PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-8 (8)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Echinococcus granulosus Antigen B binds to monocytes and macrophages modulating cell response to inflammation 
Parasites & Vectors  2016;9:69.
Background
Antigen B (EgAgB) is an abundant lipoprotein released by the larva of the cestode Echinococcus granulosus into the host tissues. Its protein moiety belongs to the cestode-specific family known as hydrophobic ligand binding protein (HLBP), and is encoded by five gene subfamilies (EgAgB8/1-EgAgB8/5). The functions of EgAgB in parasite biology remain unclear. It may play a role in the parasite’s lipid metabolism since it carries host lipids that E. granulosus is unable to synthesise. On the other hand, there is evidence supporting immuno-modulating activities in EgAgB, particularly on innate immune cells. Both hypothetical functions might involve EgAgB interactions with monocytes and macrophages, which have not been formally analysed yet.
Methods
EgAgB binding to monocytes and macrophages was studied by flow cytometry using inflammation-recruited peritoneal cells and the THP-1 cell line. Involvement of the protein and phospholipid moieties in EgAgB binding to cells was analysed employing lipid-free recombinant EgAgB subunits and phospholipase D treated-EgAgB (lacking the polar head of phospholipids). Competition binding assays with plasma lipoproteins and ligands for lipoprotein receptors were performed to gain information about the putative EgAgB receptor(s) in these cells. Arginase-I induction and PMA/LPS-triggered IL-1β, TNF-α and IL-10 secretion were examined to investigate the outcome of EgAgB binding on macrophage response.
Results
Monocytes and macrophages bound native EgAgB specifically; this binding was also found with lipid-free rEgAgB8/1 and rEgAgB8/3, but not rEgAgB8/2 subunits. EgAgB phospholipase D-treatment, but not the competition with phospholipid vesicles, caused a strong inhibition of EgAgB binding activity, suggesting an indirect contribution of phospholipids to EgAgB-cell interaction. Furthermore, competition binding assays indicated that this interaction may involve receptors with affinity for plasma lipoproteins. At functional level, the exposure of macrophages to EgAgB induced a very modest arginase-I response and inhibited PMA/LPS-mediated IL-1β and TNF-α secretion in an IL-10-independent manner.
Conclusion
EgAgB and, particularly its predominant EgAgB8/1 apolipoprotein, are potential ligands for monocyte and macrophage receptors. These receptors may also be involved in plasma lipoprotein recognition and induce an anti-inflammatory phenotype in macrophages upon recognition of EgAgB.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-016-1350-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s13071-016-1350-7
PMCID: PMC4743400  PMID: 26846700
Echinococcus granulosus; Antigen B; HLBP; Cell binding; Lipoproteins; Monocytes; Macrophages
2.  Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus 
Biochemical Journal  2015;471(Pt 3):403-414.
Necator americanus fatty acid and retinol-binding protein-1 (Na-FAR-1) is an abundantly expressed FAR from a parasitic hookworm. The present work describes its tissue distribution, structure and ligand-binding characteristics and shows that Na-FAR-1 expands to transport multiple FA molecules in its internal cavity.
Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein–ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male.
doi:10.1042/BJ20150068
PMCID: PMC4613501  PMID: 26318523
fatty acid-binding protein; Necator americanus; nematode; nuclear magnetic resonance (NMR); parasite; protein structure; retinol-binding protein; X-ray
3.  Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy 
Mediators of Inflammation  2015;2015:738563.
Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa.
doi:10.1155/2015/738563
PMCID: PMC4540995  PMID: 26346822
4.  Lipid-Free Antigen B Subunits from Echinococcus granulosus: Oligomerization, Ligand Binding, and Membrane Interaction Properties 
PLoS Neglected Tropical Diseases  2015;9(3):e0003552.
Background
The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied.
Methodology/Principal Findings
Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles.
Conclusions/Significance
We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.
Author Summary
Echinococcus granulosus is a causative agent of hydatidosis, a parasitic disease that affects humans and livestock with significant economic and public health impact worldwide. Antigen B (EgAgB), an abundant product of E. granulosus larvae, is a lipoprotein that carries a wide variety of lipids, including fatty acids and cholesterol. As E. granulosus is unable to synthesize these lipids, EgAgB likely plays an important role in parasite metabolism, participating in both the acquisition of host lipids and their distribution between parasite tissues. The protein component of EgAgB consists of 8 kDa subunits encoded by separate genes. However, the biochemical properties of EgAgB subunits, particularly their ability to bind and transfer lipids, are poorly known. Herein, using in vitro assays, we found that EgAgB subunits were capable of oligomerizing in the absence of lipids and to bind fatty acids, but not cholesterol. Moreover, EgAgB subunits showed the ability to transfer fatty acids to artificial phospholipid membranes. These results indicate new points of attack at which the parasite might be vulnerable to drugs.
doi:10.1371/journal.pntd.0003552
PMCID: PMC4358968  PMID: 25768648
5.  Resonance assignment of As-p18, a fatty acid binding protein secreted by developing larvae of the parasitic nematode Ascaris suum 
Biomolecular Nmr Assignments  2012;8(1):33-36.
