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Vivian Hook - One of the best experts on this subject based on the ideXlab platform.
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cysteine cathepsins in the Secretory vesicle produce active peptides cathepsin l generates peptide neurotransmitters and cathepsin b produces beta amyloid of alzheimer s disease
Biochimica et Biophysica Acta, 2012Co-Authors: Vivian Hook, Steven J Bark, Lydiane Funkelstein, Jill L Wegrzyn, Mark S Kindy, Gregory HookAbstract:Abstract Recent new findings indicate significant biological roles of cysteine cathepsin proteases in Secretory Vesicles for production of biologically active peptides. Notably, cathepsin L in Secretory Vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell–cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in Secretory Vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These Secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique Secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the Secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
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mass spectrometry based neuropeptidomics of Secretory Vesicles from human adrenal medullary pheochromocytoma reveals novel peptide products of prohormone processing
Journal of Proteome Research, 2010Co-Authors: Nitin Gupta, Steven J Bark, Laurent Taupenot, Daniel T Oconnor, Pavel A Pevzner, Vivian HookAbstract:Neuropeptides are required for cell-cell communication for the regulation of physiological and pathological processes. While selected neuropeptides of known biological activities have been studied, global analyses of the endogenous profile of human peptide products derived from prohormones by proteolytic processing in vivo is largely unknown. Therefore, this study utilized the global, unbiased approach of mass spectrometry-based neuropeptidomics to define peptide profiles in Secretory Vesicles, isolated from human adrenal medullary pheochromocytoma of the sympathetic nervous system. The low molecular weight pool of Secretory vesicle peptides was subjected to nano-LC-MS/MS with ion trap and QTOF mass spectrometry analyzed by different database search tools (InsPecT and Spectrum Mill). Peptides were generated by processing of prohormones at dibasic cleavage sites as well as at non-basic residues. Significantly, peptide profiling provided novel insight into newly identified peptide products derived from proenkephalin, pro-NPY, proSAAS, CgA, CgB, and SCG2 prohormones. Previously unidentified intervening peptide domains of prohormones were observed, thus, providing new knowledge of human neuropeptidomes generated from precursors. The global peptidomic approach of this study demonstrates the complexity of diverse neuropeptides present in human Secretory Vesicles for cell-cell communication.
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proteolytic fragments of chromogranins a and b represent major soluble components of chromaffin granules illustrated by two dimensional proteomics with nh 2 terminal edman peptide sequencing and maldi tof ms
Biochemistry, 2009Co-Authors: Jean C Lee, Vivian HookAbstract:Neuroendocrine chromaffin granules of adrenal medulla represent regulated Secretory Vesicles that secrete neuropeptides and catecholamines which mediate cell-cell communication for physiological functions. This study addressed the identification of the major proteins in these Secretory Vesicles that provide dynamic storage and secretion of bioactive molecules. Proteins of the soluble compartment of the Vesicles were separated by two-dimensional gels and subjected to NH(2)-terminal Edman sequencing for identification and determination of NH(2)-termini. Results showed that proteolytic fragments of chromogranin A (CgA) and chromogranin B (CgB) represent the major proteins of these Secretory Vesicles. These fragments resulted from cleavage of their respective precursor proteins at dibasic and monobasic sites, which is consistent with the known cleavage specificities of prohormone processing enzymes. MALDI-TOF MS analyses of protein spots similar in molecular weight that possessed a range of pI values were represented by molecular forms of CgA and CgB proteins. These findings indicate the high prevalence of endogenous CgA and CgB proteolytic fragments that function in chromaffin Secretory Vesicles for release of bioactive molecules for cell-cell communication.
