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)

Human beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is subject to receptor-mediated endocytosis by fibroblasts. Prior work demonstrated charge heterogeneity in beta-glucuronidase and showed that high-uptake forms are more acidic than slowly internalized forms. Considerable indirect evidence implicated mannose 6-phosphate as an essential part of the recognition marker on high-uptake enzyme forms. Here we report the purification of beta-glucuronidase from human spleen and demonstrate enzymatically that mannose 6-phosphate is released on acid hydrolysis of pure enzyme varies directly with its susceptibility to pinocytosis by fibroblasts. Enzyme forms resolved by CM-Sephadex chromatography differed over an 18-fold range in uptake rate and in mannose 6-phosphate content. The most acidic forms had 4.4 mol of mannose 6-phosphate per mol of enzyme. The mannose 6-phosphate was released from the enzyme by treatment with endoglycosidase H with concomitant loss of susceptibility to adsorptive endocytosis. Thus, these studies provide direct evidence that mannose 6-phosphate is present on high-uptake enzyme forms, that it is present in the recognition marker for uptake, and that it is present on oligosaccharide that is released by endoglycosidase H.
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PMID:Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts. 29 66

Cathepsin L, a lysosomal cysteine protease, is the major excreted protein of transformed mouse NIH 3T3 cells. Previous studies have shown that asparagine-linked oligosaccharides associated with the secreted hydrolase contain mannose 6-phosphate (Man 6-P), the recognition marker for transport of newly synthesized acid hydrolases to lysosomes. To investigate the mechanism by which cathepsin L evades targeting to lysosomes, we determined the structure of the enzyme's oligosaccharides and analyzed its interaction with the cation-independent mannose 6-phosphate (Man 6-PCl) receptor. Oligosaccharides associated with procathepsin L isolated from the medium of [3H]mannose-labeled J774 cells were remarkably homogeneous; all of the radiolabeled structures were high mannose-type units that contained two phosphomonoesters and 7 mannose residues. Both the alpha 1,3- and alpha 1,6-branches of the oligosaccharides were phosphorylated. Oligosaccharides released by endoglycosidase H from [3H]mannose-labeled procathepsin L bound to a Man 6-PCl receptor affinity column. Despite the high affinity binding of these oligosaccharides, the intact glycoprotein was not a good ligand for the Man 6-PCl receptor. Procathepsin L was internalized poorly by Man 6-P receptor-mediated endocytosis and the purified acid protease interacted weakly with a Man 6-PCl affinity column. In contrast, pro-beta-glucuronidase (another acid hydrolase produced by J774 cells) was an excellent ligand for the Man 6-PCl receptor as judged by the endocytosis and affinity chromatographic assays. Phosphorylated oligosaccharides associated with the J774-secreted pro-beta-glucuronidase were heterogeneous and contained both mono- and diphosphorylated species. Tryptic glycopeptides generated from [3H]mannose-labeled procathepsin L, unlike the intact protein, were excellent ligands for the Man 6-PCl receptor. The results indicate that oligosaccharides associated with procathepsin L are processed uniformly to diphosphorylated species that bind with high affinity to the Man 6-PCl receptor. Protein determinants inherent within the intact acid hydrolase, however, inhibit the high affinity binding of these oligosaccharides and, as a result, impair the interaction of procathepsin L with the receptor.
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PMID:Protein determinants impair recognition of procathepsin L phosphorylated oligosaccharides by the cation-independent mannose 6-phosphate receptor. 216 20

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

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.
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PMID:Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins. 282 Oct 12