As-p18 is produced and secreted by larvae of the parasitic nematode Ascaris suum as they develop within their eggs. The protein is a member of the fatty acid binding protein (FABP) family found in a wide range of eukaryotes, but is distinctive in that it is secreted from the synthesizing cell and has predicted additional structural features not previously seen in other FABPs. As-p18 and similar proteins found only in nematodes have therefore been designated ‘nemFABPs’. Sequence-specific 1H, 13C and 15N resonance assignments were established for the 155 amino acid recombinant protein (18.3 kDa) in complex with oleic acid, using a series of three-dimensional triple-resonance heteronuclear NMR experiments. The secondary structure of As-p18 is predicted to be very similar to other FABPs, but the protein has extended loops that have not been observed in other FABPs whose structures have so far been solved.
doi:10.1007/s12104-012-9447-1
PMCID: PMC3955487  PMID: 23225165
As-p18; Fatty acid binding protein; nemFABP; Nematode; Parasite; Ascaris suum
6.  1H, 13C and 15N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus 
Biomolecular Nmr Assignments  2012;8(1):19-21.
The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific 1H, 13C and 15N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3 % of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9 % of backbone resonances were identified, while 97.7 % assignment of amino acid sidechain protons is complete. All Hα(166), Hβ(250) and Hγ(160) and 98.4 % of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4 % Cα (154 out of 155) and 99.3 % Cβ (143 out of 144) resonances have been assigned. No resonances were observed for the NHn groups of R93 NεHε, arginine, Nη1H2, Nη2H2, histidine Nδ1Hδ1, Nε1Hε1 and lysine Nζ3H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix.
doi:10.1007/s12104-012-9444-4
PMCID: PMC3955486  PMID: 23179061
Parasitic nematode; Necator americanus; Fatty-acid and retinol-binding protein; Na-FAR-1; NMR
7.  Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline 
As-p18, an unusual fatty-acid-binding protein from a parasitic nematode, was expressed in bacteria, purified and crystallized. The use of a microfocus beamline was essential for data collection.
As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum. Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystals were obtained, but initial diffraction data analysis revealed that they were composed of a number of ‘microdomains’. Interpretable data could only be collected using a microfocus beamline with a beam size of 12 × 8 µm.
doi:10.1107/S1744309112026553
PMCID: PMC3412778  PMID: 22869127
fatty-acid-binding proteins; parasitic nematodes; Ascaris suum; microfocus beamlines
8.  Interaction of Enterocyte FABPs with Phospholipid Membranes: Clues for Specific Physiological Roles 
Biochimica et biophysica acta  2011;1811(7-8):452-459.
Intestinal and liver fatty acid binding proteins (IFABP and LFABP, respectively), are cytosolic soluble proteins with the capacity to bind and transport hydrophobic ligands between different sub-cellular compartments. Their functions are still not clear but they are supposed to be involved in lipid trafficking and metabolism, cell growth, and regulation of several other processes, like cell differentiation. Here we investigated the interaction of these proteins with different models of phospholipid membrane vesicles in order to achieve further insight into their specificity within the enterocyte. A combination of biophysical and biochemical techniques allowed us to determine affinities of these proteins to membranes, the way phospholipid composition and vesicle size and curvature modulate such interaction, as well as the effect of protein binding on the integrity of the membrane structure. We demonstrate here that, beside their apparently opposite ligand transfer mechanisms, both LFABP and IFABP are able to interact with phospholipid membranes, but the factors that modulate such interactions are different for each protein, further implying different roles for IFABP and LFABP in the intracellular context. These results contribute to the proposed central role of intestinal FABPs in the lipid traffic within enterocytes as well as in the regulation of more complex cellular processes.
doi:10.1016/j.bbalip.2011.04.005
PMCID: PMC3143005  PMID: 21539932
Fatty Acid Binding Proteins; Membrane Interaction; Intracellular Fatty Acid Traffic; Model Membranes

Results 1-8 (8)