Glycosylation of catalase inhibitor necessary for activity

Tsaftaris, Athanasios; Sorenson, John C.; Scandalios, John G.

doi:10.1016/0006-291x(80)90786-x

Cited by 11 publications

(5 citation statements)

References 11 publications

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“…For example, glycans may play an informational role and contain active sorting information, as in the case of the mannose-6-phosphate groups of mammalian lysosomal enzymes (Sly and Fisher 1982); they may alter the physicochemical properties of proteins so that they are more soluble or fold correctly allowing the proteins to be transported more efficiently (Gibson et al 1981); they may play a role in the correct posttranslational proteolytic processing of polypeptides ; they may be required for the biological activity of glycoproteins (Tsaftari et al 1980), or they may stabilize proteins against proteolytic degradation (Prives and Olden 1980;Winkler and Segal 1984). Each of these roles has been documented by several examples.…”

Section: Introductionmentioning

confidence: 98%

The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins

Driouich

Gonnet

Makkie

et al. 1989

Planta

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Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) secrete a number of acid hydrolases and other proteins that have both highmannose and complex asparagine-linked glycans. We used affinity chromatography with concanavalin A and an antiserum specific for complex glycans in conjunction with in vivo-labeling studies to show that all of the secreted proteins carry glycans. The presence of complex glycans on secretory proteins indicates that they are passing through the Golgi complex on the way to the extracellular compartment. The sodium ionophore, monensin, did not block the transport of proteins to the extracellular medium, even though monensin efficiently inhibited the Golgi-mediated processing of complex glycans. The inhibition of N-glycosylation by tunicamycin reduced by 76% to 84% the accumulation of newly synthesized (i.e. radioactively labeled) protein that was secreted by the sycamore cells, while cytoplasmic protein biosynthesis was not affected by this antibiotic. However, in the presence of glycoprotein-processing inhibitors, such as castanospermine and deoxymannojirimycin, the formation of complex glycans was prevented but glycoprotein secretion was unchanged. These results support the conclusion that N-linked glycan processing is not necessary for sorting, but glycosylation is required for accumulation of secreted proteins in the extracellular compartment.

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Section: Introductionmentioning

confidence: 98%

The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins

Driouich

Gonnet

Makkie

et al. 1989

Planta

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“…Catalase contains no detectable carbohydrate and is not inhibited by several by several lectins tested (19). The inhibitor does contain about 12% carbohydrate in the form ofgalactose and its inhibitory effect on catalase can be blocked by galactose-specific lectins or by treatment with fl-galactosidase (19).…”

Section: Resultsmentioning

confidence: 99%

Biochemical Characterization of a Catalase Inhibitor from Maize

Sorenson¹,

Scandalios²

1980

Plant Physiol.

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Some biochemical properties of the catalase inhibitor purified from maize scutella are described. The inhibitor is heat-labile and its activity is destroyed by trypsin, indicating that it is a protein. It does not appear to be a lectin nor does the inhibition involve proteolysis. The active inhibitor is a dimer with each subunit having a molecular weight of 5600 as determined by sodium dodecyl sulfate electrophoresis. A kinetic analysis performed in the presence of increasing levels of inhibitor gave unusual Lineweaver-Burk patterns. Possible explanations for these patterns are discussed. The inhibitor is active against all catalases tested from a wide variety of organisms.A substance in maize scutella which inhibits the enzyme catalase (H202:H202 oxidoreductase, EC 1.11.1.6) has been reported; this substance has been purified by affmity chromatography using immobilized catalase (16). The activity of the inhibitor varies inversely with catalase activity in the scutellum of the germinating seed (17) and constitutes one of several mechanisms regulating catalase activity in that tissue (10,12,18). This report describes some of the biochemical properties of this inhibitor. MATERIALS AND METHODSMaterial. Seeds from the highly inbred lines W64A and 229 were used in all studies. Seeds were imbibed in tap water for 12 to 18 h, transferred to plastic trays lined with moist germination papers, and maintained in the dark at 27 C until use. Seedlings were watered daily.Inhibitor Preparation and Assay. The inhibitor was purified by a modification of the affinity chromatography method previously described (16). A scutellar extract was passed through a column containing bovine liver catalase-Sepharose. Following extensive washing with 0.5 M galactose and 0.1 M NaCl, the bound inhibitor was eluted with 1 M NaCl. The galactose wash removed several proteins which apparently adsorbed to the Sepharose matrix. The I M NaCl wash was dialyzed against 10 mM Tris-HCl buffer (pH 7.4) and was stored at 4 C for up to I week. Such preparations lost less than 15% of their original activity during that storage period. The inhibitor was assayed against partially purified maize catalase as previously described (17).Trypsin Digestion. to conduct the digestion. Following incubation, the trypsin was removed by centrifugation at l0,OOOg and the aliquots were assayed for inhibitory activity.After the initial inhibitor action period (approximately 5 min; Fig. 3), no further decrease in catalase activity was observed. This suggests that the centrifugation effectively removed the insolubilized trypsin. Incubation of catalase with insolubilized trypsin under the inhibitor assay times and conditions did not result in a significant decrease in catalase activity.Mol Wt Determination. The mol wt of the native protein was determined by electrophoresis of purified inhibitor under nondenaturing conditions in gels composed of 9.5, 10.0, 10.5, and 11.0% acrylamide as described by Hendrick and Smith (3). The inhibitor does not stain well on gels (see "Dis...

