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Stable Protein Sialylation in Physcomitrella

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...

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Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775405/
https://www.ncbi.nlm.nih.gov/pubmed/33391325
http://dx.doi.org/10.3389/fpls.2020.610032
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author 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.
author_facet 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.
author_sort Bohlender, Lennard L.
collection PubMed
description 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.
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spelling pubmed-77754052021-01-02 Stable Protein Sialylation in Physcomitrella 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. Front Plant Sci Plant Science 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. Frontiers Media S.A. 2020-12-18 /pmc/articles/PMC7775405/ /pubmed/33391325 http://dx.doi.org/10.3389/fpls.2020.610032 Text en Copyright © 2020 Bohlender, Parsons, Hoernstein, Rempfer, Ruiz-Molina, Lorenz, Rodríguez Jahnke, Figl, Fode, Altmann, Reski and Decker. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
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.
Stable Protein Sialylation in Physcomitrella
title Stable Protein Sialylation in Physcomitrella
title_full Stable Protein Sialylation in Physcomitrella
title_fullStr Stable Protein Sialylation in Physcomitrella
title_full_unstemmed Stable Protein Sialylation in Physcomitrella
title_short Stable Protein Sialylation in Physcomitrella
title_sort stable protein sialylation in physcomitrella
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775405/
https://www.ncbi.nlm.nih.gov/pubmed/33391325
http://dx.doi.org/10.3389/fpls.2020.610032
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