Entamoeba histolytica: deletion of the GPI anchor signal sequence on the Gal/GalNAc lectin light subunit prevents its assembly into the lectin heterodimer

Adherence and cytotoxicity of Entamoeba histolytica require the function of a heterodimeric galactose and N-acetylgalactosamine (Gal/GalNAc)-specific lectin. The lectin heavy subunit (Hgl) contains a carbohydrate recognition domain and mediates inside-out cell signaling via its cytoplasmic tail. The function of the lectin light subunit (Lgl) is unknown. The lectin has a unique mechanism of membrane association: Hgl is transmembrane but Lgl is glycosylphosphatidylinositol (GPI) anchored. The role of the GPI anchor signal sequence in heterodimer assembly was tested. Epitope-tagged Lgl with or without the GPI anchor addition signal was expressed in E. histolytica trophozoites. Tagged Lgl did not assemble with Hgl into a lectin heterodimer in the absence of the GPI addition signal. Consistent with previous results that only the Hgl subunit mediates adherence, the monomeric Lgl without the GPI anchor signal lacked Gal/GalNAc-binding activity.     

Entamoeba histolytica: sequence conservation of the Gal/GalNAc lectin from clinical isolates

The Gal/GalNAc lectin gene of Entamoeba histolytica is a major amebic virulence protein responsible for interaction with host tissues. We investigated sequence differences in the Gal/GalNAc lectin heavy subunit in three isolates from Bangladesh and one isolate from Georgia, each of which was determined to be genetically distinct by SREHP AluI digestion. Interestingly, we observed only slight genetic diversity in the lectin gene as compared with the HM1:IMSS laboratory strain, originally a clinical isolate from Mexico. Genetic conservation of the Gal/GalNAc lectin between isolates may reflect that the lectin is under strong functional selection or possibly, that E. histolytica is a clonal population. Sequence conservation of the lectin indicates that immune responses against it should be cross-protective.     

Binding of N-linked bovine fetuin glycopeptides to isolated rabbit hepatocytes: Gal/GalNAc hepatic lectin discrimination between Gal beta(1,4)GlcNAc and Gal beta(1,3)GlcNAc in a triantennary structure

Glycopeptides were isolated from bovine fetuin after digestion with Pronase, aminopeptidase M, and carboxypeptidase Y. The glycopeptides were derivatized with tert-butyloxycarbonyltyrosine and separated on the basis of peptide by using reverse-phase high-performance liquid chromatography. Using 400-MHz 1H NMR, the asialotriantennary oligosaccharides at each of the three N-linked glycosylation sites were found to be combinations of the following two structures in which the third branch is either Gal beta(1,4)GlcNAc or Gal beta(1,3)GlcNAc: (formula; see text) The asialotriantennary glycopeptides containing all beta(1,4)-lactosamine as the branches were designated Gal beta(1,4)GlcNAc-TRI while triantennary glycopeptides containing beta(1,3)-lactosamine as branch III were termed Gal beta(1,3)GlcNAc-TRI. The Gal beta(1,3)GlcNAc unit was localized predominantly to the branch III arm on the basis of a downfield shift (-0.027 ppm) in the H-1 and upfield shift (0.01 ppm) in the NAc methyl signals from the branch III GlcNAc resulting from Gal beta(1,3) instead of Gal beta(1,4) substitution. Revised assignments are proposed for the H-1's of Gal residues 6 (delta 4.464) and 8 (delta 4.471) [Vliegenthart, J. F. G., Dorland, L., & van Halbeek, H. (1983) Adv. Carbohydr. Chem. Biochem. 41, 209-373] in a Gal beta(1,4)GlcNAc-TRI. The proportion of Gal beta(1,3)GlcNAc-TRI glycopeptides from the Asn-Asp, Asn-Gly, and Asn-Cys sites was found to be 40%, 60%, and 20%, respectively. Analysis of the binding of these glycopeptides, containing from 20% to 60% Gal beta(1,3)GlcNAc as branch III, to rabbit hepatocytes revealed that the greater the proportion of Gal beta(1,3)GlcNAc, the lower the affinity of the mixture. The Kd for Gal beta(1,4)GlcNAc-TRI was found to be between 3.6 and 5.4 nM (P = 0.10) with a mean of 4.4 nM from binding data analyzed by using the LIGAND program [Munson, P. J., & Rodbard, D. (1980) Anal. Biochem. 107, 220-239] and computer simulations of the binding of two ligands as a mixture to one receptor site. The Kd of Gal beta(1,3)GlcNAc-TRI oligosaccharide, prepared by hydrazinolysis, was found to be 305 nM from inhibition studies.     

