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1.
Parasite Immunol ; 38(1): 53-63, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26609699

ABSTRACT

This study investigated the details of the innate and Th1/Treg-type-associated host immune responses in Trichuris suis and Oesophagostomum dentatum mono- and co-infected pigs and in vitro in stimulated porcine dendritic cell cultures. Forty-eight pigs were allocated into a 2-factorial design with two groups trickle-inoculated with 10 T. suis eggs/kg/day (Group T) or 20 O. dentatum L3/kg/day (O). Another group (OT) was infected with both parasites. Group C remained uninfected. Expression of innate and Th1/Treg-cell-associated genes in gut mucosa and associated lymph nodes was determined by qPCR at necropsy day 35 and 71. Gene expression showed suppressed/inhibited Th1 and Treg-type immune reactions, in accordance with previous findings of a predominant Th2-type immune response to both nematodes. The in vitro part examined the production of TNF-α in porcine dendritic cells (DC) exposed to T. suis and/or O. dentatum excretory/secretory (E/S) products. Further, binding capacity and structure of E/S products were characterized. Glycan and lectin-binding capacity were generally lower in O. dentatum E/S products compared to T. suis which may explain the earlier found weaker Th2 response to the former. Surprisingly, O. dentatum E/S products induced a significant (P < 0·0001) increase in TNF-α DC production, potentially indicating a new mode of helminth-host immune response interaction.


Subject(s)
Intestinal Mucosa/immunology , Oesophagostomum/immunology , Swine Diseases/parasitology , Th1 Cells/immunology , Trichuris/immunology , Animals , Cells, Cultured , Dendritic Cells/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Swine , Swine Diseases/immunology , T-Lymphocytes, Regulatory/immunology , Th2 Cells/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/immunology
2.
Parasite Immunol ; 37(9): 453-69, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26173941

ABSTRACT

Functional impairment of dendritic cells (DCs) is part of a survival strategy evolved by Leishmania and Plasmodium parasites to evade host immune responses. Here, the effects of co-exposing human monocyte-derived DCs to Leishmania donovani promastigotes and Plasmodium falciparum-infected erythrocytes were investigated. Co-stimulation resulted in a dual, dose-dependent effect on DC differentiation which ranged from semi-mature cells, secreting low interleukin(-12p70 levels to a complete lack of phenotypic maturation in the presence of high parasite amounts. The effect was mainly triggered by the Leishmania parasites, as illustrated by their ability to induce semi-mature, interleukin-10-producing DCs, that poorly responded to lipopolysaccharide stimulation. Conversely, P. falciparum blood-stage forms failed to activate DCs and only slightly interfered with lipopolysaccharide effects. Stimulation with high L. donovani concentrations triggered phosphatidylserine translocation, whose onset presented after initiating the maturation impairment process. When added in combination, the two parasites could co-localize in the same DCs, confirming that the leading effects of Leishmania over Plasmodium may not be due to mutual exclusion. Altogether, these results suggest that in the presence of visceral leishmaniasis-malaria co-infections, Leishmania-driven effects may overrule the more silent response elicited by P. falciparum, shaping host immunity towards a regulatory pattern and possibly delaying disease resolution.


Subject(s)
Coinfection/immunology , Dendritic Cells/immunology , Leishmania donovani/physiology , Leishmaniasis, Visceral/immunology , Malaria, Falciparum/immunology , Plasmodium falciparum/physiology , Cell Differentiation , Erythrocytes/parasitology , Humans , Immune System Phenomena , Leishmania donovani/growth & development , Lipopolysaccharides/immunology , Monocytes/cytology
3.
Genes Immun ; 16(6): 378-87, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25996526

ABSTRACT

Inflammatory immune disorders such as inflammatory bowel disease and multiple sclerosis are major health problems. Currently, the intestinal whipworm Trichuris suis is being explored in clinical trials to reduce inflammation in these diseases; however, the mechanisms by which the parasite affects the host immune system are not known. Here we determined the effects of T. suis soluble products (SPs) on Toll-like receptor-4 (TLR4)-stimulated human dendritic cells (DCs) using Illumina bead chip gene arrays. Pathway analysis of lipopolysaccharide-stimulated DCs with or without T. suis treatment showed that co-stimulation with T. suis SPs resulted in a downregulation of both the myeloid differentiation primary response gene 88-dependent and the TIR-domain-containing adaptor-inducing interferon-ß-dependent signalling pathways triggered by TLR4. These data were verified using quantitative real-time PCR of several key genes within these pathways and/or defining their protein levels. In addition, T. suis SPs induce Rab7b, a negative regulator of TLR4 signalling that interferes with its trafficking, which coincided with a reduced surface expression of TLR4. These data indicate that the mechanism by which T. suis SPs reduce inflammatory responses is through suppression of both TLR4 signalling and surface expression on DCs.


