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Stable Protein Sialylation in Physcomitrella.

Authors :
Bohlender, Lennard L.
Parsons, Juliana
Hoernstein, Sebastian N. W.
Rempfer, Christine
Ruiz-Molina, Natalia
Lorenz, Timo
Rodríguez Jahnke, Fernando
Figl, Rudolf
Fode, Benjamin
Altmann, Friedrich
Reski, Ralf
Decker, Eva L.
Source :
Frontiers in Plant Science; 12/18/2020, Vol. 11, pN.PAG-N.PAG, 21p
Publication Year :
2020

Abstract

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N -glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N -glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N -glycan sialylation. Since sialic acids are a common terminal modification on human N- glycans, the property to perform N -glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N -glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of β1,4-galactosylated acceptor N -glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-type N- glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
1664462X
Volume :
11
Database :
Complementary Index
Journal :
Frontiers in Plant Science
Publication Type :
Academic Journal
Accession number :
147715070
Full Text :
https://doi.org/10.3389/fpls.2020.610032