Use of monoclonal anti-light subunit antibodies to study the structure and function of theEntamoeba histolytica Gal/GalNAc adherence lectin

Adherence ofEntamoeba histolytiea trophozoites to host cells is medicated by a galactose (Gal) andN-acetylgalactosamine (GalNAc)-specific surface lectin. The lectin is a heterodimeric protein composed of heavy (170kDa) and light (35-31 kDa) subunits linked by disulfide bonds. Polyclonal and monoclonal antibodies (mAb) raised against a light subunit-glutathione-S-transferase fusion protein were used to probe its structure and function. Four light subunit-specific mAb were produced which recognized distinct epitopes on five different light subunit isoforms. Immunoblots with these mAb demonstrated co-migration of light and heavy subunits when nonreduced trophozoite proteins were analysed by SDS-PAGE, indicating that the subunits do not exist free of the heterodimer in significant quantities. While anti-heavy subunit antibodies had previously been shown to alter adherence, anti-light subunit antibodies did not, suggesting that the heavy subunit contains the carbohydrate recognition domain.     

Elderberry bark lectins evolved to recognize Neu5Ac alpha2,6Gal/GalNAc sequence from a Gal/GalNAc binding lectin through the substitution of amino-acid residues critical for the binding to sialic acid

Bark lectins from the elderberry plants belonging to the genus Sambucus specifically bind to Neu5Acalpha2,6Gal/GalNAc sequence and have long been used for the analysis of sialoglycoconjugates that play important roles in many biological phenomena. However, molecular basis of such a unique carbohydrate binding specificity has not been understood. To answer these questions, we tried to identify the amino-acid residues in the Japanese elderberry bark lectin, Sambucus sieboldiana agglutinin that enabled the lectin to recognize sialic acid by using in silico docking simulation and site-directed mutagenesis. These studies showed that three amino-acid residues, S(197), A(233) and Q(234), in the C-terminal subdomain of SSA-B chain are critical for the binding to the sialic acid in Neu5Acalpha2,6Gal/GalNAc sequence. Replacement of one of these residues to the one in the corresponding position of ricin B-chain completely abolished the binding to a sialoglycoprotein, fetuin. Conserved presence of these amino acid residues in the corresponding sequences of two other elderberry lectins with similar binding specificity further supported the conclusion. These findings indicated that the replacement of the corresponding amino-acid residues in a putative Gal/GalNAc-specific ancestral lectin to these amino-acid residues generated the unique Neu5Acalpha2,6Gal/GalNAc-specific elderberry lectins in the course of molecular evolution.     

Cell polarization and adhesion in a motile pathogenic protozoan: role and fate of the Entamoeba histolytica Gal/GalNAc lectin

The human pathogenic protozoan Entamoeba histolytica is a motile cell polarized into a front pseudopod and a rear uroid. The amoebic Gal/GalNAc surface lectin is a major adhesion molecule composed of an immunodominant 170-kDa heavy subunit, mostly extracellular except for a short cytoplasmic tail, and of an extracellular light subunit. The binding of multivalent ligands triggers lectin capping and recruitment to the uroid. The properties of the Gal/GalNAc lectin and its role in amoeba adhesion and uroid polarization are reviewed in the context of the molecular mechanisms underlying cell polarization and locomotion.     

