Generation of Biologically Active Multi-Sialylated Recombinant Human EPOFc in Plants

<div><p>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 <em>N</em>-glycoengineering of non-mammalian expression hosts resulted in <em>in vivo</em> protein sialylation at great homogeneity. However the synthesis of multi-sialylated <em>N-</em>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 <em>Nicotiana benthamiana</em> 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 <em>N-</em>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. <em>In vitro</em> receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the <em>in planta</em> 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.</p> </div

Generation of tetra-sialylated structures in rhEPOFc.

<p>Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc co-expressed in <i>N. benthamiana</i> ΔXTFT with mammalian genes for synthesis of tetra-sialylated <i>N-</i>glycans (rhEPO_TetraSia). The analysis was performed on rhEPOFc_TetraSia present on fraction A of the 55kDa band (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone-0054836-g002" target="_blank">Figure 2B</a>, lane 4). Glycosylation patterns of rhEPO Gp1: E/A²²E<u>NIT</u>TGCAE³¹; Gp2: E/H³²CSLNE<u>NIT</u>VPDTK⁴⁵ and Gp3: R/G⁷⁷QALLV<u>NSS</u>QPWEPLQHLVDK⁹⁷ are shown. <i>N-</i>glycosylation profile of the Fc glycopeptide is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s001" target="_blank">Figure S1</a>. Glycosylation profile of rhEPOFc present on fraction B of the 55kDa band is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s003" target="_blank">Figure S3</a>. Peak labels were made according to the ProGlycAn system (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a> Illustrations display <i>N</i>-glycans on assigned peaks, for interpretation of other assigned glycoforms see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s005" target="_blank">Figure S5</a>.</p

Generation of tri-sialylated structures in rhEPOFc.

<p>Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc co-expressed in <i>N. benthamiana</i> ΔXTFT with mammalian genes for synthesis of tri-antennary sialylated <i>N-</i>glycans (rhEPO_TriSia). The analysis was performed on rhEPOFc_TriSia present on fraction A of the 55kDa band (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone-0054836-g002" target="_blank">Figure 2B</a>, lane 3). Glycosylation patterns of rhEPO Gp1: E/A²²E<u>NIT</u>TGCAE³¹; Gp2: E/H³²CSLNE<u>NIT</u>VPDTK⁴⁵ and Gp3: R/G⁷⁷QALLV<u>NSS</u>QPWEPLQHLVDK⁹⁷ are shown. <i>N-</i>glycosylation profile of the Fc glycopeptide is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s001" target="_blank">Figure S1</a>. Glycosylation profile of rhEPOFc present on fraction B of the 55 kDa band is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s002" target="_blank">Figure S2</a>. Peak labels were made according to the ProGlycAn system (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a>). Illustrations display <i>N</i>-glycans on assigned peaks, for interpretation of other assigned glycoforms see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s005" target="_blank">Figure S5</a>.</p

Generation of bi-sialylated structures in rhEPOFc.

<p>Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc co-expressed in <i>N. benthamiana</i> ΔXTFT with mammalian genes for protein sialylation (GNE, NANS, CMAS, CST, ^STGalT and ST) (rhEPOFc_Sia; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone-0054836-g002" target="_blank">Figure 2B</a>, lane 2). Glycosylation patterns of rhEPO Gp1: E/A²²E<u>NIT</u>TGCAE³¹; Gp2: E/H³²CSLNE<u>NIT</u>VPDTK⁴⁵ and Gp3: R/G⁷⁷QALLV<u>NSS</u>QPWEPLQHLVDK⁹⁷ are shown. <i>N-</i>glycosylation profile of the Fc glycopeptide is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s001" target="_blank">Figure S1</a>. Peak labels were made according to the ProGlycAn system (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a>). Illustrations display <i>N</i>-glycans on assigned peaks, for interpretation of other assigned glycoforms see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s005" target="_blank">Figure S5</a>.</p

Generation of GnGn structures in rhEPOFc.

