Mucin-type O-glycosylation in helminth parasites from major taxonomic groups: evidence for widespread distribution of the Tn antigen (GalNAc-Ser/Thr) and identification of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity

This article focuses on the initiation pathway of mucin-type O-glycosylation in helminth parasites. The presence of the GalNAc-O-Ser/Thr structure, also known as Tn antigen, a truncated determinant related to aberrant glycosylation in mammal cells, and the activity of the UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferase (ppGaNTase), the enzyme responsible for its synthesis, were studied in species from major taxonomic groups. Tn reactivity was determined in extracts from Taenia hydatigena, Mesocestoides corti, Fasciola hepatica, Nippostrongylus brasiliensis, and Toxocara canis using the monoclonal antibody 83D4. The Tn determinant was revealed in all preparations, and multiple patterns of Tn-bearing glycoproteins were observed by immunoblotting. Additionally, the first evidence that helminth parasites express ppGaNTase activity was obtained. This enzyme was studied in extracts from Echinococcus granulosus, F. hepatica, and T. canis by measuring the incorporation of UDP-(3H)GalNAc to both deglycosylated ovine syalomucin (dOSM) and synthetic peptide sequences derived from tandem repeats of human mucins. Whereas significant levels of ppGaNTase activity were detected in all the extracts when dOSM was used as a multisite acceptor, it was only observed in F. hepatica and E. granulosus extracts when mucin-derived peptides were used, suggesting that T. canis ppGaNTase enzyme(s) may represent a member of the gene family with a more restricted specificity for worm O-glycosylation motifs. The widespread expression of Tn antigen, capable of evoking both humoral and cellular immunity, strongly suggests that simple mucin-type O-glycosylation does not constitute an aberrant phenomenon in helminth parasites.     

Mucin-type O-glycosylation in Fasciola hepatica: characterisation of carcinoma-associated Tn and sialyl-Tn antigens and evaluation of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity

Simple mucin-type cancer-associated O-glycan structures, such as the Tn antigen (GalNAc-O-Ser/Thr), are expressed by certain helminth parasites. These antigens are involved in several types of receptor-ligand interactions, and they are potential targets for immunotherapy. The aim of this work was to study the initiation pathway of mucin-type O-glycosylation in Fasciola hepatica, performing a biochemical and immunohistochemical characterisation of Tn and sialyl-Tn antigens, and evaluating the ppGaNTase activity, which catalyses the first step in O-glycan biosynthesis. Using ELISA, both Tn and sialyl-Tn antigens were detected predominantly in the somatic and deoxycholate extracts. Immunofluorescence analysis revealed that Tn antigen is preferentially expressed in testis, while sialyl-Tn glycoproteins were more widely distributed, being present in parenchymal cells, basal membrane of the tegument, and apical surface of epithelial cells lining the caeca. On the basis of their electrophoretic mobility, Tn glycoproteins were resolved as six components of 10, 37, 76, 125, 170 and 205 kDa, and sialyl-Tn components showed an apparent molecular mass of 28 and 32 kDa, and two broad bands of 90-110 and 170-190 kDa. The observation that only the 76 kDa Tn-glycoprotein remained in the 0.6 N perchloric acid-soluble fraction suggests that it could be a good candidate for mucin characterisation in this parasite. The ppGaNTase activity showed its maximal activity at pH 7-7.5 and 37 degrees C, showing that Mn(2+) was the best divalent cation activator. Using a panel of nine synthetic peptides as acceptor substrates, we found that F. hepatica ppGaNTase was able to glycosylate both threonines and serines, the best substrates being the peptides derived from the tandem repeat region of human mucins (MUC2 and MUC6), and from Trypanosoma cruzi and Trypanosoma brucei glycoproteins. The results reported here constitute the first evidence on O-glycosylation pathways in F. hepatica, and may help to identify new biological characteristics of this parasite as well as of the host-parasite relationship.     