Identification and gene expression analysis of a large family of transmembrane kinases related to the Gal/GalNAc lectin in Entamoeba histolytica

We identified in the Entamoeba histolytica genome a family of over 80 putative transmembrane kinases (TMKs). The TMK extracellular domains had significant similarity to the intermediate subunit (Igl) of the parasite Gal/GalNAc lectin. The closest homolog to the E. histolytica TMK kinase domain was a cytoplasmic dual-specificity kinase, SplA, from Dictyostelium discoideum. Sequence analysis of the TMK family demonstrated similarities to both serine/threonine and tyrosine kinases. TMK genes from each of six phylogenetic groups were expressed as mRNA in trophozoites, as assessed by spotted oligoarray and real-time PCR assays, suggesting nonredundant functions of the TMK groups for sensing and responding to extracellular stimuli. Additionally, we observed changes in the expression profile of the TMKs in continuous culture. Antisera produced against the conserved kinase domain identified proteins of the expected molecular masses of the expressed TMKs. Confocal microscopy with anti-TMK kinase antibodies revealed a focal distribution of the TMKs on the cytoplasmic face of the trophozoite plasma membrane. We conclude that E. histolytica expresses members of each subgroup of TMKs. The presence of multiple receptor kinases in the plasma membrane offers for the first time a potential explanation of the ability of the parasite to respond to the changing environment of the host.     

Preparation of cluster glycosides ofN-acetylgalactosamine that have subnanomolar binding constants towards the mammalian hepatic Gal/GalNAc-specific receptor

Preparation of di-and tri-valent cluster glycosides containingN-acetyl-d-galactosamine (GalNAc) is described. Oligopeptides that contain a protected amino group and two or three free carboxyl groups are activated by methyl chloroformate and then coupled to 6-aminohexyl 2-acetamido-2-deoxy--d-galactopyranoside. The concentrations of the divalent GalNAc glycosides needed to produce 50% inhibition of the binding of asialoorosomucoid to the isolated, purified rat liver receptor specific for galactose and GalNAc and to the receptor on the hepatocyte surface were of the order of 10^–8 M and 10^–9 M, respectively. The binding affinity of the trivalent glycoside was 10-to 20-fold stronger than the divalent glycosides towards both the soluble receptor and intact hepatocyte.Abbreviations Z benzyloxycarbonyl - EDAC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride - AH 6-aminohexyl - ASOR aslaloorosomucoid - DMF N-dimethylformamide - DMSO dimethylsulfoxide - Lac lactosyl     

In vitro and in vivo interaction of Entamoeba histolytica Gal/GalNAc lectin with various target cells: an immunocytochemical analysis

The Gal/GalNAc lectin of Entamoeba histolytica trophozoites plays an important role in adhesion. The distribution and final destiny of the lectin during the interaction with host cells are poorly understood. Using monoclonal and polyclonal antibodies against the lectin we studied by immunocytochemistry the in vitro and in vivo interaction of E. histolytica trophozoites with human and hamster hepatocytes. We also analyzed the presence and distribution of the lectin in a mouse model of intestinal amoebiasis. In all cases, trophozoites were highly labeled by anti-lectin antibodies. Cultured human and hamster hepatocytes in contact with, or localized at the vicinity of parasites were also labeled by anti-lectin antibodies. Most of the labeled hepatocytes showed variable degrees of cell damage. Hepatocytes distantly localized from the parasites were also stained with the anti-lectin antibodies. Immunolabeling of tissue sections from different stages of the development of experimental amoebic liver abscess in hamsters showed inflammatory foci containing lectin-labeled trophozoites, hepatocytes, and sinusoidal and inflammatory cells. Lectin-containing hepatocytes had vacuolated cytoplasm with some nuclei with a condensed appearance. Damaged intestinal epithelium also was labeled with anti-lectin antibodies in a mouse model of intestinal amoebiasis. Electron microscopy of axenically cultured trophozoites using gold-labeled monoclonal and polyclonal anti-lectin antibody showed that plasma membrane, vacuole membranes and areas of cell cytosol were labeled. Higher deposits of gold particles in plasma membrane suggestive of cell secretion were observed. Our results demonstrated that Gal/GalNAc lectin was bound and captured by different target cells, and that host cells containing the lectin showed signs of cell damage. The contribution of lectin transfer to host cells in adherence and cell injury remains to be determined.     

