by transfection. Overexpression of ST8SiaII/STX wild type as well as of a mutant lacking enzymatic activity affected neuronal viability, leading to cell death. However, this phenomenon was abolished by a double mutation in the ST8SiaII/STX that prevents formation of its three-dimentional structure. Interestingly, the overexpressed polysialyltransferase accumulates not only in the perinuclear region but also in the plasma membrane. Thus, overexpression of an ST8SiaII/STX that conserves its structure leads to abnormal accumulation of the protein, probably on the neuronal surface, affecting cell viability. This result explains the importance of an accurate regulation of polysialyltransferase expression during development.

RNA-binding proteins and mRNA turnover in trypanosomes.

Trypanosomes, protozoan parasites of the order Kinetoplastida, control gene expression essentially through post-transcriptional mechanisms. Several motifs located mainly in the 3' untranslated region, such as AU-rich elements (AREs), were recently shown to modulate mRNA half-life, and are able to modify mRNA abundance in vivo through the interaction with specific RNA-binding proteins. Along with the detection of an active exosome, decapping activities and a regulated 3' to 5' exonuclease activity stimulated by AREs, these results suggest that modulation of mRNA stability is essential in trypanosomes. These regulatory processes are specific for different developmental stages and thus relevant for allowing trypanosomes to adapt to variable environmental conditions.

RNA recognition motif-type RNA-binding proteins in Trypanosoma cruzi form a family involved in the interaction with specific transcripts in vivo.

Trypanosomes, protozoan parasites from the order Kinetoplastida, have to deal with environmental changes during the interaction with their hosts. Trypanosoma cruzi, the causative agent of Chagas' disease, uses post-transcriptional mechanisms to regulate gene expression. However, few RNA-binding proteins involved in mRNA turnover control have been identified to date. In this work, an RNA recognition motif (RRM)-type RNA-binding protein family named T. cruzi RNA-binding protein (TcRBP) and composed of at least six members was identified. The genomic organization of four members revealed a head to tail arrangement within a region of 15 kilobase pairs. TcRBP members have a common RRM and different auxiliary domains with a high content of glycine, glutamine, and histidine residues within their N- and C-terminal regions. TcRBPs differ in their expression patterns as well as in their homoribopolymer binding interaction in vitro, although they preferentially recognize poly(U) and poly(G) RNAs. An interesting observation was the relaxed RNA-binding interactions with several trypanosome transcripts in vitro. In contrast, co-immunoprecipitation experiments of TcRBP-containing ribonucleoprotein complexes formed in vivo revealed a highly restricted binding interaction with specific RNAs. Several TcRBP-containing complexes are stage-specific and, in some cases, bear the poly(A)-binding protein TcPABP1. Altogether, these results suggest that TcRBPs might be modulated in vivo, to favor or preclude the interaction with specific transcripts in a developmentally regulated manner.

TcUBP-1, an mRNA destabilizing factor from trypanosomes, homodimerizes and interacts with novel AU-rich element- and Poly(A)-binding proteins forming a ribonucleoprotein complex.

Trypanosomes, protozoan parasites causing worldwide infections in human and animals, mostly regulate protein expression through post-transcriptional mechanisms and not at the transcription initiation level. We have previously identified a Trypanosoma cruzi RNA-binding protein named TcUBP-1. This protein is involved in mRNA destabilization in vivo through binding to AU-rich elements in the 3'-untranslated region of SMUG mucin mRNAs (D'Orso, I., and Frasch, A. C. (2001) J. Biol. Chem. 276, 34801-34809). In this work we show that TcUBP-1 is part of an approximately 450-kDa ribonucleoprotein complex with a poly(A)-binding protein and a novel 18-kDa RNA-binding protein, named TcUBP-2. Recombinant TcUBP-1 and TcUBP-2 proteins recognize U-rich RNAs with similar specificity and affinity through the approximately 92-amino acid RNA recognition motif. TcUBPs can homo- and heterodimerize in vitro through the glycine-rich C-terminal region. This interaction was also detected in vivo by co-immunoprecipitation of the ribonucleoprotein complex and using yeast two-hybrid assay. The poly(A)-binding protein identified was shown to disrupt the formation of TcUBP-1, but not TcUBP-2, homodimers in vitro. The possible role of TcUBP-1 ligands in the pathways that govern mRNA-stability and stage-specific expression in trypanosomes is discussed.

The trans-sialidase from the african trypanosome Trypanosoma brucei.

Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T. brucei ssp. gambiense and ssp. rhodesiense) and ngana in domestic animals (T. brucei brucei) in Africa. Procyclic trypomastigotes, the tsetse vector stage, express a surface-bound trans-sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface. Trans-sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host. The only enzyme extensively characterized is that of the American trypanosome T. cruzi (TcTS). In this work we identified and characterized the gene encoding the trans-sialidase from T. brucei brucei (TbTS). TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present. The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid. Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis. The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical. As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both alpha-(2,3) and alpha-(2,6)-linked sialoconjugates.