Advances and challenges in plant N-glycoengineering: when fucosylation matters

Plant-based expression systems have emerged as promising platforms to produce recombinant glycoproteins. Transient recombinant protein production is a promising alternative to stable transgenic systems, particularly for emergency situations in which rapid production of novel therapeutics is needed. A potential barrier for plant-based production of therapeutic proteins is that different glycosylation patterns are found on plants. Nevertheless, advances in glycoengineering, particularly in the generation of glycoproteins bearing human- and helminth-like N-glycans, further support the use of plants as valuable systems for biopharmaceutical manufacturing. Glyco-design, including methods to control glycan structures and distributions in plants, is a powerful tool for optimizing the efficacy of therapeutic glycoproteins. However, glycoengineering is not merely a matter of gene knock-in or knock-out and it often requires precise fine-tuning to prevent the formation of aberrant glycan structures. Strategies to address these challenges include: (i) identifying and modulating the activity of proteins/enzymes involved in aberrant glycosylation, (ii) optimizing the subcellular localization and expression levels of glyco-modifying enzymes, (iii) inhibiting glycosidases that trim terminal sugar residues, and (iv) enhancing the accessibility of glycosylation sites to processing enzymes. This review summarizes key developments and challenges in plant N-glycoengineering. Within this broad framework, we highlight core α1,3-fucosylation as a representative case illustrating how a single glycan modification can alter structural accessibility, enzyme activity, and overall glycan maturation.

1 Introduction

Protein production in plants can be scaled-up using cell suspension cultures in GMP-compliant bioreactors or whole plants cultivated in greenhouses (Fischer and Buyel, 2020; Huebbers and Buyel, 2021). Plant molecular farming (PMF) has emerged as a powerful platform that harnesses plants as biofactories for recombinant protein production. PMF offers several advantages, including cost-effectiveness, scalability, and inherent biosafety, making it a sustainable and attractive alternative for large-scale protein production. This technology has been successfully applied in the production of industrial enzymes, biopharmaceuticals, and vaccines (Margolin et al., 2020; Shanmugaraj et al., 2020; Goritzer and Strasser, 2021; Eidenberger et al., 2023; Fukuzawa et al., 2024; Yuen et al., 2024). Most therapeutic proteins are glycoproteins. These glycoproteins are decorated with carbohydrate structures (glycans) at asparagine residues within the sequon Asn-X-Ser/Thr, (X≠P)(glycosylation site), through a process called glycosylation. N-glycosylation is a critical post-translational modification that influences protein folding, stability, solubility, and intracellular localization. It is essential for the proper function and pharmacokinetics of glycoproteins and plays a key role in numerous biological processes (Zhou and Qiu, 2019; He et al., 2024).

Unlike DNA or proteins, glycans are not made from a template but are built step by step by specialized enzymes (glycosyltransferases and glycosidases), transporters, and chaperones expressed in different parts of the cell. Protein glycosylation processes involve sequentially orchestrated modification reactions in the networks of the endoplasmic reticulum (ER) and the Golgi during protein trafficking (Dünser and Schoberer, 2025). In addition, the local cellular environment and the structural accessibility of glycosylation sites further influence the activity and substrate specificity of these enzymes. From the ER to the cis Golgi apparatus, the N-glycan processing steps are highly conserved between plants and animals. Significant differences in N-glycan maturation start emerging in the medial Golgi and along the secretory pathway, where the activity of plant-specific glycosyltransferases gives rise to complex N-glycans with unique structural features (Figure 1). The predominant complex N-glycan in plants is GnGnXF which may be further modified by the addition of Lewis-A epitopes [FA] or by trimming terminal N-acetyl-glucosamine (GlcNAc) residues to produce paucimannosidic glycans, (MMXF) (Strasser, 2016) (Figure 1, Supplementary Table S1). Interestingly, a recent study showed that N. tabacum contains two types α1,3-fucosyltransferases, one of which is able to catalyze core fucosylation of high-mannose N-glycans generating Man5F glycans (Supplementary Table S1) (Navarre et al., 2025).

