The trans-sialidase, the major Trypanosoma cruzi virulence factor: Three decades of studies.

Chagas' disease is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi. Since the description of Chagas'disease in 1909 extensive research has identified important events in the disease in order to understand the biochemical mechanism that modulates T. cruzi-host cell interactions and the ability of the parasite to ensure its survival in the infected host. Exactly 30 years ago, we presented evidence for the first time of a trans-sialidase activity in T. cruzi (T. cruzi-TS). This enzyme transfers sialic acid from the host glycoconjugates to the terminal ß-galactopyranosyl residues of mucin-like molecules on the parasite's cell surface. Thenceforth, many articles have provided convincing data showing that T. cruzi-TS is able to govern relevant mechanisms involved in the parasite's survival in the mammalian host, such as invasion, escape from the phagolysosomal vacuole, differentiation, down-modulation of host immune responses, among others. The aim of this review is to cover the history of the discovery of T. cruzi-TS, as well as some well-documented biological effects encompassed by this parasite's virulence factor, an enzyme with potential attributes to become a drug target against Chagas disease.

Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi.

The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha-mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.

Biosynthesis of O-N-acetylglucosamine-linked glycans in Trypanosoma cruzi. Characterization of the novel uridine diphospho-N-acetylglucosamine:polypeptide N-acetylglucosaminyltransferase-catalyzing formation of N-acetylglucosamine alpha1-->O-threonine.

In this study, we have characterized the activity of a uridine diphospho-N-acetylglucosamine:polypeptide-alpha-N-acetylglucosaminylt ransferase (O-alpha-GlcNAc-transferase) from Trypanosoma cruzi. The activity is present in microsomal membranes and is responsible for the addition of O-linked alpha-N-acetylglucosamine to cell surface proteins. This preparation adds N-acetylglucosamine to a synthetic peptide KPPTTTTTTTTKPP containing the consensus threonine-rich dodecapeptide encoded by T. cruzi MUC gene (Di Noia, J. M., Sánchez D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146-24149). Incorporation of N-[3H]acetylglucosamine is linearly dependent on incubation time and concentration of enzyme and substrate. The transferase activity has an optimal pH of 7.5- 8.5, requires Mn2+, is unaffected by tunicamycin or amphomycin, and is strongly inhibited by UDP. The optimized synthetic peptide acceptor for the cytosolic O-GlcNAc-transferase (YSDSPSTST) (Haltiwanger, R. S., Holt, G. D., and Hart, G. W. (1990) J. Biol. Chem. 265, 2563-2568) is not a substrate for this enzyme. The glycosylated KPPTTTTTTTTKPP product is susceptible to base-catalyzed beta-elimination, and the presence of N-acetylglucosamine alpha-linked to threonine is supported by enzymatic digestion and nuclear magnetic resonance data. These results describe a unique biosynthetic pathway for T. cruzi surface mucin-like molecules, with potential chemotherapeutic implications.

Development of a chitin assay for the quantification of fungus.

Chitin, a unique structural polysaccharide found in fungi and arthropods, is not produced by vertebrates. Thus, the potential applications of a specific and sensitive assay for chitin are numerous, including the evaluation of the extent of fungal keratitis. Chitin is a homopolymer of beta (1, 4) linked D-N-acetylglucosamine. We have developed a simple and reproducible assay for chitin and applied it to Candida albicans cultures. The assay involves homogenization of the culture and treatment with 21.1 M KOH to remove soluble materials, including proteins. This base treatment also deacetylates the chitin to the glucosamine polymer, chitosan. Chitosan is hydrolyzed by 0.5 M H2SO4 to glucosamine monomers which are then deaminated by the addition of NaNO2 to the acid solution. The resulting 2,5-anhydromannose is reduced by NaB[3H]4 to 1-[3H] 2,5-anhydromannitol. This radiolabelled sugar is isolated by paper chromatography and quantified via liquid scintillation. The sensitivity of this assay is assessed by comparison of colony forming units (CFU's) with a glucosamine standard. A typical run of the assay detects 53.1 CFU/c.p.m., and 356,000 c.p.m. per nanomole of N-acetylglucosamine. The specificity of the assay is very high because of the unique nature of chitin. This method of chitin determination may be a useful alternative method for future investigations involving the study of fungal infections in mammalian tissues.

