Stahl B, Klabunde T, Witzel H, Krebs B, Steup M, Karas M, Hillenkamp F
Institut für Botanik, Westfälische Wilhelms-Universität, Münster, Germany.
Eur J Biochem. 1994 Mar 1;220(2):321-30. doi: 10.1111/j.1432-1033.1994.tb18628.x.
Purple acid phosphatase of the common bean Phaseolus vulgaris (KBPase), a dimeric 110-kDa glycoprotein related to the mammalian purple acid phosphatases with a two-metal cluster at the active site contains five oligosaccharide side chains/monomer. The N-linked glycan structures were characterized by selective enzymic degradation in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The purified protein was cleaved by cyanogen bromide. One 30-kDa large methionine-free fragment required a further tryptic digest. The peptides were separated by HPLC and the glycosylated species were identified both by their heterogeneous mass spectra and by an immunoassay. None of the glycopeptides proved to have more than one glycosylation site. The composition of the carbohydrate moieties were calculated by comparing the mass spectra of the glycopeptides before and after enzymic deglycosylation. These results were complemented by data from a carbohydrate composition analysis. In four of the five peptides an alpha 1-3 fucose attached to the asparagine-linked N-acetylglucosamine prevented removal of the glycan by peptide N-glycosidase F; peptide N-glycosidase A removed all carbohydrates from the peptides. To reveal the sequence of the carbohydrate moiety including the linkage positions between the different saccharides, one of the glycopeptides was degraded by specific exoglycosidases. The enzymic degradations by these hydrolases were monitored by mass spectrometry of small aliquots taken at intervals during the reaction. The detailed structure of this one glycan in conjunction with the respective mass spectra and the composition analysis were used to infer the structure of the other four glycans. All glycans of the KBPase have a complex-type xylose-containing structure with four of the five having an additional fucose.
菜豆（Phaseolus vulgaris）的紫色酸性磷酸酶（KBPase）是一种二聚体110 kDa糖蛋白，与哺乳动物的紫色酸性磷酸酶相关，其活性位点有一个双金属簇，每个单体含有五条寡糖侧链。通过选择性酶解结合基质辅助激光解吸/电离质谱（MALDI-MS）对N-连接聚糖结构进行了表征。纯化后的蛋白用溴化氰裂解。一个30 kDa的无甲硫氨酸大片段需要进一步用胰蛋白酶消化。肽段通过高效液相色谱分离，糖基化产物通过其异质质谱和免疫测定进行鉴定。没有一个糖肽被证明有多个糖基化位点。通过比较酶解去糖基化前后糖肽的质谱来计算碳水化合物部分的组成。碳水化合物组成分析的数据对这些结果起到了补充作用。在五条肽段中的四条中，连接在天冬酰胺连接的N-乙酰葡糖胺上的α1-3岩藻糖阻止了肽N-糖苷酶F去除聚糖；肽N-糖苷酶A去除了肽段上的所有碳水化合物。为了揭示碳水化合物部分的序列，包括不同糖类之间的连接位置，其中一个糖肽用特定的外切糖苷酶进行了降解。在反应过程中，每隔一段时间取少量样品进行质谱分析，监测这些水解酶的酶解过程。结合各自的质谱和组成分析，利用这一个聚糖的详细结构推断其他四个聚糖的结构。KBPase的所有聚糖都具有复杂型含木糖结构，五条中有四条还含有额外的岩藻糖。
