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PLOS ONE
- Source :
- PLoS ONE, PLoS ONE, Vol 8, Iss 10, p e76468 (2013)
- Publication Year :
- 2013
- Publisher :
- PLOS, 2013.
-
Abstract
- 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
- Subjects :
- 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
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- PLoS ONE, PLoS ONE, Vol 8, Iss 10, p e76468 (2013)
- Accession number :
- edsair.doi.dedup.....97e15b6c6fc419e4ed19f8b25e5b0511