Figure 1

Figure 1. Overview of N-glycosylation pathways in plants. Schematic representation of the sequential steps involved in N-glycan processing within the plant secretory pathway. Following transfer of the oligosaccharide precursor to nascent proteins in the endoplasmic reticulum (ER), glycan maturation proceeds through a series of trimming and extension reactions in the ER and Golgi apparatus. Enzymes are localized to distinct compartments, ensuring the ordered progression from oligomannosidic to complex N-glycans.Abbreviations: MNS3, ER α-mannosidase I; MNS1/2, Golgi α-mannosidase I; GnTI, α1,3-mannosyl-β1,2-N-acetylglucosaminyltransferase I; GMII, Golgi α-mannosidase II; GnTII, α1,6-mannosyl-β1,2-N-acetylglucosaminyltransferase II; XYLT, β1,2-xylosyltransferase; FUT11/12, core α1,3-fucosyltransferases; GALT1, β1,3-galactosyltransferase 1; FUT13, α1,4-fucosyltransferase; HEXO3, β-N-acetylhexosaminidase 3.

Compared to mammalian cells, plants have a more limited glycosylation repertoire, often resulting in greater glycan homogeneity but lacking the ability to perform more complex glycan structures. As a result, considerable efforts are being made to overcome the limitations of plant N-glycosylation machinery and to optimize glycosylation pathways for biopharmaceutical production. N-glycoengineering has become a vital strategy to ensure the production of defined and homogeneous glycoforms (Yang et al., 2015; Katoh and Yamamoto, 2021; Nomura et al., 2022), and plants are highly amenable to glycoengineering, enabling the production of recombinant proteins with tailored glycosylation profiles.

The distinct glycan features of plants, such as core α1,3-fucosylation and β1,2-xylosylation, and the absence of certain glycosyltransferases, nucleotide sugar transporters, and interconversion enzymes necessitate customized engineering strategies for therapeutic applications. Notably, transgenic Nicotiana benthamiana plants, the most widely used plant species in PMF, have been successfully engineered to (i) eliminate plant-specific β1,2-xylose and core α1,3-fucose residues, (ii) introduce branched GlcNAc residues, and (iii) incorporate terminal β1,4-galactose and α2,6-sialic acid residues (Eidenberger et al., 2023; Kao et al., 2024; Goritzer et al., 2025). Beyond "humanizing" plant glycosylation, there is growing interest in engineering non-human glycoforms such as Helminth-type N-glycans, onto plant-derived glycoproteins with potential therapeutic applications and vaccine development (Wilbers et al., 2017; Bunte et al., 2022; van der Kaaij et al., 2022).

Numerous glycoengineering strategies have been developed, especially for the production of immunoglobulins (IgGs) and derivatives (e.g., Fcabs, scFv-Fc, Fc-fusion proteins) with homogeneous glycosylation profiles (Montero-Morales and Steinkellner, 2018; Castilho and Strasser, 2018; Komarova et al., 2019; Goritzer and Strasser, 2021; Goritzer et al., 2025). However, achieving complete and homogeneous human-like glycosylation in other important glycoproteins, such as hormones, cytokines, receptor extracellular domains, and viral antigens, remains challenging. The success of these efforts largely depends on the number and accessibility of N-glycosylation sites (Loos et al., 2014; Montero-Morales et al., 2019; Castilho et al., 2013; Castilho et al., 2014; Goritzer et al., 2017; Izadi et al., 2025). Here, we highlight advances and persisting challenges in plant N-glycoengineering, redefining the role of plant-specific core α1,3-fucosylation from a perceived obstacle to a strategic engineering handle for optimizing N-glycan conformation, accessibility, and immune-modulating potential.

2 Glycoengineering the plant host

Efforts to glycoengineering plant hosts focus on a combination of strategies aimed at suppressing the biosynthesis of plant-specific glyco-epitopes and introducing mammalian glycosyltransferase genes into the plant genome. These approaches enable the glycosylation of recombinant proteins in plant expression systems to more closely resemble that of human cells.

