Cell Biology, Genetic engineering of the N-glycosylation pathway in plants. Glyco-modulation towards the optimization of glycan structures decorating therapeutically important recombinant proteins expressed in plants. Modulation of gene expression.
Leader: Dr. Alexandra Castilho
(from Mai 2018)
Plant Molecular Farming (PMF) refers to the production of valuable proteins through the use of plant biotechnology. Despite great successes, expression of recombinant proteins in plants still presents several bottlenecks. Protein glycosylation is one of the most important post-translational modifications and while N-glycan synthesis in the endoplasmic reticulum (ER) is relatively well conserved in eukaryotes, processing of N-glycans in the Golgi apparatus is kingdom specific and major differences in the glycosylation pathway of plants and animals, hampers the production of relevant glycoproteins in plants. Glyco-design is a powerful tool to with optimize glyco-profiles of therapeutic glycoproteins for efficacy. Our research is directed to develop, optimize and evaluate methods to tailor the structures of glycans on relevant recombinant proteins produced in Nicotiana benthamiana, currently the most appropriate host to be used in PMF. We use a variety of approaches for the genetic manipulation of glycosyltransferases (GTs), glycohydrolases (GH) and other glyco-genes and thus control the glycan biosynthesis in N. benthamiana. The outcome of our research can be applied to identify individual structures fundamental for glycan-mediated processes and facilitated the development of ‘biobetter’ products with customized glycan profiles that enhance their activities.
N. benthamiana plants are the most suitable platform for the transient expression of recombinant proteins and can tolerate considerable alterations of their N-glycan structures without any negative effect on growth or reproduction. However, glyco-engineering is not a simple process of Knock IN/OUT a gene, and often requires fine-tuning of various parameters to prevent the generation of aberrant glyco-structures inconsistent with the targeted glyco-design. Strategies to overcome challenges include:
- Identification and characterization of possible proteins(s) involved in the process of aberrant glycosylation
- Correct the subcellular localization and expression level of endogenous or recombinant protein(s)
- Prevent the activity of glycosidases involved in trimming terminal residues from N-glycans
- Increase the accessibility of the glycosite to processing enzymes
Figure 1: Trouble shooting plant glycoengineering.
Characterization of N. benthamiana β-galactosidases acting on glycoproteins.
Significant progress has been made towards the humanization of protein N-glycosylation in plants either by removing undesirable sugar residues or by introducing the ability to generate non-native N-glycan structures. However, homogenous human-like β1,4-galactosylation is very hard to achieve in recombinant glycoproteins accumulating in the plant-apoplast. Despite the vast efforts to optimize the expression of β1,4-galactosyltransferase (GalT), many glycoproteins still exhibit heterogeneous terminal galactosylation. Here we aim uncover different players that constrict the efficiency of β1,4-galactosylation in plant-derived glycoproteins.
The overall goal of this project is to elucidate the so far uncharacterized mechanisms leading to the trimming of terminal β1,4-galactose residues from N-glycans of secreted proteins. We aim to identify and fully characterize N. benthamiana β-galactosidases (BGALs) from the glycosyl-hydrolase (GH) family 35 active in the apoplast. Particularly, studies on their subcellular localization, substrates and specific biological function are not available. Ultimately, suppression of BGALs expression by RNAi interference or gene editing can be applied to increase human-like galactosylation of several plant-produced glycoproteins.
Figure 2: Protein N-glycosylation pathway in plant cells.
Schematic illustration shows important Golgi modifications that occur only in plants. Naturally occurring glycoforms can be modified through glyco-engineering either by knockOUT/down expression of endogenous glycosyltransferases and glycosidases or by knockIN glyco-modulating proteins.
- Castilho A, Pabst M, Altmann F, Mach L, Glössl J, Strasser R, and Steinkellner H. 2008. Engineering CMP-sialic acid (CMP-Neu5Ac) biosynthesis into plants. Plant Physiology: 147: 331-339.
- Strasser R, Castilho A, Stadlmann J, Kunert R, Quendler H, Gattinger P, Jez J, Rademacher T, Altmann F, Mach L, and Steinkellner H. 2009. Improved virus neutralization by plant-produced anti-HIV antibodies with a homogeneous beta1,4-galactosylated N-glycan profile. J. Biol. Chem. 284: 20479-85.
