Vaccination with Trypomastigote Surface Antigen 1-Encoding Plasmid DNA Confers Protection against Lethal Trypanosoma cruzi Infection

DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8⁺ cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2^b and H-2^d mice with a plasmid DNA encoding an N-terminally truncated TSA-1 lacking or containing the C-terminal nonapeptide tandem repeats, the antibody level, CTL response, and protection against challenge with T. cruzi were assessed. In H-2^b mice, antiparasite antibodies were induced only by immunization with the DNA construct encoding TSA-1 containing the C-terminal repeats. However, both DNA constructs were efficient in eliciting long-lasting CTL responses against the protective H-2K^b-restricted TSA-1_515–522 epitope. In H-2^d mice, inoculation with either of the two TSA-1-expressing vectors effectively generated antiparasite antibodies and primed CTLs that lysed T. cruzi-infected cells in an antigen-specific, MHC class I-restricted, and CD8⁺-T-cell-dependent manner. When TSA-1 DNA-vaccinated animals were challenged with T. cruzi, 14 of 22 (64%) H-2^b and 16 of 18 (89%) H-2^d mice survived the infection. The ability to induce significant murine anti-T. cruzi protective immunity by immunization with plasmid DNA expressing TSA-1 provides the basis for the application of this technology in the design of optimal DNA multicomponent anti-T. cruzi vaccines which may ultimately be used for the prevention or treatment of Chagas’ disease.

Chagas’ disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a lifelong health problem in Central and South America, where an estimated 18 million people are infected with this parasite and 90 million are at risk of infection (35, 65). Following a short-lived acute-phase illness characterized by fever and a patent parasitemia, infected individuals enter a nearly aparasitemic asymptomatic chronic phase, where most remain for the remainder of their lifetime. However, at 10 to 20 years postinfection nearly 30% of infected individuals develop severe cardiomyopathy, which is responsible for most of the 50,000 deaths caused by Chagas’ disease each year (45). Although reduviid vector control and blood bank screening measures have had a major impact in reducing transmission of T. cruzi (65), the operational costs to maintain such control programs, behavioral differences among vector species, existence of animal reservoirs, persistence of parasites in chronically infected patients, and lack of adequate chemotherapies to treat the infection will likely prevent these control measures alone from completely eradicating T. cruzi. An additional approach that could contribute significantly to control the transmission of Chagas’ disease is the development of anti-T. cruzi vaccines. To date, however, vaccine production for T. cruzi has been a low priority despite the current knowledge about the protective roles that antibodies, type 1 cytokines, and CD8⁺ T cells play in resistance to experimental T. cruzi infections (53).During T. cruzi infection, both chagasic patients and experimental animals produce strong immune responses to molecules from the infective nonreplicative trypomastigote stage and the replicative amastigote forms (3, 4, 14, 29). Among these, trypomastigote surface antigen 1 (TSA-1) (15, 38), a major trypomastigote surface antigen and the first identified member of the trans-sialidase gene superfamily (48), is a target of protective immune responses in mice (61, 66). Immunization with an amino-proximal fragment of TSA-1 induces a strong antibody response and protects mice against an otherwise lethal challenge with T. cruzi (66). Our studies have recently identified TSA-1 as the first bona fide target of CD8⁺ cytotoxic T lymphocytes (CTL) in T. cruzi-infected mice and demonstrated that the adoptive transfer of TSA-1-specific gamma interferon (IFN-γ)- and tumor necrosis factor alpha-producing CTL lines protects naive animals against lethal T. cruzi infection (61). Moreover, we have recently determined that TSA-1 and amastigote surface protein-1 and -2 (33, 44), which are also recognized by murine CTL (32), represent three target molecules of T. cruzi-specific human CD8⁺ CTL (62). These studies demonstrated the validity of the mouse model to identify target antigens of protective anti-T. cruzi immune responses and provide a strong incentive for the development of vaccines as a potential control measure against Chagas’ disease. For this purpose, and given the success of plasmid DNA vaccination in specifically stimulating a broad spectrum of immune responses to the vector-encoded target antigen (12), we have chosen to investigate DNA-based immunization as a system to generate vaccine-induced resistance against T. cruzi and have used TSA-1 as a model antigen for its initial evaluation. In this report we document that intramuscular injection of BALB/c and C57BL/6J mice with TSA-1-encoding plasmid DNA induces antibodies, CTL, and significant protection against lethal challenge with T. cruzi.     

Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules.     

Trypanosoma cruzi: Surface antigenic determinants

A fraction (FAd) capable of inhibiting specific agglutination reactions of anti-epimastigote sera (anti-LE) was obtained by extracting the sediment of lyophilized epimastigote lysates (LE) with 0.05 M phosphate buffered saline, at 37° C for 1 h. These conditions favored the action of parasite proteinase whose presence was detected by tandemcrossed immunoelectrophoresis experiments. As expected from the proteinase properties, the addition of 2-mercaptoethanol or sodium iodoacetate to the extracting solution resulted, respectively, in either increased or decreased amounts of protein in the resulting FAd. FAd components could be precipitated by the addition of Concanavalin A, methylated albumins or 0.1 N HCl. This fraction presented a single component when subjected to electrophoresis in 1% agarose gel with an electrophoretic mobility 1.2 times higher than that of human albumin. FAd component(s) were unable to penetrate 15% polycrylamide gel matrix unless 1% SDS was used. Under this condition four glycopeptide components, with Rm of 0.5, 0.55, 0.6 and 0.86, were detected. The antigenic determinants present in FAd resisted heating at 100° C for 30 min and the prolonged action of pronase. However, these determinants were completely destroyed by the action of 25 mM sodium periodate, thus suggesting polysaccharide characteristics. Immunization of rabbits with FAd induced the production of antibodies that were unable to precipitate with either FAd or with parasite proteinase. These antibodies exhibited positive agglutination reactions with epimastigote forms and positive immunofluorescence and immunoperoxidase reactions with trypomastigote and amastigote forms of the different strains tested. FAd was able to inhibit these reactions as well as those obtained with anti-LE and anti-FA immune sera, whereas purified proteinase was unable to inhibit any of these reactions.     

Antigenic determinants of Trypanosoma cruzi defined by cloning of parasite DNA

A genomic DNA library from Trypanosoma cruzi, the agent of Chagas' disease, was constructed in the gt11 lambda vector and was screened with serum from a Chagasic patient. Out of 53 positive clones, 23 plaques were purified to homogeneity and 10 different groups were defined by cross-hybridization experiments and by reaction of antibodies selected with products from each recombinant clone. Native T. cruzi proteins of molecular mass ranging from 85 to larger than 205 kDa that share antigenic determinants with products of the recombinant clones were observed in Western blots of parasite extracts. Some of the native proteins were detected in the trypomastigote stage of the parasite, while others were present in epimastigotes as well. The latter result was confirmed for some recombinant clones by hybridization of the cloned DNA with Northern blots of parasite RNA. Clones from each group reacted differently with nine sera from rabbits infected with several T. cruzi strains as well as with eight sera from human patients. Clone 7 was detected by all rabbit sera but not by three human sera. Conversely, clones 1, 2 and 30 were detected by all human sera but failed to be detected by most rabbit sera. We conclude that several proteins from T. cruzi are antigenically active during infection and that some of them differ in their ability to generate antibodies in rabbit or human infections.     

Transformation of Trypomastigote Forms of Trypanosoma cruzi into Activators of Alternative Complement Pathway by Immune IgG Fragments

In this report we examined the capacity of immune IgG fragments to prepare trypomastigote bloodstream forms (TBF) of Trypanosoma cruzi for lysis. F(ab')₂, fragments were capable of presensitizingTBF For complement (C) lysis, thus excluding the participation of Fc domains in the C activation process. An intact hinge region of the IgG molecule was not involved either, since the corresponding Fab' were almost as active as the original molecules in preparing TBF for lysis. Fab also retained such activity even after further reduction and alkylation. These findings indicate that neither the portions of heavy chains that make up the hinge region nor the intrachain disulphide bonds are involved in the process. The IgG fragments promoted lysis through the activation of the alternative C pathway (ACP). These results suggest that the immune IgG transforms TBF into ACP activators by blocking the capacity of some parasite cell surface components that are known inhibitors of C activation.     

