Modeling the Trypanosoma cruzi Tc85-11 protein and mapping the laminin-binding site

Trypanosoma cruzi expresses a set of glycoproteins encoded by the gp85/trans-sialidase gene superfamily. In this report a structure model is proposed for a cloned member of the superfamily, the Tc85-11 protein. The structure consists of an N-terminus beta-propeller and a C-terminus beta-sandwich interconnected by an alpha-helix. The recombinant protein, corresponding to the N-domain (Tc85-N), binds to laminin in a selective manner. Six synthetic 20-mer peptides from the N-domain adhere onto the surface of LLC-MK(2) cells and two of these peptides specifically inhibit the Tc85-N/laminin interaction, indicating that they are the laminin-binding sites of the molecule. Thus, Tc85-11 and other related members of the family appear to be good candidates to play an important role in T. cruzi infection via a laminin mediated host-parasite interaction.     

Mapping of surface glycoproteins of Trypanosoma cruzi by two-dimensional electrophoresis. A correlation with the cell invasion capacity

The cell-surface iodinatable proteins of Trypanosoma cruzi have been analyzed by two-dimensional polyacrylamide gel electrophoresis under equilibrium conditions. Antigenic polypeptides were characterized after immunoprecipitation and glycoproteins were identified by means of lectin-affinity chromatography. Two glycoproteins, with affinity for concanavalin A, were found to be common to both infective (trypomastigote) and non-infective (epimastigote) forms: protein 1 (90 kDa, pI 5.5-6.5) and protein 2 (80 kDa, pI 5.3-6.3). In epimastigotes a specific concanavalin-A-binding surface glycoprotein (70 kDa, pI 5.5) was identified. Trypomastigote forms, on the other hand, presented several specific iodinatable surface components: glycoproteins 3(85 kDa, pI 5.5), 4 (85 kDa, pI 5.0), 6 (100 kDa, pI 6.5), 7 (120 kDa, pI 6.3), 8 (68 kDa, pI 6.7) and several minor high-molecular-mass acid proteins, all containing glucose and/or mannose, and glycoprotein 5 (85 kDa, pI 6.3-7.5), containing N-acetyl-D-glucosamine (Tc-85). Proteins 1, 2 and 5 were the only ones which gave clear evidence of charge heterogeneity. Most of the surface proteins of trypomastigote forms, the exception being proteins 3, 4 and 8, were removed by treatment with trypsin. This proteolytic treatment results in 90% inhibition of the in vitro vertebrate-cell-invasion capacity of the parasites. Upon reincubation in culture medium for 4 h, the trypsin-removed glycoproteins are again detected, an observation that correlates well with the recovery of the cell-penetration capacity observed in the same period.     

Protein kinase A catalytic subunit interacts and phosphorylates members of trans-sialidase super-family in Trypanosoma cruzi

Protein kinase A (PKA) has been suggested as a regulator of stage differentiation in Trypanosoma cruzi. Using a yeast two-hybrid system we have begun to characterize the downstream substrates of T. cruzi PKA. We identified several members of the trans-sialidase super family by this approach. Immunoprecitation demonstrated that a TcPKAc monoclonal antibody was able to pull-down proteins recognized by trans-sialidase antibodies as well as a SA85-1.1 antibody and vice versa. An in vitro phosphorylation assay demonstrated that PKA phosphorylated the recombinant protein of an active trans-sialidase. In addition, a phospho-(Ser/Thr) PKA substrate antibody detected bands on immunoblot analysis of trans-sialidase antibody precipitated proteins from parasite lysate and the media of L₆E₉ myoblasts infected with trypomastigotes as well as from a SA85-1.1 antibody precipitated proteins from parasite lysate. Immunofluorescence analysis suggested that some TcPKAc localizes to the plasma membrane surface of trypomastigotes. The identified trans-sialidases have PKA consensus phosphorylation sites located near the endoplasmic reticulum retention motif in the N-terminal. These data support that PKA phosphorylates trans-sialidase super family members in vivo.     