A major achievement in this field has been the downregulation or knockout of genes responsible for the addition of core α1,3-fucose and β1,2-xylose residues. This has led to the establishment of so-called ΔXF plant (cell) lines, which predominantly produce GnGn-type N-glycans on secreted glycoproteins produced in different plant hosts (Table 1). Individual knock-out plants for either core α1,3-fucosylation (ΔF) or β1,2-xylosylation (ΔX) genes (Strasser et al., 2008) can also be useful. For example, specific helminths, such as S. mansoni, synthesize N-glycans that carry core α1,3-fucose with or without β1,2-xylose (van der Kaaij et al., 2022). Beyond the complete removal of plant-specific glycans, several studies have demonstrated the potential to modulate or reintroduce fucosylation to obtain specific glycan configurations. Heterologous expression of fucosyltransferases from Zea mays or S. mansoni has been used to alter the linkage specificity and position of core or antennary fucose residues (Castilho et al., 2015; Wilbers et al., 2017; van Noort et al., 2020). These strategies illustrate that glycoengineering can involve not only the elimination of α1,3-fucosylation but also its selective reinstatement to create defined and functionally optimized N-glycan structures.

Table 1

Table 1. Overview of glycoengineered plant host lines used to modulate complex N-glycan processing. The table summarizes commonly used knockout and overexpression lines together with their underlying genetic modifications, characteristic N-glycan profiles, known developmental or physiological phenotypes, and relevant references.

While plant-specific glycan structures have been associated with potential immunogenicity and allergic reactions, there is currently no clinical evidence of adverse effects in humans (Shaaltiel and Tekoah, 2016; Rup et al., 2017).

An alternative strategy to eliminate plant-specific glycans is the use of hosts lacking GNTI activity, such as the complex glycan 1 (cgl1) mutant (Table 1) that predominantly produce oligomannosidic N-glycans (Man5, Supplementary Table S1). Arabidopsis cgl1 mutants show no visible growth or developmental phenotypes (Vonschaewen et al., 1993; Strasser, 2014), in contrast to Oryza sativa (rice) and Lotus japonicus GNTI mutants, which exhibit severe developmental and reproductive defects (Fanata et al., 2013; Harmoko et al., 2016).

N. benthamiana tolerates the removal of plant-specific complex N-glycans well; no obvious phenotypic abnormalities have been reported in lines lacking xylosyltransferase and fucosyltransferase activity (Strasser et al., 2008; Kogelmann et al., 2024). Furthermore, the stable expression of mammalian glycosyltransferases such as N-acetylglucosaminyltransferases III, IV, and V to produce glycoproteins with bisected and branched N-glycans does not significantly impact plant growth or morphology (Rouwendal et al., 2007; Nagels et al., 2011) (Table 1). However, developmental phenotypes have been observed in transgenic N. benthamiana plants expressing α1,3/4-Lewis fucosyltransferase (Joly et al., 2004), β1,4-galactosyltransferase (ΔXF^GALT) (Schneider et al., 2015; Kogelmann et al., 2024), and α2,6-sialyltransferase (ΔXF^SIA) (Kallolimath et al., 2016). Notably, plants expressing human β1,4-galactosyltransferase and genes required for sialylation display pronounced growth defects and a marked reduction in seed production (Schneider et al., 2015; Kallolimath et al., 2016; Kogelmann et al., 2024) (Table 1). To overcome this shortcoming a recent study has established plant cell packs (PCPs) derived from ΔXF^SIA transgenic plants that enable transient expression of recombinant proteins carrying sialylated N-glycans (Dianatkhah et al., 2025).

Overall, beyond their applications in biopharmaceutical production, glycoengineered plant hosts provide valuable tools for elucidating the functional roles of protein glycosylation in plant growth, development, and stress responses (Kang et al., 2008; Liu et al., 2018; Strasser, 2014; Strasser et al, 2021; Strasser, 2022).

3 Transient glycoengineering

Despite advances in generating transgenic plant lines with engineered glycosylation pathways, PMF still predominantly relies on transient expression systems to modulate N-glycosylation of recombinant proteins.

