ABSTRACT
Zebrin II/aldolase C expression in the normal cerebellum is restricted to a Purkinje cell subset and is the canonical marker for stripes and zones. This spatial restriction has been confirmed in over 30 species of mammals, birds, fish, etc. In a transgenic mouse model in which the Neurogenin 2 gene has been disrupted (Neurog2-/-), the cerebellum is smaller than normal and Purkinje cell dendrites are disordered, but the basic zone and stripe architecture is preserved. Here, we show that in the Neurog2-/- mouse, in addition to the normal Purkinje cell expression, zebrin II is also expressed in a population of cells with a morphology characteristic of microglia. This identity was confirmed by double immunohistochemistry for zebrin II and the microglial marker, Iba1. The expression of zebrin II in cerebellar microglia is not restricted by zone or stripe or lamina. A second zone and stripe marker, PLCß4, does not show the same ectopic expression. When microglia are compared in control vs. Neurog2-/- mice, no difference is seen in apparent number or distribution, suggesting that the ectopic zebrin II immunoreactivity in Neurog2-/- cerebellum reflects an ectopic expression rather than the invasion of a new population of microglia from the periphery. This ectopic expression of zebrin II in microglia is unique as it is not seen in numerous other models of cerebellar disruption, such as in Acp2-/- mice and in human pontocerebellar hypoplasia. The upregulation of zebrin II in microglia is thus specific to the disruption of Neurog2 downstream pathways, rather than a generic response to a cerebellar disruption.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/deficiency , Cerebellum/metabolism , Microglia/metabolism , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/metabolism , Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Calcium-Binding Proteins/metabolism , Cerebellum/pathology , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/metabolism , Microglia/pathology , Nerve Tissue Proteins/genetics , Phospholipase C beta/metabolismABSTRACT
Different Francisella spp. produce five or six predicted acid phosphatases (AcpA, AcpB, AcpC, AcpD, HapA and HapB). The genes encoding the histidine acid phosphatases (hapA, hapB) and acpD of F. tularensis subsp. Schu S4 strain are truncated or disrupted. However, deletion of HapA (FTT1064) in F. tularensis Schu S4 resulted in a 33% reduction in acid phosphatase activity and loss of the four functional acid phosphatases in F. tularensis Schu S4 (ΔABCH) resulted in a>99% reduction in acid phosphatase activity compared to the wild type strain. All single, double and triple mutants tested, demonstrated a moderate decrease in mouse virulence and survival and growth within human and murine phagocytes, whereas the ΔABCH mutant showed >3.5-fold decrease in intramacrophage survival and 100% attenuation of virulence in mouse. While the Schu S4 ΔABCH strain was attenuated in the mouse model, it showed only limited protection against wild type challenge. F. tularensis Schu S4 failed to stimulate reactive oxygen species production in phagocytes, whereas infection by the ΔABCH strain stimulated 5- and 56-fold increase in reactive oxygen species production in neutrophils and human monocyte-derived macrophages, respectively. The ΔABCH mutant but not the wild type strain strongly co-localized with p47 (phox) and replicated in macrophages isolated from p47 (phox) knockout mice. Thus, F. tularensis Schu S4 acid phosphatases, including the truncated HapA, play a major role in intramacrophage survival and virulence of this human pathogen.