The accumulation of the relatively large amounts of beta-glucuronidase in microsomal fractions of normal mice depends on formation of complexes with the protein egasyn. Unexpectedly, it was found that the egasyn gene also affects the processing of beta-glucuronidase, which is segregated to lysosomes. In egasyn-positive mice lysosomal beta-glucuronidase from liver has a mean pI of 5.9 with a minor proportion at pI 5.4, whereas in egasyn-negative mice the proportion of the two lysosomal forms is reversed. Combined experiments measuring susceptibility to neuraminidase and to endoglycosidase H and specific binding to Ricinus communis lectin-agarose columns showed that the alterations in isoelectric point were associated with a decrease in complex oligosaccharides of lysosomal beta-glucuronidase in egasyn-positive mice. Since this alteration occurs not only in a congenic strain carrying the Eg0 gene but also in several other inbred strains that are homozygous for this gene, it is considered to be a genuine effect of the Eg gene rather than other genes that might regulate oligosaccharide processing. Also, the alteration is likely to be a result of direct physical interaction of the egasyn protein and lysosomal beta-glucuronidase, since a second lysosomal enzyme, beta-galactosidase, which does not form complexes with egasyn, is unaffected. The results suggest a model in which egasyn not only causes accumulation of beta-glucuronidase in the microsomal compartment but also acts upon the precursor to lysosomal beta-glucuronidase to alter its interaction with trans-Golgi-apparatus processing enzymes.
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PMID:The egasyn gene affects the processing of oligosaccharides of lysosomal beta-glucuronidase in liver. 310 73

beta-Glucuronidase from human maxillary sinus and lung cancers and from uninvolved tissues was studied. An elevation of beta-glucuronidase activity was observed in cancerous tissues as compared with the corresponding uninvolved tissues, and this increase was significant in adenocarcinoma and squamous cell carcinoma of the lung (p less than 0.01). beta-glucuronidase preparations purified from adenocarcinoma and large cell carcinoma of lung and from normal lung showed similar kinetic properties and antigenicity. beta-Glucuronidase from lung adenocarcinoma showed considerable negative charge heterogeneity in the pI range from 4.2 to 6.2 in an experiment involving isoelectric focusing on polyacrylamide gel. Similar charge heterogeneity was observed in the enzyme from lung large cell carcinoma. Upon treatment of the adenocarcinoma enzyme with exogenous alkaline phosphatase or endoglycosidase H, the heterogeneous variant forms of the tumor enzyme appeared to partly or completely lose their negative charge and to be converted into forms similar to those of the normal lung enzyme. An experiment on the labeling of beta-glucuronidase with [32P]-phosphoric acid provided further evidence that the acidic variants found in lung cancers are extensively phosphorylated forms of the enzyme.
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PMID:[beta-Glucuronidase in human maxillary sinus and lung cancers: elevation of activity level and appearance of phosphorylated variant forms]. 609 43

During pulse-chase experiments in cultured porcine kidney cells, an early 75-kilodalton (kDa) form of beta-glucuronidase is converted to a late 72-kDa form. The relative molecular weight difference between the two forms is maintained on removal of high-mannose carbohydrate with endoglycosidase H. Both forms have the same partial NH2-terminal sequence, and both migrate as single polypeptide chains following reduction, alkylation, and electrophoresis under denaturing conditions. On treatment with carboxypeptidase Y, the early form released [35S]Met faster than the late form. Thus, the late form of beta-glucuronidase is generated by COOH-terminal proteolytic processing of the early form. During similar experiments, the mass of the 30-kDa heavy chain of porcine cathepsin D decreased by about 1 kDa. The heavy chain of the two-chain enzyme is derived from the COOH terminus of a 44-kDa single-chain enzyme. On treatment with carboxypeptidase Y, the early single-chain enzyme released COOH-terminal [35S]Met and [3H]Lys faster than the later 29-kDa heavy chain. Like beta-glucuronidase, cathepsin D evidently undergoes COOH-terminal proteolytic processing during biosynthesis.
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PMID:Carboxyl-terminal proteolytic processing during biosynthesis of the lysosomal enzymes beta-glucuronidase and cathepsin D. 636 Feb 5

