Determination of glycosylation sites, disulfide bridges, and the C‐terminus of Stereum purpureum mature endopolygalacturonase I by electrospray ionization mass spectrometry

Abstract

Stereum purpureum endopolygalacturonase (endoPG; EC 3.2.1.15) is a causal protein for silver-leaf disease in apple trees. Endopolygalacturonase I, is a mixture of three components (Ia, Ib, and Ic) that produce three bands on SDS/PAGE but have the same polypeptide and sugar chains. Electrospray ionization mass spectrometry (ESI-MS) analysis of three endoPG I proteins and deglycosylated endoPG Ia revealed a molecular mass of 37 068, 38 285, and 39 503 for Ia, Ib, and Ic, respectively; the number of N-binding sugar chains matches that of a high-mannose type of sugar chain. Two, three, and four sugar chains are present in endoPG Ia, Ib, and Ic, respectively. Deletion of 44 amino acids from the deduced sequence occurred in the C-terminal region. Positions of the glycosylation sites and disulfide bridges were decided by tryptic digestion followed by liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) analysis of reductive and nonreductive pyridylethylated endoPG I proteins. The glycosylated asparagines were determined to be Asn92 and 161; Asn92, 161, 279, or 302; and Asn92, 161, 279, and 302 in Ia, Ib, and Ic, respectively. Three disulfide bridges were noted at Cys3±Cys17, Cys175±Cys191, and Cys300±Cys303. These results are the first findings for fungal endoPG and may contribute to clarification of the relationship between stereostructure and catalytic activity.

Purification, Characterization and Amino Acid Sequence of Endopolygalacturonases IVa and IVb from Fungus Stereum purpureum

“…With results of protein sequencing from the N terminal amino acid and cDNA cloning in agreement, the C terminus was judged to have differed due to C terminal analysis showing a leucine to be in place of a glycine. 4) This result was supported by ESI MS analysis showing 44 amino acid residues to be missing from the C terminal region of the mature enzyme. 4) For EndoPGs, this was the first report of its kind.…”

“…4) This result was supported by ESI MS analysis showing 44 amino acid residues to be missing from the C terminal region of the mature enzyme. 4) For EndoPGs, this was the first report of its kind. The reasons for such an absence are yet unknown.…”

“…4) Enzymatic properties were found common between EndoPG Is except for the difference in number of sugar chains. A common characteristic found of EndoPG Is is their extreme thermostability, with no decrease in activity on heating at 70 C for 30 min.…”

“…3,4) EndoPG I has been separated into three components (Ia, Ib and Ic) by CM52 column chromatography in spite of an identical isoelectric point.…”

“…The position of three disulphide bonds in EndoPG I has also been clarified based on ESI MS analysis of peptides from tryptic digestion of the enzyme. 4) Structural analysis by X ray crystallography has given results at resolutions up to 0.96 A . 8) Part of the enzyme s catalytic mechanism has also been clarified by analysis of the crystal complex (binary and ternary) occurring in reaction between enzyme and substrate.…”