Secretion Of Lysosomal Hydrolases Research Articles

A lysosomal hydrolase receptor, CPBF2, is associated with motility and invasion of the enteric protozoan parasite Entamoeba histolytica

Proper targeting and secretion of lysosomal hydrolases are regulated by transporting receptors. Entamoeba histolytica, the enteric protozoan parasite responsible for human amebiasis, has a unique family of lysosomal hydrolase receptors, cysteine protease binding protein family, CPBF. CPBFs, consisting of 11 members with conserved domain organization, bind to a wide range of cargos including cysteine proteases and glycosidases, which are also known to be involved in pathogenesis of this parasite. In this study, we characterized one of CPBFs, CPBF2, which is involved in cell motility and extracellular matrix invasion. Unexpectedly, these roles of CPBF were not related to its cargo, α-amylase. This is the first demonstration that a putative hydrolase receptor is involved in cell motility and invasion in parasitic protozoa.

Clathrin heavy chain is required for spore cell but not stalk cell differentiation in Dictyostelium discoideum.

Previous studies of a clathrin-minus Dictyostelium cell line revealed important roles for clathrin heavy chain (clathrin) in endocytosis, secretion of lysosomal hydrolases and osmoregulation. In this paper, we examine the contribution of clathrin-mediated membrane traffic to development in Dictyostelium discoideum. Clathrin-minus cells were delayed in early development. When exposed to starvation conditions, clathrin-minus cells streamed and aggregated more slowly than wild-type cells. Although clathrin-minus cells displayed only 40% the level of extracellular cyclic AMP binding normally found in wild-type cells, they responded chemotactically to extracellular cyclic AMP. Clathrin-minus cells down-regulated cyclic AMP receptors, but only to half the extent of wild-type cells. We found that the extent of development of clathrin-minus cells was variable and influenced by environmental conditions. Although the mutant cells always progressed beyond the tipped mound stage, the final structure varied from a finger-like projection to a short, irregular fruiting body. Microscopic examination of these terminal structures revealed the presence of intact stalks but a complete absence of spores. Clathrin-minus cells expressed prestalk (ecmA and ecmB) and prespore (psA and cotB) genes normally, but were blocked in expression of the sporulation gene spiA. Using clathrin-minus cells that had been transformed with various promoter-lacZ reporter constructs, we saw only partial sorting of clathrin-minus prestalk and prespore cells. Even when mixed with wild-type cells, clathrin-minus cells failed to sort correctly and never constructed functional spores. These results suggest three roles for clathrin during Dictyostelium development. First, clathrin increases the efficiency of early development. Second, clathrin enables proper and efficient patterning of prestalk and prespore cells during culmination. Third, clathrin is essential for differentiation of mature spore cells.

Cloning and characterization of seven novel Dictyostelium discoideum rac-related genes belonging to the rho family of GTPases

Cellular processes including proliferation, organization of the actin cytoskeleton, vesicular traffic and secretion of proteins comprising the lysosomal/endosomal system are regulated by low-molecular-weight GTP-binding proteins of the Ras superfamily. However, to date only three Dictyostelium discoideum ras-like genes and two ypt-1/sec4-like genes have been identified and characterized. We report here the identification (using an oligodeoxyribonucleotide probe) of seven additional cDNAs coding for members highly related to the Rac proteins (Ras-related-C3 botulinum toxin substrate) which belong to the Rho (Ras homologous) family of GTPases. Three of these rac-related genes ( rac1A, rac1B and rac1C) predict proteins with > 90% amino acid (aa) sequence identity with each other and > 80% identity to the human rac1 gene product, whereas the other members ( racA, racB, racC and racD) predict proteins with 46–74% identity to the rac1 and rhoA gene products and to each other. The D. discoideum proteins were entirely conserved over the four regions known to be important for GTP binding and all contained the C-terminal CAAX aa motifs shared by other Rho proteins. Interestingly, the D. discoideum rac-related genes revealed unique patterns of expression during growth and development. For instance, the steady-state level of rac1 mRNA, encoded by three highly related genes, increased transiently during aggregation and then rapidly decreased. In contrast, the cellular abundance of mRNAs encoded by the other rac-like genes decreased at different rates and to different levels during development from the peak levels observed during growth. This suggests that the GTP-binding proteins encoded by these genes may play unique roles during the different stages of the D. discoideum life cycle. This study forms the foundation of future investigations to determine the role of these small GTPases in cell proliferation, organization of the actin cytoskeleton, vesicular trafficking during growth and development, and secretion of lysosomal hydrolases.