Entamoeba histolytica: Expression and localization of Gal/GalNAc lectin in virulent and non-virulent variants from HM1:IMSS strain

We have purified Gal/GalNAc lectin from Entamoeba histolytica by electroelution. The purified protein was used to immunize rabbits and obtain polyclonal IgG’s anti-lectin. These antibodies were used as tools to analyze the expression and localization of the amoebic lectin in both virulent (vEh) and non-virulent (nvEh) variants of axenically cultured HM1:IMSS strain. vEh is able to induce liver abscesses in hamsters, whereas nvEh has lost this ability. In vitro, amoebic trophozoites from both variants equally express this protein as shown by densitometric analysis of the corresponding band in Western blots from lysates. In both types of trophozoites, the pattern of distribution of the lectin was mainly on the surface. We have also compared by immunohistochemistry the presence and distribution of lectin in the in vivo liver lesions produced in hamsters. In order to prolong the survival of nvEh to analyze both variants in an in vivo model, hamsters inoculated with nvEh were treated with methyl prednisolone. Our results suggest that the Gal/GalNAc lectin is equally expressed in both nvEh and vEh.     

Stable expression of Gal/GalNAc lectin of Entamoeba histolytica in transgenic chloroplasts and immunogenicity in mice towards vaccine development for amoebiasis

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multigene engineering in a single transformation event. Entamoeba histolytica infects 50 million people, causing about 100 000 deaths annually, but there is no approved vaccine against this pathogen. LecA , a potential target for blocking amoebiasis, was expressed for the first time in transgenic plants. Stable transgene integration into chloroplast genomes and homoplasmy were confirmed by polymerase chain reaction and Southern blot analyses. LecA expression was evaluated by Western blots and quantified by enzyme-linked immunosorbent assay (up to 6.3% of total soluble protein or 2.3 mg LecA/g leaf tissue). Subcutaneous immunization of mice with crude extract of transgenic leaves resulted in higher immunoglobulin G titres (up to 1 : 10 000) than in previous reports. An average yield of 24 mg of LecA per plant should produce 29 million doses of vaccine antigen per acre of transgenic plants. Such high levels of expression and immunogenicity should facilitate the development of a less expensive amoebiasis vaccine.     

Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica

A new galactose-specific lectin has been purified from the extracts of Trichosanthes dioica seeds by affinity chromatography on cross-linked guar gum. The purified lectin (T. dioica seed lectin, TDSL) moved as a single symmetrical peak on gel filtration on Superose-12 in the presence of 0.1 M lactose with an M(r) of 55 kDa. In the absence of ligand, the movement was retarded, indicating a possible interaction of the lectin with the column matrix. In SDS-PAGE, in the presence of beta-mercaptoethanol, two non-identical bands of M(r) 24 and 37 kDa were observed, whereas in the absence of beta-mercaptoethanol, the lectin yielded a single band corresponding to approximately 55,000 Da, indicating that the two subunits of TDSL are connected by one or more disulfide bridges. TDSL is a glycoprotein with about 4.9% covalently bound neutral sugar. Analysis of near-UV CD spectrum by three different methods (CDSSTR, CONTINLL, and SELCON3) shows that TDSL contains 13.3% alpha-helix, 36.7% beta-sheet, 19.4% beta-turns, and 31.6% unordered structure. Among a battery of sugars investigated, TDSL was inhibited strongly by beta-d-galactopyranosides, with 4-methylumbelliferyl-beta-d-galactopyranoside being the best ligand. Chemical modification studies indicate that tyrosine residues are important for the carbohydrate-binding and hemagglutinating activities of the lectin. A partial protection was observed when the tyrosine modification was performed in the presence of 0.2 M lactose. The tryptophan residues of TDSL appear to be buried in the protein interior as they could not be modified under native conditions, whereas upon denaturation with 8 M urea two Trp residues could be selectively modified by N-bromosuccinimide. The subunit composition and size, secondary structure, and sugar specificity of this lectin are similar to those of type-2 ribosome inactivating proteins, suggesting that TDSL may belong to this protein family.     

