title: N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin.
abstract: Erythropoietin (EPO) is a glycoprotein hormone that displays both hematopoietic and tissue-protective functions by binding to two distinct receptors. Recombinant human EPO (rhuEPO) is widely used for the treatment of anemia, but its use for tissue protection is limited because of potentially harmful increases in red blood cell mass when higher doses of rhuEPO are used. Recent studies have shown that asialoerythropoietin (asialo-rhuEPO), a desialylated form of rhuEPO, lacks hematopoietic activity, but retains cytoprotective activity. Currently, a small amount of asialo-rhuEPO is produced by enzymatic desialylation of rhuEPO. The prohibitive cost of rhuEPO, however, is a major limitation of this method. Plants have the ability to synthesize complex N-glycans, but lack enzymatic activities to add sialic acid and β1,4-galactose to N-glycan chains. Plants could be genetically engineered to produce asialo-rhuEPO by introducing human β1,4-galactosyltransferase. The penultimate β1,4-linked galactose residues are important for in vivo biological activity. In this proof of concept study, we show that tobacco plants co-expressing human β1,4-galactosyltransferase and EPO genes accumulated asialo-rhuEPO. Purified asialo-rhuEPO binds to an Erythrina cristagalli lectin column, indicating that its N-glycan chains bear terminal β1,4-galactose residues and that the co-expressed GalT is functionally active. Asialo-rhuEPO interacted with the EPO receptor (EPOR) with similar affinity as rhuEPO, implying that it was properly folded. The strategy described here provides a straightforward way to produce asialo-rhuEPO for research and therapeutic purposes.
  
  
  CONCLUSIONS
  N-glycosylation pathway in tobacco plants could be genetically engineered to produce a tissue-protective cytokine, asialoerythropoietin (a desialylated form of human hormone erythropoietin).
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA.
authors.forename: Farooqahmed S
authors.initials: FS
authors.lastname: Kittur
authors.forename: Chiu-Yueh
authors.initials: CY
authors.lastname: Hung
authors.forename: Diane E
authors.initials: DE
authors.lastname: Darlington
authors.forename: David C
authors.initials: DC
authors.lastname: Sane
authors.forename: Jiahua
authors.initials: J
authors.lastname: Xie
mesh_terms: D001212:Asialoglycoproteins / Q000096:biosynthesis*; D004921:Erythropoietin / Q000031:analogs & derivatives* / Q000096:biosynthesis; D006031:Glycosylation; D006801:Humans; D060847:Metabolic Engineering; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017467:Receptors, Erythropoietin / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D001212:Asialoglycoproteins; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; C500605:asialoerythropoietin; D004921:Erythropoietin