Figure 1.
Schematic representation of the multi-gene vectors used in this investigation.
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 AgeI-NotI 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 [20] 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 in planta protein sialylation, pG371. STGalT, CST and ST [20] 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-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase; NANS: Homo sapiens N-acetylneuraminic acid phosphate synthase; CMAS: Homo sapiens CMP-N-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.
Figure 2.
Expression of rhEPOFc in N. benthamiana.
A. Western blot analysis of total soluble proteins extracted from N. benthamiana 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 N. benthamiana mutants lacking plant specific β1,2-xylose and α1,3-fucose (rhEPOFcΔXTFT); lane 2: rhEPOFc co-expressed with mammalian genes for protein sialylation (GNE, NANS, CMAS, CST, STGalT and ST) (rhEPOFcSia,); lane 3: rhEPOFc co-expressed with mammalian genes necessary for sialylation and synthesis of tri-antennary N-glycans GnTIV or GnTV, (rhEPOTriSia,); lane 4: rhEPOFc co-expressed with mammalian genes for sialylation and synthesis of tetra-antennary N-glycans, GnTIV and GnTV (rhEPOTetraSia). A and B represent distinct protein fractions from the 55 kDa band of rhEpoFcTriSia and rhEPOTetraSia, 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 N. benthamiana ΔXTFT mutants (control; lane 1) and of purified rhEPOFcΔXTFT (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 N-glycans with Lewis-a epitopes. (M) protein marker.
Table 1.
Expression of rhEPOFc in N. benthamiana.
Figure 3.
Generation of GnGn structures in rhEPOFc.
Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc expressed in N. benthamiana ΔXTFT (rhEPOFcΔXTFT; Figure 2B, lane 1). Glycosylation patterns of rhEPO glycopeptide 1 (Gp1): E/A22ENITTGCAE31; glycopeptide 2 (Gp2): E/H32CSLNENITVPDTK45 and glycopeptide 3 (Gp3): R/G77QALLVNSSQPWEPLQHLVDK97 are shown. The corresponding N-glycosylation profile of the Fc glycopeptide (R/EEQYNSTYR) is shown in Figure S1. Peak labels were made according to the ProGlycAn system (www.proglycan.com). Illustrations display N-glycans on assigned peaks, for interpretation of other assigned glycoforms see Figure S5.
Figure 4.
Generation of bi-sialylated structures in rhEPOFc.
Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc co-expressed in N. benthamiana ΔXTFT with mammalian genes for protein sialylation (GNE, NANS, CMAS, CST, STGalT and ST) (rhEPOFcSia; Figure 2B, lane 2). Glycosylation patterns of rhEPO Gp1: E/A22ENITTGCAE31; Gp2: E/H32CSLNENITVPDTK45 and Gp3: R/G77QALLVNSSQPWEPLQHLVDK97 are shown. N-glycosylation profile of the Fc glycopeptide is shown in Figure S1. Peak labels were made according to the ProGlycAn system (www.proglycan.com). Illustrations display N-glycans on assigned peaks, for interpretation of other assigned glycoforms see Figure S5.
Table 2.
Relative abundance of different complex glycoforms detected in rhEPOFc. (oligomannosidic structures that are present in all samples are not included).
Figure 5.
Generation of tri-sialylated structures in rhEPOFc.
Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc co-expressed in N. benthamiana ΔXTFT with mammalian genes for synthesis of tri-antennary sialylated N-glycans (rhEPOTriSia). The analysis was performed on rhEPOFcTriSia present on fraction A of the 55kDa band (Figure 2B, lane 3). Glycosylation patterns of rhEPO Gp1: E/A22ENITTGCAE31; Gp2: E/H32CSLNENITVPDTK45 and Gp3: R/G77QALLVNSSQPWEPLQHLVDK97 are shown. N-glycosylation profile of the Fc glycopeptide is shown in Figure S1. Glycosylation profile of rhEPOFc present on fraction B of the 55 kDa band is shown in Figure S2. Peak labels were made according to the ProGlycAn system (www.proglycan.com). Illustrations display N-glycans on assigned peaks, for interpretation of other assigned glycoforms see Figure S5.
Figure 6.
Generation of tetra-sialylated structures in rhEPOFc.
Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc co-expressed in N. benthamiana ΔXTFT with mammalian genes for synthesis of tetra-sialylated N-glycans (rhEPOTetraSia). The analysis was performed on rhEPOFcTetraSia present on fraction A of the 55kDa band (Figure 2B, lane 4). Glycosylation patterns of rhEPO Gp1: E/A22ENITTGCAE31; Gp2: E/H32CSLNENITVPDTK45 and Gp3: R/G77QALLVNSSQPWEPLQHLVDK97 are shown. N-glycosylation profile of the Fc glycopeptide is shown in Figure S1. Glycosylation profile of rhEPOFc present on fraction B of the 55kDa band is shown in Figure S3. Peak labels were made according to the ProGlycAn system (www.proglycan.com Illustrations display N-glycans on assigned peaks, for interpretation of other assigned glycoforms see Figure S5.
Table 3.
in vitro activity of CHO- and plant-derived rhEPOFC.