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

Schematic representation of the multi-gene vectors used in this investigation.

<p>A. Structural features of the pSAT series of vectors (pSATn) suitable for the expression of target genes under the control of various constitutive promoters and terminators (pSAT1A, pSAT3A, pSAT4A and pSAT7A). Expression cassettes are interchangeable within pSATn as <i>Age</i>I-<i>Not</i>I fragments. Rare-cutting enzymes flanking each pSAT vector are used to transfer the expression cassettes into the expression vector pPZP-RCS2. B. Outline of the cloning strategy to assemble the mammalian genes necessary for the synthesis of sialic acid, pC144. The GNE, NANS and CMAS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836-Castilho2" target="_blank">[20]</a> open reading frames were subcloned into pSAT auxiliary vectors and were then sequentially assembled in pPZP-RCS2 using specific rare-cutting enzymes. C. Outline of the cloning strategy to assemble the mammalian genes acting in the Golgi apparatus for <i>in planta</i> protein sialylation, pG371. ^STGalT, CST and ST <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836-Castilho2" target="_blank">[20]</a> genes were put under control of different promoters and terminators in pSAT vectors. These were then sequentially assembled into pPZP-RCS2 vector using appropriate rare-cutting enzymes. 35SP: cauliflower mosaic virus (CaMV) 35S promoter; TL: translational enhancer 5′-UTR from tobacco etch; 35ST: CaMV 35S terminator; OcsP: octopine synthase promoter; OcsT: octopine synthase terminator; actP: actin promoter; agsT: agropin synthase terminator; masP: manopine synthase promoter masT: manopine synthase terminator; GNE: mouse UDP-<i>N-</i>acetylglucosamine-2-epimerase/<i>N-</i>acetylmannosamine kinase; NANS: Homo sapiens <i>N-</i>acetylneuraminic acid phosphate synthase; CMAS: Homo sapiens CMP-<i>N-</i>acetylneuraminic acid synthase; ^STGalT: β1,4-galactosyltransfease fused to the cytoplasmic tail, transmembrane domain and stem region of the rat α2,6-sialyltransferase; CST: Mouse CMP-sialic acid transporter; ST: rat α2,6-sialyltransferase; LB: left border; RB: right border.</p

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

<p>Concentration of transiently expressed rhEPOFc was determined using a commercially available immunoassay. For each sample the concentration is given in mg/kg of fresh leaf. The percentage of the total soluble protein (TSP) was also calculated. rhEPOFc was expressed 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

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

<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

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

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 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