Localisation to lipid rafts correlates with increased function of the Gal/GalNAc lectin in the human protozoan parasite, Entamoeba histolytica

Entamoeba histolytica is the causative agent of dysentery and liver abscess and is prevalent in developing countries. Adhesion to the host is critical to infection and is mediated by amoebic surface receptors. One such receptor, the Gal/GalNAc lectin, binds to galactose or N-acetylgalactosamine residues on host components and consists of heavy (Hgl), light (Lgl) and intermediate (Igl) subunits. The mechanism by which the lectin assembles into a functional complex is not known. The parasite also relies on cholesterol-rich domains (lipid rafts) for adhesion. Therefore, it is conceivable that rafts regulate the assembly or function of the lectin. To test this, amoebae were loaded with cholesterol and lipid rafts were purified and characterised. Western blotting showed that cholesterol loading resulted in co-compartmentalisation of all three subunits in rafts. This co-compartmentalisation was accompanied by an increase in the ability of the amoebae to bind to host cells in a galactose-specific manner, suggesting that there is a correlation between location and function of the Gal/GalNAc lectin. Cholesterol loading did not increase the surface levels of the lectin subunits. Therefore, the cholesterol-induced increase in adhesion was not the result of externalisation of an internal pool of subunits. A mutant cell line that modestly responded to cholesterol with a slight increase in adhesion exhibited only a slight enrichment of Hgl and Lgl in rafts. This supports the connection between location and function of the Gal/GalNAc lectin. Actin can also influence the interaction of proteins with rafts. Therefore, the sub-membrane distribution of the lectin subunits was also assessed after treatment with an actin depolymerising agent, cytochalasin D. Cytochalasin D-treatment had no effect on the submembrane distribution of the subunits, suggesting that actin does not prevent the association of lectin subunits with rafts in this system. Together, these data provide insight into the molecular mechanisms regulating the location and function of this adhesin.     

Binding characteristics of N-acetylglucosamine-specific lectin of the isolated chicken hepatocytes: similarities to mammalian hepatic galactose/N-acetylgalactosamine-specific lectin

Binding characteristics of N-acetylglucosamine- (GlcNAc) specific lectin on the chicken hepatocyte surface were probed by an inhibition assay using various sugars and glycosides as inhibitors. Results indicated that the binding area of the lectin is small, interacting only with GlcNAc residues whose 3- and 4-OH's are open. The combining site is probably of trough-type, since substitution with as large a group as monosaccharide is permitted on the C-6 side of GlcNAc, and on the C-1 side, the aglycon of GlcNAc can be very large (e.g., a glycoprotein). These binding characteristics are shared with the homologous mammalian lectin specific for galactose/N-acetylgalactosamine, suggesting that tertiary structure of the combining area of these two lectins is similar. This is understandable, since there is approximately 40% amino acid sequence identity in the carbohydrate recognition domain of these two lectins [Drickamer, K., Mannon, J. F., Binns, G., & Leung, J. O. (1984) J. Biol. Chem. 259, 770-778]. A series of glycosides, each containing two GlcNAc residues separated by different distances (from 0.8 to 4.7 nm), were synthesized. Inhibition assay with these and other cluster glycosides indicated that clustering of two or more GlcNAc residues increased the affinity toward the chicken lectin tremendously. Among the ligands containing two GlcNAc residues, the structure which allows a maximal inter-GlcNAc distance of 3.3 nm had the strongest affinity, its affinity increase over GlcNAc (monosaccharide) amounting to 100-fold. Longer distances slightly diminished the affinity, while shortening the distance caused substantial decrease in the affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recent developments in amoebiasis:the Gal/GalNAc lectins of Entamoeba histolytica and Entamoeba dispar

Amoebiasis is responsible for 50000-100000 deaths annually. Invasive amoebic disease begins with the attachment of Entamoeba histolytica trophozoites to colonic mucin, a process mediated by the amoebic Gal/GalNAc lectin. The non-pathogenic counterpart, E. dispar, is morphologically identical but genetically distinct. Investigations comparing the Gal/GalNac lectin from these two organisms are under way.     

