N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin.

UNLABELLED: 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 ß1,4-galactose to N-glycan chains. Plants could be genetically engineered to produce asialo-rhuEPO by introducing human ß1,4-galactosyltransferase. The penultimate ß1,4-linked galactose residues are important for in vivo biological activity. In this proof of concept study, we show that tobacco plants co-expressing human ß1,4-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 ß1,4-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. KEY MESSAGE: N-glycosylation pathway in tobacco plants could be genetically engineered to produce a tissue-protective cytokine, asialoerythropoietin (a desialylated form of human hormone erythropoietin).

Identification of a Mg-protoporphyrin IX monomethyl ester cyclase homologue, EaZIP, differentially expressed in variegated Epipremnum aureum 'Golden Pothos' is achieved through a unique method of comparative study using tissue regenerated plants.

Variegated plants provide a valuable tool for studying chloroplast biogenesis by allowing direct comparison between green and white/yellow sectors within the same leaf. While variegated plants are abundant in nature, the mechanism of leaf variegation remains largely unknown. Current studies are limited to a few mutants in model plant species, and are complicated by the potential for cross-contamination during dissection of leaf tissue into contrasting sectors. To overcome these obstacles, an alternative approach was explored using tissue-culture techniques to regenerate plantlets from unique sectors. Stable green and pale yellow plants were developed from a naturally variegated Epipremnum aureum 'Golden Pothos'. By comparing the gene expression between green and pale yellow plants using suppression subtractive hybridization in conjunction with homologous sequence search, nine down-regulated and 18 up-regulated genes were identified in pale yellow plants. Transcript abundance for EaZIP (Epipremnum aureum leucine zipper), a nuclear gene homologue of tobacco NTZIP and Arabidopsis CHL27, was reduced more than 4000-fold in qRT-PCR analysis. EaZIP encodes the Mg-protoporphyrin IX monomethyl ester cyclase, one of the key enzymes in the chlorophyll biosynthesis pathway. Examination of EaZIP expression in naturally variegated 'Golden Pothos' confirmed that EaZIP transcript levels were correlated with leaf chlorophyll contents, suggesting that this gene plays a major role in the loss of chlorophyll in the pale yellow sectors of E. aureum 'Golden Pothos'. This study further suggests that tissue-culture regeneration of plantlets from different coloured sectors of variegated leaves can be used to investigate the underlying mechanisms of variegation.