Determination of glycosylation sites, disulfide bridges, and the C-terminus of Stereum purpureum mature endopolygalacturonase I by electrospray ionization mass spectrometry.
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.
Shimizu
T
T
Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, City of Hirosaki, Aomori Prefecture, Japan.
Miyairi
K
K
Okuno
T
T
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D005658:Fungi / Q000201:enzymology* / Q000472:pathogenicity; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010935:Plant Diseases / Q000382:microbiology; D011096:Polygalacturonase / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D014197:Trees / Q000382:microbiology
D004220:Disulfides; D005656:Fungal Proteins; D009844:Oligosaccharides; D010446:Peptide Fragments; D011096:Polygalacturonase