Subject(s)
Dendritic Cells/parasitology , Toll-Like Receptor 4/metabolism , Trichuris/immunology , rab GTP-Binding Proteins/metabolism , Animals , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Down-Regulation , Humans , Inflammation/immunology , Inflammation/parasitology , Inflammation/therapy , Lipopolysaccharides/pharmacology , Real-Time Polymerase Chain Reaction , Signal Transduction , Transcriptome , rab7 GTP-Binding Proteins
4.
Genes Immun ; 15(7): 477-86, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25008860

ABSTRACT

Recent clinical trials in patients with inflammatory diseases like multiple sclerosis (MS) or inflammatory bowel disease (IBD) have shown the beneficial effects of probiotic helminth administration, although the underlying mechanism of action remains largely unknown. Potential cellular targets may include innate immune cells that propagate inflammation in these diseases, like pro-inflammatory macrophages. We here investigated the effects of the helminth Trichuris suis soluble products (SPs) on the phenotype and function of human inflammatory (granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated) macrophages. Interestingly, we here show that T. suis SPs potently skew inflammatory macrophages into a more anti-inflammatory state in a Toll-like receptor 4 (TLR4)-dependent manner, and less effects are seen when stimulating macrophages with TLR2 or -3 ligands. Gene microarray analysis of GM-CSF-differentiated macrophages further revealed that many TLR4-induced inflammatory mediators, including interleukin (IL)-12B, CCL1 and CXCL9, are downregulated by T. suis SPs. In particular, we observed a strong reduction in the expression and function of P2RX7, a purinergic receptor involved in macrophage inflammation, leading to reduced IL-1ß secretion. In conclusion, we show that T. suis SPs suppress a broad range of inflammatory pathways in GM-CSF-differentiated macrophages in a TLR4-dependent manner, thereby providing enhanced mechanistic insight into the therapeutic potential of this helminth for patients with inflammatory diseases.


Subject(s)
Helminth Proteins/pharmacology , Macrophages/drug effects , Toll-Like Receptor 4/metabolism , Trichuris/immunology , Animals , Cells, Cultured , Chemokine CCL1/genetics , Chemokine CCL1/metabolism , Chemokine CXCL9/genetics , Chemokine CXCL9/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Helminth Proteins/immunology , Humans , Immunity, Innate , Inflammation/immunology , Inflammation/metabolism , Interleukin-12 Subunit p40/genetics , Interleukin-12 Subunit p40/metabolism , Macrophages/immunology , Receptors, Purinergic P2X7/genetics , Receptors, Purinergic P2X7/metabolism , Trichuris/chemistry
5.
Mol Immunol ; 60(1): 1-7, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24705296

ABSTRACT

The administration of helminths is considered a promising strategy for the treatment of autoimmune diseases due to their immunomodulatory properties. Currently, the application of the helminth Trichuris suis as a treatment for Crohn's disease is being studied in large multi-center clinical trials. The intestinal epithelium forms an efficient barrier between the intestinal lumen containing the microbial flora and helminths, and dendritic cells (DCs) present in the lamina propria that determine the TH response. Here, we investigated how excreted/secreted (E/S) products of T. suis affect the barrier function of intestinal epithelial cells (IECs) in order to reach the DCs and modulate the immune response. We show that T. suis E/S products reduce the barrier function and the expression of the tight junction proteins EMP-1 and claudin-4 in IEC CMT93/69 monolayers in a glycan-dependent manner. This resulted in an increased passage of soluble compounds to the basolateral side that affected DC function. In addition, T. suis E/S suppressed LPS-induced pro-inflammatory cytokine production by CMT93/69 cells, whereas the production of the TH2 response-inducing cytokine thymic stromal lymphopoietin (TSLP) was induced. Our studies indicate that T. suis E/S glycans affect the function of the intestinal epithelium in order to modulate DC function. Identification of the T. suis E/S glycans that modulate IEC and DC function may lead to a strategy to reduce symptoms of autoimmune and allergic immune diseases by orally administrated helminth-derived factors without the need of infection with live helminths.