Subject(s)
Acid Phosphatase/metabolism , Francisella tularensis/enzymology , Francisella tularensis/physiology , Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Animals , Female , Francisella tularensis/genetics , Gene Deletion , Humans , Macrophages/cytology , Macrophages/metabolism , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Monocytes/cytology , Monocytes/microbiology , NADPH Oxidases/metabolism , Neutrophils/cytology , Neutrophils/microbiology , Phagosomes/microbiology , Protein Transport , Reactive Oxygen Species/metabolismABSTRACT
PURPOSE OF REVIEW: Type I interferon (IFN-I) is strongly implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we focus on new developments in pathways of IFN-I stimulation, the role of IFN-I in syndromes associated with lupus-like diseases, the utility of IFN-I signatures as biomarkers, and the progress of therapeutic agents targeting IFN-I pathways in SLE. RECENT FINDINGS: Immune complexesimmune complex are a dominant driver of IFN-I production by activating toll-like receptors (TLRs) in plasmacytoid dendritic cells (pDC) in SLE. The level of IFN-I production is attenuated by C1q in immune complexes and enhanced by natural killer (NK) cells as well as by activated platelets that express CD40L. In addition to immune complexs, cell-intrinsic activation pathways utilize recently described non-TLR RNA and DNA sensors. Some modules or clusters of IFN-I stimulated genes or proteins correlate with disease activity, whereas IFN-I biomarkers of disease flare or specific clinical manifestations need further study. IFN-I blocking studies have reached phase II clinical trials. SUMMARY: Significant progress has been made in defining both TLR as well as non-TLR mediated stimulation of IFN-I. This has helped elucidate the mechanisms of several mutations and common genetic variations in predisposing to SLE. Challenges remain in the establishing the utility of biomarkers and the role of IFN-I blockade in the clinical management of patients with this disease.
Subject(s)
Interferon Type I/metabolism , Lupus Erythematosus, Systemic/etiology , Lupus Erythematosus, Systemic/immunology , Acid Phosphatase/deficiency , Antigen-Antibody Complex/metabolism , Biomarkers/metabolism , Blood Platelets/immunology , Complement C1q/metabolism , Exodeoxyribonucleases/deficiency , Humans , Immunity, Innate , Isoenzymes/deficiency , Killer Cells, Natural/immunology , Lupus Erythematosus, Systemic/metabolism , Lupus Erythematosus, Systemic/therapy , Neutrophils/immunology , Phosphoproteins/deficiency , Signal Transduction , Tartrate-Resistant Acid Phosphatase , Toll-Like Receptors/metabolismABSTRACT
Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5(-/-)) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5(-/-) mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products.
Subject(s)
Acid Phosphatase/metabolism , Isoenzymes/metabolism , Mannosephosphates/metabolism , Proteins/metabolism , Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Animals , Cholesterol/metabolism , Electrophoresis, Gel, Two-Dimensional , Hepatocytes/metabolism , Isoenzymes/deficiency , Isoenzymes/genetics , Mice , Mice, Knockout , Phosphorylation , Proteins/chemistry , Tandem Mass Spectrometry , Tartrate-Resistant Acid Phosphatase , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/metabolismABSTRACT
Vertebral and metaphyseal dysplasia, spasticity with cerebral calcifications, and strong predisposition to autoimmune diseases are the hallmarks of the genetic disorder spondyloenchondrodysplasia. We mapped a locus in five consanguineous families to chromosome 19p13 and identified mutations in ACP5, which encodes tartrate-resistant phosphatase (TRAP), in 14 affected individuals and showed that these mutations abolish enzyme function in the serum and cells of affected individuals. Phosphorylated osteopontin, a protein involved in bone reabsorption and in immune regulation, accumulates in serum, urine and cells cultured from TRAP-deficient individuals. Case-derived dendritic cells exhibit an altered cytokine profile and are more potent than matched control cells in stimulating allogeneic T cell proliferation in mixed lymphocyte reactions. These findings shed new light on the role of osteopontin and its regulation by TRAP in the pathogenesis of common autoimmune disorders.