Antigenic polymorphism of Trypanosoma cruzi: clonal analysis of trypomastigote surface antigens

The surface membrane antigens of infectious Trypanosoma cruzi trypomastigotes were studied at the levels of the strain and of individual trypomastigote clones. Blood trypomastigotes from three T. cruzi strains, Y, CL and Tehuantepec (“Teh”), were grown in vitro by weekly infection of J774 mouse macrophage tumor cells. Each T. cruzi strain was subsequently cloned by infection of J774 cells at limited trypomastigote dilution, and antisera were produced in mice against a selection of trypomastigote clones. Criss-cross panel analyses indicated the existence of a large degree of polymorphism among trypomastigote surface antigens. Various trypomastigote surface antigens were cross-reactive, appeared to be highly conserved, and were common to the three strains considered and to all the clones derived from each strain. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that numerous trypomastigote antigenic proteins were precipitated by mouse antisera generated against cloned trypomastigotes. Some of these proteins were commonly distributed, while others were polymorphic. Finally, a state of cross-reactive immunity could be induced in C3H/He mice by infection with a cloned T. cruzi trypomastigote population. Immune mice resisted subsequent infections with lethal doses of wild-type bloodstream trypomastigotes from any one of the three T. cruzi strains.     

Mapping of a B-cell epitope present in the neuraminidase of Trypanosoma cruzi.

We have previously shown that a polyclonal (rabbit anti-TCNA) and a mouse monoclonal antibody (TCN-2) against the neuraminidase of Trypanosoma cruzi (TCNA) inhibit enzyme activity, immunoprecipitate active enzyme, enhance in vitro infection, and identify a subpopulation of extracellular trypomastigotes. We now report on the identification of a synthetic peptide that contains the epitope recognized by these antibodies. The synthetic peptide (TR) is a dodecamer (D-S-S-A-H-G-T-P-S-T-P-A) deduced from the DNA sequence of the long tandem repeat (LTR) domain present in the TCNA carboxyterminus. By ELISA, rabbit anti-TCNA bound to TR coupled to ovalbumin, and the binding was inhibited by soluble TR but not by BR (Y-S-V-D-D-G-E-T-W-E), a peptide derived from the N-terminal domain of the enzyme. TCN-2 recognized TR, and this reaction as well as TCN-2 binding to endogenous TCNA could be inhibited by soluble TR but not by BR. These results indicate that the rabbit anti-TCNA and TCN-2 react with the LTR region of TCNA. Antibodies to TR reacted by immunoblot with the TCNA of the Silvio X-10/4, MV-13 and Y-H6 strains, identifying the same molecular polymorphism previously observed with the rabbit anti-TCNA and TCN-2. Furthermore, anti-TR antibodies immunoprecipitated active enzyme and immunofluorescence analysis revealed that anti-TR and TCN-2 antibodies detected equally well the differential expression of their epitopes in intra- and extracellular trypomastigotes. Moreover, expression of TR and TCN-2 epitopes on the different stages of T. cruzi paralleled the stage-specificity of TCNA activity. TCN-2 prevented desialylation by TCNA of intact cells but not of soluble glycoconjugates, indicating that TCN-2 epitope is probably not associated with the enzyme catalytic site, in agreement with the predicted sequence of the TCNA gene. Finally, analysis of the humoral response of a Chagasic patient to different areas of the TCNA molecule indicated that the antibody response is predominantly against TR suggesting that the tandem repeat is the immunodominant domain of TCNA.     

