Engineering of Sialylated Mucin-type O-Glycosylation in Plants

• Published in: The Journal of biological chemistry Vol. 287; no. 43; pp. 36518 - 36526
• Main Authors: Castilho, Alexandra, Neumann, Laura, Daskalova, Sasha, Mason, Hugh S., Steinkellner, Herta, Altmann, Friedrich, Strasser, Richard
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.10.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Caenorhabditis elegans - genetics; Caenorhabditis elegans Proteins - biosynthesis; Caenorhabditis elegans Proteins - genetics; Drosophila melanogaster; Drosophila Proteins - biosynthesis; Drosophila Proteins - genetics; Erythropoietin; Erythropoietin - biosynthesis; Erythropoietin - genetics; Genetic Engineering; Glycobiology and Extracellular Matrices; Glycosylation; Humans; Immunoglobulin G - biosynthesis; Immunoglobulin G - genetics; Mice; Mucins; Nicotiana - genetics; Nicotiana - metabolism; Plant; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Post Translational Modification; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Protein Expression; Sialic Acid; Sialyltransferases - biosynthesis; Sialyltransferases - genetics; Post Translational Modification; Sialic Acid; Recombinant Protein Expression; Plant; Glycosylation; Erythropoietin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.402685

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. Background: Plants lack the machinery for mucin-type O-glycosylation. Results: Transient expression of the mammalian O-glycosylation pathway in Nicotiana benthamiana resulted in the formation of sialylated mucin-type O-glycans on recombinant erythropoietin. Conclusion: Therapeutic proteins with engineered N- and O-glycosylation can be produced in plants. Significance: Plants are attractive hosts for the production of glycosylated recombinant proteins with defined glycan structures.