Only some members of a gene family in Trypanosoma cruzi encode proteins that express both trans-sialidase and neuraminidase activities.

Trypomastigotes, the blood stage form of the human parasite Trypanosoma cruzi, contain an enzyme on their surface, trans-sialidase, which catalyses the transfer of sialic acid from host glycoconjugates to acceptors on its own cell surface. At least a subset of the sialic acid-bearing acceptor molecules are involved in parasite invasion of host cells, an essential step in the life cycle of the parasite. Another trypomastigote surface enzyme that affects host cell invasion is neuraminidase and recent evidence suggests that both trans-sialidase and neuraminidase activities may be expressed by the same proteins on the parasite surface. We describe here the isolation and expression of several members of a trans-sialidase--neuraminidase gene family from T.cruzi. One of the isolated genes does indeed encode a protein with both trans-sialidase and neuraminidase activities, while other members of the gene family encode closely related proteins that express neither enzymatic activity. Chimeric protein constructs combining different portions of active and inactive genes identified a region of the gene necessary for enzymatic activity. Sequence analysis of this portion of the gene revealed a limited number of amino acid differences between the predicted active and inactive gene products.     

Expression of fasciclin I and II glycoproteins on subsets of axon pathways during neuronal development in the grasshopper

The "labeled pathways" hypothesis predicts that axon fascicles in the embryonic neuropil are differentially labeled by surface recognition molecules used for growth cone guidance. To identify candidates for such recognition molecules, we generated monoclonal antibodies (MAbs) that recognize surface antigens expressed on subsets of axon fascicles in the grasshopper embryo. The 3B11 and 8C6 MAbs immunoprecipitate 70- and 95-kd membrane glycoproteins called fasciclin I and II, respectively, which are expressed on different subsets of axon fascicles during development. These two glycoproteins are expressed regionally on particular portions of embryonic axons in correlation with their patterns of fasciculation, dynamically during the period of axon outgrowth in a manner consistent with a role in growth cone guidance, and at other times and places during embryogenesis, suggesting multiple developmental roles.     

Prominent 85-kDa oligomannosidic glycoproteins of rat brain are signal regulatory proteins and include the SHP substrate-1 for tyrosine phosphatases

The glycoprotein component in rat brain reacting most strongly with Galanthus nivalis agglutinin (GNA) on western blots migrates as an 85-kDa band. GNA identifies mannose-rich oligosaccharides because it is highly specific for terminal alpha-mannose residues. After purification of this 85-kDa glycoprotein band by chromatography on GNA-agarose and preparative gel electrophoresis, binding of other lectins demonstrated the presence of fucose and a trace of galactose, but no sialic acid. Treatment with N-Glycanase or endoglycosidase H produced a 65-kDa band, indicating that it consisted of about one-fourth N-linked oligomannosidic carbohydrate moieties. High-performance anion-exchange chromatography and fluorescence-assisted carbohydrate electrophoresis indicated that the major carbohydrate moiety is a heptasaccharide with the structure Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4Glc-NAcbeta1-4GlcNAc (Man5GlcNAc2). Determination of amino acid sequences of peptides produced by endoproteinase digestion demonstrated that this 85-kDa mannose-rich glycoprotein component contained the SHP substrate-1 for phosphotyrosine phosphatases and at least one other member of the signal-regulatory protein (SIRP) family. The unusually high content of oligomannosidic carbohydrate moieties on these receptor-like members of the immunoglobulin superfamily in neural tissue could be of functional significance for intercellular adhesion or signaling.     