Subject(s)
Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Autoimmunity , Bone Diseases, Developmental/metabolism , Brain/metabolism , Calcium/chemistry , Genetic Predisposition to Disease , Isoenzymes/deficiency , Isoenzymes/genetics , Autoimmune Diseases/enzymology , Autoimmune Diseases/genetics , Bone and Bones/diagnostic imaging , Child , Homozygote , Humans , Male , Mutation , Osteochondrodysplasias/enzymology , Osteochondrodysplasias/genetics , Osteopontin/metabolism , Phosphorylation , Radiography , Tartrate-Resistant Acid PhosphataseABSTRACT
We studied ten individuals from eight families showing features consistent with the immuno-osseous dysplasia spondyloenchondrodysplasia. Of particular note was the diverse spectrum of autoimmune phenotypes observed in these individuals (cases), including systemic lupus erythematosus, Sjögren's syndrome, hemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynaud's disease and vitiligo. Haplotype data indicated the disease gene to be on chromosome 19p13, and linkage analysis yielded a combined multipoint log(10) odds (LOD) score of 3.6. Sequencing of ACP5, encoding tartrate-resistant acid phosphatase, identified biallelic mutations in each of the cases studied, and in vivo testing confirmed a loss of expressed protein. All eight cases assayed showed elevated serum interferon alpha activity, and gene expression profiling in whole blood defined a type I interferon signature. Our findings reveal a previously unrecognized link between tartrate-resistant acid phosphatase activity and interferon metabolism and highlight the importance of type I interferon in the genesis of autoimmunity.
Subject(s)
Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Bone Diseases, Developmental/genetics , Bone Diseases, Developmental/pathology , Gene Expression Regulation , Interferon Type I/metabolism , Isoenzymes/deficiency , Isoenzymes/genetics , Animals , Autoimmunity , Bone Diseases, Developmental/enzymology , Cattle , Chromosomes, Human, Pair 19 , Female , Humans , Inflammation , Lupus Erythematosus, Systemic/metabolism , Male , Models, Molecular , Mutation , Mutation, Missense , Phenotype , Sclerosis/pathology , Tartrate-Resistant Acid PhosphataseABSTRACT
Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.
Subject(s)
Acid Phosphatase/biosynthesis , Arm Bones/enzymology , Isoenzymes/biosynthesis , Leg Bones/enzymology , Osteoclasts/enzymology , Skull/metabolism , Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Animals , Cathepsin K , Cathepsin L , Cathepsins/metabolism , Cysteine Endopeptidases/metabolism , Enzyme Activation , Isoenzymes/deficiency , Isoenzymes/genetics , Mice , Mice, Knockout , Microscopy, Electron, Transmission , Reverse Transcriptase Polymerase Chain Reaction , Tartrate-Resistant Acid PhosphataseABSTRACT
Tartrate-resistant acid phosphatase (TRAP) is an enzyme highly expressed in osteoclasts (OC) and chondroclasts. As an approach to pinpoint the function of TRAP in bone-resorbing osteoclasts, the morphological phenotypic alterations of bone and osteoclasts in mice with targeted disruption of the TRAP gene were assessed by quantitative histomorphometry and immunocytochemistry at the light microscopic and ultrastructural levels. TRAP-deficient mice display alterations in the epiphyseal growth plates as evidenced by increased height with disorganized columns of chondrocytes, in particular affecting the zone of hypertrophic chondrocytes, consistent with a disturbance of chondrocyte maturation and chondroclastic resorption at the epiphyseal/metaphyseal junction. TRAP -/- mice express an early onset osteopetrotic bone phenotype, apparent already at 4 weeks of age. The differentiation of OCs was apparently normal; however, the osteoclasts in TRAP-deficient mice were less active in terms of degradation or release of the resorption marker C-terminal type I collagen cross-linked peptide, indicative of an intrinsic defect. Ultrastructural morphometry disclosed that OCs from TRAP-deficient young mice exhibited an increased relative area of ruffled borders. Moreover, mutant OC accumulated cytoplasmic vesicles 200-500 nm in size in both ruffled border and basolateral parts of the cytoplasm, reflecting disturbed intracellular transport. The accumulated vesicles were not likely derived from the secretory pathway, since cathepsin K was detected at normal levels in the ruffled border area and matrix in TRAP -/- mice. In summary, the resorptive defect in TRAP-deficient OCs is reflected by a disturbance at the level of ruffled borders and intracellular transport vesicles. Consequently, accumulation of vesicles in the cytoplasm of mutant OCs indicates a novel function for TRAP in modulating intracellular vesicular transport in osteoclasts.