Multiple Overlapping Epitopes in the Repetitive Unit of the Shed Acute-Phase Antigen from Trypanosoma cruzi Enhance Its Immunogenic Properties

The repetitive shed acute-phase antigen (SAPA) from Trypanosoma cruzi was thoroughly mapped by SPOT peptides and phage display strategies, showing that a single SAPA repeat is composed of multiple overlapping B-cell epitopes. We propose that this intricate antigenic structure constitutes an alternative device to repetitiveness in order to improve its immunogenicity.     

Surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi.

The surface antigen makeup of metacyclic trypomastigote forms of strain G of Trypanosoma cruzi, which produce a subpatent infection in mice, differed from those of the virulent strains Y and CL. A 100,000-molecular-weight protein, barely detectable on the Y or CL cell surface, appeared as the main surface antigen of the G metacyclic trypomastigotes. In addition, the G metacyclic forms differed from those of the virulent strains in their susceptibility to complement-mediated immunolysis.     

Characterization of an Immunodominant Antigenic Epitope from Trypanosoma cruzi as a Biomarker of Chronic Chagas' Disease Pathology

Nowadays, the techniques available for chronic Chagas'' disease diagnosis are very sensitive; however, they do not allow discrimination of the patient''s clinical stages of the disease. The present paper describes that three out of the five different repeats contained in the Trypanosoma cruzi TcCA-2 membrane protein (3972-FGQAAAGDKPPP, 6303-FGQAAAGDKPAP, and 3973-FGQAAAGDKPSL) are recognized with high sensitivity (>90%) by sera from chronic Chagas'' disease patients and that they are not recognized by sera from patients in the acute phase of the disease. A total of 133 serum samples from chagasic patients and 50 serum samples from healthy donors were tested. In addition, sera from 15 patients with different autoimmune diseases, 43 serum samples from patients suffering an infectious disease other than Chagas'' disease, and 38 serum samples from patients with nonchagasic cardiac disorders were also included in this study. The residue 3973 peptide shows a specificity of >98%, as it is not recognized by individuals with autoimmune and inflammatory processes or by patients with a nonchagasic cardiomyopathy. Remarkably, the levels of antibody against the 3973 epitope detected by the sera from Chagas'' disease patients in the symptomatic chronic phase, involving cardiac or digestive alterations, are higher than those detected by the sera from Chagas'' disease patients in the indeterminate phase of the disease. It is suggested that the diagnostic technique described could also be used to indicate the degree of pathology. The amino acids F, Q, and DKP located in the peptide at positions 1, 3, and 8 to 10, respectively, are essential to conform to the immunodominant antigenic epitope.     

Mapping of B-cell epitopes in a Trypanosoma cruzi immunodominant antigen expressed in natural infections

Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules.     

Antigenic variation in Trypanosoma equiperdum.

Trypanosoma equiperdum is an African trypanosome that causes dourine in horses. Like the other African trypanosomes, T. equiperdum escapes elimination by the immune system of its host by using an elaborate system of antigenic variant. The trypanosomes are covered by a coat consisting of a single protein called the variable surface glycoprotein (VSG) that acts as the major trypanosome immunogen. As the host responds to one VSG, trypanosomes covered with another VSG become dominant. There is a loose order of appearance of these VSG during the infection. The factors that affect the timing of VSG expression and the effective size of the VSG repertoire in T. equiperdum are reviewed. The VSG genes are generally activated by a process of duplicative transposition involving the duplication of a silent VSG gene and inserting a copy of the gene into an expression site. The order of VSG expression is related to the amount of homology between the silent gene and the expression site. The genes expressed late in infection lack extensive homology with the expression site and depend on homology with the gene in the expression site. The genes coding for VSG expressed late in infection are hybrid genes because of this mode of transfer. This transfer mechanism allows the trypanosome to create complex VSG genes from parts of several different silent genes that are each pseudogenes. Additionally, data are presented showing that only a limited portion of the VSG is actually seen by the host immune system. These factors indicate that the effective VSG repertoire is greater than the number of VSG genes in the trypanosome genome.     