Metabolic fate of cell surface glycoproteins during immunoglobulin-induced internalization

Goat antibodies directed against a subset of the externally oriented plasma membrane glycoproteins of hepatoma tissue culture (HTC) cells were used to follow the metabolic fate of the membrane antigens and the specifically bound immunoglobulin molecules in this cell type in cultures. Analyses of the immunoprecipitates from cells labeled in situ with neuraminidase and galactose oxidase, followed by reduction with tritiated sodium borohydride, indicate that about 40% of the galactose-labeled plasma membrane glycoproteins are recognized by the antiserum. Fluorescent microscopic analyses of cells treated with fluorescein-conjugated immunoglobulins and analyses of trypsin accessibility indicate that probably all of the antibodies bound to the cell surface are patched and internalized within about 4 hr when the cells are subsequently cultured at 37 degrees C in the presence of rabbit anti-goat immunoglobulins. At the same time, the antigens are also interiorized. Analyses of the cellular localization of the interiorized antigens and antibodies by cell fractionation on Percoll gradients show that the immunoglobulins to the cell surface antigens and the antigens themselves migrate to the same region of the Percoll gradient as lysosomal hydrolases. Although the antibodies bind to the cell surface glycoproteins and bring about patching and interiorization, there is no effect on the degradation of the plasma membrane antigens labeled via the galactose oxidase/borohydride reduction method. Furthermore, the iodinated antibodies directed against these membrane glycoproteins behave in their turnover properties like membrane antigens; the cell-bound specific immunoglobulins have the same half-life as the membrane glycoproteins. When the cells that had been reacted with the goat antibodies to membrane glycoprotein were cultured in the presence of rabbit anti-goat immunoglobulins, degradation of the former antibodies was effectively decreased. Similar results were obtained with concanavalin A and antibodies directed against this plant lectin.     

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

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

Two distinct TL-like molecular subsets defined by monoclonal antibodies on the surface of human thymocytes with different expression on leukemia lines

Monoclonal antibodies reacting with TL-like class I antigens expressed on the surface of human thymocytes and some T leukemia lines were found to define three independent epitopic clusters, two of which could be shown to reside on serologically distinct molecular subsets by a solid-phase radioimmunoassay as well as by sequential immunoprecipitation. Both molecular subsets consist of a 49-K heavy chain associated with a beta-2 microglobulin light chain. Thymocytes expressed similar amounts of the two molecular subsets, while on T leukemia lines the amount of these two molecular subsets varied from line to line.     

The SRS superfamily of Toxoplasma surface proteins

The surface of the protozoan parasite Toxoplasma gondii is coated with developmentally expressed, glycosylphosphatidylinositol-linked proteins structurally related to the highly immunogenic surface antigen SAG1. Collectively, these surface antigens are known as the SRS (SAG1-related sequences) superfamily of proteins. SRS proteins are thought to mediate attachment to host cells and activate host immunity to regulate the parasite's virulence. To better understand the number, evolution and developmental expression of SRS genes, this study has bioinformatically identified 161 unique SRS DNA sequences present in the T. gondii type II Me49 genome. The SRS superfamily of sequences phylogenetically bifurcates into two subfamilies, the prototypic members being SAG1 and SAG2A, respectively. Paralogous SRS sequences are 24-99% identical, are tandemly arrayed throughout the genome, and are present on most, if not all, chromosomes. All 11 SRS sequences on chromosomes Ia and Ib are clustered at sub-telomeric expression sites. Messenger RNA expression in the majority of SRS sequences for which multiple Expressed Sequence Tags exist is developmentally regulated. A consensus nucleotide sequence surrounding both the splice acceptor and donor sites was identified in those SRS sequences possessing an intron. Genotypic differences among SRS sequences are present at several loci (e.g. the absence of SAG5B, the truncation of SAG2D in Me49 compared with RH) indicating that different genotypes possess distinct sets of SRS sequences. Orthologous genes are restricted to tissue-dwelling coccidia (Neospora, Sarcocystis) with no related sequences present in other more distant apicomplexa such as Eimeria, Cryptosporidia, and Plasmodium spp.     

