Structural and immunological characterization of sulphatides: relevance of sulphate moieties in Trypanosoma cruzi glycoconjugates.

Sulphoglycosphingolipids, present on the surface of diverse cells, participate in the regulation of various cellular events. However, little is known about the structure and the role of sulphoglycosphingolipids in trypanosomatids. Herein, sulphated dihexosylceramide structures - composed mainly of sphingosine as the long chain base acylated with stearic acid - have been determined for the first time in Trypanosoma cruzi epimastigotes by UV-MALDI-TOF-MS analysis. Interestingly, inhibition ELISA assays using cruzipain as antigen and polyclonal rabbit antibodies specific for cruzipain, the major cysteine proteinase of T. cruzi, or for its C-terminal domain, have demonstrated (i) that sulphate epitopes are shared between cruzipain and sulphatides of T. cruzi, (ii) that cross-reactivity maps to the C-terminal domain and (iii) the existence of other antigenic determinants in the glycolipidic structures. These features provide evidence that sulphate groups are antigenic in sulphate-containing parasite glycoconjugates. Furthermore, IgG2 antibody levels inversely correlate with disease severity in chronic Chagas disease patients, suggesting that IgG2 antibodies specific for sulphated epitopes might be associated with protective immunity and might be considered as potential surrogates of the course of chronic Chagas disease.

A striking common O-linked N-acetylglucosaminyl moiety between cruzipain and myosin.

Single units of O-linked N-acetylglucosamine (GlcNAc), usually components of nuclear and cytoplasmatic proteins, are present at the C-terminal domain of cruzipain (Cz), a lysosomal major antigen from Trypanosoma cruzi. On the other hand, antibodies directed against some self-antigens like myosin are associated with Chagas heart disease. The participation of O-GlcNAc moieties in the molecular antigenicity of Cz was determined using GlcNAc linked to aprotinin by ELISA. The immune cross-reactivity between Cz and myosin is mainly focused in the C-T domain. ELISA inhibition assays using rabbit sera specific for Cz and C-T in conjunction with immune-gold electron microscopy analysis of heart tissues from mice immunized with C-T confronted with polyclonal rabbit sera specific for Cz and C-T prior and after myosin adsorption provided evidence which indicates that O-GlcNAc moieties constitute a common epitope between Cz and either myosin or other cardiac O-GlcNAc-containing proteins, showing a new insight into the molecular immune pathogenesis of Chagas heart disease.

MALDI-TOF MS analysis of labile Lolium perenne major allergens in mixes.

BACKGROUND: It is well known that allergen extracts used for specific therapy of allergic disorders are commonly stored as mixtures, causing an alteration of its stability. OBJECTIVE: The aim of this report is to identify pollen allergens susceptible to degradation during storage of mixtures containing different sources of proteases in the absence of glycerol as a preserving agent. METHODS: Mixes containing Lolium perenne (Lol p) pollen extract with either Aspergillus fumigatus or Periplaneta americana extracts were prepared and co-incubated for 90 days at 4 degrees C. Samples were taken off at fixed times and comparatively tested by in vitro and in vivo assays with atopic patients. Selected pollinic allergens were subjected to MALDI-TOF MS analysis. RESULTS: ELISA inhibition evidenced the loss of potency from ryegrass extract, and immunoblotting assays showed the degradation of specific pollinic allergens during storage of mixtures containing protease-rich sources. An in vivo intradermal skin assay confirmed the gradual loss of the biological activity of L. perenne pollen extract co-incubated with non-related protease-rich extracts in comparison with that of the control pollen extract. MALDI-TOF MS analysis allowed us to determine that Lol p 1 and Lol p 5 are susceptible to proteolysis whereas Lol p 4 was found to be resistant to degradation during storage. CONCLUSIONS: Lol p 1 and Lol p 5 degradation is responsible for the loss of the biological activity of L. perenne pollen extract when co-incubated with protease-rich fungal and cockroach extracts in the same vial for months in the absence of glycerol as a preserving agent. The integrity of these major allergens must be preserved to increase the vaccine stability and to assure efficacy when mixes are used for immunotherapy.

Novel cysteine proteinase in Trypanosoma cruzi metacyclogenesis.

With the aim to study proteinases released to the culture medium during Trypanosoma cruzi metacyclogenesis, the presence of cysteine proteinases (CPs) was analysed in culture supernatants obtained throughout the differentiation induced by stimulation of epimastigotes with Triatoma infestans hindgut homogenate. In SDS-gelatin containing gels, an important endopeptidase activity with apparent molecular weight range between 97 and 116 kDa was encountered at pH 6, which was abolished by the specific cysteine proteinase inhibitor E-64 and TLCK, but not by pepstatin, 1,10 phenantroline or PMSF. This novel CP, named TcCPmet, showed affinity to cystatin-Sepharose, denoting its thiol-proteinase character as well as to ConA-Sepharose, indicating it contains N-linked oligosaccharides. However, it presented a different elution pattern on ConA-Sepharose than cruzipain and, in addition, it was not recognized by anti-cruzipain serum, facts that strongly suggest the different nature of both CPs. Moroever, evidence is presented indicating that TcCPmet was able to hydrolyse the same chromogenic peptides as cruzipain at optimal alkaline pH values, although with a different order of effectiveness. Our results indicate the presence of a novel CP secreted by metacyclic trypomastigotes and reinforces the important role of these enzymes in metacyclogenesis.

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.

Humoral immune response to cruzipain and cardiac dysfunction in chronic Chagas disease.