- Castilho A, Strasser, R, Stadlmann J, Grass J, Jez J, Gattinger P, Kunert, R., Quendler, H., Pabst, M., Leonard, R., Altmann, F., Steinkellner H. 2010. In planta protein sialylation through over-expression of the respective mammalian pathway. J Biol Chem. 285: 15923-30. F1000 Biology (must read) http://www.f1000biology.com/article/id/3063956
- Castilho A, Gattinger P, Grass J, Jez J, Pabst M, Altmann F, Gorfer M, Strasser R, Steinkellner H 2011. N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans. Glycobiology. 21:813-23.
- Castilho A, Bohorova N, Gras J, Bohorov O, Zeitlin L, Whaley K, Altmann F and Steinkellner H. 2011. Rapid high yield production of different glycoforms of Ebola virus monoclonal antibody. PLoS One. 6(10):e26040.
- Loos A, Van Droogenbroeck B, Hillmer S, Grass J, Pabst M, Castilho A, Kunert R, Liang M, Arcalis E, Robinson DG, Depicker A, Steinkellner H. 2011. Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. Plant Physiol. 155:2036-48.
- Nagels B, Van Damme E J.M, Callewaert N, Zabeau L, Tavernier J, Delanghe JR, Boets A, Castilho A, Weterings K 2012. EPO-Fc expression in Nicotiana benthamiana plants presenting tetra-antennary N-glycan structures. J Biotechnol. Epub 2012 Mar 16
- Jez J, Antes B, Castilho A, Kainer M, Wiederkum S, Grass J, Rüker F, Woisetschläger M, Steinkellner H. 2012. Significant impact of single N-glycan residues on the biological activity of Fc-based antibody-like fragments. J Biol Chem. 287:24313-9
- Castilho A and Steinkellner, H. 2012. Glyco-engineering in plants to produce human-like N-glycan structures. Biotechnology journal 7: 1088-1098.
- Castilho A, Neumann L, Daskalova S, Mason HS, Steinkellner H, Altmann F, Strasser R. 2012. Engineering of sialylated mucin-type O-glycosylation in plants. J Biol Chem.287:36518-26.
- Jez J, Castilho A, Grass J, Vorauer-Uhl K, Sterovsky T, Altmann F, Steinkellner H. 2013. Expression of functionally active sialylated human erythropoietin in plants. Biotechnol J. 8:371-82.
- Castilho A, Neumann L, Gattinger P, Strasser R, et al., 2013. Generation of biologically active multi-sialylated recombinant human EPOFc in plants. PloS one 2013, 8, e54836.
- Bosch D, Castilho A, Loos A, Schots A, Steinkellner H. 2013. N-Glycosylation of plant-produced recombinant proteins. Curr Pharm Des. 19(31):5503-12. Review.
- Schneider J, Castilho A, Neumann L, Altmann F, Loos A, Kannan L., Tsafrir M, Steinkellner H. 2013. Expression of human butyrylcholinesterase with a glycosylation profile resembling the plasma derived orthologue. Biotechnol J 8, doi: 10.1002/biot.201300229.
- Schneider J , Marillonnet S, Castilho A, Gruber C, Werner S, Mach L, Klimyuk V, Tsafrir S. Mor S, and Steinkellner H. 2014. Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana. Plant Biotechnol J. 12(7):832-9. doi: 10.1111/pbi.12184.
- Schoberer J, Liebminger E, Vavra U, Veit C, Castilho A, Dicker M, Maresch D, Altmann F, Hawes C, Botchway SW, Strasser R. 2014. The transmembrane domain of N-acetylglucosaminyltransferase I is the key determinant for its Golgi sub-compartmentation. Plant J. doi: 10.1111/tpj.12671.
- Castilho A, Windwarder M, Gattinger P, Mach L, Strasser R, Altmann F, Steinkellner H. 2014. Proteolytic and N-glycan processing of human α1-antitrypsin expressed in Nicotiana benthamiana. Plant Physiol. 166(4):1839-51. doi: 10.1104/pp.114.250720.
