PLOS ONE
| Autor: | David C. Sane, Stephanie A. Archer-Hartmann, Mamudou Bah, Jiahua Xie, Chiu-Yueh Hung, Parastoo Azadi, Farooqahmed S. Kittur, Mayumi Ishihara |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Programmed cell death
Glycosylation Transgene Genetic Vectors lcsh:Medicine Gene Expression law.invention Mice 03 medical and health sciences 0302 clinical medicine Tandem Mass Spectrometry law Cell Line Tumor Gene Order Tobacco Gene expression medicine Animals Humans Phosphorylation lcsh:Science Erythropoietin 030304 developmental biology Neurons 0303 health sciences Multidisciplinary Janus kinase 2 Cell Death biology Caspase 3 lcsh:R Plants Genetically Modified Staurosporine Cytoprotection Recombinant Proteins Cell biology Biochemistry Cell culture biology.protein Recombinant DNA lcsh:Q 030217 neurology & neurosurgery Research Article medicine.drug |
| Zdroj: | PLoS ONE PLoS ONE, Vol 8, Iss 10, p e76468 (2013) |
| Popis: | Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPOM) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPOP) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPOP bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPOP (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPOM (21%). The cytoprotective effect of the asialo-rhuEPOP was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production. Published version |
| Databáze: | OpenAIRE |
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