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Stable Protein Sialylation in Physcomitrella

2020; Frontiers Media; Volume: 11; Linguagem: Inglês

10.3389/fpls.2020.610032

1664-462X

Lennard L. Bohlender, Juliana Parsons, Sebastian N. W. Hoernstein, Christine Rempfer, Natalia Ruiz-Molina, Timo Lorenz, Fernando Rodríguez Jahnke, Rudolf Figl, Benjamin Fode, Friedrich Altmann, Ralf Reski, Eva L. Decker,

Viral Infectious Diseases and Gene Expression in Insects

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.