- Schneider J, Castilho A, Pabst M, Altmann F, Gruber C, Strasser R, Gattinger P, Seifert GJ, Steinkellner H. 2015. Characterization of plants expressing the human β1,4-galactosyltrasferase gene. Plant Physiol Biochem. 92:39-47. doi: 10.1016/j.plaphy.2015.04.010.
- Wilbers R, Westerhof L, Reuter L, Castilho A, Raaij van D, Nguyen D, Lozano-Torres J, Smant G, Hokke C, Bakker J, Schots A. 2015. The N-glycan on Asn54 affects the atypical N-glycan composition of plant-produced interleukin-22, but does not influence its activity. Plant Biotechnol J Jun 8. doi: 10.1111/pbi.12414.
- Castilho A, Gruber C, Thader A, Oostenbrink C, Pechlaner M, Steinkellner H, Altmann F. 2015. Processing of complex N-glycans in IgG Fc-region is affected by core-fucosylation. mAb DOI:10.1080/19420862.2015.1053683
- Shin YJ, Castilho A, Dicker M, Sádio F, Grünwald-Gruber C, Kwon TH, Altmann F, Steinkellner H, Strasser R. 2016. Reduced paucimannosidic N-glycan formation by suppression of a specific β-hexosaminidase from Nicotiana benthamiana. Plant Biotechnol J. doi: 10.1111/pbi.12602.
- Kallolimath S, Castilho A, Strasser R, Grünwald-Gruber C, Altmann F, Galuska CE, Galuska SP, Werner S, Werneburg S, Hildebrandt H, Gerardy-Schahn R, Steinkellner H. 2016. Engineering of Complex Protein Sialylation in Plants. Proc Natl Acad Sci U S A. 113:9498-9503. doi: 10.1073/pnas.1604371113.
- Montero-Morales L, Maresch D, Castilho A, Turupcu A, Ilieva K, Crescioli S, Karagiannis S, Lupinek C, Oostenbrink C, Altmann F, Steinkellner H. Recombinant plant-derived human IgE glycoproteomics. J Proteomics. 2017. Apr 8. pii: S1874-3919(17)30121-5. doi: 10.1016/j.jprot.2017.04.002
- Kallolimath S, Gruber C, Steinkellner H, Castilho A. 2018. Promoter choice impacts the efficiency of plant glyco-engineering. Biotechnol J. DOI: 10.1002/biot.201700380
- Castilho A, Beihammer G, Pfeiffer C, Kathrin Göritzer K, Montero-Morales L, Christiane Veit C, Ulrike Vavra U, Maresch D, Grünwald-Gruber C, Altmann A, Steinkellner H, Strasser R. 2018. Leishmania major STT3D improves the N-glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana. Plant Biotechnol J. 2018 Feb 26. doi: 10.1111/pbi.12906.
- Steinkellner H and Castilho A (2015) N-Glyco-engineering in plants: update on strategies and major achievements. In: Methods Mol. Biol. (Castilho A, ed). Springer Humana Press. Vol 1321 pp 195-212. DOI: 10.1007/978-1-4939-2760-9_14.
- Loos A and Castilho A (2015) Transient engineering of the N-glycosylation pathway in N. benthamiana plants towards the synthesis of tetra-sialylated Glycans. In: Methods Mol. Biol. (Castilho A, ed). Springer, Humana Press. Vol 1321 pp 233-248. DOI: 10.1007/978-1-4939-2760-9_17.
- Castilho A and Steinkellner H (2015) Transient expression of mammalian glyco-genes in N. benthamiana plants to modulate N-glycosylation. In: Methods Mol. Biol. (MacDonald J, ed). Springer, Humana Press. Vol 1385 pp 99-113. doi: 10.1007/978-1-4939-3289-4_7.
- Castilho A and Strasser R. (2018) Production of functionally active recombinant proteins in plants: Manipulating N-and O-glycosylation. Molecular Pharming: Applications, Challenges and Emerging Areas (Allison Kermode, ed).
- Glyco-Engineering: Methods and Protocols (2015) Methods Mol. Biol. (Castilho, Alexandra Editor). Springer, Humana Press. Vol 1321
Dr. Sylvestre Marillonnet
Department of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, Halle, Germany
Dr. Victor Klimyuk
Icon Genetics, Halle, Germany
Prof. Renier van der Hoorn
Department of Plant Sciences, University of Oxford, Oxford, UK