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:3.5.1.52 (PNGase F)
1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monensin, an inhibitor of Golgi function, was used to investigate the role of this cell compartment in the glycosylation of Leishmania donovani promastigote secretory acid phosphatase (EC 3.1.3.2). Monensin-treated cells demonstrated morphological changes in the Golgi complex and secreted enzyme with an altered electrophoretic mobility: two discrete bands of approximately 95 and 110 kDa were found, as compared to the heterodisperse nature of the enzyme from untreated controls. Chemical deglycosylation by mild acid hydrolysis resulted in a similar effect on the electrophoretic mobility of purified extracellular enzyme. Acid phosphatase was also treated with N-glycosidase F (EC 3.5.1.52) to remove N-linked oligosaccharides. The altered lectin-binding properties of the enzyme after these two treatments demonstrated that an unusual type of galactose-containing acid-labile carbohydrate was present in secretory acid phosphatase in addition to the N-linked oligosaccharides. Further, experiments with 32P-labelled enzyme indicated that phosphodiester bonds were the structural component responsible for the sensitivity of this carbohydrate to mild acid hydrolysis. Cumulatively, these results demonstrated that a novel form of Golgi-mediated posttranslational modification had occurred to the secretory acid phosphatase presumably by the addition of an acid-labile phosphoglycan.
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PMID:Golgi-mediated post-translational processing of secretory acid phosphatase by Leishmania donovani promastigotes. 232 58

The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of the abundant surface lipophosphoglycan, but differ in having much shorter phosphoglycan chains and a more diverse series of mannose cap oligosaccharides. These data suggest that there are marked differences in the ability of different glycosyltransferases to utilize peptide-linked versus glycolipid-linked acceptors.
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PMID:O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans. 792 59

We previously described the purification of an 83-kDa phosphatidic acid phosphatase (PAP) from the porcine thymus membranes (Kanoh, H., Imai, S.-i., Yamada, K. and Sakane, F.(1992) J. Biol. Chem. 267, 25309-25314). However, we found that a minor 35-kDa protein could account for the PAP activity when the purified enzyme preparation was further analyzed. We thus determined the N-terminal sequence of the 35-kDa candidate protein and prepared antipeptide antibody against the determined sequence, MFDKTRLPYVALDVL. The antibody almost completely precipitated the purified enzyme activity. Furthermore, the antibody precipitated from the radioiodinated enzyme preparation a single 35-kDa protein, which was converted to a 29-kDa form when treated with N-glycanase. We also found that the immunoprecipitable PAP activity was exclusively associated with the plasma membranes of porcine thymocytes. These results indicated that the 35-kDa glycosylated protein represents the plasma membrane-bound (type 2) PAP. We surprisingly noted that the N-terminal sequence of the porcine PAP was almost completely conserved in the internal sequence encoded by a mouse partial cDNA clone, hic53, reported as a H2O2-inducible gene (Egawa, K., Yoshiwara, M., Shibanuma, M., and Nose, K.(1995) FEBS Lett. 372, 74-77). We thus amplified from the mouse kidney RNA the hic53 clone by polymerase chain reaction, and obtained a cDNA encoding a novel protein of 283 amino acid residues with a calculated Mr of 31,894. Methionine reported as an internal residue was found to serve as an initiator, and the C-terminal 64 residues were lacking in hic53. The protein contains several putative membrane-spanning domains and two N-glycosylation sites. When transfected into 293 cells, the cDNA gave more than 10-fold increase of the membrane-bound PAP activity, which could be precipitated by the antipeptide antibody. In [35S]methionine-labeled cells, the translational product was confirmed to be a 35-kDa protein, which became 30 kDa in cells treated with tunicamycin, an inhibitor of N-glycosylation. We thus succeeded first in identifying the porcine type 2 PAP and subsequently in determining the primary structure of a mouse homolog of the PAP.
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PMID:Identification and cDNA cloning of 35-kDa phosphatidic acid phosphatase (type 2) bound to plasma membranes. Polymerase chain reaction amplification of mouse H2O2-inducible hic53 clone yielded the cDNA encoding phosphatidic acid phosphatase. 870 56