Subject(s)
Acid Phosphatase/physiology , Cell Surface Extensions/metabolism , Isoenzymes/physiology , Osteoclasts/metabolism , Protein Transport/physiology , Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Animals , Cathepsin K , Cathepsins/metabolism , Femur/anatomy & histology , Femur/physiology , Growth Plate/cytology , Growth Plate/metabolism , Isoenzymes/deficiency , Isoenzymes/genetics , Mice , Mice, Knockout , Osteoclasts/ultrastructure , Tartrate-Resistant Acid PhosphataseABSTRACT
Acid phosphatases (APs) are a family of enzymes that are widespread in nature, and can be found in many animal and plant species. Mystery surrounds the precise functional role of these molecular facilitators, despite much research. Yet, paradoxically, human APs have had considerable impact as tools of clinical investigation and intervention. One particular example is tartrate resistant acid phosphatase, which is detected in the serum in raised amounts accompanying pathological bone resorption. This article seeks to explore the identity and diversity of APs, and to demonstrate the relation between APs, human disease, and clinical diagnosis.
Subject(s)
Acid Phosphatase/metabolism , Bone Resorption/metabolism , Isoenzymes/metabolism , Osteoclasts/enzymology , Osteoporosis/enzymology , Acid Phosphatase/analysis , Acid Phosphatase/deficiency , Biomarkers/analysis , Biomarkers/blood , Favism/diagnosis , Gaucher Disease/diagnosis , Humans , Intracellular Fluid/enzymology , Isoenzymes/analysis , Isoenzymes/deficiency , Leukemia, Hairy Cell/diagnosis , Male , Prostate , Prostatic Neoplasms/diagnosis , Protein Binding , Reactive Oxygen Species/metabolism , Tartrate-Resistant Acid Phosphatase , alpha-Macroglobulins/metabolismABSTRACT
To date, two lysosomal acid phosphatases are known to be expressed in cells of the monocyte/phagocyte lineage: the ubiquitously expressed lysosomal acid phosphatase (LAP) and the tartrate-resistant acid phosphatase-type 5 (Acp5). Deficiency of either acid phosphatase results in relatively mild phenotypes, suggesting that these enzymes may be capable of mutual complementation. This prompted us to generate LAP/Acp5 doubly deficient mice. LAP/Acp5 doubly deficient mice are viable and fertile but display marked alterations in soft and mineralised tissues. They are characterised by a progressive hepatosplenomegaly, gait disturbances and exaggerated foreshortening of long bones. Histologically, these animals are distinguished by an excessive lysosomal storage in macrophages of the liver, spleen, bone marrow, kidney and by altered growth plates. Microscopic analyses showed an accumulation of osteopontin adjacent to actively resorbing osteoclasts of Acp5- and LAP/Acp5-deficient mice. In osteoclasts of phosphatase-deficient mice, vacuoles were frequently found which contained fine filamentous material. The vacuoles in Acp5- and LAP/Acp5 doubly-deficient osteoclasts also contained crystallite-like features, as well as osteopontin, suggesting that Acp5 is important for processing of this protein. This is further supported by biochemical analyses that demonstrate strongly reduced dephosphorylation of osteopontin incubated with LAP/Acp5-deficient bone extracts. Fibroblasts derived from LAP/Acp5 deficient embryos were still able to dephosphorylate mannose 6-phosphate residues of endocytosed arylsulfatase A. We conclude that for several substrates LAP and Acp5 can substitute for each other and that these acid phosphatases are essential for processing of non-collagenous proteins, including osteopontin, by osteoclasts.