Macrophage surface glycoproteins binding to galectin-3 (Mac-2-antigen)

Galectin-3 (formerly called Mac-2 antigen) is a ∼30 kDa carbohydrate-binding protein expressed on the surface of inflammatory macrophages and several macrophage cell lines. We have purified from lysates of the murine macrophage cell line WEHI-3 glycoproteins that bind to a galectin-3 affinity column. Several of these receptors are labelled after biotinylation of intact cells showing their location at the cell surface. N-terminal aminoacid sequencing of intact galectin-3-binding glycoproteins isolated from preparative SDS-gels or of chemically derived fragments showed several homologies with known proteins and identification was confirmed by immunoprecipitation with specific antibodies. The glycoproteins were shown to be: the α-subunit(CD11b) of the CD11b/CD18 integrin(Mac-1 antigen); the lysosomal membrane glycoproteins LAMPs 1 and 2 which are known in part to be expressed at cell surfaces; the Mac-3 antigen, a mouse macrophage differentiation antigen defined by the M3/84 monoclonal antibody and related immunochemically to LAMP-2; the heavy chain of CD98, a 125 kDa heterodimeric glycoprotein identified by the 4F2/RL388 monoclonal antibodies respectively on human and mouse monocytes/macrophages and on activated T cells. Further studies showed that CD11b/CD18, CD98 and Mac-3 are major surface receptors for galectin-3 on murine peritoneal macrophages elicited by thioglycollate. Abbreviations: PBS, phosphate buffered saline; CNBR, cyanogen bromide; PMSF, phenyl methyl sulphonyl fluoride This revised version was published online in November 2006 with corrections to the Cover Date.     

The surface protein superfamily of Trypanosoma cruzi stimulates a polarized Th1 response that becomes anergic

Trypanosoma cruzi is an obligate intracellular parasite that chronically infects mammals. Extracellular mammalian stage trypomastigotes simultaneously express and release multiple members of the parasite's surface protein superfamily; these extracellular proteins should stimulate MHC class II-restricted CD4 T cells. The surface protein superfamily, however, encodes variant epitopes that may inhibit this CD4 response. In this report the surface protein-specific CD4 response was investigated. CD4 cells isolated from acutely and chronically infected mice did not proliferate when stimulated with surface proteins. Adoptive transfer of surface protein-specific CD4 clones or immunization with a peptide encoding a surface protein T cell epitope protected mice during T. cruzi infection. These data strongly suggested that surface proteins were expressed and presented to CD4 cells during infection. Limiting dilution analysis identified an expanded population of surface protein-specific CD4 cells during the acute and chronic infection. These surface protein-specific CD4 cells did not produce IL-2 or IL-4, but did produce IFN-gamma. Enzyme-linked immunospot analyses confirmed that many of the surface protein-specific CD4 cells produce IFN-gamma. Together these results suggest that during T. cruzi infection a potentially protective CD4 response becomes anergic. It is possible that this anergy is induced by variant T cell epitopes encoded by the surface protein superfamily.     

Limited and selective localization of the lysosomal membrane glycoproteins LGP85 and LGP96 in rat osteoclasts

 Monospecific antibodies against two major glycoproteins of rat lysosomal membranes with apparent molecular masses of 96 and 85 kDa, termed LGP96 and LGP85, respectively, were used as probes to determine the expression and distribution of lysosomal membranes in rat osteoclasts. At the light microscopic level, the preferential immunoreactivity for both proteins was found at high levels at the side facing bone of actively bone-resorbing osteoclasts. Osteoclasts detached from bone surface were devoid of immunoreactivity for each protein. At the electron microscopic level, both proteins were exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts with well-developed ruffled border membrane. No immunolabeling for both proteins was observed in the basolateral membrane and the clear zone of bone-resorbing osteoclasts. The plasma membrane of preosteoclasts and post- and/or resting osteoclasts showed little or no reactivity against these two antibodies. The results indicate that lysosomal membrane glycoproteins are actively synthesized in active osteoclasts, rapidly transported to the ruffled border area, and contribute to the formation and maintenance of the acidic resorption lacuna of osteoclasts. Accepted: 9 December 1998     

Trypanosoma cruzi: differential expression and distribution of an 85-kDa polypeptide epitope by in vitro developmental stages.