The IgE and IgG subclass responses of mice to four helminth parasites

To investigate whether the formation of IgE is linked in vivo to an IgG subclass, mice were infected with four helminth parasites, Nippostrongylus brasiliensis (Nbr), Mesocestoides corti, Taenia crassiceps and Trichinella spiralis, and the changes in the serum levels of the different Ig isotypes as well as the antibody response to M. corti and T. crassiceps antigen extracts were determined by radioimmunoassays. All four parasites induced a concomitant increase of the IgE and IgG1 serum levels and usually a decrease of the IgG2a level. They also induced an increase of the IgM level but had little effect on the IgG2b, IgG3, and IgA serum levels. The specific antibodies to an M. corti antigen extract were mainly of the IgG1 subclass, whereas it was of both IgG1 and IgG2a subclasses to T. crassiceps. Injections of dead M. corti induced an increase of all IgG subclasses and similar levels of IgG1 and IgG2a anti-parasite antibodies. Subcutaneous instead of intraperitoneal infection with T. crassiceps induced higher IgG2a than IgG1 levels and 10-fold lower IgE levels than the natural ip infection; however, despite the greater IgG2a polyclonal response, anti-parasite antibodies were predominantly of the IgG1 subclass. The data demonstrate that natural infection with four different helminth parasites induces a concomitant polyclonal IgG1 and IgE response. These in vivo observations corroborate the recent in vitro findings demonstrating that interleukin-4 induces lipopolysaccharide-activated murine B cells to secrete both IgG1 and IgE, suggesting that the regulation of these two isotypes is linked.     

O-methylated glycans from Toxocara are specific targets for antibody binding in human and animal infections

The parasitic helminth Toxocara canis is a widely distributed nematode of mammals. Larval parasites, which infect a wide range of hosts including mice and humans, export glycosylated macromolecules bearing novel methylated oligosaccharide structures, similar to the mammalian blood group antigen H but bearing one or two O-methylated substitutions on the terminal fucose and subterminal galactose residues. We have studied the reactivity of synthetic forms of these glycans to parasite-specific antibodies and mammalian immune system lectins. Murine antibodies, generated to T. canis infection, predominantly recognise the mono-O-methylated form with the beta-configuration of the GalNAc residue (MoMbeta), and antibodies are entirely IgM isotype. The mAb Tcn-2 reproduces this pattern, and shows little reactivity to either the alpha isomer (MoMalpha) or the di-O-methylated form (DiM). Antibodies generated to helminth infections other than T. canis were unreactive with the glycans, except antibodies to other members of the Toxocara genus. Hence, the carbohydrate structures represent immunogenic, genus-specific antigens. Antibodies from human toxocariasis patients are reactive with the same sugars, although preferentially towards DiM. Sera from unrelated helminth infections do not react, confirming the status of these structures as Toxocara-specific glycans. The human dendritic cell lectin, DC-SIGN, was found to bind both Toxocara excretory/secretory products and mammalian blood group antigen H3. However, DC-SIGN did not bind the synthetic glycans, indicating additional non-methylated carbohydrates may also play a role in the interaction between T. canis and its host.     

The 10 kDa protein of Taenia solium metacestodes shows genus specific antigenicity

Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species.     

Expression of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 in gastric mucosa, intestinal metaplasia, and gastric carcinoma

Aberrant mucin O-glycosylation is often observed in cancer and is characterized by the expression of immature simple mucin-type carbohydrate antigens. UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 (ppGalNAc-T6) is one of the enzymes responsible for the initial step in O-glycosylation. This study evaluated the expression of ppGalNAc-T6 in human gastric mucosa, intestinal metaplasia, and gastric carcinomas. Our results showed that ppGalNAc-T6 is expressed in normal gastric mucosa and in intestinal metaplasia. A heterogeneous expression and staining pattern for this enzyme was observed in gastric carcinomas. ppGalNAc-T6 was expressed in 79% of the cases, and its expression level was associated with the presence of venous invasion. Our results provide evidence that ppGalNAc-T6 is an IHC marker associated with venous invasion in gastric carcinoma and may contribute to the understanding of the molecular mechanisms that underlie aberrant glycosylation in gastric carcinogenesis and in gastric carcinoma.     

Sialyl-Tn antigen expression and O-linked GalNAc-Thr synthesis by Trypanosoma cruzi

Most Trypanosoma cruzi O-glycans are linked to Thr/Ser residues via N-acetylglucosamine. We report that the mucin-type carcinoma-associated sialyl-Tn antigen (NeuAc-GalNAc-O-Ser/Thr) is expressed by T. cruzi. A specific MAb allowed us to localize the antigen on the surface of epimastigotes and to identify reactive components in parasite lysates (32, 60, and 94kDa). In addition, ppGalNAc-T activity was characterized in epimastigotes, and direct evidence was obtained for the in vitro incorporation of GalNAc to a synthetic peptide derived from a T. cruzi mucin. These results add an as yet unknown complexity to the pathways of O-glycan biosynthesis in this protozoan parasite.     

Taenia solium and Taenia saginata: identification of sequence characterized amplified region (SCAR) markers

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies.     

Facile preparation of the alpha-Gal-recognizing Griffonia simplicifolia I-B4 isolectin

The B4 isolectin from Griffonia simplicifolia is of great utility as a reagent for the identification of alpha-D-galactopyranosyl end groups. Its separation from isolectins containing A subunits has been greatly improved by a simple, rapid procedure using a column of N-acetylgalactosamine coupled to vinyl sulfone-activated Sepharose 4B to selectively retain the A subunit-containing isolectins. The procedure has the advantages over previous affinity procedures of speed (the isolation of B4 isolectin can be achieved in one day), simplicity, and high degree of resolution of the B4 isolectin.     

