Efficient N-Glycosylation of the Heavy Chain Tailpiece Promotes the Formation of Plant-Produced Dimeric IgA

Göritzer, Kathrin; Goet, Iris; Duric, Stella; Maresch, Daniel; Altmann, Friedrich; Obinger, Christian; Strasser, Richard

doi:10.3389/fchem.2020.00346

Cited by 19 publications

(39 citation statements)

References 49 publications

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“…However, there are subtle differences in protein folding and modifications in the ER that cause, for example, underglycosylation of recombinant human proteins expressed in plants ( Castilho et al, 2018 ) or affect the assembly of protein subunits. Co-expression of folding assistants, including the chaperone BIP or the human thioredoxin family protein ERp44 increased the overall yield of a recombinant dimeric IgA produced in N. benthamiana ( Göritzer et al, 2020 ). Similarly, co-expression of human CRT improved the expression of viral glycoproteins like HIV gp140 ( Margolin et al, 2020a ) and the full-length SARS-CoV-2 spike ectodomain ( Margolin et al, 2020c ).…”

Section: Discussionmentioning

confidence: 99%

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

et al. 2021

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Nicotiana benthamiana is used worldwide as production host for recombinant proteins. Many recombinant proteins such as monoclonal antibodies, growth factors or viral antigens require posttranslational modifications like glycosylation for their function. Here, we transiently expressed different variants of the glycosylated receptor binding domain (RBD) from the SARS-CoV-2 spike protein in N. benthamiana. We characterized the impact of variations in RBD-length and posttranslational modifications on protein expression, yield and functionality. We found that a truncated RBD variant (RBD-215) consisting of amino acids Arg319-Leu533 can be efficiently expressed as a secreted soluble protein. Purified RBD-215 was mainly present as a monomer and showed binding to the conformation-dependent antibody CR3022, the cellular receptor angiotensin converting enzyme 2 (ACE2) and to antibodies present in convalescent sera. Expression of RBD-215 in glycoengineered ΔXT/FT plants resulted in the generation of complex N-glycans on both N-glycosylation sites. While site-directed mutagenesis showed that the N-glycans are important for proper RBD folding, differences in N-glycan processing had no effect on protein expression and function.

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Section: Discussionmentioning

confidence: 99%

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

et al. 2021

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“…Additional constraints, such as the host chaperone machinery or differences in the plant oligosaccaryltransferase complex, may also complicate the production of, and glycosylation of, certain viral glycoproteins in planta [11][12][13]. Recently, it has been proposed that engineering the plant secretory pathway to support the production of viral glycoproteins could improve their accumulation in plants, and support their maturation during synthesis [9].…”

Section: Introductionmentioning

confidence: 99%

Calreticulin co-expression supports high level production of a recombinant SARS-CoV-2 spike mimetic inNicotiana benthamiana

Margolin

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Meyers

et al. 2020

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15An effective prophylactic vaccine is urgently needed to protect against SARS-CoV-2 16 infection. The viral spike, which mediates entry into cells by interacting with the host 17 angiotensin-converting enzyme 2, is the primary target of most vaccines in development. 18These vaccines aim to elicit protective immunity against the glycoprotein by use of 19 inactivated virus, vector-mediated delivery of the antigen in vivo, or by direct immunization 20 with the purified antigen following expression in a heterologous system. These approaches 21 are mostly dependent on the growth of mammalian or insect cells, which requires a 22 mammalian cells despite including a modified site for improved processing [17]. Co-126 expression of furin did however improve processing, yielding the expected cleavage product, 127 which increased in intensity with higher levels of furin expression. 128 129 130 131 132 133 134 135 136 137 138 139

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“…Given the role of glycans in directing glycoprotein folding, lower levels of glycan occupancy could be expected to compromise chaperone-mediated folding and to result in increased aggregation or impaired oligomerization. The latter was recently illustrated for recombinant IgA produced in N. benthamiana , where under-glycosylation in the heavy chain tail piece resulted in inefficient dimerization ( Göritzer et al, 2020 ).…”