Subject(s)
Cytokines/antagonists & inhibitors , Dendritic Cells/immunology , Helminth Proteins/immunology , Intestinal Mucosa/immunology , Therapy with Helminths/methods , Trichuris/immunology , Animals , Biological Transport , Cell Line , Chemokine CXCL1/biosynthesis , Claudin-4/biosynthesis , Crohn Disease/therapy , Cytokines/biosynthesis , Cytokines/immunology , Helminth Proteins/administration & dosage , Humans , Lipopolysaccharides , Mice , Neoplasm Proteins/biosynthesis , Polysaccharides/administration & dosage , Polysaccharides/metabolism , Receptors, Cell Surface/biosynthesis , Th2 Cells/immunology , Tight Junctions/immunology , Trichuris/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Thymic Stromal Lymphopoietin
6.
Anal Biochem ; 329(2): 293-9, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-15158490

ABSTRACT

The quantification of gene expression by real-time polymerase chain reaction (PCR) has revolutionized the field of gene expression analysis. Due to its sensitivity and flexibility it is becoming the method of choice for many investigators. However, good normalization protocols still have to be implemented to facilitate data exchange and comparison. We have designed primers for 10 unrelated genes and developed a simple protocol to detect genes with stable expression that are suitable for use as endogenous reference genes for further use in the normalization of gene expression data obtained by real-time PCR. Using this protocol, we were able to identify human proteosome subunit Y as a reliable endogenous reference gene for human umbilical vein endothelial cells treated for up to 18 h with TNFalpha, IL-4, or IFNgamma and for B cells isolated from healthy controls and patients suffering from IgA nephropathy. Other optional endogenous reference genes that can be considered are phosphomannomutase (PPMM) and actin for endothelial cells and glyceraldehyde-3-phosphate dehydrogenase and PPMM for B cells.


Subject(s)
Gene Expression Profiling/methods , Gene Expression , Genes, Reporter , Endothelial Cells/metabolism , Humans
7.
Exp Parasitol ; 105(3-4): 219-25, 2003.
Article in English | MEDLINE | ID: mdl-14990315

ABSTRACT

The development of the humoral anti-glycan immune response of chimpanzees, either or not vaccinated with radiation-attenuated Schistosoma mansoni cercariae, was followed during 1 year after infection with S. mansoni. During the acute phase of infection both the vaccinated and the control chimpanzees produce high levels of immunoglobulin G (IgG) antibodies against carbohydrate structures that are characteristic for schistosomes carrying the Fucalpha1-3GalNAc and Fucalpha1-2Fucalpha1-3GlcNAc motifs, but not to the more widespread occurring structures GalNAcbeta1-4GlcNAc, GalNAcbeta1-4(Fucalpha1-3)GlcNAc, and Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis(x)). In addition, high levels of IgM antibodies were found against the trimeric Lewis(x) epitope. Apparently, the schistosome-characteristic carbohydrate structures are dominant epitopes in the anti-glycan humoral immune response of the chimpanzees. All chimpanzees showed an increase in the level of antibodies against most of the carbohydrate structures tested directly after vaccination, peaking at challenge time and during the acute phase of infection. With the exception of anti-F-LDN antibody responses, the anti-carbohydrate antibody responses upon schistosome infection of the vaccinated animals were muted in comparison to the control animals.


Subject(s)
Antibodies, Helminth/biosynthesis , Disaccharides/immunology , Epitopes/immunology , Schistosoma mansoni/immunology , Trisaccharides/immunology , Animals , Carbohydrate Sequence , Disaccharides/chemical synthesis , Disaccharides/chemistry , Immunoglobulin G/biosynthesis , Immunoglobulin M/biosynthesis , Longitudinal Studies , Male , Molecular Sequence Data , Pan troglodytes , Polysaccharides/chemical synthesis , Polysaccharides/chemistry , Polysaccharides/immunology , Schistosomiasis mansoni/immunology , Spectrum Analysis/methods , Trisaccharides/chemical synthesis , Trisaccharides/chemistry , Vaccination
8.
Parasite Immunol ; 24(4): 189-201, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12010484