Supernatants prepared from disrupted Coxiella burnetii possess acid phosphatase (ACP) activity that apparently accounts for the inhibition of the metabolic burst of formyl-Met-Leu-Phe(fMLP)-stimulated human neutrophils. Results are presented regarding purification and biochemical-biological characterization of the ACP. The highly purified enzyme, which exhibited an apparent M(r) of 91 K and optimal activity at pH 5.0, also inhibited neutrophils. The enzyme retained full activity at pH 4.5, 5.5, and 7.4, when incubated overnight at 0 degrees C and room temperature; at pH 5.5, it retained full activity after overnight incubation at 37 degrees C. Apparently, the enzyme contains asparagine-linked but not serine- or threonine-linked glycan residues since its treatment with N-glycosidase F (PNGase F) decreased its M(r) to 87 K and no changes were detected with O-glycosidase. The enzyme's capacity to hydrolyze phosphate from a number of phosphate-containing compounds was examined; five phosphocompounds were significantly hydrolyzed: 5'-CMP > fructose 1,6-diphosphate > tyrosine phosphate > 3'-AMP > 5'-AMP. The ACP also dephosphorylated (32)P-Raytide, a phosphotyrosine-containing peptide. Dephosphorylation of Raytide was inhibited by the following phosphatase inhibitors: sodium molybdate, potassium fluoride, sodium ortho-vanadate and D2, a heteropolymolybdate compound. These results indicate that C. burnetii ACP may play a role in disrupting tyrosine phosphorylation/dephosphorylation reactions associated with the signal transduction pathway culminating in the metabolic burst. Interestingly, Western blot analysis of ACP-inhibited neutrophils showed a marked increase in tyrosine phosphorylation of a 44 K protein as compared to uninhibited cells.
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PMID:Protein-tyrosine phosphatase activity of Coxiella burnetii that inhibits human neutrophils. 917 54

We obtained two human cDNA clones encoding phosphatidic acid phosphatase (PAP) isozymes named PAP-2a (Mr = 32,158) and -2b (Mr = 35, 119), both of which contained six putative transmembrane domains. Both enzymes were glycosylated and cleaved by N-glycanase and endo-beta-galactosidase, thus suggesting their post-Golgi localization. PAP-2a and -2b shared 47% identical sequence and were judged to be the human counterparts of the previously sequenced mouse 35-kDa PAP(83% identity) and rat Dri42 protein (94% identity), respectively. Furthermore, the sequences of both PAPs were 34-39% identical to that of Drosophila Wunen protein. In view of the functions ascribed to Wunen and Dri42 in germ cell migration and epithelial differentiation, respectively, these findings unexpectedly suggest critical roles of PAP isoforms in cell growth and differentiation. Although the two PAPs hydrolyzed lysophosphatidate and ceramide-1-phosphate in addition to phosphatidate, the hydrolysis of sphingosine-1-phosphate was detected only for PAP-2b. PAP-2b was expressed almost ubiquitously in all human tissues examined, whereas the expression of PAP-2a was relatively variable, being extremely low in the placenta and thymus. In HeLa cells, the transcription of PAP-2a was not affected by different stimuli, whereas PAP-2b was induced (up to 3-fold) by epidermal growth factor. These findings indicate that despite structural similarities, the two PAP isozymes may play distinct functions through their different patterns of substrate utilization and transcriptional regulation.
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PMID:Cloning and characterization of two human isozymes of Mg2+-independent phosphatidic acid phosphatase. 930 23

We investigated the effect of the carbohydrate chain and two phosphate moieties on heat-induced aggregation of hen ovalbumin. The dephosphorylated form of ovalbumin was obtained by treating the original protein with acid phosphatase. The single carbohydrate chain was removed by digestion of heat-denatured ovalbumin with glycopeptidase F, and the resulting polypeptide without this carbohydrate chain was correctly refolded to acquire protease-resistance. Thermal unfolding can be approximated by a mechanism involving a two-state transition between the folded and unfolded states with a midpoint temperature of 76 degrees C for the original form, of 74 degrees C for the dephosphorylated form, and of 71 degrees C for the carbohydrate-free form. The conformational stability of the original form was higher than that of the carbohydrate-free form. When the three forms of ovalbumin were heated to 80 degrees C and then cooled rapidly in an ice bath, the polypeptide chains were compactly collapsed to metastable intermediates with secondary structures whose properties were indistinguishable. Upon incubation at 60 degrees C, renaturation was possible for a large portion of the intermediates of the original form, but for only a small portion of those of the carbohydrate-free form. Light scattering experiments showed that in the presence of sulfate anions, the intermediates of the carbohydrate-free form aggregated to a greater extent than did those of the original form. The intermediates of the carbohydrate-free form bound to the chaperonin GroEL with about 10-fold higher affinity than those of the original form. It follows that the carbohydrate chain and the two phosphate moieties do not affect hydrophobic collapse in the kinetic refolding of hen ovalbumin but play an important role in the slow rearrangement. They block the off-pathway reaction that competes with correct refolding by effectively decreasing surface hydrophobicity.
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PMID:Role of the carbohydrate chain and two phosphate moieties in the heat-induced aggregation of hen ovalbumin. 1561 16

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