Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kinetic properties of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) partially purified from the soil amoeba Acanthamoeba castellanii have been studied. The transferase phosphorylated the lysosomal enzymes uteroferrin and cathepsin D 3-90-fold better than nonlysosomal glycoproteins and 16-83-fold better than a Man9GlcNAc oligosaccharide. Deglycosylated uteroferrin was a potent competitive inhibitor of the phosphorylation of intact uteroferrin (Ki of 48 microM) but did not inhibit the phosphorylation of RNase B or the simple sugar alpha-methylmannoside. Deglycosylated RNase (RNase A) did not inhibit the phosphorylation of RNase B or uteroferrin. These results indicate that purified amoeba GlcNAc-phosphotransferase recognizes a protein domain present on lysosomal enzymes but absent in most nonlysosomal glycoproteins. The transferase also exhibited a marked preference for oligosaccharides containing mannose alpha 1,2-mannose sequences, but this cannot account for the high affinity binding to lysosomal enzymes. A. castellanii extracts do not contain detectable levels of N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, the second enzyme in the biosynthetic pathway for the mannose 6-phosphate recognition marker. We conclude that A. castellanii does not utilize the phosphomannosyl sorting pathway despite expression of very high levels of GlcNAc-phosphotransferase.
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PMID:Characterization of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii. 131 74

The mannose 6-phosphate (Man6P)-dependent pathway for routing lysosomal enzymes was characterized in the hepatopancreas of the estuary crab Chasmagnatus granulata: (a) an acid alpha-L-fucosidase was purified to homogeneity from the above-mentioned organ and was shown to contain mannose-linked phosphate residues; (b) high-mannose-type oligosaccharides isolated from a protein fraction enriched in acid hydrolases were found to contain acid-labile N-acetylglucosamine (GlcNAc) residues; (c) a membrane-bound UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase was detected that phosphorylated the estuary-crab alpha-L-fucosidase and bovine uteroferrin but not bovine pancreas ribonuclease B; (d) a GlcNAc-1-phosphodiester alpha-N-acetylglucosaminidase that released GlcNAc units from GlcNAc alpha 1-P6Man alpha 1-methyl was detected in microsomal membranes of the hepatopancreas; (e) two detergent-solubilized microsomal proteins having molecular masses of 205 and 215 kDa that were retained by a Man6P-rich mannan-Sepharose column, from where they were eluted with Man6P but not with glucose 6-phosphate, were recognized by antisera raised against bovine large (215 kDa) and small (46 kDa) Man6P receptors. This is the first description of all the components of the Man6P-dependent mechanism for routing lysosomal enzymes in an invertebrate.
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PMID:Characterization of the mannose 6-phosphate-dependent pathway of lysosomal enzyme routing in an invertebrate. 765 99

The kinetic properties of UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) purified to homogeneity from lactating bovine mammary gland have been investigated. GlcNAc-phosphotransferase transferred GlcNAc 1-phosphate from UDP-GlcNAc to the synthetic acceptor alpha-methylmannoside, generating GlcNAc-1-phospho-6-mannose alpha-methyl, the structure of which was confirmed by mass spectroscopy. GlcNAc-phosphotransferase was active between pH 5.7 and 9.3, with optimal activity between pH 6.6 and 7.5. Activity was strictly dependent on Mg2+ or Mn2+. The Km for Mn2+ was 185 microM. The Km for UDP-GlcNAc was 30 microM, and that for alpha-methylmannoside was 63 mM. The enzyme was competitively inhibited by UDP-Glc, with a Ki of 733 microM. The 166-kDa subunit was identified as the catalytic subunit by photoaffinity labeling with azido-[beta-32P]UDP-Glc. Purified GlcNAc-phosphotransferase utilizes the lysosomal enzyme uteroferrin approximately 163-fold more effectively than the non-lysosomal glycoprotein ribonuclease B. Antibodies to GlcNAc-phosphotransferase blocked the transfer to cathepsin D, but not to alpha-methylmannoside, suggesting that protein-protein interactions are required for the efficient utilization of glycoprotein acceptors. These results indicate that the purified bovine GlcNAc-phosphotransferase retains the specificity for lysosomal enzymes as acceptors previously observed with crude preparations.
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PMID:Bovine UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase. II. Enzymatic characterization and identification of the catalytic subunit. 894 Jan 56

Increasing evidence suggests that estrogen deficiency in women promotes the expansion of populations of bone marrow cells that differentiate into osteoclasts under the influence of osteotropic hormones and local factors. A progressive cytoplasmic accumulation of osteoclastic bone resorbing enzymes, such as tartrate-resistant acid phosphatase (TRACP) and carbonic anhydrase II (CA II), characterizes osteoclast differentiation. To evaluate the possibility that estrogen may have a direct effect on osteoclast precursor cells, we investigated the mRNA levels of estrogen receptor a (ERa), TRACP and CA II genes in neonatal rat bone imprints by fluorescence in situ hybridization and confocal microscopy. Morphological assessment of bone imprints has shown that the putative mononuclear osteoclast precursor cells (MOPC) display strongly basophilic cytoplasm and a low nuclear/cytoplasmic ratio, while some of these cells possess pale-staining ruffled border regions similar to those observed in osteoclasts. Both CA II and TRACP mRNAs were detected in putative MOPC as well as multinuclear osteoclasts. The gene transcripts were mainly located in the cytoplasm of these cells. To determine whether these putative MOPC possess ER mRNA, a 637 base pair antisense ER riboprobe was used. The results indicated that MOPC which show TRACP reactivity express high levels of ER gene transcripts in their cytoplasm. In contrast, only a few multinuclear osteoclasts in the bone imprints possessed ER gene transcripts. Interestingly, the levels of ER mRNA in these multinuclear osteoclasts were very low compared with those in the putative MOPC. Treatment with RNase prior to hybridization resulted in a significant loss of signal in these cells. The results of these studies suggest that estrogen may have a direct role in modulating the recruitment of osteoclast precursor cells during osteoclastogenesis.
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PMID:Detection of estrogen receptor alpha, carbonic anhydrase II and tartrate-resistant acid phosphatase mRNAs in putative mononuclear osteoclast precursor cells of neonatal rats by fluorescence in situ hybridization. 958 36

Legionella pneumophila, the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase. By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans-complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L. pneumophila infection of both amoebae and human macrophages. Based upon a complete analysis of lspDE, lspF, and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies. Examination of the developing L. pneumophila genome database indicated that the organism has two other loci (lspC and lspLM) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria. In contrast to lsp mutants, L. pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication. L. pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes. Upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays. The lspF mutant was also defective in an in vivo kinetic assay. Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo. Thus, the L. pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.
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PMID:Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires' disease pneumonia. 1468 10