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proteomics of neuroendocrine Secretory Vesicles reveal distinct functional systems for biosynthesis and exocytosis of peptide hormones and neurotransmitters
Journal of Proteome Research, 2007Co-Authors: Jill L Wegrzyn, John M Neveu, William S Lane, Jean Lee, Vivian HookAbstract:Regulated Secretory Vesicles produce, store, and secrete active peptide hormones and neurotransmitters that function in cell-cell communication. To gain knowledge of the protein systems involved in such Secretory vesicle functions, we analyzed proteins in the soluble and membrane fractions of dense core Secretory Vesicles purified from neuroendocrine chromaffin cells. Soluble and membrane fractions of these Vesicles were subjected to SDS-PAGE separation, and proteins from systematically sectioned gel lanes were identified by microcapillary LC-MS/MS (microLC-MS/MS) of tryptic peptides. The identified proteins revealed functional categories of prohormones, proteases, catecholamine neurotransmitter metabolism, protein folding, redox regulation, ATPases, calcium regulation, signaling components, exocytotic mechanisms, and related functions. Several novel Secretory vesicle components involved in proteolysis were identified consisting of cathepsin B, cathepsin D, cystatin C, ubiquitin, and TIMP, as well carboxypeptidase E/H and proprotein convertases that are known to participate in prohormone processing. Significantly, the membrane fraction exclusively contained an extensive number of GTP nucleotide-binding proteins related to Rab, Rho, and Ras signaling molecules, together with SNARE-related proteins and annexins that are involved in trafficking and exocytosis of Secretory vesicle components. Membranes also preferentially contained ATPases that regulate proton translocation. These results implicate membrane-specific functions for signaling and exocytosis that allow these Secretory Vesicles to produce, store, and secrete active peptide hormones and neurotransmitters released from adrenal medulla for the control of physiological functions in health and disease. In summary, this proteomic study illustrates Secretory vesicle protein systems utilized for the production and secretion of regulatory factors that control neuroendocrine functions.
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Secretory vesicle aminopeptidase b related to neuropeptide processing molecular identification and subcellular localization to enkephalin and npy containing chromaffin granules
Journal of Neurochemistry, 2007Co-Authors: Shinrong Hwang, Thierry Foulon, Vivian Hook, Audrey K Oneill, Steven J BarkAbstract:Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by Secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH2-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in Secretory Vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in ‘model’ neuropeptide-containing Secretory Vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in Secretory Vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5–6.5 that is similar to the internal pH of Secretory Vesicles. The significant finding of the Secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.
John Samuelson - One of the best experts on this subject based on the ideXlab platform.
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chitinase secretion by encysting entamoeba invadens and transfected entamoeba histolytica trophozoites localization of Secretory Vesicles endoplasmic reticulum and golgi apparatus
Infection and Immunity, 1999Co-Authors: Sudip Kumar Ghosh, Jessica Field, Marta Frisardi, Benjamin M Rosenthal, Rick A Rogers, John SamuelsonAbstract:Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small Secretory Vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small Secretory Vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few Vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear Vesicles, was identified with antibodies to ADP-ribosylating factor and to ɛ-COP. We conclude that the amebic Secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.
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chitinase secretion by encysting entamoeba invadens and transfected entamoeba histolytica trophozoites localization of Secretory Vesicles endoplasmic reticulum and golgi apparatus
Infection and Immunity, 1999Co-Authors: Sudip Kumar Ghosh, Jessica Field, Marta Frisardi, Benjamin M Rosenthal, Rick A Rogers, Zhiming Mai, John SamuelsonAbstract:Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small Secretory Vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small Secretory Vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few Vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear Vesicles, was identified with antibodies to ADP-ribosylating factor and to epsilon-COP. We conclude that the amebic Secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.
Niels Borregaard - One of the best experts on this subject based on the ideXlab platform.
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extracellular superoxide dismutase is present in Secretory Vesicles of human neutrophils and released upon stimulation
Free Radical Biology and Medicine, 2016Co-Authors: Marie B Iversen, Jan J Enghild, Randi H Gottfredsen, Ulrike G Larsen, Jeppe Praetorius, Niels Borregaard, Steen V PetersenAbstract:Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within Secretory Vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When Secretory Vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils.