The expression by Trypanosoma cruzi developmental stages of an 85-kDa polypeptide epitope defined by the 155D3 monoclonal antibody (mAb) has been investigated. Immunoprecipitation revealed the presence of an 85-kDa antigen in the NP-40 soluble extract of parasites freshly released from infected fibroblasts; this antigen was not found in epimastigote and Leishmania infantum promastigote. Indirect immunofluorescence revealed that the mAb 155D3 failed to react with trypomastigotes, whereas extracellular amastigotes were heavily stained. Positive organisms displayed either surface or polar fluorescence. Since the same mAb immunoprecipitated the 85-kDa antigen in both radioactive iodine- and methionine-labeled trypomastigote detergent soluble extracts, the reactive epitope is likely to be hidden in a cryptic site in trypomastigotes. An alternative explanation for the negative immunofluorescence on trypomastigotes and the positive immunoprecipitation is the presence, in the extracts, of a small population of parasites already expressing the 155D3 epitope. Immunoelectron microscopy revealed that the target epitope is heterogenously distributed among the populations of differentiating parasites. Two types of immunogold labeling were observed: (a) mAb revealed a high amount of reactive material associated with the periphery of the parasites and (b) a label was observed on the inner surface of peripheral vacuoles that might correspond to cross sections of inflated flagellar pockets and in association with vesicles which were released by the parasites. The surface expression of the epitope recognized by the 155D3 mAb was followed by fluorescence-activated cell-sorting analysis. The results showed that the epitope is increasingly accessible during trypomastigote differentiation in vitro. Taken together, these results suggest that the epitope reacting with the 155D3 mAb is heavily expressed on extracellular amastigotes after the transformation process and, thus, appears to be developmentally regulated.     

Extension of neurites on axons is impaired by antibodies against specific neural cell surface glycoproteins

We have developed an in vitro assay which measures the ability of growth cones to extend on an axonal substrate. Neurite lengths were compared in the presence or absence of monovalent antibodies against specific neural cell surface glycoproteins. Fab fragments of antibodies against the neural cell adhesion molecule, NCAM, have an insignificant effect on the lengths of neurites elongating on either an axonal substrate or a laminin substrate. Fab fragments of polyclonal antibodies against two new neural cell surface antigens, defined by mAb G4 and mAb F11, decrease the lengths of neurites elongating on an axonal substrate, but have no effect on the lengths of neurites elongating on a laminin substrate. G4 antigen is related to mouse L1, while F11 antigen appears to be distinct from all known neural cell surface glycoproteins. Our results suggest that the G4 and F11 antigens help to promote the extension of growth cones on axons.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 2: Unique carbohydrate antigens are topographic markers for selective projection patterns of olfactory axons.

