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.
Highlights
Erythropoietin (EPO) is a glyco-hormone consisting of 166 amino acid long polypeptide chain containing one O- and three Nglycan chains [1]
EPO is best known for its regulatory role in the production of red blood cells, and is widely used to treat anemia resulting from chronic renal failure, AIDS, rheumatoid arthritis, malignancies and many other types of anemia [1,2]
The EPO cDNA was fused with the TEV protease cutting site ENLYFQG, StrepII tag, and an endoplasmic reticulum (ER) retention signal KDEL at its 39 end (Figure 1A)
Summary
Erythropoietin (EPO) is a glyco-hormone consisting of 166 amino acid long polypeptide chain containing one O- and three Nglycan chains [1]. A number of studies using mammalian cellmade recombinant human EPO (rhuEPOM) have revealed remarkable cytoprotective activities occurring independent of its hematopoietic activity in preclinical models of ischemic injury involving neuronal, cardiac and kidney cells [4,5,6]. The therapeutic application of rhuEPOM for tissue protection was tempered by the observations that EPO greatly amplifies brain injury [14], increases thrombotic events [15] and decreases survival rates [16] because of its hematopoietic activity. The cytoprotective doses of EPO are much higher than those required for stimulation of erythropoiesis and its hematopoietic activity at these high doses can stimulate mass production of red blood cells causing more damage [3]. Cytoprotective EPO derivatives lacking hematopoietic activity are highly desired
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