Pathogenic bacteria prime the induction of Toll-like receptor signalling in human colonic cells by the Gal/GalNAc lectin Carbohydrate Recognition Domain of Entamoeba histolytica

In mixed intestinal infections with Entamoeba histolytica trophozoites and enteropathogenic bacteria, which are wide-spread in areas of endemic amoebiasis, interaction between the pathogens could be an important factor in the occurrence of invasive disease. It has been reported that exposure of human colonic cells to enteropathogenic bacteria increased trophozoite adherence to the cells and their subsequent damage. We report here that the Carbohydrate Recognition Domain (CRD) of the amoebic Gal/GalNAc lectin binds to Toll-like receptors TLR-2 and TLR-4 in human colonic cells, activating the “classic” signalling pathway of these receptors. Activation induced expression of TLR-2 and TLR-4 mRNAs and the mRNAs of pro-inflammatory cytokines, as well as an increase in the corresponding proteins. Direct correlation was observed between the increased expression of TLRs and pro-inflammatory cytokines, the enhanced adhesion of trophozoites to the cells and the inflicted cell damage. When cells were exposed to pathogenic bacteria Staphylococcus aureus (Gram+) or Shigella dysenteriae (Gram−), elements of an innate immune response were induced. CRD by itself elicited a similar cell response, while exposure to a commensal Escherichia coli had a null effect. Pre-exposure of the cells to pathogenic bacteria and then to CRD rendered an inflammatory-like microenvironment that after addition of trophozoites facilitated greater cell destruction. Our results suggest that CRD is recognised by human colonic cells as a pathogen-associated-molecular-pattern-like molecule and as such can induce the expression of elements of an innate immune response. In the human host, an exacerbated inflammatory environment, derived from pathogen interplay, may be an important factor for development of invasive disease.     

Different modes of ligand binding to the hepatic galactose/N-acetylgalactosamine lectin on the surface of rabbit hepatocytes

A study of the binding of three different 125I-labeled, galactose-terminated ligands to the hepatic galactose/N-acetylgalactosamine-specific lectin found on the surface of rabbit hepatocytes revealed that the different ligands manifest different physical parameters of binding. Asialoorosomucoid (125I-ASOR) binding was best described as involving two independent classes of binding sites on rabbit hepatocytes, with 161 000 sites/cell with a dissociation constant of 0.44 nM and 292 000 sites/cell with a Kd of 9.7 nM. Asialotriantennary glycopeptide purified from human alpha-1 protease inhibitor and modified with tyrosine at the N-terminus to permit radioiodination (TRI) [Lee, Y. C., Townsend, R. R., Hardy, M. R., L?nngren, J., Arnarp, J., Haraldsson, M., & L?nn, H. (1983) J. Biol. Chem. 258, 199-202] was also found to bind to two apparent classes of binding sites but with different binding parameters: 292 000 sites/cell of Kd = 1.47 nM and 982 000 sites/cell of Kd = 25.3 nM. A synthetic ligand, alpha,beta-diaspartamide of tris[(beta-lactosyloxy)methyl](6-aminohexanamido)methane (di-tris-lac) containing six nonreducing galactose residues [Lee, R. T., Lin, P., & Lee, Y. C. (1984) Biochemistry 23, 4255-4261], was found to bind to 817 000 sites/cell of Kd = 0.63 nM and 1.23 X 10(6) sites/cell of Kd = 25.3 nM. Thus, there were many more total binding sites for TRI or di-tris-lac on the surface of rabbit hepatocytes than there were for asialoorosomucoid, although the dissociation constants were similar for all three ligands.(ABSTRACT TRUNCATED AT 250 WORDS)