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human neutrophil granules and Secretory Vesicles
European Journal of Haematology, 2009Co-Authors: Niels Borregaard, Lars Kjeldsen, Karsten Lollike, H Sengelov, Morten H Nielsen, Lone Bastholm, Dorothy F BaintonAbstract:Abstract: The traditional classification of neutrophil granules as peroxidase-positive (azurophil, or primary) and peroxidase-negative (specific or secondary) has proven to be too simple to explain the differential exocytosis of granule proteins and incorporation of granule membrane into the plasma membrane which is an important aspect of neutrophil activation. Combined subcellular fractionation and immunoelectron microscopy has revealed heterogeneity among both peroxidase-positive and peroxidase-negative granules with regard to their content, mobilization and time of formation. Peroxidase-negative granules may be classified according to their content of lactoferrin and gelatinase: 15% of peroxidase-negative granules contain lactoferrin, but no gelatinase. 60% contain both lactoferrin and gelatinase. The term specific or secondary granule should be reserved for these two subsets. In addition, 25% of peroxidase-negative granules contain gelatinase but no lactoferrin. These should be termed gelatinase granules or tertiary granules. Gelatinase granules are formed later than specific granules and mobilized more readily. In addition, a distinct, highly mobilizable intracellular compartment, the Secretory vesicle, has now been recognized as an important store of surface membrane-bound receptors. This compartment is formed in band cells and segmented cells by endocytosis. This heterogeneity among the neutrophil granules is of functional significance, and may also be reflected in the dysmaturation which is an important feature of myeloproliferative and myelodysplastic disorders.
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neutrophil granules and Secretory Vesicles in inflammation
Microbes and Infection, 2003Co-Authors: Mikkel Faurschou, Niels BorregaardAbstract:The neutrophil is a major effector cell of innate immunity. Exocytosis of granules and Secretory Vesicles plays a pivotal role in most neutrophil functions from early activation to the destruction of phagocytosed microorganisms. Neutrophil granules contain a multitude of antimicrobial and potentially cytotoxic substances that are delivered to the phagosome or to the exterior of the cell following degranulation. This review summarises current knowledge of granule biology and highlights the effects of neutrophil degranulation in the acute inflammatory response.
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mobilization of granules and Secretory Vesicles during in vivo exudation of human neutrophils
Journal of Immunology, 1995Co-Authors: Henrik Sengelov, Claes Dahlgren, Per Follin, Lars Kjeldsen, Karsten Lollike, Niels BorregaardAbstract:The extent of mobilization of four different intracellular compartments was measured during in vivo exudation of neutrophils into skin chambers and compared with resting neutrophils obtained from blood. Exudation of neutrophils induced increased surface expression of alkaline phosphatase, complement receptor 1, and Mac-1, and a complete loss of L-selectin. The increase in the content of surface molecules in the plasma membrane is in accordance with complete mobilization of Secretory Vesicles. Granule matrix proteins were secreted into the chamber fluid by the exudated neutrophils and the exocytosed proteins were recovered in the skin chamber fluid. Release of gelatinase from gelatinase granules was 38.1%, lactoferrin release from specific granules was 21.9%, and myeloperoxidase release from azurophil granules was 7.0%, clearly illustrating a hierarchy in mobilization among granules. When exudate neutrophils were stimulated with FMLP, additional mobilization of granules was observed and the rank order regarding release was preserved. This is the first report to evaluate the mobilization of Secretory Vesicles during in vivo exudation of human neutrophils. It is shown that Secretory Vesicles are regulated exocytotic Vesicles that are fully mobilized during in vivo exudation. Once exocytosed, Secretory Vesicles are not re-formed within a period of 6 h.