An Immunoaffinity-Purified Trypanosoma cruzi Antigen Suppresses Cellular Proliferation through a TGF-β-Mediated Mechanism

Two subfractions with opposite immunological properties were obtained from the flagellar antigens (FF) of Trypanosoma cruzi epimastigotes by immunoaffinity chromatography. The ligand-bound material (Ag 123) contained four polypeptide bands of 97, 55, 38 and 14 kDa. The nonretained flow-through (FT), induced a potent proliferation of murine naive splenocytes. In contrast, Ag 123 inhibited the proliferative capacity of the FT as well as the proliferation mediated by the mitogen Concanavalin A (Con A). The suppressive effect of Ag 123 on the Con A-mediated proliferation was neutralized by an anti-TGF-β monoclonal antibody. Both Ag 123 and FF stimulated high serum levels of TGF-β in injected mice. Ag 123 also induced in vitro secretion of TGF-β by murine splenocytes. These results demonstrate that Ag 123 is a potent stimulator of TGF-β both in vivo and in vitro . Oligopeptides derived from the 38 kDa protein present in Ag 123 showed homology with human and rat alpha-fetoproteins (AFP). Ag 123 seems to have a key role in the immunosuppression that develops during early stages in the infection with T. cruzi .     

Molecular and biochemical characterization of hexokinase from Trypanosoma cruzi

The Trypanosoma cruzi hexokinase gene has been cloned, sequenced, and expressed as an active enzyme in Escherichia coli. Sequence analysis revealed 67% identity with its counterpart in Trypanosoma brucei but low similarity with all other available hexokinase sequences including those of human. It contains an N-terminal peroxisome-targeting signal (PTS-2) and has a calculated basic isoelectric point (pI = 9.67), a feature often associated with glycosomal proteins. The polypeptide has a predicted mass of approximately 50 kDa similar to that of many non-vertebrate hexokinases and the vertebrate hexokinase isoenzyme IV. The natural enzyme was purified to homogeneity from T. cruzi epimastigotes and appeared to exist in several aggregation states, an apparent tetramer being the predominant form. Its kinetic properties were compared with those of the purified recombinant protein. Higher K(m) values for glucose and ATP were found for the (His)(6)-tag-containing recombinant hexokinase. However, removal of the tag produced an enzyme displaying similar values as the natural enzyme (K(m) for glucose = 43 and 60 microM for the natural and the recombinant protein, respectively). None of these enzymes presented activity with fructose. As reported previously for hexokinases from several trypanosomatids, no inhibition was exerted by glucose 6-phosphate (G6-P). In contrast, a mixed-type inhibition was observed with inorganic pyrophosphate (PPi, K(i) = 0.5mM).     

Characterization of a lysosomal serine carboxypeptidase from Trypanosoma cruzi

Trypanosoma cruzi, the flagellate protozoan which is the causative agent of the American trypanosomiasis, Chagas disease has carboxypeptidase activity. The enzyme has been purified to protein homogeneity, and shown to be a lysosomal monomeric glycoprotein with a molecular mass of about 54kDa. The enzyme has an optimum acidic pH (4.5 with furyl acryloyl-Phe-Phe as substrate), is highly specific for hydrophobic C-terminal amino acid residues, and is strongly inhibited by 3,4-dichloroisocoumarin (IC(50) value 0.3 microM). The enzyme is encoded by a number of genes arrayed in head-to-tail tandems; one of these genes has been cloned and sequenced. Sequence comparisons indicate that the enzyme belongs to the C group of serine carboxypeptidases, within the S10 serine peptidase family, and shows the higher similarity to plant and yeast enzymes. The residues involved in catalysis and most of those involved in substrate binding are conserved in the T. cruzi enzyme as well as 8 out of 10 Cys residues known to be involved in disulfide bridges in the yeast enzyme. This is the first report of an S10 family enzyme in trypanosomatids. The presence of serine carboxypeptidases is not restricted to T. cruzi, being possibly a general character of trypanosomatids.     