Rhizobium radiobacter (formerly Agrobacterium tumefaciens) is used as a gene delivery vector by agroinfiltration, enabling high-efficient protein expression without stable genetic transformation (Chincinska, 2021). The remarkable plasticity of the plant glycosylation machinery allows for the de novo design and synthesis of novel glycans, difficult to achieve via chemoenzymatic methods or alternative expression systems.

A particularly innovative approach is the multiple transient expression system (MUTE) in N. benthamiana, where agrobacteria carrying different expression constructs are co-infiltrated to deliver reporter glycoproteins along with glycosyltransferases, nucleotide sugar interconversion enzymes, and transporters required to reprogram the plant glycosylation pathway (Loos and Castilho, 2015). This modular system enables the in-planta assembly of synthetic glycosylation pathways in a controlled and flexible manner. MUTE has been successfully applied to produce recombinant proteins with tailored glycosylation (Figure 2A). Engineering certain glycoforms can be relatively straightforward, requiring only the overexpression of enzymes that are absent or "missing" in plants. For example, overexpression of the core α1,6-fucosyltransferase (FUT8); β1,4-galactosyltransferase (B4GALT1); β1,4-N-acetyl-glucosaminyltransferase III (GNTIII); α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GNTIV) or α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GNTV) has enabled the synthesis of human-like core-fucosylated, galactosylated, bisected and tri-antennary N-glycans, respectively (Figure 2A). In contrast, other modifications require configuration of the plant N-glycosylation machinery to reconstruct entire biosynthetic pathways, such as those needed for human-type protein sialylation (Castilho et al., 2010; Castilho et al., 2012; Bohlender et al., 2020; Izadi et al., 2023a); for the synthesis of helminth-like mono- and bi-fucosylated β14GalNAc-GlcNAc (LacdiNAc or LDN) motifs (LDN-F/F-LDN-F, Figure 2A, Supplementary Table S1) (Wilbers et al., 2017; Bunte et al., 2022; van Noort et al., 2020; van der Kaaij et al., 2022) and for blood group antigens (Beihammer et al., 2021). One striking example is the coordinated transient co-expression of eleven mammalian proteins involved in glycan branching, galactosylation, and sialylation in an N. benthamiana ΔXF background, which enabled the production of recombinant human erythropoietin (EPO) carrying bi-, tri-, and tetra-sialylated complex N-glycans as well as sialylated mucin-type O-glycans, devoid of plant-specific residues (Castilho et al., 2012; Castilho et al., 2013). Despite the remarkable achievements, multiple transient expression methodology can be prone to batch-to-batch inconsistencies. Therefore, binary expression vectors tailored to accommodate multiple genes encoding glycosyltransferases and/or glycosylhydrolases, along with various genetic elements are being optimized, hereby enhancing the flexibility to manipulate N-glycan structures in plant transient expression systems (Castilho et al., 2013; Schneider et al., 2014; Bohlender et al., 2020; Kittur et al., 2020; Mukherjee et al., 2021; Izadi et al., 2023b; Kao et al., 2024) (Figure 2B).

Figure 2

Figure 2. Strategies for generating helminth- and human-like N-glycans in plants. (A) Targeted knockout of XYLT and FUT (red arrows) produces the ΔXF plant line, which synthesizes complex N-glycans of the GnGn type. This “glycan template” provides a foundation for further modification through the transient single or multiple expression of heterologous mammalian or helminth glycosyltransferases (blue arrows). Such engineered enzymes introduce branching, bisecting, fucosylation, galactosylated or sialylation patterns, enabling the production of highly customized glycoforms. Glycan structures enclosed in dashed boxes represent glycan structures that have also been obtained in stably transformed plant lines. N-glycan structures were labelled according to the ProGlycAn nomenclature (Proglycan_nomenclature_2023.pdf) (B) Multiple transient expression (MUTE) of glycosylation enzymes in plants can be implemented by: (i) co-infiltrating several Agrobacterium strains, each carrying a single expression cassette; (ii) assembling multiple expression constructs within one Agrobacterium strain; or (iii) introducing a single vector containing multiple gene expression cassettes. Abbreviations: GNTIII, β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase III; GNTIV, α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase IVa; GNTV, α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase V; FUT8, α1,6-fucosyltransferase; FUT1/2, α1,2-fucosyltransferases; FUT9a, α1,3-fucosyltransferase IXa; B4GALT1, β1,4-galactosyltransferase; ST6GAL1, α2,6-sialyltransferase; ST3GAL4, α2,3-sialyltransferase; ABO-A, α1,3-N-acetylgalactosaminyltransferase; GalNAcT, α1,4-N-acetylgalactosaminyltransferase; ST8Sia II/IV, α2,8-sialyltransferases II and IV; SmFucT D/E/F, Schistosoma mansoni α1,3-fucosyltransferases D, E, and F.