The alpha-mannosidase of Trypanosoma cruzi: structure and function.

Trypanosoma cruzi alpha-mannosidase has been purified to apparent homogeneity. It is a 240,000-Da tetramer composed of four identical subunits (58,000 Da). Each subunit contains one N-linked high-mannose oligosaccharide. Based on pH optimum and sensitivity to inhibition by swainsonine, we suggest it to be lysosomal, but this has yet to be demonstrated directly. The enzyme appears to be developmentally regulated and may be a key enzyme in the degradation of the lipopeptidophosphoglycan (LPPG) during transformation from epimastigote to trypomastigote. Preliminary experiments suggest T. cruzi does not utilize the mannose 6-phosphate recognition system for sorting alpha-mannosidase (or other acid hydrolases) to the lysosome. To clone the alpha-mannosidase from T. cruzi we have used the same approach that has been used for other alpha-mannosidases. The cDNA amplification product was subcloned and sequenced. Comparison of the T. cruzi alpha-mannosidase sequence with the alpha-mannosidases that were used in the original primer design demonstrated a greater similarity to murine lysosomal and Dictyostelium alpha-mannosidases than to Golgi alpha-mannosidases.

Identification and characterization of an alpha-mannosidase from Trypanosoma cruzi.

In this report we describe the first purification and characterization of the acid alpha-mannosidase from the human parasite Trypanosoma cruzi. The purified enzyme exhibited a native mol. wt of 240,000 Da and is apparently composed of four identical subunits of mol. wt 58,000 Da. Each of the four subunits contains one N-linked high-mannose-type oligosaccharide. The alpha-mannosidase exhibited a pH optimum of 3.5 and a pI of 5.9. This low pH optimum and the ability of swainsonine to inhibit its activity suggest that the alpha-mannosidase is a lysosomal enzyme. Antibodies against the T.cruzi enzyme did not react with mammalian lysosomal alpha-mannosidase and, conversely, antibody against a rat lysosomal alpha-mannosidase did not react with the T.cruzi enzyme. Thus, the T.cruzi enzyme appears to be distinct from its mammalian counterpart.

Immunolesioning: selective destruction of neurons using immunotoxin to rat NGF receptor.

192 IgG, a monoclonal antibody to the rat nerve growth factor (NGF) receptor, was disulfide-coupled to saporin, a ribosome-inactivating protein. Systemic injection of 192 IgG-saporin destroyed sympathetic postganglionic neurons and some sensory neurons. Injection of 192 IgG-saporin into the lateral ventricle destroyed cholinergic neurons of the basal forebrain. These results show that antineuronal immunotoxins are a powerful approach that may prove useful in a variety of neurobiological applications.

Advances in immunotoxins.

The field of immunotoxins is continuing to grow, as shown by the increase in attendance (175 vs. 145) and abstracts (110 vs. 60) at the symposium. Advances in the molecular biology of the toxins and clinical applications were notable at this meeting. The next 2 years should see better definition of the mechanism of action of peptide toxins as well as improvements in the design of targeted toxins for potential therapy of a wide variety of human diseases.

Alpha-mannosidase treatment of NK cells does not inhibit NK cell lytic function.

Alpha-mannosidase was tested for its ability to inhibit lytic activity of nonadherent, mononuclear peripheral blood leukocytes (PBL) against K-562 target cells. Pretreatment of effector cells (60 min., 37 degrees C, pH 7.3) with this enzyme, prior to and after exhaustive dialysis, was examined. Nondialyzed enzyme preparations completely inhibited NK lytic function at all concentrations tested (1.0, 0.5, and 0.25 units/ml). On the other hand, dialyzed enzyme preparations had no inhibitory effect on NK lytic function over the same range of concentrations. The inhibitory effects of the nondialyzed enzyme were due to the presence of (NH4)2SO4, which could be removed by dialysis. Studies were also performed to determine whether enzyme treatment of effector cells resulted in hexose release from cell surface structures. Treatment of effector cells with alpha-mannosidase (dialyzed preparation) resulted in a dose dependent release of mannose. These data demonstrate that NK cell lytic function is not inhibited by pretreatment of effector cells with alpha-mannosidase even though mannose is quantitatively released from cell surface oligosaccharide structures. These results suggest that NK lytic function does not involve an effector cell surface structure bearing terminal alpha-linked mannose residues as previously reported.