ABSTRACT

The efficacy of two recombinant proteins of Haemonchus contortus was studied in both adult sheep and young lambs. These 15 and 24 kDa excretory/secretory proteins were given combined, either supplemented or not with a glycan-rich insect cell extract. In 9-month-old sheep (trial 1), faecal egg output and worm burden were reduced by 49% and 55%, respectively, after vaccination with rec15/24, and by 46% and 65% after vaccination with rec15/24 and glycan extract. No reduction in egg output or number of worms was found in young lambs using the above recombinant proteins plus glycan-rich extract (trial 2). When trial 1 was repeated (trial 3), the protection could not be reproduced, possibly due to differences in batches of recombinant proteins. In all sheep, independent of their age, rec15/24-specific immunoglobulin (Ig)G1 and IgA titres were present, but 9-month-old protected sheep had significantly higher IgA titres than the lambs. Addition of glycans resulted in lower rec15/24-specific IgG1 and IgA in 9-month-old sheep after challenge. This did not affect the level of protection. A significant negative correlation was found between IgA and worm numbers in protected sheep immunized with rec15/24 supplemented with glycans. Total IgE and rec15/24 specific IgE titres were low. The number of eosinophils, mast cells, sheep mast cell protease (SMCP)+ cells and IgA+ cells did not differ between the protected and unprotected sheep, but the lambs had significantly fewer mast cells independent of their immunization.


Subject(s)
Antigens, Helminth/immunology , Haemonchus/immunology , Sheep Diseases/prevention & control , Animals , Haemonchus/chemistry , Immunoglobulin A/analysis , Immunoglobulin E/analysis , Immunoglobulin G/analysis , Immunohistochemistry , Lymphocyte Activation/immunology , Recombinant Proteins/immunology , Sheep , Sheep Diseases/parasitology , Vaccination/veterinary
9.
FEBS Lett ; 507(3): 307-12, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11696361

ABSTRACT

Three putative alpha1-->3/4-fucosyltransferase (alpha1-->3/4-FucT) genes have been detected in the Arabidopsis thaliana genome. The products of two of these genes have been identified in vivo as core alpha1-->3-FucTs involved in N-glycosylation. An orthologue of the third gene was isolated from a Beta vulgaris cDNA library. The encoded enzyme efficiently fucosylates Galbeta1-->3GlcNAcbeta1-->3Galbeta1-->4Glc. Analysis of the product by 400 MHz (1)H-nuclear magnetic resonance spectroscopy showed that the product is alpha1-->4-fucosylated at the N-acetylglucosamine residue. In vitro, the recombinant B. vulgaris alpha1-->4-FucT acts efficiently only on neutral type 1 chain-based glycan structures. In plants the enzyme is expected to be involved in Lewis(a) formation on N-linked glycans.


Subject(s)
Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Acetylglucosamine/chemistry , Acetylglucosamine/metabolism , Amino Acid Sequence , Animals , Arabidopsis/genetics , Arabidopsis/metabolism , Beta vulgaris/genetics , CHO Cells , Carbohydrate Sequence , Cloning, Molecular , Cricetinae , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Multigene Family , Plant Proteins/genetics , Plant Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
10.
Infect Immun ; 69(4): 2396-401, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11254599

ABSTRACT

We report here that sera of children and adults infected with Schistosoma mansoni, S. haematobium, or S. japonicum contain antibodies against GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) and to a lesser extent to Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis(x)) and GalNAcbeta1-4GlcNAc (LDN). Surface plasmon resonance (SPR) spectroscopy was used to monitor the presence of serum antibodies to neoglycoconjugates containing these carbohydrate epitopes and to define the immunoglobulin M (IgM) and IgG subclass distribution of the antibodies. The serum levels of antibodies to LDN-DF are high related to LDN and Lewis(x) for all examined groups of Schistosoma-infected individuals. A higher antibody response to the LDN-DF epitope was found in sera of infected children than in sera of infected adults regardless of the schistosome species. With respect to the subclasses, we found surprisingly that individuals infected with S. japonicum have predominantly IgG antibodies, while individuals infected with S. mansoni mainly show an IgM response; high levels of both isotypes were measured in sera of individuals infected with S. haematobium. These data provide new insights in the human humoral immune response to schistosome-derived glycans.