Finally, studies evaluating the impact of glycosylation on protein function traditionally relied on site-directed mutagenesis to remove N-glycosylation sites, thereby preventing glycan attachment. However, such mutations can alter protein folding, conformation, or stability, making it difficult to distinguish the direct effects of glycosylation from those arising due to structural perturbations. As an alternative approach, the synthesis of mannosidic glycans can be induced by treatment with kifunensine (KIF), a potent α-mannosidase I inhibitor, which blocks N-glycan processing at the Man₉GlcNAc₂ stage. When combined with the overexpression of an Endo H enzyme targeted to the late Golgi, this strategy enables the production of deglycoproteins, without the need for mutating the protein sequence (Izadi et al., 2023a; Izadi et al., 2025. This method provides a powerful tool to investigate the functional consequences of glycosylation while preserving native protein structure and folding.

4 Fine-tuning the N-glycan processing

Glycosylation is tightly regulated across multiple levels, including gene expression, protein folding, intracellular trafficking, and the activity and localization of glycosyltransferases and glycosidases. Engineering recombinant protein glycosylation is often complicated by the number and accessibility of glycosylation sites. “Humanization” and "helminthization" of N-glycans in plants by glycoengineering seems to be hampered by the formation of aberrant glycan structures and often requires fine-tuning to (i) optimize the expression and subcellular localization of glyco-modifying enzymes (Strasser et al., 2009; Castilho et al., 2011b; Wilbers et al., 2017; Kallolimath et al., 2018; Konig-Beihammer et al., 2021), (ii) inhibit glycosidases that trim terminal sugar residues (Shin et al., 2017; Kriechbaum et al., 2020; Blumberg et al., 2025) (iii) modulate enzyme activity, and (iv) improve the glycosite accessibility to processing enzymes (Castilho et al., 2015; Wilbers et al., 2017).

N-glycan processing enzymes are arranged along the plant secretory pathway, where type II membrane-bound glycosyltransferases/glycosidases act in a stepwise manner (Schoberer and Strasser, 2011). These enzymes contain a CTS (cytoplasmic-transmembrane-stem) domain, that governs their sub-cellular localization, and an enzymatic domain (Sasai et al., 2001; Tu and Banfield, 2010; Welch and Munro, 2019) (Figure 3A). While the exact size of the stem region of the CTS domain can vary, it generally spans a significant portion of the protein between the transmembrane domain and the catalytic domain. Besides regulating the localization, the stem region is also required for multimer formation and important for homophilic interactions with other glycosyltransferases (Welch and Munro, 2019; Tomida et al., 2022). Localization within Golgi cisternae (cis, medial, trans/TGN) reflects the enzyme sequential role in glycan maturation, although overlap between sub-compartments is common (Schoberer and Strasser, 2011). Recent insights identified molecular codes within CTS regions as key determinants of this localization (Yagi et al., 2024).

Figure 3

Figure 3. Domain swapping of glycosyltransferases (GTs) to control sub-Golgi localization. (A) Glycosyltransferases (GTs) are typically type II membrane proteins composed of an N-terminal cytosolic tail, a transmembrane domain, and a luminal stem region (collectively referred to as the CTS domain). The CTS region is a key determinant of the enzyme’s sub-Golgi localization, directing it to the cis-, medial-, or trans-Golgi compartments. (B) Swapping CTS regions between different GTs enables the rational reorganization of the glycosylation pathway. The resulting chimeric GT localizes to the Golgi subcompartment defined by the CTS region of the donor enzyme, thereby allowing fine-tuned control over glycan maturation.

