Determination of glycosylation sites, disulfide bridges, and the C-terminus of Stereum purpureum mature endopolygalacturonase I by electrospray ionization mass spectrometry.
Determination of glycosylation sites, disulfide bridges, and the C-terminus of Stereum purpureum mature endopolygalacturonase I by electrospray ionization mass spectrometry.
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通过电喷雾电离质谱法测定 Stereum purpureum 成熟内聚半乳糖醛酸酶 I 的糖基化位点、二硫桥和 C 末端。
DOI：
10.1046/j.1432-1327.2000.01249.x
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
T. Okuno
中科院分区：
文献类型：
--
作者：
T. Shimizu;K. Miyairi;T. Okuno
紫革菌内切多聚半乳糖醛酸酶（endoPG; EC 3.2.1.15）是苹果树中银叶病的致病蛋白。内聚半乳糖醛酸酶I是三种组分（Ia、Ib和Ic）的混合物，在SDS/PAGE上产生三条带，但具有相同的多肽和糖链。三种endoPG I蛋白和去糖基化endoPG Ia的电喷雾电离质谱（ESI-MS）分析显示Ia、Ib和Ic的分子量分别为37 068、38 285和39 503; N-结合糖链的数量与高甘露糖型糖链的数量相匹配。endoPG Ia、Ib和Ic中分别存在两条、三条和四条糖链。在C-末端区域发生了44个氨基酸的缺失。糖基化位点和二硫键的位置通过胰蛋白酶消化，然后通过液相色谱-电喷雾质谱（LC-ESI-MS）分析还原性和非还原性吡啶乙基化endoPG I蛋白来确定。经测定，Ia、Ib和Ic中的糖基化天冬酰胺分别为Asn 92和161; Asn 92、161、279或302; Asn 92、161、279和302。在Cys 3-Cys 17、Cys 175-Cys 191和Cys 300-Cys 303处观察到三个二硫键。这些结果是真菌endoPG的首次发现，可能有助于澄清立体结构和催化活性之间的关系。
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.