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...

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Autores principales:	Alexandra Castilho, Laura Neumann, Pia Gattinger, Richard Strasser, Karola Vorauer-Uhl, Thomas Sterovsky, Friedrich Altmann, Herta Steinkellner
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b5f06cfea02040d2973c071052b72e7a
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spelling	oai:doaj.org-article:b5f06cfea02040d2973c071052b72e7a2021-11-18T07:59:53ZGeneration of biologically active multi-sialylated recombinant human EPOFc in plants.1932-620310.1371/journal.pone.0054836https://doaj.org/article/b5f06cfea02040d2973c071052b72e7a2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23372778/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Hyperglycosylated 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.Alexandra CastilhoLaura NeumannPia GattingerRichard StrasserKarola Vorauer-UhlThomas SterovskyFriedrich AltmannHerta SteinkellnerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 1, p e54836 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Alexandra Castilho Laura Neumann Pia Gattinger Richard Strasser Karola Vorauer-Uhl Thomas Sterovsky Friedrich Altmann Herta Steinkellner Generation of biologically active multi-sialylated recombinant human EPOFc in plants.
description	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.
format	article
author	Alexandra Castilho Laura Neumann Pia Gattinger Richard Strasser Karola Vorauer-Uhl Thomas Sterovsky Friedrich Altmann Herta Steinkellner
author_facet	Alexandra Castilho Laura Neumann Pia Gattinger Richard Strasser Karola Vorauer-Uhl Thomas Sterovsky Friedrich Altmann Herta Steinkellner
author_sort	Alexandra Castilho
title	Generation of biologically active multi-sialylated recombinant human EPOFc in plants.
title_short	Generation of biologically active multi-sialylated recombinant human EPOFc in plants.
title_full	Generation of biologically active multi-sialylated recombinant human EPOFc in plants.
title_fullStr	Generation of biologically active multi-sialylated recombinant human EPOFc in plants.
title_full_unstemmed	Generation of biologically active multi-sialylated recombinant human EPOFc in plants.
title_sort	generation of biologically active multi-sialylated recombinant human epofc in plants.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/b5f06cfea02040d2973c071052b72e7a
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Generation of biologically active multi-sialylated recombinant human EPOFc in plants.

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