Engineering of sialylated mucin-type O-glycosylation in plants

Abstract

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.

FIGURE 1.

Schematic representation of the pathway for the formation of disialylated O-glycans in plants. UDP-GalNAc formation and CMP-NeuAc biosynthesis start from the nucleotide sugar UDP-GlcNAc. The steps for conversion of UDP-GlcNAc to CMP-NeuAc and transport of CMP-NeuAc to the Golgi have been engineered in plants previously (25, 34). The specific steps that seem absolutely required for sialylated mucin-type O-glycan biosynthesis are depicted in bold. GNE, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase; NANS, N-acetylneuraminic acid phosphate synthase; CMAS, CMP-N-acetylneuraminic acid synthetase; NANP, N-acetylneuraminate-9-phosphate phosphatase. Conversion of NeuAc-9-P to NeuAc is very likely carried out by an endogenous plant enzyme.

FIGURE 2.

Schematic representation of newly generated vectors. A, binary vector for the expression of the Drosophila C1GALT1 is shown. B, shown are structural features of the pSAT series of auxiliary vectors (pSAT1A, pSAT3A, and pSAT6A) for the assembly of promoter-gene-terminator cassettes. Rare-cutting enzymes flanking each pSAT vector are used to transfer the expression cassettes into the expression vector pPZP-RCS2. C, shown is the outline of the cloning strategy for expression of human C1GALT1 and its chaperone COSMC. D, shown is a schematic representation of the cloning strategy for the multiple gene expression vector. The CST, ST3Gal-I, and ST6GALNAc-III/IV open reading frames were cloned into different pSAT auxiliary vectors and were then sequentially assembled in pPZP-RCS2 using specific rare-cutting enzymes. In the final constructs all three proteins are expressed under different promoter and terminator sequences. 35SP, cauliflower mosaic virus 35S promoter; g7T, Agrobacterium gene 7 terminator; ocsP, octopine synthase promoter; ocsT, octopine synthase terminator; rbcP, rubisco small subunit promoter; rbcT, rubisco small subunit terminator; masP, manopine synthase promoter; masT, manopine synthase terminator; LB, left border sequence; RB, right border sequence.

FIGURE 3.

SDS-PAGE and immunoblot analysis of EPO-Fc expressed in N. benthamiana. Total soluble protein extracts were subjected to SDS-PAGE followed by immunoblotting with anti-EPO antibodies (α-EPO) (1). Eluates from the protein A purification were separated by SDS-PAGE and analyzed by immunoblotting with α-EPO (2) with anti human IgG antibodies (α-Fc) (3) or by Coomassie Brilliant Blue staining (4). Representative images are shown.

FIGURE 4.

Initiation of O-GalNAc formation on Ser-126 of recombinant plant-produced EPO-Fc. Mass spectra of trypsin and endoproteinase Glu-C double-digested EPO-Fc expressed in N. benthamiana ΔXTFT line are shown. A, shown is a spectrum of the EPO-Fc Ser-126-containing peptide(s) in the absence of any O-glycan machinery; due to partial miscleavage, two peptides containing Ser-126 are generated, pep1 (AISPPDAASAAPLR) and pep2 (EAISPPDAASAAPLR). B, shown is a spectrum of the EPO-Fc Ser-126 peptides from plants co-expressing GalNAc-T2, UPD-GlcNAc 4-epimerase, and UDP-GlcNAc/UDP-GalNAc transporter with EPO-Fc. The presence of glycosylated peptides is indicated (+An indicates the presence of GalNAc residues). The presence of peptides with hydroxyproline residues (Pro to Hyp conversion: +16 Da, e.g. pep2ox) is indicated by arrows. The glycosylated versions of this peptide as well as the double- hydroxylated peptide eluted outside of the displayed time window. Asterisks denote the presence of co-eluting peptides or contaminations. C, shown is the Y-ion series of LC-ESI-MS/MS fragmentation experiment of the O-glycosylated EPO-Fc peptide (+An indicates the presence of a single GalNAc residue).

FIGURE 5.

Generation of T-antigen (Galβ1–3GalNAc) by co-expression of C1GALT1. A, co-expression of EPO-Fc with the machinery for O-GalNAc formation is shown. B, co-expression of EPO-Fc with the machinery for O-GalNAc formation, human C1GALT1, and its specific chaperone COSMC is shown. C, co-expression of EPO-Fc with the machinery for O-GalNAc formation and Drosophila C1GALT1 is shown. The spectra show the O-glycosylated EPO-Fc Ser-126-containing peptide (¹¹⁷EAISPPDAASAAPLR¹³¹). The arrow points at the peptide with one hydroxyproline residue. Unrelated peaks are denoted by an asterisk.

FIGURE 6.

Generation of disialylated O-glycans on EPO-Fc. A, shown are co-expression of EPO-Fc with the mammalian pathway for CMP-sialic acid synthesis, its transport to the Golgi, and the O-glycosylation machinery including UDP-GlcNAc 4-epimerase, UDP-GlcNAc/UDP-GalNAc transporter, GalNAc-T2, Drosophila C1GALT1, ST3Gal-I, and ST6GalNAc-III/IV. The LC-ESI-MS spectrum depicts the presence of all possible glycosylation variants up to a doubly sialylated core-1 O-glycan structure. The presence of a peptide corresponding to the hydroxylation of a single proline residue is indicated by an arrow. The asterisk denotes the presence of a contamination. B, analysis of the three EPO-Fc peptides containing N-linked glycans is shown. N-Glycan analysis was carried out by LC-ESI-MS of tryptic/Glu-C double-digested EPO-Fc.