Subject(s)
Acid Phosphatase/physiology , Isoenzymes/physiology , Lysosomes/enzymology , Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Animals , Bone and Bones/abnormalities , Bone and Bones/enzymology , Bone and Bones/pathology , Hepatomegaly/genetics , Isoenzymes/deficiency , Isoenzymes/genetics , Kidney/enzymology , Kidney/pathology , Liver/enzymology , Liver/pathology , Lysosomal Storage Diseases/enzymology , Lysosomal Storage Diseases/genetics , Lysosomal Storage Diseases/pathology , Lysosomes/ultrastructure , Mannosephosphates/metabolism , Mice , Mice, Knockout , Microscopy, Electron , Osteopontin , Phenotype , Phosphorylation , Sialoglycoproteins/metabolism , Spleen/enzymology , Spleen/pathology , Splenomegaly/genetics , Tartrate-Resistant Acid PhosphataseABSTRACT
Tartrate-resistant acid phosphatase (TRAP) is a lysosomal di-iron protein of mononuclear phagocytes and osteoclasts. Hitherto, no role for the enzyme in immunity has been identified; however, knockout mice lacking TRAP have a skeletal phenotype caused by an intrinsic osteoclast defect. To investigate a putative function for TRAP in macrophages (Mphi), we investigated proinflammatory responses and systemic microbial clearance in knockout mice compared with age- and gender-matched congenic wild-type mice. Phorbol 12-myristate 13-acetate (PMA)-stimulated and interferon-gamma (IFN-gamma)-induced superoxide formation was enhanced in peritoneal Mphi lacking TRAP; nitrite production in response to stimulation with lipopolysaccharide (LPS) and IFN-gamma was also increased. In addition, secretion of the proinflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-12, was significantly greater in TRAP-deficient Mphi when stimulated with LPS, with or without addition of either TNF-alpha or IFN-gamma. The activity of tartrate-sensitive (lysosomal) acid phosphatase was increased in Mphi from the knockout mice but activities of the lysosomal hydrolases N-acetyl beta-glucosaminidase and acid beta-glucuronidase were unchanged, indicating selective activation of compensatory acid phosphatase activity. Evidence of impaired Mphi function in vivo was obtained in TRAP knockout mice, which showed delayed clearance of the microbial pathogen, Staphylococcus aureus, after sublethal intraperitoneal inoculation. After microbial challenge, peritoneal exudates obtained from TRAP knockout mice had a reduced population of Mphi. As peritoneal Mphi and neutrophils lacking TRAP were able to phagocytose and kill S. aureus normally in vitro, TRAP may directly or indirectly influence recruitment of Mphi to sites of microbial invasion. Our study shows that TRAP participates in the inflammatory response of the Mphi and influences effector signalling pathways in innate immunity.
Subject(s)
Acid Phosphatase/deficiency , Inflammation/immunology , Isoenzymes/deficiency , Macrophages/immunology , Staphylococcal Infections/immunology , Acid Phosphatase/immunology , Animals , Bone Marrow/immunology , Cytokines/metabolism , Female , Free Radicals/metabolism , Immunophenotyping , Isoenzymes/immunology , Lysosomes/enzymology , Lysosomes/ultrastructure , Macrophages/enzymology , Macrophages/ultrastructure , Macrophages, Peritoneal/immunology , Male , Mice , Mice, Knockout , Phagocytosis/immunology , Staphylococcus aureus/isolation & purification , Superoxides/metabolism , Tartrate-Resistant Acid PhosphataseABSTRACT
Lysosomal acid phosphatase (LAP) is a tartrate-sensitive enzyme with ubiquitous expression. Neither the physiological substrates nor the functional significance is known. Mice with a deficiency of LAP generated by targeted disruption of the LAP gene are fertile and develop normally. Microscopic examination of various peripheral organs revealed progredient lysosomal storage in podocytes and tubular epithelial cells of the kidney, with regionally different ultrastructural appearance of the stored material. Within the central nervous system, lysosomal storage was detected to a regionally different extent in microglia, ependymal cells, and astroglia concomitant with the development of a progressive astrogliosis and microglial activation. Whereas behavioral and neuromotor analyses were unable to distinguish between control and deficient mice, approximately 7% of the deficient animals developed generalized seizures. From the age of 6 months onward, conspicuous alterations of bone structure became apparent, resulting in a kyphoscoliotic malformation of the lower thoracic vertebral column. We conclude from these findings that LAP has a unique function in only a subset of cells, where its deficiency causes the storage of a heterogeneously appearing material in lysosomes. The causal relationship of the enzyme defect to the clinical manifestations remains to be determined.