The humoral immune response to epitopes expressed on cruzipain was evaluated in 31 Chagas disease patients (CDP) with different degrees of cardiac dysfunction. We took advantage of the availability of anti-Trypanosoma cruzi microsomal fraction monoclonal antibodies (MoAbs) reactive with epitopes that are recognized (5A9B11) or not recognized (1A10C11) by CDP sera. The 5A9B11- and 1A10C11-like epitopes are expressed on cruzipain. The reactivity of 5A9B11 against cruzipain was completely inhibited by sera of severe cardiopathy patients while a partial inhibition was found with sera from chagasic patients with mild disease. CDP sera did not block cruzipain recognition by 1A10C11. The antigenic determinants recognized by CDP sera appeared to be linear and carbohydrate free. When the overall anti-cruzipain immune response was evaluated, 70% of CDP with severe disease showed cruzipain titers higher than 1/800 while none of them displayed titers lower that 1/400. This report shows for the first time that the humoral immune response against epitopes expressed on cruzipain appeared to be related with the severity of chronic Chagas disease.

Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii.

A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host.

Proteinase and gelatinolytic activities of house dust mite and cockroach extracts.

The presence of gelatinolytic activity in dust mite and Periplaneta americana allergenic crude extracts were studied. The former presented major activity in a broad band between 45 and 66 kDa and minor activity at 32 kDa, while the latter showed a more complex pattern with gelatinolytic activity at 90, 78, 65, 34, 32 and 24 kDa. When the proteolytic activity patterns of dust mites and cockroach crude extracts were analyzed at three different pH levels, the proteases in both cases were optimally active at pH 6, showed no activity at pH 3.5 and little activity at pH 8.5. The susceptibility of both extracts to a set of well-known protease inhibitors suggested that they are composed of cysteine and serine proteinases, the latter probably being a trypsin-like type. When immunochemical properties were studied, dust mite bands of about 200, 110, 65, 60 and 43 kDa showed immunoreactivity against a polyclonal human anti-dust mite serum, with the band of approximately 200 kDa presenting the highest antigenicity. A similar analysis was applied to the cockroach extract, which exhibited immunoreactive bands at 90, 78, 65 and 34 kDa when incubated with a polyclonal rabbit anti-Blatta serum. Only those of 90, 78 and 65 kDa reacted against a polyclonal human anti-Blatta serum. These results suggested a correlation between some proteases with gelatinolytic activity and the allergenicity of both extracts.

Membrane-bound cysteine proteinase isoforms in different developmental stages of Trypanosoma cruzi.

Cysteine proteinase isoforms, immunologically cross-reactive with cruzipain and with a similar apparent molecular mass, have been identified in epimastigotes of Trypanosoma cruzi by extraction and phase partition using the detergent Triton X-114. These isoforms are concentrated in the microsomal fraction obtained after differential centrifugation, which is known to consist essentially of plasma membrane, can be labelled by incubation of live parasites with sulfo-NHS-biotin, and bind to cystatin-sepharose affinity columns. They are present, albeit with a different electrophoretic pattern, in the epimastigote, amastigote and trypomastigote stages of the parasite.

Subcellular localization of glutamate dehydrogenases and alanine aminotransferase in epimastigotes of Trypanosoma cruzi.

The subcellular localization of NAD- and NADP-linked glutamate dehydrogenases (GDH-NAD and GDH-NADP), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in epimastigotes of Trypanosoma cruzi was studied by digitonin extraction from whole cells, subcellular fractionation by differential centrifugation and isopycnic ultracentrifugation. All enzymes presented both a cytosolic and a mitochondrial form; in addition, GDH-NADP seems to have a third, still undefined, localization. The results are compatible with the existence of two pathways for the production of L-alanine linked to the reoxidation of glycolytic NADH, one operative in the mitochondrion and the other in the cytosol, and perhaps responsible for the existence of the two alanine pools detected by 13C-nuclear magnetic resonance (B. Frydman et al., Eur. J. Biochem. 192 (1990) 363-368).

The histones of the insect trypanosomatid, Crithidia fasciculata.

The histone-like proteins of the monogenetic parasite Crithidia fasciculata were extracted with 0.2 M sulfuric acid either from purified nuclei, or from purified chromatin, in both cases in the presence of 1 mM tosyl lysylchloromethylketone and 2 mM phenyl methyl sulfonyl fluoride as proteinase inhibitors. The presence of histones in the flagellate, nonidentical with those from calf thymus used as controls, was shown by their electrophoretic patterns in three different polyacrylamide gel systems; their staining with Alkaline fast green, specific for basic proteins; their global amino acid composition and absorption spectrum and their molecular weights. The protein showing the slower mobility in SDS gels and the fastest mobility in the urea-acetic acid-Triton gels, seems to be an H1 histone, because of its metachromatic staining with Coomassie brilliant blue, solubility characteristics, differential destaining properties and amino acid composition. Band 5 in Triton-urea-acetic acid gels is probably an HMG protein. We conclude that C. fasciculata has a complete set of histones and that the lack of chromosome condensation during mitosis is not due to lack of histone H1.

Effects of DNA gyrase inhibitors in a polyamine-auxotrophic strain of Escherichia coli.

The polyamine content of the Escherichia coli polyamine-auxotrophic strain BGA 8 seemed to influence the effects of nalidixic acid, an antibiotic acting on subunit A of DNA gyrase. The growth rate was more affected under conditions of putrescine depletion and the inhibition could be partially relieved if the polycation was added back to the culture. DNA synthesis was likewise more sensitive to nalidixic acid in cultures grown without polyamine. The expression of some proteins characteristic of the heat-shock response, evoked by the antibiotic, showed a different persistence according to the presence or absence of polyamines. Novobiocin, acting on subunit B of gyrase, also promoted a differential effect depending on the polyamine content, but in this case putrescine-supplemented cells were more sensitive. The described findings suggest a role of polyamines in all the reactions carried out by gyrase, perhaps due to the influence of the polycations on the state of DNA aggregation.