Three monoclonal antibodies specific for different carbohydrate antigens were used to analyze the development of the olfactory system in rats. CC2 antibodies react with a subset of main olfactory neurons, their axons, and terminals in the olfactory bulb. CC2 antigens are expressed on dorsomedial neurons in the olfactory epithelium (OE) from embryonic (E) day 15 to adults. In the olfactory bulb (OB), only dorsomedially located glomeruli express CC2 glycoconjugates from postnatal day (P) 2 to adults. Thus CC2 defines a dorsomedially organized projection that is established early in embryonic development and continues in adults. P-Path antibodies react with antigens that are expressed on the olfactory nerve in embryos, and are also detected on cell bodies in the neuroepithelium and in glomeruli of the OB at P2. At P14, P-Path staining is weaker, but remains present on many cells in the epithelium and in many glomeruli in the bulb. Postnatally, P-Path immunostaining continues to decrease in most regions of the OE and OB. At P35 and afterwards, only a few P-Path-positive neuronal cells can be detected in the OE. Furthermore, after P35 only two groups of glomeruli in the OB are P-Path immunoreactive. One is situated adjacent to the accessory olfactory bulb (AOB) at the dorsocaudal surface of the OB. The other is adjacent to the AOB at the ventrocaudal surface of the OB. Thus, in adults, P-Path glycoconjugates are expressed in neurons and axons that project only to a specific subset of caudal glomeruli of the OB. Monoclonal antibody 1B2, reacts with beta-galactose-terminating glycolipids and glycoproteins. At P2, 1B2 immunoreactivity is seen on a subset of cell bodies that are distributed throughout the OE and is expressed in most glomeruli in the OB at this age. By P35 and in adults, 1B2 continues to be expressed on a subset of neurons in the OE that project to only a small subset of glomeruli in the OB. Unlike CC2 and P-Path antigens that define specific groups of glomeruli, 1B2-immunoreactive glomeruli do not have a detectable spatial pattern. It is more likely that 1B2 antigens define a specific stage in the maturation of connections between the OE and OB.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 2: Unique carbohydrate antigens are topographic markers for selective projection patterns of olfactory axons

Three monoclonal antibodies specific for different carbohydrate antigens were used to analyze the development of the olfactory system in rats. CC2 antibodies react with a subset of main olfactory neurons, their axons, and terminals in the olfactory bulb. CC2 antigens are expressed on dorsomedial neurons in the olfactory epithelium (OE) from embryonic (E) day 15 to adults. In the olfactory bulb (OB), only dorsomedially located glomeruli express CC2 glycoconjugates from postnatal day (P) 2 to adults. Thus CC2 defines a dorsomedially organized projection that is established early in embryonic development and continues in adults. P-Path antibodies react with antigens that are expressed on the olfactory nerve in embryos, and are also detected on cell bodies in the neuroepithelium and in glomeruli of the OB at P2. At P14, P-Path staining is weaker, but remains present on many cells in the epithelium and in many glomeruli in the bulb. Postnatally, P-Path immunostaining continues to decrease in most regions of the OE and OB. At P35 and afterwards, only a few P-Path-positive neuronal cells can be detected in the OE. Furthermore, after P35 only two groups of glomeruli in the OB are P-Path immunoreactive. One is situated adjacent to the accessory olfactory bulb (AOB) at the dorsocaudal surface of the OB. The other is adjacent to the AOB at the ventrocaudal surface of the OB. Thus, in adults, P-Path glycoconjugates are expressed in neurons and axons that project only to a specific subset of caudal glomeruli of the OB. Monoclonal antibody 1B2, reacts with β-galactose-terminating glycolipids and glycoproteins. At P2, 1B2 immunoreactivity is seen on a subset of cell bodies that are distributed throughout the OE and is expressed in most glomeruli in the OB at this age. By P35 and in adults, 1B2 continues to be expressed on a subset of neurons in the OE that project to only a small subset of glomeruli in the OB. Unlike CC2 and P-Path antigens that define specific groups of glomeruli, 1B2-immunoreactive glomeruli do not have a detectable spatial pattern. It is more likely that 1B2 antigens define a specific stage in the maturation of connections between the OE and OB.     

Selective cleavage of variant surface glycoproteins from Trypanosoma brucei.

Two conformationally distinct regions were revealed by tryptic cleavage of six undenatured variant surface glycoproteins purified from clones of Trypanosoma brucei. Within 5 min, the native glycoproteins (65,000 mol.wt.) were cleaved, yielding a large N-terminal fragment (48,000-55,000 mol.wt. depending on the variant) together with one or more C-terminal fragments. After 30-60 min incubation, further breakdown of the large fragment occurred in some variants. The ultimate large product (40,000-52,000 mol.wt.) was very resistant to further degradation by trypsin (in the absence of denaturation). The distinction between N-terminal and C-terminal domains may be significant in relation to the organization and function of these glycoproteins on the trypanosome surface.