GLYCAN STRUCTURE & SITE OF GLYCOSYLATION IN RECOMBINANT ALPHA1 ANTITRYPSIN

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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.