Subject(s)
Antibodies, Helminth/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Lewis X Antigen/immunology , Polysaccharides/immunology , Schistosoma/immunology , Adult , Animals , Child , Epitopes , Humans , Surface Plasmon Resonance
11.
J Cell Sci ; 114(Pt 7): 1321-9, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11256998

ABSTRACT

Signal regulatory protein-alpha (SIRP alpha) is a member of the Ig superfamily selectively expressed by neuronal and myeloid cells. The molecule mediates functional interactions with CD47/integrin-associated protein. Here we provide evidence for the tissue-specific glycosylation of neuronal and haematopoietic SIRP alpha. We demonstrate a major difference in the galactosylation of N-linked glycans isolated from neuronal (i.e. brain-derived) SIRP alpha as compared to myeloid (i.e. spleen-derived) SIRP alpha, with neuronal SIRP alpha almost completely lacking galactose. beta 4-galactosyltransferase assays demonstrated that this is most likely due to a low galactosylation capacity of the brain. In order to investigate the role of galactosylation of SIRP alpha in cellular interactions, soluble recombinant SIRP alpha glycoforms containing galactose (SIRP alpha-Fc) or lacking galactose (SIRP alpha(Delta Gal)-Fc) were produced. Binding studies demonstrated superior binding of SIRP alpha(Delta Gal)-Fc to cerebellar neurons and isolated lymphocytes. In contrast, SIRP alpha-Fc bound relatively strong to macrophages. These data show that the galactosylation of SIRP alpha determines its cellular binding specificity.


Subject(s)
Antigens, Differentiation , Galactose/metabolism , Membrane Glycoproteins/metabolism , Neural Cell Adhesion Molecule L1 , Neural Cell Adhesion Molecules/metabolism , Neurons/metabolism , Receptors, Immunologic , Animals , CHO Cells , Cricetinae , Glycosylation , Humans , Immunoglobulin Fc Fragments/biosynthesis , Immunoglobulin Fc Fragments/genetics , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , N-Acetyllactosamine Synthase/metabolism , Neural Cell Adhesion Molecules/biosynthesis , Neural Cell Adhesion Molecules/genetics , PC12 Cells , Polysaccharides/metabolism , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics
12.
J Immunol ; 166(6): 3637-40, 2001 Mar 15.
Article in English | MEDLINE | ID: mdl-11238599

ABSTRACT

Sialoadhesin (Siglec-1) is a macrophage-restricted sialic acid-binding receptor that mediates interactions with hemopoietic cells, including lymphocytes. In this study, we identify sialoadhesin counterreceptors on T lymphocytes. Several major glycoproteins (85, 130, 240 kDa) were precipitated by sialoadhesin-Fc fusion proteins from a murine T cell line (TK-1). Binding of sialoadhesin to these glycoproteins was sialic acid dependent and was abolished by mutation of a critical residue (R97A) of the sialic acid binding site in the membrane distal Ig-like domain of sialoadhesin. The 130- and 240-kDa sialoadhesin-binding glycoproteins were identified as the sialomucins CD43 and P-selectin glycoprotein ligand 1 (CD162), respectively. CD43 expressed in COS cells supported increased binding to immobilized sialoadhesin. Finally, sialoadhesin bound different glycoforms of CD43 expressed in Chinese hamster ovary cells, including unbranched (core 1) and branched (core 2) O:-linked glycans, that are normally found on CD43 in resting and activated T cells, respectively. These results identify CD43 as a T cell counterreceptor for sialoadhesin and suggest that in addition to its anti-adhesive role CD43 may promote cell-cell interactions.


Subject(s)
Antigens, CD/metabolism , Membrane Glycoproteins/metabolism , Receptors, Immunologic/metabolism , Sialoglycoproteins/metabolism , T-Lymphocytes/metabolism , Animals , CHO Cells , COS Cells , Cell Adhesion Molecules/metabolism , Cricetinae , Humans , Interphase/immunology , Leukosialin , Lymphocyte Activation , Mice , Protein Binding/immunology , Sialic Acid Binding Ig-like Lectin 1 , T-Lymphocytes/immunology , Tumor Cells, Cultured
13.
Eur J Biochem ; 267(15): 4753-62, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10903509

ABSTRACT

We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO-type glycans and is devoid of any of the N, N'-diacetyllactosediamine (lacdiNAc)-based chains previously identified in glycodelin-A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a beta1-->4-N-acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc-based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc-based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form.


