Woosley Bryan, Xie Min, Wells Lance, Orlando Ron, Garrison Derek, King Daniel, Bergmann Carl
Complex Carbohydrate Research Center and Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
Anal Biochem. 2006 Jul 1;354(1):43-53. doi: 10.1016/j.ab.2006.02.002. Epub 2006 Feb 21.
The enzyme PGC is produced by the fungus Aspergillus niger during invasion of plant cell walls. The enzyme has been homologously overexpressed to provide sufficient quantities of purified enzyme for biological studies. We have characterized this enzyme in terms of its posttranslational modifications (PTMs) and found it to be both N- and O-glycosylated. The glycosyl moieties have also been characterized. This has involved a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), liquid chromatography (LC)-ion trap, and LC-electrospray ionization (ESI) mass spectrometries in conjunction with trypsin degradation and beta-elimination, followed by Michael addition with dithiothreitol (BEMAD). This is the first demonstration of the ability of BEMAD to map glycosylation sites other than O-GlcNAc sites. The complete characterization of all PTMs on PGC allows us to model them on the peptide backbone, revealing potential roles played by the glycans in modulating the interaction of the enzyme with other macromolecules.
真菌黑曲霉在侵入植物细胞壁的过程中会产生PGC酶。该酶已通过同源过表达来提供足够数量的纯化酶用于生物学研究。我们已从其翻译后修饰（PTM）方面对这种酶进行了表征，发现它同时进行了N-糖基化和O-糖基化。糖基部分也已得到表征。这涉及将基质辅助激光解吸/电离飞行时间（MALDI-TOF）、液相色谱（LC）-离子阱和LC-电喷雾电离（ESI）质谱法与胰蛋白酶降解和β-消除相结合，随后用二硫苏糖醇进行迈克尔加成（BEMAD）。这是首次证明BEMAD能够绘制除O-GlcNAc位点以外的糖基化位点。对PGC上所有PTM的完整表征使我们能够在肽主链上对它们进行建模，揭示聚糖在调节该酶与其他大分子相互作用中所起的潜在作用。
