GLYCAN STRUCTURE & SITE OF GLYCOSYLATION IN RECOMBINANT ALPHA1 ANTITRYPSIN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本研究的目的是确定重组α1抗胰蛋白酶β1PI的糖链结构和糖基化位点(S)的位置。 在本研究中，A1PI在黑曲霉中得到了表达，并负载于SDS-PAGE中，考马斯亮蓝染色。从凝胶中分离出两条主带，并分别研究了每一组分(带)的糖链结构。用HPAEC、MALDI/TOF-MS、NSI-MSN和GC-MS对释放的多糖进行了分析。我们发现，在两个池中，重组的a1PI中都含有由几个末端的呋喃半乳糖苷取代的高甘露糖型N-聚糖的异质混合物，但比例不同。B池含有较高比例的较长多聚糖，而D池含有较高比例的较短多聚糖。 2008年，我们研究了a1PI蛋白的N-糖基化位点定位。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The aim of the present study is to determine the glycan structure and the positions of glycosylation site(s) on the recombinant alpha1 antitrypsin, ¿1PI. In this study, A1PI was expressed in Aspergillus niger and loaded in SDS-PAGE and stained with Coomassie Blue. The two main bands were excised from the gel, and the glycan structure of each fraction (band) was investigated separately. The released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MSn, and GC-MS. We found that the recombinant a1PI, in both pools, contained heterogeneous mixture of high-mannose type N-glycans substituted with several terminal Galactofuranoses but the proportions were different. Pool B contains higher ratio of the longer glycans, whereas pool D contained higher portions of shorter glycans. In 2008, we studied the N-glycosylation site mapping of a1PI protein.