Subject(s)
Contraceptive Agents/metabolism , Glycoproteins/biosynthesis , Pregnancy Proteins/biosynthesis , Recombinant Proteins/biosynthesis , Animals , CHO Cells , Carbohydrate Sequence , Cell Culture Techniques/methods , Cell Line , Contraceptive Agents/chemistry , Cricetinae , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Glucosamine/pharmacology , Glucose/pharmacology , Glycodelin , Glycoproteins/chemistry , Glycosylation , Humans , Immunoassay , Immunoblotting , Lectins/chemistry , Molecular Sequence Data , Monosaccharides/chemistry , Pregnancy Proteins/chemistry , Recombinant Proteins/chemistry , Spectrometry, Mass, Fast Atom Bombardment , Transfection
14.
Infect Immun ; 68(6): 3541-7, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10816509

ABSTRACT

F1C fimbriae are correlated with uropathogenic Escherichia coli strains. Although F1C fimbriae mediate binding to kidney tubular cells, their receptor is not known. In this paper, we demonstrate for the first time specific carbohydrate residues as receptor structure for F1C-fimbria-expressing E. coli. The binding of the F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) and purified F1C fimbriae to reference glycolipids of different carbohydrate compositions was evaluated by using thin-layer chromatography (TLC) overlay and solid-phase binding assays. TLC fimbrial overlay analysis revealed the binding ability of purified F1C fimbriae only to glucosylceramide (GlcCer), beta1-linked galactosylceramide 2 (GalCer2) with nonhydroxy fatty acids, lactosylceramide, globotriaosylceramide, paragloboside (nLc(4)Cer), lactotriaosylceramide, gangliotriaosylceramide (asialo-GM(2) [GgO(3)Cer]) and gangliotetraosylceramide (asialo-GM(1) [GgO(4)Cer]). The binding of purified F1C fimbriae as well as F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) was optimal to microtiter plates coated with asialo-GM(2) (GgO(3)Cer). The bacterial interaction with asialo-GM(1) (GgO(4)Cer) and asialo-GM(2) (GgO(3)Cer) was strongly inhibited only by disaccharide GalNAcbeta1-4Galbeta linked to bovine serum albumin. We observed no binding to globotetraosylceramide or Forssman antigen (Gb(5)Cer) glycosphingolipids or to sialic-acid-containing gangliosides. It was demonstrated that the presence of a GalCer or GlcCer residue alone is not sufficient for optimal binding, and additional carbohydrate residues are required for high-affinity adherence. Indeed, the binding efficiency of F1C fimbriated recombinant bacteria increased by 19-fold when disaccharide sequence GalNAcbeta1-4Galbeta is linked to glucosylceramide as in asialo-GM(2) (GgO(3)Cer). Thus, it is suggested that the disaccharide sequence GalNAcbeta1-4Galbeta of asialo-GM(2) (GgO(3)Cer) which is positioned internally in asialo-GM(1) (GgO(4)Cer) is the high-affinity binding epitope for the F1C fimbriae of uropathogenic E. coli.


Subject(s)
Adhesins, Escherichia coli/metabolism , Escherichia coli/pathogenicity , Fimbriae, Bacterial/metabolism , Glycolipids/metabolism , Receptors, Cell Surface/metabolism , Urinary Tract Infections/microbiology , Binding, Competitive , Glycoside Hydrolases/pharmacology , Neuraminidase/pharmacology
15.
Glycobiology ; 10(6): 601-9, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10814702

ABSTRACT

We report here that fucosylated epitopes such as Lewis(x), LacdiNAc, fucosylated LacdiNAc (LDN-F) and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) are expressed by schistosomes throughout their life cycle. These four epitopes were enzymatically synthesized and coupled to bovine serum albumin to yield neoglycoproteins. Subsequently these neoglycoproteins were used to probe a panel of 188 monoclonal antibodies obtained from infected or immunized mice, in ELISA and surface plasmon resonance analysis. Of these antibodies, 25 recognized one of the fucosylated structures synthesized, indicating that these structures are immunogenic during infection. The MAbs identified could be subdivided in four different groups based on the recognition of either the Lewis(x)-, the LacdiNAc-, the LDN-DF-, or both the LDN-F- and LDN-DF epitope. These monoclonal antibodies were then used to investigate the localization of the fucosylated epitopes in various stages of Schistosoma mansoni using indirect immunofluorescence. Lewis(x)epitopes were mainly found in the gut and on the tegument of adult worms, on egg shells, and on the oral sucker of cercariae. The LacdiNAc epitope was expressed on the tegument of adult worms, on miracidia, and on the oral sucker of cercariae. In contrast, LDN-DF epitopes were mainly present in the excretory system of adult worms, on miracidia and on whole cercariae. These also stained positive with the LDN-F/LDN-DF epitope antibodies, while whole parenchyma reacted characteristically only with the latter antibodies. The identification of different carbohydrate structures in various stages of schistosomes may lead to a better understanding of the function of glycans in the immune response during infection.