In plants, a clear separation of early- (MNS1, GNTI, GMII, GNTII, XYLT, and FUT11/12) from late-acting enzymes (GALT1, FUT13) has been established but the existence of a distinct medial-Golgi or intermediate location is questionable (Schoberer and Strasser, 2011).

Despite conserved localization signals across species, subtle differences in sub-Golgi targeting have been observed for heterologous glycosyltransferases expressed in plants (Saint-Jore-Dupas et al., 2006). Miss localization of heterologous enzymes can disrupt native pathways, leading to hybrid or incomplete glycan structures. For instance, overexpressing full-length GNTIII, B4GALT1, or α1,3-fucosyltransferase IXa (FUT9a) disrupted the activity of endogenous GNTII, GMII and XYLT (Bakker et al., 2001; Rouwendal et al., 2007, Rouwendal et al., 2009; Wilbers et al., 2017; Nguyen et al., 2023). To overcome this, domain-swapped chimeric enzymes with Golgi-targeting CTS domains have been engineered (Figure 3B, Table 2) (Loos and Steinkellner, 2014; Strasser et al., 2014), enabling precise sub-Golgi localization and control over glycosylation (Strasser et al., 2009; Castilho et al., 2011a; Castilho et al., 2011b; Dicker et al., 2015; Wilbers et al., 2017; Bohlender et al., 2020; van Noort et al., 2020).

Table 2

Table 2. Representative type II glycosyltransferases and glycosidases whose cytoplasmic, transmembrane, and stem (CTS) domains serve as targeting signals for specific subcellular localization of heterologous enzymes. UniProt accession numbers and amino acid ranges defining the CTS or CT domains are indicated.

Multiple examples of CTS swapping are available in the literature (Table 3) (McGinness et al., 2025). An important one was the replacement of the endogenous CTS of the human B4GALT1 by the rat α2,6-sialyltransferase (ST6GAL1) or GALT1 instead of the medial-Golgi targeting CTS domains previously used (Bakker et al., 2001, Bakker et al., 2006; Rouwendal et al., 2009) to avoid synthesis of hybrid structures. Targeting B4GALT1 to the late Golgi compartment (^ST6B4GALT1, ^GALT1B4GALT1 and ^FT4B4GALT1) is required for the synthesis of biantennary galactosylated (AA)_; sialylated (NaNa); Lewis-X (Le^X, [AF]) and blood type antigen epitopes (Figure 2A, Supplementary Table S1) (Castilho et al., 2010; Strasser et al., 2009; Wilbers et al., 2017; Goritzer et al., 2019; Kallolimath et al., 2020; Kriechbaum et al., 2020; Bohlender et al., 2020; Beihammer et al., 2021). Also, accurate mimicry of Schistosoma mansoni N-glycosylation in plants requires a strict sequential order of core fucosylation, wherein α1,6-fucosylation must precede α1,3-fucosylation to prevent interference with the activity of the introduced fucosyltransferases (FucTs) (van Noort et al., 2020). While this strategy has been effective, species-specific differences must be considered (Hesselink et al., 2014; Dicker et al., 2015).

Table 3

Table 3. Chimeric glycosyltransferases with swapped cytoplasmic–transmembrane–stem (CTS) regions used to achieve specific target glycoforms, either individually or in co-expression experiments. (“^CTSGT”).

Besides swapping CTS regions, the expression level of the heterologous glycosyltransferase is also an important factor and often fine-tuning the enzyme expression is required to increase the homogeneity of glycosylation on recombinant glycoproteins (Dicker et al., 2015; Wilbers et al., 2017; Kallolimath et al., 2018). It has become clear that even with the appropriate CTS region, expression levels over a certain threshold can miss target the enzyme and negatively impact the generation of fully processed N-glycans. Indeed, studies conducted with B4GALT1 targeted to the required late Golgi compartment (^ST6B4GALT1) still showed the importance of choosing the right promoter to optimize expression and avoid aberrant glycosylation (Wilbers et al., 2017; Kallolimath et al., 2018; Bohlender et al., 2020).

