Construction of a Functional CMP-Sialic Acid Biosynthesis Pathway in Arabidopsis

Previous studies have reported that plants contain negligible amounts of free or protein-bound N-acetylneuraminic acid (Neu5Ac). This is a major disadvantage for the use of plants as a biopharmaceutical expression system, since N-glycans with terminal Neu5Ac residues are important for the biological...

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Published in:Plant physiology (Bethesda) 2008-05, Vol.147 (1), p.331-339
Main Authors: Castilho, Alexandra, Pabst, Martin, Leonard, Renaud, Veit, Christiane, Altmann, Friedrich, Mach, Lukas, Glössl, Josef, Strasser, Richard, Steinkellner, Herta
Format: Article
Language:English
Subjects:
Animals
Antibodies
Arabidopsis - enzymology
Arabidopsis - genetics
Arabidopsis thaliana
Biochemical Processes and Macromolecular Structures
Biological and medical sciences
Biosynthesis
Carbohydrate Epimerases - metabolism
Cytidine Monophosphate N-Acetylneuraminic Acid - metabolism
Enzymes
Fundamental and applied biological sciences. Psychology
Genes. Genome
Glycoproteins
Humans
Leaves
Mice
Molecular and cellular biology
Molecular genetics
N-Acetylneuraminic Acid - biosynthesis
N-Acylneuraminate Cytidylyltransferase - metabolism
Oxo-Acid-Lyases - metabolism
Plants
Plants, Genetically Modified - enzymology
Polymerase chain reaction
Polysaccharides - metabolism
Seedlings
Transgenic animals
Transgenic plants
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Summary:Previous studies have reported that plants contain negligible amounts of free or protein-bound N-acetylneuraminic acid (Neu5Ac). This is a major disadvantage for the use of plants as a biopharmaceutical expression system, since N-glycans with terminal Neu5Ac residues are important for the biological activities and half-lives of recombinant therapeutic glycoproteins in humans. For the synthesis of Neu5Ac-containing N-glycans, plants have to acquire the ability to synthesize Neu5Ac and its nucleotide-activated derivative, cytidine monophospho-N-acetylneuraminic acid. In this study, we have generated transgenic Arabidopsis (Arabidopsis thaliana) plants expressing three key enzymes of the mammalian Neu5Ac biosynthesis pathway: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, and CMP-N-acetylneuraminic acid synthetase. Simultaneous expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase and N-acetylneuraminic acid phosphate synthase resulted in the generation of significant Neu5Ac amounts (1,275 nmol g⁻¹ fresh weight in leaves) in planta, which could be further converted to cytidine monophospho-N-acetylneuraminic acid (2.4 nmol g⁻¹ fresh weight in leaves) by coexpression of CMP-N-acetylneuraminic acid synthetase. These findings are a major step toward the production of Neu5Ac-containing glycoproteins in plants.
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.108.117572