Subject(s)
Disaccharides/analysis , Epitopes/analysis , Fucose/metabolism , Lactose/analogs & derivatives , Lewis X Antigen/analysis , Schistosoma mansoni/growth & development , Schistosoma mansoni/immunology , Animals , Antibodies, Monoclonal , Cricetinae , Disaccharides/metabolism , Fluorescent Antibody Technique, Indirect , Glycoproteins/analysis , Glycoproteins/chemical synthesis , Glycoproteins/immunology , Lactose/analysis , Lactose/metabolism , Mice , Schistosomiasis mansoni/immunology , Surface Plasmon Resonance , Tissue Distribution
16.
Glycobiology ; 10(3): 263-71, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10704525

ABSTRACT

Several studies suggest, that the snail Lymnaea stagnalis contains glycoproteins whose oligosaccharide side chains have structural features not commonly found in mammalian glycoproteins. In this study, prostate glands of L. stagnalis were incubated in media containing either [(3)H]-mannose, [(3)H]-glucosamine, or [(3)H]-galactose, and the metabolically radiolabeled protein-bound oligosaccharides were analyzed. The newly synthesized diantennary-like complex-type asparagine-linked chains contained a considerable amount of glucose, next to mannose, GlcNAc, fucose, galactose, and traces of GalNAc. Since glucose has not been found before as a constituent of diantennary N-linked glycans as far as we know, we assayed the prostate gland of L. stagnalis for a potential glucosyltransferase activity involved in the biosynthesis of such structures. We report here, that the prostate gland of L. stagnalis contains a beta1-->4-glucosyltransferase activity that transfers glucose from UDP-glucose to acceptor substrates carrying a terminal N-acetylglucosamine. The enzyme prefers substrates carrying a terminal GlcNAc that is beta6 linked to a Gal or a GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is beta2 linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymatic data, we propose that the novel beta1-->4-gluco-syltransferase present in the prostate gland may be involved in the biosynthesis of Glcbeta1-->4GlcNAc units in complex-type glycans, in particular in N-linked diantennary glycans.


Subject(s)
Glucosyltransferases/metabolism , Lymnaea/enzymology , Oligosaccharides/metabolism , Prostate/enzymology , Animals , Carbohydrate Sequence , Chromatography, Agarose , Concanavalin A/chemistry , Electrophoresis, Polyacrylamide Gel , Fluorometry , Galactose/metabolism , Glucosyltransferases/isolation & purification , Glycoproteins/biosynthesis , Glycoproteins/isolation & purification , Male , Molecular Sequence Data , Polysaccharides/biosynthesis , Polysaccharides/metabolism , Prostate/metabolism , Substrate Specificity , Tritium
17.
FEBS Lett ; 463(1-2): 189-93, 1999 Dec 10.
Article in English | MEDLINE | ID: mdl-10601665

ABSTRACT

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex-type N-glycans from Arabidopsis thaliana, which contain R-GlcNAcbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-Asn (core alpha1-->3-Fuc) and Xylbeta1-->2Manbeta1-->4GlcNAcbeta1-R (core beta1-->2-Xyl) modifications, and honeybee phospholipase A2, which carries N-glycans that contain the core alpha1-->3-Fuc epitope. Evidence is presented that core alpha1-->3-fucosylated N-glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core alpha1-->3-Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N-glycans carrying core alpha1-->3-Fuc and beta1-->2-Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N-glycans containing the core alpha1-->3-Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.