Section: Pit Stop 2: Implications Of Plant-specific N-glycans For Virmentioning

confidence: 97%

“…The recent observation that some proteins may be under-glycosylated in plants raises concerns that this may be an important constraint for the production of heavily glycosylated biologics in the system ( Jarczowski et al, 2016 ; Castilho et al, 2018 ; Montero-Morales et al, 2019 ; Göritzer et al, 2020 ; Margolin et al, 2020c ; Singh et al, 2020 ). It is presently unclear how widespread this phenomenon is, particularly in the context of viral glycoproteins, as few published reports have described the quantitative glycosylation analysis of plant-produced proteins.…”

Section: Destination 1: Approaches To Produce Viral Glycoproteins In mentioning

confidence: 99%

“…These include glycoproteins from Rift Valley fever virus, chikungunya virus and Epstein-Barr virus ( Meyers et al, 2018 ; Margolin et al, 2020b ). This approach has also been applied to producing recombinant antibodies, although the impact was more modest ( Meyers et al, 2018 ; Göritzer et al, 2020 ).…”

Section: Destination 2: Glycosylation-directed Folding Of Viral Glycomentioning

confidence: 99%

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A Roadmap for the Molecular Farming of Viral Glycoprotein Vaccines: Engineering Glycosylation and Glycosylation-Directed Folding

et al. 2020

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Immunization with recombinant glycoprotein-based vaccines is a promising approach to induce protective immunity against viruses. However, the complex biosynthetic maturation requirements of these glycoproteins typically necessitate their production in mammalian cells to support their folding and post-translational modification. Despite these clear advantages, the incumbent costs and infrastructure requirements with this approach can be prohibitive in developing countries, and the production scales and timelines may prove limiting when applying these production systems to the control of pandemic viral outbreaks. Plant molecular farming of viral glycoproteins has been suggested as a cheap and rapidly scalable alternative production system, with the potential to perform post-translational modifications that are comparable to mammalian cells. Consequently, plant-produced glycoprotein vaccines for seasonal and pandemic influenza have shown promise in clinical trials, and vaccine candidates against the newly emergent severe acute respiratory syndrome coronavirus-2 have entered into late stage preclinical and clinical testing. However, many other viral glycoproteins accumulate poorly in plants, and are not appropriately processed along the secretory pathway due to differences in the host cellular machinery. Furthermore, plant-derived glycoproteins often contain glycoforms that are antigenically distinct from those present on the native virus, and may also be under-glycosylated in some instances. Recent advances in the field have increased the complexity and yields of biologics that can be produced in plants, and have now enabled the expression of many viral glycoproteins which could not previously be produced in plant systems. In contrast to the empirical optimization that predominated during the early years of molecular farming, the next generation of plant-made products are being produced by developing rational, tailor-made approaches to support their production. This has involved the elimination of plant-specific glycoforms and the introduction into plants of elements of the biosynthetic machinery from different expression hosts. These approaches have resulted in the production of mammalian N-linked glycans and the formation of O-glycan moieties in planta. More recently, plant molecular engineering approaches have also been applied to improve the glycan occupancy of proteins which are not appropriately glycosylated, and to support the folding and processing of viral glycoproteins where the cellular machinery differs from the usual expression host of the protein. Here we highlight recent achievements and remaining challenges in glycoengineering and the engineering of glycosylation-directed folding pathways in plants, and discuss how these can be applied to produce recombinant viral glycoproteins vaccines.

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Efficient N-Glycosylation of the Heavy Chain Tailpiece Promotes the Formation of Plant-Produced Dimeric IgA

Cited by 19 publications

References 49 publications

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

Calreticulin co-expression supports high level production of a recombinant SARS-CoV-2 spike mimetic inNicotiana benthamiana

A Roadmap for the Molecular Farming of Viral Glycoprotein Vaccines: Engineering Glycosylation and Glycosylation-Directed Folding

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