Altered T helper responses in CD40 and interleukin-12 deficient mice reveal a critical role for Th1 responses in eliminating the helminth parasite Taenia crassiceps

A key feature of helminth infections is the induction of strong Th2-biased immune responses in their hosts. We have previously found that Th2-like responses mediate susceptibility to the helminth parasite Taenia crassiceps, probably by inhibiting Th1 responses required for the development of protective immunity against this parasite. Here we show that mice lacking interleukin-12p35 (IL-12p35-/-) following T. crassiceps infection, failed to mount a Th1 response, but developed a strong Th2-type response, produced higher levels of IgG1, IgE, interleukin-4, interleukin-5 as well as interleukin-13 than wild-type mice, and became highly susceptible to the larval stage of this cestode. In contrast, similarly-infected CD40 deficient BALB/c mice (CD40-/-) displayed impairment of both Th1 and Th2-type responses associated with low levels of interferon-gamma as well as IgE, interleukin-4, interleukin-5 and interleukin-13, but efficiently controlled T. crassiceps infection. Together, these findings suggest a detrimental role for Th2-biased responses during the larval stage of T. crassiceps infection. Furthermore, they also suggest a pivotal role for CD40 in developing Th2-type responses.     

Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.     

Engineering neoglycoproteins with multiple O-glycans using repetitive pentapeptide glycosylation units

Controlled protein remodeling with O-linked glycans has been limited by our incomplete understanding of the process of glycosylation. Here we describe a secretable fibroblast growth factor (FGF) with multiple mucin-type O-glycans produced by introducing a minimum pentapeptide glycosylation unit in a decarepeat format at its N- or C-terminus. Expressed in Chinese hamster ovary cells, chemical and biochemical analyses of the resultant proteins (Nm10-FGF and Cm10-FGF, respectively) demonstrated that all O-glycosylation units were glycosylated and the dominant structure was sialylated Gal[1–3]GalNAc. This indicates that minimum O-glycosylation unit in multirepeat format serves as a remarkably efficient acceptor in CHO cells. The Nm10-FGF and Cm10-FGF proteins maintained the mitogenic activity to vascular endothelial cells. In addition, intact Cm10-FGF and its desialylated form interacted with several lectins in the same way as mucin-type glycoproteins. The intact Cm10-FGF with multiple sialylated O-glycans exhibited a longer lifetime in circulating blood, whereas the Cm10-FGF with desialylated O-glycans exhibited a shorter lifetime than the deglycosylated form of Cm10-FGF. Our approach would thus appear to be highly effective for engineering neoglycoproteins, the characteristics of which are determined by their multiple mucin-type O-glycans.     

Mucin-type O-glycosylation and its potential use in drug and vaccine development

Mucin-type O-glycans are found on mucins as well as many other glycoproteins. The initiation step in synthesis is catalyzed by a large family of polypeptide GalNAc-transferases attaching the first carbohydrate residue, GalNAc, to selected serine and threonine residues in proteins. During the last decade an increasing number of GalNAc-transferase isoforms have been cloned and their substrate-specificities partly characterized. These differences in substrate specificities have been exploited for in vitro site-directed O-glycosylation. In GlycoPEGylation™, polyehylene glycol (PEG) is transferred to recombinant therapeutics to specific acceptor sites directed by GalNAc-transferases. GalNAc-transferases have also been used to control density of glycosylation in the development of glycopeptide-based cancer vaccines. The membrane-associated mucin-1 (MUC1) has long been considered a target for immunotherapeutic and immunodiagnostic measures, since it is highly overexpressed and aberrantly O-glycosylated in most adenocarcinomas, including breast, ovarian, and pancreatic cancers. By using vaccines mimicking the glycosylation pattern of cancer-cells, it is possible to overcome tolerance in transgenic animals expressing the human MUC1 protein as a self-antigen providing important clues for an improved MUC1 vaccine design. The present review will highlight some of the potential applications of site-directed O-glycosylation.     

Metagonimus yokogawai: a 100-kDa Somatic Antigen Commonly Reacting with Other Trematodes

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients'' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.     

