Post‐translational modifications of recombinant B. cinerea EPG 6

Abstract

The fungus Botrytis cinerea is a ubiquitous plant pathogen that infects more than 200 different plant species and causes substantial economic losses in a wide range of agricultural crops and harvested products. Endopolygalacturonases (EPGs) are among the first array of cell-wall-degrading enzymes secreted by fungi during infection. Up to 13 EPG glycoforms have been described for B. cinerea. The presence of multiple N-linked glycosylation modifications in BcPG1-6 is predicted by their deduced amino acid sequences. In this work, the glycosylation sites and the attached oligosaccharide structures on BcPG6 were analyzed. The molecular mass of the intact glycoprotein was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis. BcPG6 contains seven potential N-linked glycosylation sites. Occupancy of these glycosylation sites and the attached carbohydrate structures were analyzed by tryptic digestion followed by liquid chromatography/mass spectrometry (LC/MS) using a stepped orifice voltage approach. Five out of seven potential N-linked sites present in BcPG6 were determined to be occupied by high-mannose-type oligosaccharides. Four of them were readily determined to be at Asn58 (T3 peptide), Asn198 (T7 peptide), Asn237 (T9 peptide) and Asn256 (T11 peptide), respectively. Another was located on the T8 peptide, which contained two potential N-linked sites, Asn224 and Asn227 (SNNN224VTN227ITFK). LC/MS/MS of a sample treated with N-glycanase placed the glycan in this peptide at Asn224 rather than at Asn227. The potential glycosylation site on Asn146 (T6 peptide) was not glycosylated. In addition, two disulfide bonds were observed, linking the Cys residues within the T13 and T16 peptides.

A proteomic study of pectin‐degrading enzymes secreted by Botrytis cinerea grown in liquid culture

“…There is a possibility that the BcPG1 protein was present, but not in sufficient quantity for detection via shotgun proteomics, or it is possible that it was not detected as the result of extensive post translation modifications. Post translational modifications have been reported on other endo PG isoforms, notably BcPG6 [28], which were, in fact, detected in our analysis. The controlled secretion of endo PGs and the role of the PG inhibiting proteins in the plant wall as a defense mechanism against B. cinerea infections emphasize the importance of BcPG [29, 30].…”