Genetic characterization of the secretory mutant MS-1 of Tetrahymena thermophila: vacuolarization and block in secretion of lysosomal hydrolases are caused by a single gene mutation.

The genetics and phenotypic features (light and electron microscopy) of a secretory mutant, MS-1 of Tetrahymena thermophila blocked in secretion of lysosomal acid hydrolases have been analyzed. Although blocked constitutively in secretion, MS-1 contains active lysosomal hydrolases in amounts equivalent to the wild type. The 3:1 segregation in F-2 in sib crosses and the 1:1 segregation in test crosses indicate that the block in secretion of lysosomal hydrolases is controlled by a recessive single gene locus termed sec. The sec allele of MS-1 proved also to be responsible for the highly vacuolarized phenotype the mutant developed when it was transferred from nutrient medium into buffers of low ionic strength. Deletion mapping by crossing MS-1 with nullisomic strains, all secreting lysosomal hydrolases at wild-type rates, was performed. The sec phenotype was expressed in monosomic-4 progeny only, indicating that the sec allele is located on chromosome 4 of T. thermophila.

Secretion of lysosomal hydrolases by cultured human amnion epithelial cells

Primary microcultures of human amnion epithelial cells were established, starting from sterile term placentae. Over a period of 1 week in culture, the epithelial cells release into the extracellular medium substantial amounts of some lysosomal hydrolases, such as sphingomyelinase, N-acetyl-β-glucosaminidase, α-fucosidase, β-glucuronidase, α-mannosidase, and arylsulfatase. Judging from experiments conducted with the protein synthesis inhibitor, cycloheximide, the enzymes released are not newly synthesized forms, but very likely derive from lysosomes. The constitutive secretion of lysosomal enzymes, coupled with lack of immunogenicity, makes amnion epithelial cells a convenient source of enzymes for implantation in attempts of enzyme replacement therapies.

Biosythesis Processing, and Intracellular Transport of Lysosomal Acid Phosphatase in Rat Hepatocytes1

The biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloroquine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1+2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultaneously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of macrophage lysosomal hydrolase synthesis and secretion by β-1,3-glucan

Studies were undertaken to elucidate the active component in zymosan necessary to induce the delayed-onset synthesis and secretion of representative lysosomal hydrolases, hexosaminidase, and β-glucuronidase in macrophages. Resident mouse peritoneal macrophages were challenged with zymosan particles and particulate β-1,3-glucan, the major subcomponent of zymosan. Zymosan was found to induce a rapid secretion of preformed hexosaminidase with maximal release (75%) occurring 6 hr after the addition of zymosan. By contrast, β-1,3-glucan was totally inactive in this respect. However, both zymosan and β-1,3-glucan were found to induce the delayed-onset synthesis and secretion of hexosaminidase and β-glucuronidase while maintaining constant cellular enzyme levels over a 5-day period following the addition of stimulus. These late responses were almost totally blocked by a noncytolytic concentration of cycloheximide, indicating their dependence on de novo protein synthesis. Mannan, the second major subcomponent of zymosan, had no effect on either immediate secretion or delayed-onset synthesis and secretion of hexosaminidase. These results suggest that the induction of the delayed-onset synthesis and secretion of the lysosomal hydrolases by zymosan may be dependent on the glucan subcomponent of zymosan. Moreover, it would also appear that the release of preformed lysosomal enzymes is not the trigger for the delayed-onset synthesis and secretion of hexosaminidase.

Mechanisms of mediator release from neutrophils.

