Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biosynthesis of myeloperoxidase (MPO), a myeloid lysosomal hemoprotein critical for the optimal oxygen-dependent microbicidal activity of human neutrophils, is incompletely understood. The primary translation product undergoes cotranslational N-linked glycosylation with subsequent insertion of the Fe-containing prosthetic group into the peptide backbone, thereby converting the enzymatically inactive, heme-free apoproMPO into the peroxidatively active precursor, proMPO. Eventually, proMPO undergoes proteolytic processing into native, lysosomal MPO, with subunits of 59 and 13.5 Kd. We studied three unanswered questions regarding MPO biosynthesis: (1) At what point during MPO biosynthesis is the heme moiety inserted into the apoenzyme? (2) What consequences does heme-insertion have on subsequent processing events? (3) What role does the mannose-6-phosphate receptor (M6PR) system play in the delivery of MPO to the lysosome? Disruption of Golgi by brefeldin A (BFA) produced two major changes in MPO biosynthesis: (1) processing of the 89-Kd precursor to mature MPO was blocked and (2) constitutive secretion of the MPO precursor was inhibited. Inhibition of heme synthesis with succinyl acetone (SA) reduced peroxidase activity and profoundly blocked processing of proMPO to mature MPO. This inhibition of processing was not a generalized effect on all lysosomal enzymes, because the maturation of a non-heme-containing lysosomal enzyme, beta-glucuronidase, was not altered. Electron microscopy showed that, although the normal peroxidase staining of endoplasmic reticulum was absent in SA-treated cells, there were MPO-related peptides in the ER. The role of the M6PR system was assessed by immunoprecipitating fractions obtained from M6PR affinity column chromatography. The 89-Kd proMPO failed to adhere to the M6PR affinity column, whereas the 59-Kd heavy subunit of mature MPO was specifically eluted from the column. We interpret these data to indicate that: (1) processing of proMPO to mature MPO occurs in a post-ER compartment that is itself BFA-sensitive or is distal to a BFA-sensitive compartment and (2) heme insertion into apoproMPO precedes and may be a prerequisite for proteolytic processing to enzymatically active mature MPO. Our analysis of the M6PR system in MPO biosynthesis led to the unanticipated finding that there were phosphomannosyl residues on mature MPO, but none on proMPO. We suggest that the bulk of proMPO at any time is not phosphorylated, but, when generated, the phosphorylated proMPO is quickly processed to the phosphorylated 59-Kd subunit of mature MPO. Thus, if the M6PR is important in the intracellular transport of MPO, it is the phosphorylated mature MPO that is directed to the lysosomal compartment by this system.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Roles of heme insertion and the mannose-6-phosphate receptor in processing of the human myeloid lysosomal enzyme, myeloperoxidase. 133 78

