| Human alpha-antitrypsin (AAT) is the most abundant circulating protease inhibitor in the human plasma. It is produced in the liver and exerts a primary physiological role as inhibitor for the neutrophil elastase in the lung. Individuals with one or several mutations in AAT gene causing reduction of the protein are related to lung, liver diseases and are treated lifelong with infusions of human plasma-derived AAT. Due to shortage of plasma and low expression levels of recombinant AAT in conventional gene expression systems, we explored the possibility to produce recombinant AAT in rice grains(Oryza sativa AAT, OsrAAT). And OsrAAT purification protocol, physical, biochemical features, pharmacokinetics and subcellular localization were analysed. Furthermore, the glycan modification of OsrAAT was detected by LC-MS. Due to the overexpression of OsrAAT in rice endosperm induced the detrimental seed phenotype, the molecular mechanism for this phenomenon was elucidated. In addition, we analysed the glycan modification structures of OsrAAT and endogenous glutelin among ZH11,132-10and132-17, indicating ER stress had influence on the processing of glycan modifications. The major results are following:1, A total of23independent transgenic plants were obtained using a two-strain Agrobacteriummediated transformation and12of these were fertile. Nine transgenic lines accumulating OsrAAT in rice endosperm were identified by western blotting and OsrAAT expression levels ranged from0.41-2.24g/kg brown rice.2. The molecular mass (MM) of OsrAAT was analyzed using MALDI-MS, indicating OsrAAT have four major peaks (40.1KDa,44.8KDa?49KDa and53.3KDa). The secondary structure of OsrAAT was evaluated by comparing the circular dichroism (CD) spectra of OsrAAT in the far-UV region with that of plasma AAT, showing that the secondary structure of OsrAAT was equivalent to pAAT. N-terminus analysis indicated that N-terminus of OsrAAT was processed in rice endosperm. The peptide of40.1kDa is the consequence of a loss on the recording of eleven amino acids from the N-terminus in OsrAAT3.10glycan structrues (GN2M4XFGN2, GN2M4XGN2, GNM3XFGN2,GNM3XGN2, M3XFGN2, M3XGN2, M2XFGN2, M2XGN2, M4GN2and GNM2GN2) were monitored by LC-MS. The complex glycan structures accounted for12.8%, hybrid type was18.7%and Paucimannosidic type was64.8%.4. The biological activity of OsrAAT was analyzed by the band shift and porcine elastase inhibitory activity assays. The results indicated that OsrAAT had the same biological activity to pAAT.The pharmacokinetics study indicated that OsrAAT was quickly metabolized in rat blood, but pAAT retained for more than two hours.5. The OsrAAT purification protocol from brown rice was developed by three chromatography steps:1, anion-exchange on diethylaminoethanol (DEAE) linked Sepharose Fast Flow;2, anion exchanger CHT hydroxyapatite;3, anion exchanger on N-Benzyl-N-methytlethanolamine linked Capto high flow agarose (Capto Adhere). The results from pilot-scale purification provided a recovery for OsrAAT of18.89±3.19%, equivalent to a yield of0.37g of OsrAAT per kilogram of brown rice.6. OsrAAT accumulating in rice endosperm produced abnormal seed phenotype. We analyzed seed phenotypes of132-10and132-17and AAT mRNA expression profiles, indicating the seed sizes were dependent on the OsrAAT accumulation level in rice endosperm.7. Protein body morphology observation and Quantitative PCR results indicated that ER stress induced in the both of132-10and132-17. IRE1-Osbzip50signal pathway was activated in132-10, indicating the sever ER stress was produced in132-17.8. OsrAAT accumulating in rice endosperm produced ER stress, which disturbed the rice endosperm development. The analysis of the programmed cell death (PCD)-related genes expression profiles and TUNEL assay elucidated that ER stress induced pre-mature PCD in the both of132-10and132-17, resulting in the reduction of seed size.9. The analysis of glycan modification and molecular mass between132-10and132-17indicated OsrAAT N-terminus processing in132-17decreased and the glycan modification containing ?-(1,3) fucose structure also decreased, but the ?-(1.2)-xylose structure increased in132-17. However, the complex and hybrid type glycan modification decreased and paucimannosidic type glycan structures increased in OsrAAT derived froml32-10transgenic line, indicating ER stress have influence on the processing of OsrAAT glycan modification in rice endosperm.10. The analysis of glutelin glycan modification among ZH11,132-10and132-17revealed N-glycan modifications on glutelin had few different glycan structures. High mannose typeand ?-(1.4) linked to the ternimal N-acylgluosidase glycan modifcations were detected in glutelin, but not in OsrAAT.Our research indicated rice endosperm was the effective and promising recombinant expression system. OsrAAT with the biological activity were produced in rice endosperm and the second structure of OsrAAT was equivalent to pAAT. Otherwise, the simple and effective purification protocol was developed to purify OsrAAT from the transgenic rice. The endoplasmic reticulum stress induced by highly expressed OsrAAT in rice endosperm reduces seed size via pre-mature programmed cell death. The analysis of OsrAAT and glutelin glycan modification between132-10and132-17indicated that ER stress have the influence on the processing of OsrAAT glycan modification. |
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