An antimelanoma-barley ribosome inactivating protein conjugate is cytotoxic to melanoma cells in vitro.

The ribosome inactivating protein (BRIP) from barley is a single polypeptide chain (Mr = 32,000 Dalton) and is nontoxic to intact cells. The BRIP has been purified to homogeneity by modifications of the methods of Roberts and Selitrennikoff and crosslinked to monoclonal antibodies by Succinimidyl-3 (2- Pyridyldithio) -Propionate (SPDP) and by the cystamine-EDAC methods. The resulting hybrids were purified from the free BRIP by gel filtration on a Sephadex G-75 column. The model suicide transport agents were assayed against melanoma cells; K-562 cells were used as control. The hybrids were found to be selectively toxic to melanoma cells in a dose dependent manner.

A possible role for protein kinase C activity but not cyclic nucleotide-dependent protein kinases in human natural killer cell lytic activity.

Recent evidence suggests that a Ca++, phospholipid, diacylglycerol-dependent protein kinase, protein kinase C, plays a role in the activation of cytotoxic T lymphocytes by target cells. In this investigation we have examined the role of protein kinase C in human NK cell-mediated cytolysis of K-562 cells. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibited human NK cell-mediated cytolysis in a dose dependent manner. On the other hand, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a specific inhibitor of cyclic nucleotide dependent protein kinases had no effect on human NK cell-mediated cytolysis of K562 cells. There is little or no effect on protein synthesis or N-glycosylation activity in human NK cells by H-7. The relative inhibitory ability of the two inhibitors suggest that protein kinase C, acting synergistically with Ca++ mobilization, plays a role in the early stages of human NK cell-mediated cytolysis of K562 target cells.

Neuronotoxic effects of monoclonal anti-Thy 1 antibody (OX7) coupled to the ribosome inactivating protein, saporin, as studied by suicide transport experiments in the rat.

As a first attempt to develop suicide transport agents based upon antineuronal antibodies, we studied an immunotoxin directed against the Thy 1 antigen which is on rat neurons. The immunotoxin was composed of mouse monoclonal anti-Thy 1 antibody (OX7) and the ribosome-inactivating protein, saporin, and was prepared using the heterobifunctional cross linker, SPDP, which provides a disulfide linkage between the two protein components. This immunotoxin reliably and selectively destroyed ipsilateral vagal motor and sensory neurons after injection into the cervical vagus. Injection of the immunotoxin into the caudate nucleus produced destruction of the ipsilateral substantia nigra, pars compacta and intralaminar thalamic nuclei (parafascicular and central median). Anti-mouse IgG immunoperoxidase staining confirmed axonal transport of OX7 by vagal sensory and motor neurons and by caudate afferents and efferents. Systemic toxicity was not observed with OX7-saporin. The neuronotoxic effects of OX7-saporin were specific since injections of a similarly constructed immunotoxin of irrelevant specificity or a mixture of OX7 and saporin were without suicide transport activity. These results show the feasibility of using immunotoxins as suicide transport agents.

Anti-ricin antibody protects against systemic toxicity without affecting suicide transport.

Ricin is an effective suicide transport agent which reliably destroys sensory and motor neurons in anatomically selective fashion after peripheral nerve application. In the present study, we gave subcutaneous injections of commercially available antiricin antibody (ARA) at the time of ricin application to peripheral nerves. The ARA protected rats against systemic ricin poisoning without altering the suicide transport activity of ricin. This approach removes a significant barrier to the routine use of ricin and should prove useful when ricin is employed to ablate large peripheral nerves. However, antibody protection permits use of such large ricin doses that the toxin may diffuse within the CNS destroying neurons adjacent to those that project through the injected nerve. Consequently, antibody and ricin doses must be optimized for any particular experimental situation.

Synthesis and in vitro and in vivo activity of a hybrid composed of ricin B chain-barley ribosome-inactivating protein.