Bone resorption plays an important role in bone modeling and remodeling. Osteoclasts are the cells responsible for the bone resorption. Osteoclasts are located on endosteal bone surfaces and on the periosteal surface beneath the periosteum. They are multinucleated giant cells highly polarized in their morphology and function. Among the proximal surface, the membrane and the area of the cytoplasm directly oppose to the bone surface, which are specialized into two regions. A central region consisting of many irregular cytoplasmic processes and infoldings, the ruffled border, is known to be the active site of bone resorption. Surrounding the ruffled border, a second region, the clear zone provides an area of close attachment to the mineralized bone surface. The osteoclasts secrete a large amount of protons by the action of H(+)-pump on the ruffled border into the sealed resorption cavity, resulting in the acidified microenvironment under which condition the bone matrix is dissolved. Protons are provided by the intracellular action of carbonic anhydrase. Following the secretion of the protons, several ion-transporting systems, i.e., carbonate-chloride exchanger, chloride-channel, Ca(2+)-transport systems, Na+/K(+)-ATPase, and voltage-dependent Ca(2+)-channel, are sequentially operated on both apical and basolateral cytoplasmic membranes. In addition, osteoclasts contain a large amount of lysosomal enzymes (cathepsin C, beta-glycerophosphatase, beta-glucuronidase, etc.), which contribute to degrade the bone organic matrices exposed in the resorption cavity. These enzymes bind to the mannose-6-phosphate receptor on Golgi apparatus, are transported to the ruffled border and are secreted into the extracellular compartment in an exocytotic manner. Osteoclasts also have a high tartrate-resistant acid phosphatase activity which is currently used as a marker enzyme osteoclastic differentiation. Osteoclasts are considered to develop from hematopoietic stem cells. So far, the following four different pathways of the differentiation of osteoclast are proposed: The precursors of osteoclast develop (1) from multilineage hematopoietic cells via a completely separate differentiation line, (2) from granulocyte macrophage-colony forming cells, (3) from committed but proliferative monocyte-macrophage, and (4) from mature and unproliferative monocyte-macrophage. However, the differentiation line of the osteoclasts has still to be elucidated. The formation of osteoclasts as well as that of other hematopoietic cells is strongly regulated by many cytokines [interleukin (IL)-1,IL-3,IL-6, M-colony stimulating factor (CSF), and GM-CSF]. 1,25-Dihydroxyvitamin D3 and parathyroid hormone also stimulate the differentiation of osteoclast precursors. However, the mature osteoclasts do not possess the receptors for these hormones.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Osteoclasts in bone metabolism]. 175 56

A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn-78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with alpha-2-macroglobulin at the low temperature. Thus, the process of lysosomal maturation appears to involve the progressive delivery of lysosomal enzymes to various types of endosomes that may have already received some of the lysosomal membrane proteins. Although endolyn-78 would be one of the proteins added early to endosomes, other lysosomal membrane proteins may be added only to multivesicular endosomes that represent very advanced stages of maturation.
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PMID:Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis. 265 37

The intracellular and extracellular distribution of acid hydrolases in cultured retinal pigmented epithelium (RPE) was studied. Incubation of cultured RPE in medium containing 20 mM mannose-6-phosphate resulted in the extracellular release of approximately 15% of the cell-associated activity of several acid hydrolases. This represents an approximate 120% increase over control levels after 24 hr of culture with 20 mM mannose-6-phosphate. The extracellular release is not due to cell lysis, since no release of the cytoplasmic marker lactate dehydrogenase was seen. n-Acetyl-beta-glucosaminidase, alpha-mannosidase, and beta-glucuronidase were released into the extracellular medium, while acid phosphatase and beta-glucosidase were not. The release was specific for mannose-6-phosphate, and was dose-dependent. Inhibition of protein synthesis by treatment of RPE cells with cycloheximide (100 micrograms/ml) inhibited extracellular acid hydrolase release. RPE cells exhibited n-Acetyl-beta-glucosaminidase bound to the cell surface via a mannose-6-phosphate sensitive receptor. These results demonstrate a specific extracellular release of acid hydrolases by RPE and the presence of at least one acid hydrolase on the RPE cell surface. This may represent a mechanism for control of cell surface and extracellular levels of these enzymes in RPE via the mannose-6-phosphate receptor.
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PMID:Extracellular release of acid hydrolases from cultured retinal pigmented epithelium. 310 Apr 74

We cloned and sequenced the 8767-bp full-length cDNA for the chicken cation-independent mannose-6-phosphate receptor (CI-MPR), of interest because, unlike its mammalian homologs, it does not bind insulin-like growth factor II (IGF-II). The cDNA encodes a protein of 2470 aa that includes a putative signal sequence, an extracytoplasmic domain consisting of 15 homologous repeat sequences, a 23-residue transmembrane sequence, and a 161-residue cytoplasmic sequence. Overall, it shows 60% sequence identity with human and bovine CI-MPR homologs, and all but two of 122 cysteine residues are conserved. However, it shows much less homology in the N-terminal signal sequence, in repeat 11, which is proposed to contain the IGF-II-binding site in mammalian CI-MPR homologs, and in the 14-aa residue segment in the cytoplasmic sequence that has been proposed to mediate G-protein-coupled signal transduction in response to IGF-II binding by the human CI-MPR. Transient expression in COS-7 cells produced a functional CI-MPR which exhibited mannose-6-phosphate-inhibitable binding and mediated endocytosis of recombinant human beta-glucuronidase. Expression of the functional chicken CI-MPR in mice lacking the mammalian CI-MPR should clarify the controversy over the physiological role of the IGF-II-binding site in mammalian CI-MPR homologs.
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PMID:Cloning and expression of the cDNA of chicken cation-independent mannose-6-phosphate receptor. 756 13

