Generation of biologically active multi-sialylated recombinant human EPOFc in plants.
Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity. However the synthesis of multi-sialylated N-glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc) was transiently expressed in Nicotiana benthamiana ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i) branching (ii) β1,4-galactosylation as well as for the (iii) synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex N-glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. In vitro receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the in planta synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
Alexandra
A
Castilho
Laura
L
Neumann
Pia
P
Gattinger
Richard
R
Strasser
Karola
K
Vorauer-Uhl
Thomas
T
Sterovsky
Friedrich
F
Altmann
Herta
H
Steinkellner
D004921:Erythropoietin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*
C554270:EPO protein, human; D011994:Recombinant Proteins; D004921:Erythropoietin