The encounter of neutrophils with immune complexes and complement components - in the bulk phase or on a surface - leads to their secretion of lysosomal hydrolases, especially neutral proteases, which provoke tissue injury. Secretion of lysosomal enzymes and generation of reactive oxygen species (e.g., O2-. generation are stimulus-specific and can be dissected to establish cause and effect relationships by means of: a) kinetic analysis, b) variations in the stimulus, and c) use of impermeant reagents to block discrete responses. Neutrophils also generate products of 11-cyclooxygenase (e.g., PGE2, TxA2) and of the 5- and 15-lipoxygenases (mono-, di-, and tri-HETEs, LTB4, and their isomers). But the cyclooxygenase products (save TxA2) are not phlogistic by themselves: they inhibit the functions of neutrophils, platelets, macrophages, and mast cells. The most potent pro-inflammatory agent yet identified as a product of arachidonate is LTB4. LTB4 is a potent Ca ionophore, constricts airways, is a potent chemoattractant, and induces local inflammation.

Neutrophils: release of mediators of inflammation with special reference to rheumatoid arthritis.

The encounter of neutrophils with immune complexes and complement components, in the bulk phase or on a surface, leads to their secretion of lysosomal hydrolases, especially neutral proteases, which provoke tissue injury. Secretion of lysosomal enzymes and generation of reactive oxygen species (e.g., O(2)) is part of a stimulus-secretion response to a variety of secretagogues, including immune complexes and complement components. However, the pathways of secretion and O(2) generation are stimulus-specific and can be dissected to establish cause and effect relationships by means of: a) kinetic analysis, b) variations in the stimulus, and c) use of impermeant reagents to block discrete responses. Neutrophils also generate products of 11-cyclooxygenase (e.g., PGE2, TxA2) and of the 5-and 15-lipoxygenases (mono-, di-, and tri-HETEs, LTB4, and their isomers). But the cyclooxygenase products (save TxA2) are not phlogistic by themselves: they inhibit the functions of neutrophils, platelets, macrophages, and mast cells. The most potent pro-inflammatory agent yet identified as a product of arachidonate is LTB4. LTB4 is a potent Ca ionophore, constricts airways, is a potent chemoattractant, and induces local inflammation.

Die Sekretion von Mediatoren der Entzündung und der antimikrobiellen Abwehr durch Makrophagen

Macrophages are highly differentiated mononuclear phagocytes which originate from stem cells of the bone marrow. The secretory potential of these cells has been recognized in recent years. Major secretory products comprise lysosomal enzymes, complement proteins, prostaglandins and interferon. Secretion of lysosomal hydrolases and proteinases is most prominent in macrophages stimulated in vivo or in vitro (Fig. 4). Lysosomal enzyme secretion may be an important factor in the induction and maintenance of inflammatory reactions. Complement (C) proteins secreted by macrophages belong to the classical activation unit (C1, C4 and C2), alternative activation unit (C3, B, D, P) and to the group of delayed-acting C proteins (Fig. 7). Therefore macrophages produce at local sites the C component C3 from which biologically active C3 fragments (C3a, C3b, C3e) can be generated. These C3 fragments mediate inflammatory and cytotoxic reactions and also promote phagocytic processes (Fig. 6). Cleavage of secreted C3 into the active fragments may occur by enzymes derived from both C activation units or by secreted lysosomal proteinases (Fig. 8). Stimulated macrophages also synthesize and release prostaglandins. These compounds which have inflammatory as well as antiinflammatory effects (Fig. 12) may play an important regulatory role in inflammatory processes. Interferon has been also recognized as a secretory product of macrophages. This substance supports antimicrobial resistance by its phagocytosis-increasing effect and its antiviral activity. The secretory function of macrophages as well as the biological effects of secreted mediators are highly susceptible to modulation. Thus, C3 fragments stimulate the secretion of lysosomal enzymes (Fig. 6) whereas prostaglandins inhibit their release (Fig. 12). The inflammatory reactions induced by lysosomal enzymes may be further increased by the generation of C3b which stimulates additional lysosomal enzyme release (Fig. 4). These and other examples suggest that endogenous control mechanisms may have a strong influence on the secretory function of macrophages as well as on the biological activity of secreted mediators.