The glycosylation status and the role of carbohydrate moieties in the heterogeneity of cucumber anionic virus-inducible peroxidase.
Three forms of anionic peroxidase (PRX) from hypersensitively reacting cucumber cotyledons were purified to homogeneity and different methods were used to analyze the nature of their carbohydrate chains. Immunoblot analysis with betaF1 antiserum showed that all three forms are highly glycosylated and contain asparagine N-linked glycans commonly found in other plant glycoproteins. Mobility shift analysis showed that chemical deglycosylation converted PRXs 1, 2 and 3 to the same-sized (35 K) products. Enzymatic deglycosylation with alpha-mannosidase converted PRX1 and PRX2 to immunoreactive products migrating in mobility shift polyacrylamide gels at the positions of PRX2 and PRX3, respectively. PRX3 treated with alpha-mannosidase yielded a product with Mr similar to that obtained with the chemical deglycosylation. Cleavage of the PRXs 1, 2 and 3 by formic acid at the Asp-Pro site resulted in peptide maps and the putative glycopeptide(s) were recognized using betaF1 antiserum. Only one glycopeptide was observed for each of the forms. Lectin-affinity blot analysis using biotin-conjugated lectins suggested that virus-inducible PRX contains complex-type N-glycosyl carbohydrate chain(s). These results indicate that heterogeneity of cucumber virus-inducible PRX is not caused mainly by differences in the terminal alpha-linked mannose residues.
Laboratory of Molecular Biology and Virology, Research Institute of Viticulture and Enology, Bratislava, Slovak Republic. repka@crive.axonpro.sk
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D050260:Carbohydrate Metabolism; D018548:Cotyledon / Q000201:enzymology; D018553:Cucumis sativus / Q000201:enzymology* / Q000821:virology; D004591:Electrophoresis, Polyacrylamide Gel; D005561:Formates; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D037102:Lectins; D008361:Mannosidases / Q000494:pharmacology; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D037121:Plant Lectins; D043323:alpha-Mannosidase
D005561:Formates; D006020:Glycopeptides; D007527:Isoenzymes; D037102:Lectins; D037121:Plant Lectins; C030544:formic acid; D010544:Peroxidases; C120494:anionic peroxidase; D008361:Mannosidases; D043323:alpha-Mannosidase