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“…The host factor(s) responsible for inhibition of the fungal phosphatase activity are not known, but may include the higher pH and phosphate concentration in the host cells, since these have a similar effect on enzyme activity and distribution in hyphae growing in pure culture. However, glycoproteins of eucaryotes which are rich in mannose residues , as is the LMW phosphatase of H. ericae, have a common precursor [polypeptide (N or O), (GlcNAc)2, (mannose 9)1 modifications of which are determinate for different functions such as biological activity (Tsaftari et al 1980), post-translational maturation (Fay and Chrispeels 1987), secretion and transport (Driouich et al 1989), solubility and stability (Driouich et al 1989;Prives and Olden 1980;Sly and Fischer 1982;Winkler and Segal 1984). In ericoid endomycorrhiza, the fact that immunoserological reactions of acid phosphatase antigen in the fungal wall are associated with the greatly reduced or inhibited enzyme activity of hyphae growing in host cells could be due to isozymes having conformational modifications, for example on the glycosidic part of the molecule which we have shown to be necessary for enzyme activity.…”

Section: Discussionmentioning

confidence: 97%

“…In ericoid endomycorrhiza, the fact that immunoserological reactions of acid phosphatase antigen in the fungal wall are associated with the greatly reduced or inhibited enzyme activity of hyphae growing in host cells could be due to isozymes having conformational modifications, for example on the glycosidic part of the molecule which we have shown to be necessary for enzyme activity. Removal of mannose residues from the enzyme molecule or interference with glycosylation of a precursor by the host cell would expose the N-acetyl glucosamine moiety, so inactivating the enzyme and eventually giving the protein another function (Tsaftari et al 1980). The possibility of modification of the enzyme on its glycosidic groups is supported by the observation of Bonfante et al (1987) that the plant lectin concanavalin A, which detects mannose residues, binds with external hyphae but not internal hyphae, which only react with wheat germ agglutinin for N-acetyl glucosamine detection.…”

Section: Discussionmentioning

confidence: 99%

Occurrence and expression of acid phosphatase of Hymenoscyphus ericae (Read) Korf & Kernan, in isolation or associated with plant roots

et al. 1992

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Summary. The activity of acid phosphatase produced in pure culture by the endomycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan (H. ericae LPA 2) was inhibited by high phosphorus levels, alkaline pH, fluoride, molybdate and mannosidase, and activated by concanavalin A. Over 80% of the enzyme activity was due to two wall-bound acid phosphatase isozymes with the characteristics of mannose-rich glycoproteins. Antiserum was raised against the major, low-molecularweight wall isozyme and its activity tested by immunoblotting and ELISA. The antiserum cross reacted 100~ with exocellular (excreted) and 28~ with cytoplasmic cellular fractions of H. ericae (LPA 2) cultures, and showed high reactivity with other strains of H. ericae but not with fungal isolates from Erica hispidula L. or E. mauritanica L. Ultrastructural localization of acid phosphatase by cytoenzymology and indirect immunogold labelling confirmed its association with the fungal wall in pure culture and showed that the influence of a high phosphorus level, fluoride and molybdate is through inactivation of the enzyme. Intense acid phosphatase activity, sensitive to the latter inhibitors, was also present on external hyphae growing over a host or non-host root but it was weak or absent from intracellular hyphae where these developed within a host root. Indirect immunolabelling confirmed that this acid phosphatase was of fungal origin and that the specific inhibitory effect of host cells is due to inactivation of the enzyme rather than repression of its synthesis. Possible implications of fungal acid phosphatase in ericoid endomycorrhizal infection processes are discussed together with mechanisms that may be regulating the enzyme activity.

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Glycosylation of catalase inhibitor necessary for activity

Cited by 11 publications

References 11 publications

The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins

The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins

Biochemical Characterization of a Catalase Inhibitor from Maize

Occurrence and expression of acid phosphatase of Hymenoscyphus ericae (Read) Korf & Kernan, in isolation or associated with plant roots

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