BackgroundSec18p/N-ethylmaleimide-sensitive factor (NSF) is a conserved eukaryotic ATPase, which primarily functions in vesicle membrane fusion from yeast to human. However, the function of the OsSec18 gene, a homologue of NSF in rice, remains unknown.ResultsIn the present study, we investigated the function of OsSec18 in rice and found that OsSec18 complements the temperature-sensitive phenotype and interferes with vacuolar morphogenesis in yeast. Overexpression of OsSec18 in rice decreased the plant height and 1000-grain weight and altered the morphology of the protein bodies. Further examination revealed that OsSec18 presented as a 290-kDa complex in rice endosperm cells. Moreover, Os60sP0 was identified a component of this complex, demonstrating that the OsSec18 complex contains another complex of P0(P1-P2)2 in rice endosperm cells. Furthermore, we determined that the N-terminus of OsSec18 can interact with the N- and C-termini of Os60sP0, whereas the C-terminus of OsSec18 can only interact with the C-terminus of Os60sP0.ConclusionOur results revealed that the OsSec18 regulates vacuolar morphology in both yeast and rice endosperm cell and the OsSec18 interacts with P0(P1-P2)2 complex in rice endosperm cell.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0324-1) contains supplementary material, which is available to authorized users.
Human serum albumin (HSA) is the most abundant protein in human plasma and is widely used at high doses for treating various diseases. Recombinant HSA is an alternative approach to plasma-derived HSA, providing increased safety and an unlimited supply. However, the safety of the residual host cell proteins (HCPs) co-purified with Oryza sativa HSA (OsrHSA) remains to be determined. An animal system was used to assess the immunogenicity of OsrHSA and its residual HCPs. Low immunogenicity and immunotoxicity of the residual HCPs at a dose of 25 μg/kg, equivalent to 25 times the clinical dosage of HSA, were observed. An anti-drug-antibody (ADA) analysis revealed that anti-HSA, anti-OsrHSA or anti-HCP antibodies developed with a low frequency in pHSA and OsrHSA treatments, but the titers were as low as 1.0–2.0. Furthermore, the titer and the incidence of the specific antibodies were not significantly different between the pHSA and OsrHSA groups, indicating that OsrHSA presents similar immunogenicity to that of pHSA. More importantly, no cytokines were stimulated after the administration of OsrHSA and the residual HCPs, suggesting that there was no risk of a cytokine storm. These results demonstrated that the residual HCPs from OsrHSA have low immunogenicity, indicating that the rice endosperm is one of the best hosts for plant molecular pharming.
The high accumulation of a recombinant protein in rice endosperm causes endoplasmic reticulum (ER) stress and in turn dramatically affects endogenous storage protein expression, protein body morphology and seed phenotype. To elucidate the molecular mechanisms underlying these changes in transgenic rice seeds, we analyzed the expression profiles of endogenous storage proteins, ER stress-related and programmed cell death (PCD)-related genes in transgenic lines with different levels of Oryza sativa recombinant alpha antitrypsin (OsrAAT) expression. The results indicated that OsrAAT expression induced the ER stress and that the strength of the ER stress was dependent on OsrAAT expression levels. It in turn induced upregulation of the expression of the ER stress response genes and downregulation of the expression of the endogenous storage protein genes in rice endosperm. Further experiments showed that the ER stress response upregulated the expression of PCD-related genes to disturb the rice endosperm development and induced pre-mature PCD. As consequence, it resulted in decrease of grain weight and size. The mechanisms for the detriment seed phenotype in transgenic lines with high accumulation of the recombinant protein were elucidated.
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