Subject(s)
Epitopes/immunology , Fucose/immunology , Haemonchiasis/immunology , Haemonchus/chemistry , Immunoglobulin E/immunology , Polysaccharides/immunology , Animals , Arabidopsis/chemistry , Binding, Competitive , Blotting, Western , Caenorhabditis elegans/chemistry , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Fucose/chemistry , Haemonchus/immunology , Immunoglobulin E/blood , Immunoglobulin G/blood , Immunoglobulin G/immunology , Mice , Polysaccharides/chemistry , Schistosoma mansoni/chemistry , Sheep/immunology , Sheep/parasitology , Substrate Specificity
18.
Glycoconj J ; 16(2): 99-107, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10612410

ABSTRACT

Filamentous fungi are commonly used in the fermentation industry for large scale production of glycoproteins. Several of these proteins can be produced in concentrations up to 20-40 g per litre. The production of heterologous glycoproteins is at least one or two orders of magnitude lower but research is in progress to increase the production levels. In the past years the structure of protein-linked carbohydrates of a number of fungal proteins has been elucidated, showing the presence of oligo-mannosidic and high-mannose chains, sometimes with typical fungal modifications. A start has been made to engineer the glycosylation pathway in filamentous fungi to obtain strains that show a more mammalian-like type of glycosylation. This mini review aims to cover the current knowledge of glycosylation in filamentous fungi, and to show the possibilities to produce glycoproteins with these organisms with a more mammalian-like type of glycosylation for research purposes or pharmaceutical applications.


Subject(s)
Fungi/metabolism , Glycoproteins/biosynthesis , Recombinant Proteins/biosynthesis , Biotechnology , Carbohydrate Sequence , Glycoproteins/chemistry , Glycosylation , Humans , Molecular Sequence Data , Oligosaccharides/chemistry , Protein Engineering , Recombinant Proteins/chemistry
19.
FEBS Lett ; 461(3): 311-4, 1999 Nov 19.
Article in English | MEDLINE | ID: mdl-10567717

ABSTRACT

We have identified a core alpha1-->3-fucosyltransferase activity in the albumin and prostate glands of the snail Lymnaea stagnalis. Incubation of albumin gland extracts with GDP-[(14)C]Fuc and asialo/agalacto-glycopeptides from human fibrinogen resulted in a labeled product in 50% yield. Analysis of the product by 400 MHz (1)H-NMR spectroscopy showed the presence of a Fuc residue alpha1-->3-linked to the Asn-linked GlcNAc. Therefore, the enzyme can be identified as a GDP-Fuc:GlcNAc (Asn-linked) alpha1-->3-fucosyltransferase. The enzyme acts efficiently on asialo/agalacto-glycopeptides from both human fibrinogen and core alpha1-->6-fucosylated human IgG, whereas bisected asialo/agalacto-glycopeptide could not serve as an acceptor. We propose that the enzyme functions in the synthesis of core alpha1-->3-fucosylated complex-type glycans in L. stagnalis. Core alpha1-->3-fucosylation of the asparagine-linked GlcNAc of plant- and insect-derived glycoproteins is often associated with the allergenicity of such glycoproteins. Since allergic reactions have been reported after consumption of snails, the demonstration of core alpha1-->3-fucosylation in L. stagnalis may be clinically relevant.


Subject(s)
Fucosyltransferases/isolation & purification , Lymnaea/enzymology , Polysaccharides/biosynthesis , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Fibrinogen/metabolism , Food Hypersensitivity/etiology , Fucosyltransferases/metabolism , Glycopeptides/metabolism , Glycoproteins/chemistry , Glycoproteins/immunology , Humans , Immunoglobulin G/metabolism , Lymnaea/immunology , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Polysaccharides/chemistry , Polysaccharides/immunology
20.
FEBS Lett ; 459(3): 327-31, 1999 Oct 15.
Article in English | MEDLINE | ID: mdl-10526159

ABSTRACT

A considerable amount (approximately 1.6 microg from 1 mg of dried nematode) of non-sulfated chondroitin, two orders of magnitude less yet an appreciable amount of heparan sulfate, and no hyaluronate were found in Caenorhabditis elegans nematodes. The chondroitin chains were heterogeneous in size, being shorter than that of whale cartilage chondroitin sulfate. The disaccharide composition analysis of heparan sulfate revealed diverse sulfation including glucosamine 2-N-sulfation, glucosamine 6-O-sulfation and uronate 2-O-sulfation. These results imply that chondroitin and heparan sulfate are involved in fundamental biological processes.


Subject(s)
Caenorhabditis elegans/chemistry , Glycosaminoglycans/analysis , Animals , Chondroitin/isolation & purification , Heparitin Sulfate/isolation & purification , Magnetic Resonance Spectroscopy
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