Purification and characterization of stage-specific glycoproteins from Trypanosoma cruzi

Four developmentally regulated glycoproteins were purified from detergent solubilized cell membranes of Trypanosoma cruzi. Three trypomastigote specific glycoproteins each migrated as single bands under denaturing conditions with approximate Mr of 90,000, 85,000, and 55,000 and pI values of 4.3-5.0, 8.5-9.1, and 8.2, respectively. The fourth, epimastigote specific, protein had an approximate Mr of 72,000 and a pI of 4.8-5.1. The Mr of all four glycoproteins changed by 5-50% upon endoglycosidase F treatment. The Mr 72,000 antigen was the only one that reacted strongly with anti-epimastigote sera raised in rabbits. Sera from a Chagasic patient reacted strongly with the three trypomastigote specific glycoproteins and very weakly with the Mr 72,000 glycoprotein.     

Structural characterization of neutral glycosphingolipids from Trypanosoma cruzi.

The major neutral glycosphingolipids from Trypanosoma cruzi ceramide mono- and dihexosides (CMH and CDH, respectively) were analysed after chromatographic purification using 1H 500 MHz nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The ceramide monohexoside fraction (CMH) contained both glucosyl- and galactosylceramides. After peracetylation, the CMH fraction was separated into 2 subfractions, CMH-COH and CMH-Cn, containing either hydroxy fatty acids or n-fatty acids. In the CMH-COH fraction glucose and galactose were present in a ratio of 2:1, whereas this ratio was 1:1 in the CMH-Cn fraction. The CDH fraction was identified as lactosylceramide with sphingosine as the long chain base and 16:0, 18:0, and 24:0, 24:4 fatty acids as major components.     

Cloning of a pyruvate phosphate dikinase from Trypanosoma cruzi

We have cloned and characterised a gene that encodes a putative pyruvate phosphate dikinase (PPDK) from Trypanosoma cruzi, an enzyme that catalyses the reversible conversion of phosphoenolpyruvate to pyruvate. PPDK is absent in mammalian cells, but has been found in a wide variety of other organisms, including plants and bacteria. In T. cruzi, two genes (PPDK1 and PPDK2) are present in a tandem array localised on a 1 Mbp chromosome. Northern and Western blot analyses indicates that PPDK is expressed as a 100-kDa protein in epimastigote, amastigote and trypomastigote forms. PPDK1 and PPDK2 encode an identical protein of 100.8 kDa with a C-terminal extension ending with the sequence AKL, a signal for glycosomal import. Both T. cruzi and T. brucei enzymes possess a 23-residue insertion, that is absent in other PPDKs. A three-dimensional alignment with the crystal structure of the enzyme from Clostridium symbiosum predicts that this insertion is located on the surface of the nucleotide-binding domain. Phylogenetic studies indicate that bacterial and protist PPDKs cluster as a separate group from those of plants. The evolutionary implications and possible role of this enzyme in T. cruzi is discussed.     

Subcellular localization of a cysteine proteinase from Trypanosoma cruzi

Epimastigotes of Trypanosoma cruzi, Tulahuén strain, Tul 2 stock, contain a cysteine proteinase able to degrade azocasein at pH 5. This enzyme activity was extracted from whole cells by digitonin concentrations higher than those required for cytosolic markers, lower than those required for glycosomal and mitochondrial markers, and very similar to those required for solubilization of the acidic alpha-mannosidase. Both, the azocasein-degrading proteinase and the alpha-mannosidase, showed similar latency and distribution in subcellular fractions (the large granule fraction was the most active), and the same behavior in isopycnic sucrose gradient centrifugation; a broad peak centered at an equilibrium density of about 1.15 g cm-3, with a shoulder between 1.07 and 1.10 g cm-3, was obtained for both enzymes. The results suggest that the cysteine proteinase activity is placed in the lysosomes.