Subject(s)
Acid Phosphatase/deficiency , Central Nervous System Diseases/enzymology , Kidney Diseases/enzymology , Lysosomal Storage Diseases/enzymology , Lysosomes/enzymology , Acid Phosphatase/genetics , Animals , Antigens, CD/metabolism , Bone and Bones/abnormalities , Cathepsin D/metabolism , Central Nervous System Diseases/pathology , Fibroblasts/enzymology , Kidney Diseases/pathology , Lysosomal Membrane Proteins , Membrane Glycoproteins/metabolism , Mice , Microglia/enzymology , Microglia/pathology , Phenotype , Seizures/enzymology , Tartrates/pharmacologyABSTRACT
Exogenous Ca2+ at concentrations up to 3.5 mM increases the sucrose-induced acidification of the culture medium when the mold Neurospora crassa is grown on low-phosphate (Pi) medium at pH 7.8. Induction depends on the pH of the culture medium adjusted for conidial inoculation and on the absence of carbon sources generating cytoplasmic acetyl CoA. Furthermore, the excretion of Pi-repressible acid and alkaline phosphatases was not stimulated by increasing exogenous Ca2+ levels. We also provide evidence that the extracellular pH monitoring by Neurospora crassa may be a determinant in the selective excretion of Pi-repressible acid and alkaline phosphatases.
Subject(s)
Acid Phosphatase/biosynthesis , Alkaline Phosphatase/biosynthesis , Neurospora crassa/enzymology , Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Alkaline Phosphatase/deficiency , Alkaline Phosphatase/genetics , Calcium/pharmacology , Culture Media/chemistry , Enzyme Repression , Hydrogen-Ion Concentration , Neurospora crassa/genetics , Neurospora crassa/growth & development , Sucrose/metabolismABSTRACT
Exogenous Ca2+ at concentrations up to 3.5 mM increases the sucrose-induced acidification of the culture medium when the mold Neurospora crassa is grown on low-phosphate (Pi) medium at pH 7.8. Induction depends on the pH of the culture medium adjusted for conidial inoculation and on the absence of carbon sources generating cytoplasmic acetyl CoA. Furthermore, the excretion of Pi-repressible acid and alkaline phosphatases was not stimulated by increasing exogenous Ca2+ levels. We also provide evidence that the extracellular pH monitoring by Neurospora crassa may be a determinant in the selective excretion of Pi-repressible acid and alkaline phosphatases.
Subject(s)
Alkaline Phosphatase , Acid Phosphatase/biosynthesis , Neurospora crassa , Alkaline Phosphatase , Calcium , Culture Media , Enzyme Repression , Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Hydrogen-Ion Concentration , Neurospora crassa , SucroseABSTRACT
A one-step mutant of Escherichia coli K-12 lacking both glucose-1-phosphatase (Agp) and pH 2.5 acid phosphatase (AppA) activities in the periplasmic space was isolated. The mutation which mapped close to chlB, at 87 min on the E. coli linkage map, also caused the loss of alkaline phosphatase (PhoA) activity, even when this activity was expressed from TnphoA fusions to genes encoding periplasmic or membrane proteins. A DNA fragment that complements the mutation was cloned and shown to carry the dsbA gene, which encodes a periplasmic disulphide bond-forming factor. The mutant had an ochre triplet in dsbA, truncating the protein at amino acid 70. Introduction of TnphoA fusions into a plasmid-borne dsbA gene resulted in DsbA-PhoA hybrid proteins that were all exported to the periplasmic space in both dsbA+ and dsbA strains. They belong to three different classes, depending on the length of the DsbA fragment fused to PhoA. When PhoA was fused to an amino-terminal DsbA heptapeptide, the protein was only seen in the periplasm of a dsbA+ strain, as in the case of wild-type PhoA. Hybrid proteins missing up to 29 amino acids at the carboxy-terminus of DsbA were stable and retained both the DsbA and PhoA activities. Those with shorter DsbA fragments that still carried the -Cys-Pro-His-Cys- motif were rapidly degraded (no DsbA activity). The presence is discussed of a structural domain lying around amino acid 170 of DsbA and which is probably essential for its folding into a proteolytic-resistant and enzymatically active form.