Extraintestinal Helminth Infection Reduces the Development of Colitis-Associated Tumorigenesis

Colitis-associated colorectal cancer (CAC) is one of the most common cancers and is closely related to chronic or deregulated inflammation. Helminthic infections can modulate inflammatory responses in some diseases, but their immunomodulatory role during cancer development remains completely unknown. We have analyzed the role of Taenia crassiceps-induced anti-inflammatory response in determining the outcome of CAC. We show that extraintestinal T. crassiceps infection in CAC mice inhibited colonic inflammatory responses and tumor formation and prevented goblet cell loss. There was also increased expression of IL-4 and alternatively activated macrophages markers in colonic tissue and negative immunomodulation of pro-inflammatory cytokine expression. In addition, T. crassiceps infection prevented the upregulation of β-catenin and CXCR2 expression observed in the CAC mice, which are both markers associated with CAC-tumorigenesis, and reduced the numbers of circulating and colonic CD11b⁺Ly6C^hiCCR2⁺ monocytes. Thus, immunomodulatory activities induced by helminth infections may have a role in the progression of CAC.     

In vitro testing of anthelmintic efficacy of Flemingia vestita (Fabaceae) on carbohydrate metabolism in Rallietina echinobothrida

The root tuber peel of Flemingia vestita has been in use in local traditional medicine against intestinal worm infections in Meghalaya (North-East India). In order to evaluate and authenticate the anthelminitc efficacy of the isoflavones of F. vestita, the root peel extract of this putative plant was tested against several helminth parasites, extensively on Rallietina echinobothrida, with respect to different parameters of these parasites. In this paper, we describe various methods to evaluate the anthelmintic efficacy of this medicinal plant with respect to carbohydrate metabolism in R. echinobothrida at paralytic time caused by the isoflavones of F. vestita. To meet the high energy demand by the parasite due to the anthelmintic stress, glucose breakdown follows the PEPCK-malate pathway in the parasite.     

A stimulating effect of guanyl nucleotides on the rat-liver soluble cyclic GMP high-affinity phosphodiesterase activity

The high affinity (low Km) cyclic GMP phosphodiesterase (PDE) is activated by GTP, while the cyclic AMP PDE is not. GTP and its hydrolysis-resistant analogue, guanylylimidodiphosphate (GppNHp), display a half-maximal stimulating effect at almost the same concentration (5 X 10(-6) M). The GTP stimulating effect is not observed when the socalled cyclic GMP low affinity (high Km) PDE is operative. GTP cooperates with the increase of the substrate concentration on removing the IBMX inhibitory effect. The isolation through a classical chromatographic procedure on a DEAE-cellulose column, of a PDE fraction specific for cyclic GMP, results in the loss of the GTP stimulating effect.     

State of the Art of Taenia solium as Compared to Taenia asiatica

Three species of tapeworms infect humans in their adult stage (Taenia solium, Taenia saginata and Taenia asiatica). The 3 are flat, opaque white or yellowish, and exceptional long segmented parasites, measuring 1 to 12 m in their adult stage. In this review, the development of the knowledge regarding the first species, mainly focused on understanding how the larval stage or cysticercus is transmitted to humans, is described. The second species is a cosmopolitan parasite that only causes taeniosis and not cysticercosis; therefore, it will not be included. Information on the third species, which is presently being produced, since this species was recognized as such only at the end of the 20th century, will be discussed at the end of this review.     

O-linked N,N'-diacetyllactosamine (LacdiNAc)-modified glycans in extracellular matrix glycoproteins are specifically phosphorylated at subterminal N-acetylglucosamine

The terminal modification of glycans by β4 addition of N-acetylgalactosamine to N-acetylglucosamine with formation of the N,N-diacetyllactosediamine (LacdiNAc) moiety has been well documented for a number of N-linked glycoproteins and peptides, like neurohormones. Much less is known about O-glycoproteins in this regard because only human zona pellucida glycoprotein 3 (ZP3) and bovine proopiomelanocortin were reported to be LacdiNAc-modified. In searching for mammalian proteins modified with O-linked LacdiNAc we identified six positive species among nine endogenous and recombinant O-glycoproteins, which were extracellular matrix, or matrix-related proteins. These are ZP3 and the five novel LacdiNAc-positive species ECM1, AMACO, nidogen-1, α-dystroglycan, and neurofascin. The mass spectrometric analyses revealed a core 2-based tetrasaccharide as the common structural basis of O-linked LacdiNAc that could be further modified, similar to the type 2 LacNAc termini, with fucose, sialic acid, or sulfate. Here, we provide structural evidence for a novel type of mucin-type O-glycans that is strictly specific for LacdiNAc termini: sugar phosphorylation with formation of GalNAcβ1-4(phospho-)GlcNAc. The structural details of the phosphatase-labile compound were elucidated by MS(2) analysis of tetralysine complexes and by MS(n) measurements of the permethylated glycan alditols. Phospho-LacdiNAc was detected in human HEK-293 as well as in mouse myoblast cells and in bovine brain tissue.     

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.