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separation of human neutrophil plasma membrane from intracellular Vesicles containing alkaline phosphatase and nadph oxidase activity by free flow electrophoresis
Journal of Biological Chemistry, 1992Co-Authors: H Sengelov, Morten H Nielsen, Niels BorregaardAbstract:Abstract A putative reservoir of functional plasma membrane proteins, the Secretory vesicle identified by latent alkaline phosphatase and tetranectin, has previously been demonstrated based on indirect evidence (Borregaard, N., Miller, L. J., and Springer, T. A. (1987) Science 237, 1204-1206; Borregaard, N., Christensen, L., Bjerrum, O. W., Birgens, H. S., and Clemmesen, I. (1990) J. Clin. Invest. 85, 408-416). Difficulties in separating plasma membranes from this entity by density gradient centrifugation has prohibited discriminative dynamic and quantitative studies of Secretory Vesicles and plasma membranes. By combining density centrifugation with free flow electrophoresis we overcame this obstacle. Freshly prepared unperturbed human neutrophils were subjected to nitrogen cavitation followed by density centrifugation on Percoll gradients. Light membrane fractions containing plasma membranes and Secretory Vesicles were applied to high voltage free flow electrophoresis on an Elphor VaP 22. Plasma membrane Vesicles, identified by HLA class I antigen mixed enzyme-linked immunosorbent assay (Bjerrum, O. W., and Borregaard, N. (1990) Scand. J. Immunol. 31, 305-313) and 125I applied to cells before cavitation, were clearly separated from Secretory Vesicles. Electron microscopy revealed a morphology typical of plasma membranes in the former fraction and a population of Vesicles with markedly different appearance in the latter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles demonstrated distinct differences in protein patterns between the two fractions. Superoxide generating capacity induced by sodium dodecyl sulfate and cytosol, an entity traditionally ascribed to the plasma membrane, was largely confined to fractions containing Secretory Vesicles. Thus, the majority of membrane-bound NADPH oxidase components of light membranes of human neutrophils colocalize with Secretory Vesicles.
Sudip Kumar Ghosh - One of the best experts on this subject based on the ideXlab platform.
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chitinase secretion by encysting entamoeba invadens and transfected entamoeba histolytica trophozoites localization of Secretory Vesicles endoplasmic reticulum and golgi apparatus
Infection and Immunity, 1999Co-Authors: Sudip Kumar Ghosh, Jessica Field, Marta Frisardi, Benjamin M Rosenthal, Rick A Rogers, John SamuelsonAbstract:Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small Secretory Vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small Secretory Vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few Vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear Vesicles, was identified with antibodies to ADP-ribosylating factor and to ɛ-COP. We conclude that the amebic Secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.
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chitinase secretion by encysting entamoeba invadens and transfected entamoeba histolytica trophozoites localization of Secretory Vesicles endoplasmic reticulum and golgi apparatus
Infection and Immunity, 1999Co-Authors: Sudip Kumar Ghosh, Jessica Field, Marta Frisardi, Benjamin M Rosenthal, Rick A Rogers, Zhiming Mai, John SamuelsonAbstract:Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small Secretory Vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small Secretory Vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few Vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear Vesicles, was identified with antibodies to ADP-ribosylating factor and to epsilon-COP. We conclude that the amebic Secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.
Benjamin M Rosenthal - One of the best experts on this subject based on the ideXlab platform.
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chitinase secretion by encysting entamoeba invadens and transfected entamoeba histolytica trophozoites localization of Secretory Vesicles endoplasmic reticulum and golgi apparatus
Infection and Immunity, 1999Co-Authors: Sudip Kumar Ghosh, Jessica Field, Marta Frisardi, Benjamin M Rosenthal, Rick A Rogers, John SamuelsonAbstract:Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small Secretory Vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small Secretory Vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few Vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear Vesicles, was identified with antibodies to ADP-ribosylating factor and to ɛ-COP. We conclude that the amebic Secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.
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chitinase secretion by encysting entamoeba invadens and transfected entamoeba histolytica trophozoites localization of Secretory Vesicles endoplasmic reticulum and golgi apparatus
Infection and Immunity, 1999Co-Authors: Sudip Kumar Ghosh, Jessica Field, Marta Frisardi, Benjamin M Rosenthal, Rick A Rogers, Zhiming Mai, John SamuelsonAbstract:Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small Secretory Vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small Secretory Vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few Vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear Vesicles, was identified with antibodies to ADP-ribosylating factor and to epsilon-COP. We conclude that the amebic Secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.