Another aspect that needs attention when transiently expressing recombinant proteins is the possible “overloading” of the secretory pathway which can interfere with the activity of endogenous Golgi-resident enzymes leading to incomplete glycosylation (truncated and hybrid glycans). This is well illustrated when MUTE is used to produce multi-sialylated (Castilho et al., 2014; Schneider et al., 2014; Izadi et al., 2023a) or multi-fucosylated recombinant proteins in plants (van Noort et al., 2020). Some recombinant proteins expressed in plants are decorated with mono-antennary N-glycans (MGn, MA, MNa, single Le^x and LDN-F motifs, Figure 2A, Supplementary Table S1). The presence of N-glycan structures with a single terminal GlcNAc residue has been attributed to a partial inhibition of endogenous GNTII since co-expression of recombinant GNTII results in a significant increment of fully processed glycans (Loos et al., 2014; Schneider et al., 2014; Dicker et al., 2016; Goritzer et al., 2017).

Substantial progress has been achieved in engineering plant glycosylation machinery; however, the efficacy of these approaches remains protein-specific, as outcomes are strongly influenced by the local structural context and accessibility of individual N-glycosylation sites due to protein conformation. Exposed glycosylation sites are more accessible to glycan-modifying enzymes (glycosyltransferases and glycosidases), leading to greater glycan heterogeneity. In contrast, buried glycosylation sites are more protected from glycosidases but also less accessible to glycosyltransferases, often resulting in incomplete glycan processing. For example, full antibodies, particularly IgGs, have a conserved N-glycosylation site at Asn297 buried within the CH2 domain of Fc region. Although there is no direct information comparing the exposure of the glycosylation site Fcabs (Fc fragment with engineered antigen binding sites) and in full antibodies, some relevant insights can be inferred by comparing glycoengineering of Fcabs and full antibodies (Kriechbaum et al., 2020). Site-specific N-glycan processing has been observed for several glycoproteins with multiple glycosylation sites (GS). Good examples are IgM and IgE antibodies with 5 and 7 GS, respectively. While most GS carry complex GnGn/MGn glycans (IgM : GS1–3 and IgE: GS1-5), those at the CH3 domain are decorated with oligomannosidic structures (Loos et al., 2014; Montero-Morales et al., 2019; Jugler et al., 2022). The structural polypeptide features that affect these modifications are not yet understood.

4.1 Paucimannosidic N-glycans

Proteins trafficking through the Golgi apparatus are subjected to processing by various glycosyltransferases. Most recombinant proteins expressed in plants acquire complex N-glycans, with GnGnXF or GnGn as the predominant forms, depending on whether the host is wild-type or glycoengineered ΔXF plants, respectively. However, the removal of terminal GlcNAc residues can lead to truncated N-glycans such as MMXF or MM, collectively referred to as paucimannosidic glycans (Figure 1, Supplementary Table S1).

The formation of these structures is primarily mediated by β-N-acetylhexosaminidases particularly HEXO1 and HEXO3 (Strasser et al., 2007a; Liebminger et al., 2011). Among them, HEXO3, localized at the plasma membrane, has been identified as the main enzyme responsible for trimming terminal GlcNAc and N-acetylgalactosamine (GalNAc) (Strasser et al., 2007b; Liebminger et al., 2011; Shin et al., 2017; Alvisi et al., 2021). In contrast, HEXO2 exhibits strict β-galactosaminidase activity (Alvisi et al., 2021).