beta-Glucuronidase undergoes proteolytic C-terminal processing during or after its transport to lysosomes or endosomes. We determined the C-terminal processing site for human placental beta-glucuronidase to be the peptide bond between Thr633-Arg634. To evaluate the role of the 18-amino acid peptide removed in C-terminal processing, we changed the codon for Arg634 to a stop codon by site-directed mutagenesis and studied expression of the truncated mutant enzyme in COS-7 cells. An increased fraction of newly synthesized enzyme from R634Stop cDNA was secreted. Pulse-chase experiments provided no evidence for increased degradation of the intracellular R634Stop enzyme. The total amount of catalytic activity expressed from the R634Stop mutant cDNA was only half that seen with the wild type cDNA, and the Kcat of the mutant enzyme was 52% that of wild type enzyme. These results indicate that the C-terminal propeptide in the precursor is important for beta-glucuronidase to achieve maximal activity. The truncated enzyme formed hybrid tetramers in cotransfection experiments with the cDNA for rat beta-glucuronidase. There appeared to be no decrease in stability of the R634Stop enzyme, since chaotropic agents, heat treatment, and pH had similar effects on the mutant and the wild type enzymes. The uptake rate of the truncated mutant (R634Stop) enzyme by beta-glucuronidase-deficient human fibroblast cells was only 55-60% that of the wild type enzyme. Binding to the immobilized cation-independent mannose-6-phosphate receptor and measurement of the 32P-labeled phosphorylated oligosaccharides revealed that the truncated mutant enzyme was 32-34% less phosphorylated and appeared to contain proportionately more covered phosphate groups than the wild type enzyme. These results suggest that the propeptide influences the accessibility to both processing enzymes that produce the mannose-6-phosphate recognition marker on beta-glucuronidase.
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PMID:C-terminal processing of human beta-glucuronidase. The propeptide is required for full expression of catalytic activity, intracellular retention, and proper phosphorylation. 822 71

The abundance of amyloid beta peptide (A beta) and the selective loss of neurons are characteristics of Alzheimer's disease. However, subpopulations of brain cells survive, including neurons near A beta-rich plaques. The surviving neurons may have gene expression profiles that allow them to be resistant to A beta toxicity. Here we use the differential display technique to compare the profiles of gene expression in an A beta-resistant cell line with its parental cells. Prominent among the changes are two components of the endosomal-lysosomal system, insulin growth factor II receptor/mannose-6-phosphate receptor and arylsulfatase B. Both are more highly expressed in the A beta-resistant clone, and arylsulfatase is inducible by A beta and hydrogen peroxide. Another lysosomal enzyme, beta-glucuronidase, is also up-regulated in A beta-resistant cells. These results are consistent with the observation that the endosomal-lysosomal system is highly activated in Alzheimer's disease brains, and they raise the possibility that the high expression of endosomal-lysosomal components is important for neuronal survival in the presence of A beta.
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PMID:The up-regulation of endosomal-lysosomal components in amyloid beta-resistant cells. 1050 Nov 92

Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease. A. felis can survive and multiply in several mammalian cell types, including macrophages, but the precise intracellular compartmentalization of A. felis-containing phagosomes is unknown. Here, we demonstrate that, in murine macrophages, most A. felis-containing phagosomes exclude lysosomal tracer loaded into macrophage lysosomes before, as well as endocytic tracer loaded after, establishment of an infection. Established Afipia-containing phagosomes possess neither early endosomal marker proteins [early endosome antigen 1 (EEA1), Rab5, transferrin receptor, trytophane aspartate containing coat protein (TACO)] nor late endosomal or lysosomal proteins [cathepsin D, beta-glucuronidase, vacuolar proton-pumping ATPase, rab7, mannose-6-phosphate receptor, vesicle-associated membrane protein 8, lysosome-associated membrane proteins LAMP-1 and LAMP-2]. Those bacteria that will be found in a nonendosomal compartment enter the macrophage via an EEA1-negative compartment, which remains negative for LAMP-1. The smaller subpopulation of afipiae whose phagosomes will be part of the endocytic system enters into an EEA1-positive compartment, which also subsequently acquires LAMP-1. Killing of Afipia or opsonization with immune antibodies leads to a strong increase in the percentage of A. felis-containing phagosomes that interact with the endocytic system. We conclude that most phagosomes containing A. felis are disconnected from the endosome-lysosome continuum, that their unusual compartmentalization is decided at uptake, and that this compartmentalization requires bacterial viability.
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PMID:Afipia felis induces uptake by macrophages directly into a nonendocytic compartment. 1140 61

A portion of the lysosomal enzymes produced by cells is secreted, diffuses through extracellular spaces, and can be taken up by distal cells via mannose-6-phosphate receptor-mediated endocytosis. This provides the basis for treating lysosomal storage diseases, many of which affect the CNS. Normal enzyme secreted from a cluster of genetically corrected cells has been shown to reverse storage lesions in a zone of surrounding brain tissue in mouse disease models. However, low levels of enzyme activity and reduction of storage lesions also have been observed at sites in the brain that may not be explained by a contiguous gradient of secreted enzyme diffusing away from the genetically corrected cells. No direct evidence for alternative mechanisms of enzyme transport has been shown, and little is understood about the intracellular movement of lysosomal enzymes in neurons. We investigated whether axonal transport could occur, by expressing an eukaryotic lysosomal enzyme that can be visualized in tissue sections (beta-glucuronidase) in brain structures that have defined axonal connections to other structures. This resulted in the transfer of enzyme to, and a reversal of storage lesions in, neurons that project to the gene expression site, but not in nearby structures that would have been corrected if the effect had been mediated by diffusion. In addition, transduction of cells in the subventricular zone resulted in the uptake of beta-glucuronidase by cells entering the rostral migratory stream. Gene transfer to specific neuronal circuits or cells in migratory pathways may facilitate delivery to the global brain lesions found in these disorders.
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PMID:Distribution of a lysosomal enzyme in the adult brain by axonal transport and by cells of the rostral migratory stream. 1215 23

The co-existence of two mannose-6-phosphate receptors (CD-MPR and CI-MPR) in most cell types is still a dilemma to be resolved. In this study, some parameters were measured to explore lysosomal apparatus evolution in rat liver during perinatal development, and establish a possible involvement of CD- and/or CI-MPR in lysosome maturation. Activity of four acid hydrolases was measured in the whole organ at different ages and it was found that N-acetyl-beta-D-glucosaminidase (NAG), beta-galactosidase, and beta-glucuronidase change during development, reaching a peak at the 10th day after birth. These results correlated with the expression and binding properties of CD-MPR previously reported. We also used a method that recognizes phosphomannosylated ligands by using purified biotinylated CI-MPR as a probe, and found that the highest concentrations of ligands also appear around the 10th day. Binding assays were also carried out, incubating endogenous NAG from 10-day-old and adult rats with membranes from their respective ages, and the results indicated that cation-dependent mannose-6-phosphate receptor (CD-MPR) has more impact on trafficking of the enzyme at the 10th day after birth. We concluded that lysosome maturation in the rat liver occurs around the 10th day after birth, and that the CD-MPR may participate in that event.
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PMID:Changes in phosphomannosyl ligands correlate with cation-dependent mannose-6-phosphate receptors in rat liver during perinatal development. 1663 May 51


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