Subject(s)
Acid Phosphatase/genetics , Bacterial Proteins/metabolism , Escherichia coli/enzymology , Genes, Bacterial/genetics , Recombinant Fusion Proteins/metabolism , Acid Phosphatase/deficiency , Acid Phosphatase/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Cell Membrane/enzymology , Cloning, Molecular , DNA Mutational Analysis , DNA Transposable Elements , DNA, Bacterial/genetics , DNA, Recombinant , Disulfides/metabolism , Escherichia coli/genetics , Molecular Sequence Data , Mutation , Recombinant Fusion Proteins/genetics , Restriction Mapping , Structure-Activity RelationshipABSTRACT
1. The mycelial Pi-repressible acid phosphatase presented p-nitrophenylphosphatase activity with negative cooperativity and Michaelian behavior when synthesized by the wild-type and pho-2A mutant strains of Neurospora crassa, respectively. 2. The major acid phosphatase present in cell extracts of the pho-2A mutant of N. crassa grown in low Pi medium is more thermolabile (t1/2 = 4 min at 54 degrees C, pH 5.4) than that of the wild strain (stable for at least 80 min at 54 degrees C, pH 5.4). 3. The pho-2A mutant of N. crassa secreted a more thermolabile acid phosphatase (t1/2 = 30 min at 50 degrees C, pH 5.4) than the wild strain (t1/2 of at least 80 min at 50 degrees C, pH 5.4). 4. The pho-2A mutant of N. crassa synthesized a more thermolabile acid phosphatase (t1/2 = 37 min at 54 degrees C, pH 5.4) than the wild strain in high Pi medium (t1/2 = 14 min at 54 degrees C, pH 5.4). 5. The pleiotropic nature of the pho-2 locus and its possible involvement in the mechanism of phosphatase secretion by N. crassa are proposed.
Subject(s)
Acid Phosphatase/biosynthesis , Alkaline Phosphatase/biosynthesis , Mutation/physiology , Neurospora/enzymology , 4-Nitrophenylphosphatase/metabolism , Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Alkaline Phosphatase/deficiency , Alkaline Phosphatase/genetics , Enzyme Repression , Enzyme Stability , Hydrogen-Ion Concentration , Neurospora/genetics , TemperatureABSTRACT
In 24 patients with Hodgkin's disease the activity of acid phosphatase was determined in granulocytes. Its level was only half that in the control group. The causes of this low activity of this enzyme may be increased number of cells without it, exocytosis, and possible influence of substances secreted by the neoplasm. It is suggested that the determination of this activity may be indirectly useful for verification of the efficacy of the antibacterial mechanisms, and also of antitumour resistance of the host.
Subject(s)
Acid Phosphatase/blood , Blood Bactericidal Activity/immunology , Hodgkin Disease/enzymology , Neutrophils/enzymology , Acid Phosphatase/deficiency , Female , Hodgkin Disease/immunology , Hodgkin Disease/pathology , Humans , Male , Neoplasm Staging , Neutrophils/immunologyABSTRACT
The catalytic and immunological properties of acid phosphatases (EC 3.1.3.2.) in different tissues were studied. It was demonstrated that high uptake forms of lysosomal enzymes like beta-galactosidase isolated from human platelets and bovine testis are mature enzymes, which have not lost their mannose-6phosphate marker. The results presented indicate that this phenomenon is related to a low activity or the complete absence of the lysosomal tartrate sensitive acid phosphatase activity in the tissues concerned.