In our continued studies on hybrid proteins for use as cytotoxins and possible suicide transport agents, we have begun to investigate the use of ribosome-inactivating proteins (RIP) isolated from grain. The RIP from barley has been purified to homogeneity by modifications of the methods of Roberts and Selitrennikoff and crosslinked to the binding subunit B of the seed toxin ricin (RTB). The resulting hybrid was purified by a combination of gel filtration and affinity chromatography on acid-washed Sepharose 4B. This model suicide transport agent was assayed in vitro against K-562 cells and was found to be cytotoxic in a dose-dependent manner (ID50 = 0.15 micrograms/ml). Lactose inhibited the toxicity of the hybrid, indicating that cytotoxicity was dependent on the cell binding property of the ricin B moiety. In addition, free RIP and free ricin B, either alone or in combination, were nontoxic over this concentration range. The in vivo effects of the RTB-RIP hybrid were assessed by pressure microinjection into the vagus nerves of rats. Injection of 0.18 to 6.5 micrograms of conjugate resulted in death of vagal sensory but not motor neurons after 3-17 days. The cytotoxic changes in vagal sensory neurons were identical to those previously observed with a variety of RIP toxins such as ricin.

Natural killer cell infection and inactivation in vitro by the human immunodeficiency virus.

Cytolytic activity of human mononuclear peripheral blood leukocytes from healthy donors, cultured in interleukin-2 conditioned medium, was abrogated by in vitro infection with the lymphadenopathy associated virus (LAV) isolate of the human immunodeficiency virus (HIV). Although viral antigens are not expressed in cultured cells until 14 days postinfection, cytolytic activity was lost as early as 3 days after infection. Loss of cytolytic function was not a result of the release of suppressive factors from either infected cells or uninfected CEM cells since supernatants from neither infected cultures nor CEM cell cultures had any inhibitory effects on the function of uninfected cells. Cultured lymphocytes expressing Leu 11b were also shown to express HIV antigens via immunofluorescence after 14 days in culture. These results suggest that natural killer (NK) cells, as defined by expression of Leu 11b, were infected by HIV in vitro and the loss of lytic function was likely a direct consequence of that infection.

Role of respiratory tract proteases in infectivity of influenza A virus.

Proteolytic cleavage of influenza virus hemagglutinin (HA) glycoprotein into subunits designated HA1 and HA2 is required for penetration of virus into the cell. It is generally assumed that this cleavage is an intracellular function of the host cell. Human adenoid fibroblast (HAF) lines, which support the growth of influenza A virus but release virus with an uncleaved HA, provide a model system that has allowed exploration of mechanisms of cleavage in vivo. Exposure of HAF-grown influenza virus to nasal secretions from children with respiratory tract symptoms induced HA cleavage and rendered virus fully infectious. Characterization of this proteolytic enzyme, present in the extracellular environment of the respiratory tract, suggests that it is a serine endopeptidase.

The effects of hexose 6-O-sulfate esters on human natural killer cell lytic function.

Natural cell-mediated cytotoxicity (NCMC) is inhibited by some neutral hexoses and hexose phosphates at 25 to 100 mM concentrations. In this study we describe the effects of hexose 6-O-sulfate esters on NCMC against K-562 target cells. Mannose 6-sulfate, galactose 6-sulfate, N-acetylglucosamine 6-sulfate, and N-acetylgalactosamine 6-sulfate inhibit NCMC in a dose-dependent manner at concentrations of 10 mM and below. Inhibitory effects of mannose 6-sulfate and galactose 6-sulfate were evident at concentrations as low as 1.25 mM. The neutral forms of these sugars, glucose and glucose 6-sulfate, did not inhibit NCMC over this range of concentrations. Comparison of the inhibitory effects of sulfated and phosphorylated forms of mannose and galactose indicated that the sulfated forms are much more potent inhibitors. Formation of effector cell:target cell conjugates was unaffected by the presence of sugar sulfates. Calcium pulse experiments demonstrated that inhibitory effects of sugar sulfates were exerted after the Ca++-dependent triggering step in the NK lytic process. Kinetic studies showed that addition of sugars as long as 60 min after initiation of cultures yielded potent inhibitory effects. Sugar sulfates were not toxic for effector cell populations and effectors were not refractory for lytic function after removal of sugars. Sugar sulfates were inhibitory against multiple tumor types in both human and murine NK lytic assays. These results suggest that the sugar sulfates inhibit NK cells at a postconjugation, posttriggering step involving lectin-like receptors or lectin-like molecules.