beta-Glucuronidase from human lung neoplasms of various histological types and from uninvolved tissues was studied. A significant elevation of beta-glucuronidase activity was observed in adenocarcinoma and squamous cell carcinoma of the lung as compared with the corresponding uninvolved tissues (P less than 0.01). Saccharo-1,4-lactone, a strong inhibitor of the enzyme, exhibited a substantially greater stabilizing effect on the adenocarcinoma enzyme than on the other enzymes. However, removal of the carbohydrate moiety from the adenocarcinoma enzyme by treatment with endo-beta-N-acetylglucosamidase H (endoglycosidase H) brought about a decrease in the stabilizing effect. Tumor beta-glucuronidase showed considerable negative charge heterogeneity in the pI range from 4.2 to 6.2 in isoelectric focusing on polyacrylamide gel. Upon treatment with exogenous alkaline phosphatase or endoglycosidase H, the heterogenous variant forms of the tumor enzyme appeared to partly or completely lose their negative charge and to be converted into forms similar to those of the normal lung enzyme. These data strongly suggest that the variants are highly phosphorylated on the oligosaccharide chains of the enzyme. An experiment on the labelling of beta-glucuronidase with [32P]-phosphoric acid provided further evidence that the acidic variants found in lung cancers are extensively phosphorylated forms of the enzyme.
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PMID:Cancer-associated alteration of beta-glucuronidase in human lung cancer: elevated activity and increased phosphorylation. 643 19

Human beta-glucuronidase bears 3-4 oligosaccharide moieties/subunit of Mr = 75,000. We have previously characterized the endoglycosidase H-releasable oligosaccharides of this enzyme including those which are phosphorylated and involved in targeting to lysosomes. In this study, we report the characterization of the endoglycosidase H-resistant oligosaccharides which were released from beta-glucuronidase with anhydrous hydrazine. Approximately 65% of the hydrazine-released oligosaccharides are of the high mannose type, with the predominant species containing 9 mannose residues. The remaining oligosaccharides appear to originate from incomplete complex oligosaccharides. Their basic structures are Man alpha 1,6Man beta 1,4Glc-NAc beta 1,4GlcNAcol, and Man alpha 1,3[Man alpha 1,6]Man beta 1,4Glc-NAc beta 1,4GlcNAcol with roughly half of each species containing an additional fucose linked alpha 1,6 to the N-acetylglucosaminitol (GlcNAcol) residue. The small amount of complex oligosaccharide present bearing 1 sialic acid was heterogeneous in nature with incompletion of the nonsialylated branch. In addition, there was a minor specie of high mannose-type oligosaccharide bearing 5 mannose residues with an alpha 1,6-linked fucose on the GlcNAcol. This structure was not expected since high mannose-type oligosaccharides have been reported to not be substrates for the alpha 1,6-fucosyl transferase.
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PMID:Structural studies of the endoglycosidase H-resistant oligosaccharides present on human beta-glucuronidase. 680 59

Phosphomannosyl residues present on numerous acid hydrolases serve a critical role in mediating the endocytosis and intracellular transport of these glycoproteins. Previous work established that the mannose 6-phosphate on lysosomal enzymes is present on endoglycosidase H releasable oligosaccharides and that much of the phosphate is in diester linkage. In order to determine the number and location of the phosphates as well as the precise arrangement of the neutral sugar residues, we examined the structures of the phosphorylated oligosaccharides from a single acid hydrolase, human beta-glucuronidase isolated from spleen. The beta-glucuronidase-derived phosphorylated oligosaccharides are all high mannose-type oligosaccharides whose linkages correspond to previously described prototypical high mannose structures. They contain 1 or 2 moieties of mannose 6-phosphate/oligosaccharide. The major species contains 1 phosphate in diester linkage and represents approximately 63% of the phosphorylated oligosaccharides. Only 15% of the phosphorylated oligosaccharides have their phosphate exclusively in monoester linkage. The phosphate(s) present on these molecules is heterogeneous in location, but all of the phosphate present on the branch linked to the 3-carbon of the beta-linked mannose is found on its innermost alpha-1,2-linked mannose. Analysis of the phosphate-covering moiety showed it to be alpha-linked N-acetylglucosamine in most, if not all, cases.
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PMID:Structural studies of the phosphorylated high mannose-type oligosaccharides on human beta-glucuronidase. 706 41


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