Novel Transmembrane Receptor Involved in Phagosome Transport of Lysozymes and β-Hexosaminidase in the Enteric Protozoan Entamoeba histolytica

Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes

Novel transmembrane receptor involved in phagosome transport of lysozymes and β-hexosaminidase in the enteric protozoan Entamoeba histolytica

金沢大学医薬保健研究域薬学系Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes. © 2012 Furukawa et al

Cysteine protease-binding protein family 6 mediates the trafficking of amylases to phagosomes in the enteric protozoan entamoeba histolytica

金沢大学医薬保健研究域薬学系Phagocytosis plays a pivotal role in nutrient acquisition and evasion from the host defense systems in Entamoeba histolytica, the intestinal protozoan parasite that causes amoebiasis. We previously reported that E. histolytica possesses a unique class of a hydrolase receptor family, designated the cysteine protease-binding protein family (CPBF), that is involved in trafficking of hydrolases to lysosomes and phagosomes, and we have also reported that CPBF1 and CPBF8 bind to cysteine proteases or α-hexosaminidase β-subunit and lysozymes, respectively. In this study, we showed by immunoprecipitation that CPBF6, one of the most highly expressed CPBF proteins, specifically binds to β-amylase and γ-amylase. We also found that CPBF6 is localized in lysosomes, based on immunofluorescence imaging. Immunoblot and proteome analyses of the isolated phagosomes showed that CPBF6 mediates transport of amylases to phagosomes. We also demonstrated that the carboxyl-terminal cytosolic region of CPBF6 is engaged in the regulation of the trafficking of CPBF6 to phagosomes. Our proteome analysis of phagosomes also revealed new potential phagosomal proteins. © 2013, American Society for Microbiology

The giardial VPS35 retromer subunit is necessary for multimeric complex assembly and interaction with the Vacuolar protein sorting receptor

The retromer is a pentameric protein complex that mediates the retrograde transport of acid hydrolase receptors between endosomes and the trans-Golgi network and is conserved across all eukaryotes. Unlike other eukaryotes, the endomembrane system of Giardia trophozoite is simple and is composed only of the endoplasmic reticulum and peripheral vesicles (PVs), which may represent an ancient organellar system converging compartments such as early and late endosomes and lysosomes. Sorting and trafficking of membrane proteins and soluble hydrolases from the endoplasmic reticulum to the PVs have been described as specific and conserved but whether the giardial retromer participates in receptor recycling remains elusive. Homologs of the retromer Vacuolar Protein Sorting (Vps35p, Vps26p, and Vps29p) have been identified in this parasite. Cloning the GlVPS35 subunit and antisera production enabled the localization of this protein in the PVs as well as in the cytosol. Tagged expression of the subunits was used to demonstrate their association with membranes, and immunofluorescence confocal laser scanning revealed high degrees of colabeling between the retromer subunits and also with the endoplasmic reticulum and PV compartment markers. Protein-protein interaction data revealed interaction between the subunits of GlVPS35 and the cytosolic domain of the hydrolase receptor GlVps. Altogether our data provide original information on the molecular interactions that mediate assembly of the cargo-selective retromer subcomplex and its involvement in the recycling of the acid hydrolase receptor in this parasite.Fil: Miras, Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; ArgentinaFil: Merino, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; ArgentinaFil: Gottig Schor, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ropolo, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; ArgentinaFil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; Argentin

Secreted Phospholipase A2 Involvement in Neurodegeneration: Differential Testing of Prosurvival and Anti-Inflammatory Effects of Enzyme Inhibition

There is increased interest in the contribution of secreted phospholipase A2 (sPLA2) enzymes to neurodegenerative diseases. Systemic treatment with the nonapeptide CHEC-9, a broad spectrum uncompetitive inhibitor of sPLA2, has been shown previously to inhibit neuron death and aspects of the inflammatory response in several models of neurodegeneration. A persistent question in studies of sPLA2 inhibitors, as for several other anti-inflammatory and neuroprotective compounds, is whether the cell protection is direct or due to slowing of the toxic aspects of the inflammatory response. To further explore this issue, we developed assays using SY5Y (neuronal cells) and HL-60 (monocytes) cell lines and examined the effects of sPLA2 inhibition on these homogeneous cell types in vitro. We found that the peptide inhibited sPLA2 enzyme activity in both SY5Y and HL-60 cultures. This inhibition provided direct protection to SY5Y neuronal cells and their processes in response to several forms of stress including exposure to conditioned medium from HL-60 cells. In cultures of HL-60 cells, sPLA2 inhibition had no effect on survival of the cells but attenuated their differentiation into macrophages, with regard to process development, phagocytic ability, and the expression of differentiation marker CD36, as well as the secretion of proinflammatory cytokines TNF-α and IL-6. These results suggest that sPLA2 enzyme activity organizes a cascade of changes comprising both cell degeneration and inflammation, processes that could theoretically operate independently during neurodegenerative conditions. The effectiveness of sPLA2 inhibitor CHEC-9 may be due to its ability to affect both processes in isolation. Testing potential anti-inflammatory/neuroprotective compounds with these human cell lines and their conditioned media may provide a useful screening tool prior to in vivo therapeutic applications

VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for Retromer Interaction with Accessory Proteins

VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn2+ and Zn2+ in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies

Resolving the homology-function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology

With advances in DNA sequencing technology, it is increasingly common and tractable to informatically look for genes of interest in the genomic databases of parasitic organisms and infer cellular states. Assignment of a putative gene function based on homology to functionally characterized genes in other organisms, though powerful, relies on the implicit assumption of functional homology, i.e. that orthology indicates conserved function. Eukaryotes reveal a dazzling array of cellular features and structural organization, suggesting a concomitant diversity in their underlying molecular machinery. Significantly, examples of novel functions for pre-existing or new paralogues are not uncommon. Do these examples undermine the basic assumption of functional homology, especially in parasitic protists, which are often highly derived? Here we examine the extent to which functional homology exists between organisms spanning the eukaryotic lineage. By comparing membrane trafficking proteins between parasitic protists and traditional model organisms, where direct functional evidence is available, we find that function is indeed largely conserved between orthologues, albeit with significant adaptation arising from the unique biological features within each lineage

Comparison of hemolytic activity of the intermediate subunit of Entamoeba histolytica and Entamoeba dispar lectins

Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica has roles in pathogenicity and induction of protective immunity in rodent models of amoebiasis. Recently, the intermediate subunit of the lectin, Igl1, of E. histolytica has been shown to have hemolytic activity. However, the corresponding lectin is also expressed in a non-virulent species, Entamoeba dispar, and another subunit, Igl2, is expressed in the protozoa. Therefore, in this study, we compared the activities of Igl1 and Igl2 subunits from E. histolytica and E. dispar using various regions of recombinant Igl proteins expressed in Escherichia coli. The recombinant E. dispar Igl proteins had comparable hemolytic activities with those of E. histolytica Igl proteins. Furthermore, Igl1 gene-silenced E. histolytica trophozoites showed less hemolytic activity compared with vector-transfected trophozoites, indicating that the expression level of Igl1 protein influences the activity. These results suggest that the lower hemolytic activity in E. dispar compared with E. histolytica reflects the lower expression level of Igl1 in the E. dispar parasite