<p>Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc expressed in <i>N. benthamiana</i> ΔXTFT (rhEPOFc_ΔXTFT; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone-0054836-g002" target="_blank">Figure 2B</a>, lane 1). Glycosylation patterns of rhEPO glycopeptide 1 (Gp1): E/A²²E<u>NIT</u>TGCAE³¹; glycopeptide 2 (Gp2): E/H³²CSLNE<u>NIT</u>VPDTK⁴⁵ and glycopeptide 3 (Gp3): R/G⁷⁷QALLV<u>NSS</u>QPWEPLQHLVDK⁹⁷ are shown. The corresponding <i>N-</i>glycosylation profile of the Fc glycopeptide (R/EEQY<u>NST</u>YR) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s001" target="_blank">Figure S1</a>. Peak labels were made according to the ProGlycAn system (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a>). Illustrations display <i>N</i>-glycans on assigned peaks, for interpretation of other assigned glycoforms see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s005" target="_blank">Figure S5</a>.</p

Expression of rhEPOFc in <i>N. benthamiana</i>.

<p>A. Western blot analysis of total soluble proteins extracted from <i>N. benthamiana</i> expressing rhEPOFc. 55 kDa protein reacts to both anti-EPO (α-EPO) and anti-Fc (α-Fc) antibodies while the ∼30 kDa band reacts only with α-Fc antibodies. B. Protein A purified rhEPOFc fractionated by SDS PAGE and stained with Coomassie-brilliant blue R-250. lane 1: rhEPOFc expressed in <i>N. benthamiana</i> mutants lacking plant specific β1,2-xylose and α1,3-fucose (rhEPOFc_ΔXTFT); lane 2: rhEPOFc co-expressed with mammalian genes for protein sialylation (GNE, NANS, CMAS, CST, ^STGalT and ST) (rhEPOFc_Sia,); lane 3: rhEPOFc co-expressed with mammalian genes necessary for sialylation and synthesis of tri-antennary <i>N-</i>glycans GnTIV or GnTV, (rhEPO_TriSia,); lane 4: rhEPOFc co-expressed with mammalian genes for sialylation and synthesis of tetra-antennary <i>N-</i>glycans, GnTIV and GnTV (rhEPO_TetraSia). A and B represent distinct protein fractions from the 55 kDa band of rhEpoFc_TriSia and rhEPO_TetraSia, used for N-glycan analysis; the ∼30 kDa band represent free Fc. C. Western blot analysis of total soluble proteins (5 µg TSP) extracted from <i>N. benthamiana</i> ΔXTFT mutants (control; lane 1) and of purified rhEPOFc_ΔXTFT (lane 2) using antibodies against Lewis-A epitopes (JIM 84). Several proteins in TSP and the 55 kDa protein band corresponding to intact rhEPOFc reacted to JIM 84 revealing the presence of <i>N-</i>glycans with Lewis-a epitopes. (M) protein marker.</p

<i>in vitro</i> activity of CHO- and plant-derived rhEPOFC.

<p><i>In vitro</i> activity assay of plant- and CHO- derived rhEPOFc. Half maximal effective doses (ED₅₀) are displayed. rhEPOFc was expressed in CHO cells (CHO); in <i>N. benthamiana</i> ΔXTFT mutants (ΔXTFT); co-expressed in ΔXTFT with mammalian genes for protein sialylation (Sia); co-expressed in ΔXTFT with mammalian genes for synthesis of tri-antennary sialylated <i>N-</i>glycans (TriaSia) and co-expressed in ΔXTFT with mammalian genes for synthesis of tetra-sialylated <i>N-</i>glycans (TetraSia).</p

Relative abundance of different complex glycoforms detected in rhEPOFc. (oligomannosidic structures that are present in all samples are not included).

<p>Relative abundance of complex <i>N</i>-glycans determined by LC-ESI-MS. <b>rhEPOFc_Sia</b>: rhEPOFc co-expressed with mammalian genes for protein sialylation; <b>rhEPOFc_TriaSia</b>: rhEPOFc co-expressed with mammalian genes for synthesis of tri-antennary sialylated <i>N-</i>glycans; <b>rhEPOFc_TetraSia:</b> rhEPOFc co-expressed with mammalian genes for synthesis of tetra-sialylated <i>N-</i>glycans. ΔXTFT was used as expression host. Values are in percentages. Quantifications were done for complex <i>N-</i>glycans (oligomannosidic structures were not included in calculations). Gp1: glycopeptide 1; Gp2: Glycopeptide 2; Gp3: Glycopeptide 3.</p