Determination of glycosylation sites, disulfide bridges, and the Câterminus of Stereum purpureum mature endopolygalacturonaseâI by electrospray ionization mass spectrometry
2000
Shimizu, Tetsuya | Miyairi, Kazuo | Okuno, Toshikatsu
Stereum purpureum endopolygalacturonase (endoPG; ECâ3.2.1.15) is a causal protein for silverâleaf disease in apple trees. EndopolygalacturonaseâI, 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 (ESIâMS) analysis of three endoPGâI proteins and deglycosylated endoPGâIa revealed a molecular mass of 37â068, 38â285, and 39â503 for Ia, Ib, and Ic, respectively; the number of Nâbinding sugar chains matches that of a highâmannose type of sugar chain. Two, three, and four sugar chains are present in endoPGâIa, Ib, and Ic, respectively. Deletion of 44 amino acids from the deduced sequence occurred in the Câterminal region. Positions of the glycosylation sites and disulfide bridges were decided by tryptic digestion followed by liquid chromatographyâelectrospray mass spectrometry (LCâESIâMS) analysis of reductive and nonreductive pyridylethylated endoPGâI 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.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
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