Recombinant proteins expressed in N. benthamiana exhibit varying sensitivity to β-hexosaminidase activity. For instance, in full IgG1 antibodies and Fc fragments, the N-glycosylation site buried within the CH2 domain is largely protected from enzymatic trimming. Conversely, glycoproteins such as bovine follicle-stimulating hormone (Dirnberger et al., 2001), human α1-antitrypsin (A1AT) (Castilho et al., 2014), human secreted alkaline phosphatase (SEAP) (Becerra-Arteaga and Shuler, 2007), and the helminth glycoprotein Omega-1 (Wilbers et al., 2017) have shown a high proportion of paucimannosidic glycans.

These truncated glycans are often considered undesirable in therapeutic applications due to their potential to increase immunogenicity and adverse immune responses. Consequently, several strategies have been developed to suppress their formation. These include (i) the use of Arabidopsis thaliana hexo3 knockout mutants (Castilho et al., 2014), (ii) RNA interference to silence HEXO3 (Shin et al., 2017) and (iii) CRISPR-based genome editing of β-hexosaminidase genes (Blumberg et al., 2025).

An alternative strategy involves capping terminal GlcNAc and GalNAc residues with fucose using FUT9a. This strategy was used to make glycans resistant to hexosaminidase activity and enhanced the accumulation of fucosylated LDN motifs in helminth glycoproteins (Wilbers et al., 2017). Glycan processing by endogenous glycosidases should in most cases be avoided, but sometimes the action of glycosidases can be beneficial to fine-tune a specific glycan structure. Interestingly, paucimannosidic glycans can also be advantageous. Exposed mannose residues promote rapid receptor-mediated clearance from the bloodstream (Yang et al., 2015) and have shown enhanced efficacy in mannose receptor (MR)-mediated cellular uptake of biopharmaceuticals (Limkul et al., 2016; Sariyatun et al., 2021). Thus, for certain therapeutic proteins, especially those used in enzyme replacement therapy (ERT), paucimannosidic N-glycans are desirable. To enrich these glycans in plant-derived ERT proteins (e.g., human β-glucocerebrosidase, acid α-glucosidase), proteins have been targeted to the plant storage vacuole, where terminal residues are naturally removed to produce paucimannosidic glycans (Shaaltiel et al., 2007; Liebminger et al., 2011; Tekoah et al., 2013). Additionally, glycoengineered A. thaliana mutants lacking ALG3 (an α1,3-mannosyltransferase involved in early N-glycan precursor assembly) have been used to produce recombinant human acid α-glucosidase with predominantly MM-type glycans (Sariyatun et al., 2021).

A landmark in the field is taliglucerase alfa, a β-glucocerebrosidase produced in carrot cell suspension cultures by Protalix Biotherapeutics (Carmiel, Israel). ELELYSO® was the first plant-made pharmaceutical approved by the U.S. Food and Drug Administration (FDA) for human use.

As noted, sensitivity to β-hexosaminidase trimming is highly correlated with the accessibility of glycosylation sites. While glycosylation sites on proteins such as A1AT are fully accessible to β-hexosaminidase activity, others like the Asn297 buried in the CH2 domain of antibodies are structurally shielded from enzymatic access. Interestingly, the presence of core α1,3-fucose appears to enhance the exposure of certain glycans to β-hexosaminidases. Co-expression of FUT11 significantly increases the formation of paucimannosidic glycans in Fcabs (MMF, Supplementary Table S1), but not in full antibodies. This observation suggests that structural rearrangements in Fcabs may expose the glycosylation site, rendering it more susceptible to glycan trimming, particularly when core fucosylation alters glycan conformation and enhances accessibility to glycosidases. Of note, enhancing the synthesis of paucimannosidic N-glycans by core fucosylation is not observed when the α1,6-fucosyltransferase is used (Shin et al., 2017).

4.2 Man5 and hybrid N-glycans

Man5 and hybrid N-glycans play crucial roles in both normal physiology and disease. Man5 structures are central to glycoprotein quality control, structural integrity, and therapeutic relevance, while hybrid N-glycans contribute to neuronal function, immune modulation, and protein stability. Alterations in either glycoform are linked to pathological processes, underscoring their biological and biomedical importance (Hall et al., 2016).

To promote the synthesis of high-mannose-type N-glycans (Man7-9), which resemble those found on many helminth glycoproteins and are desirable for the production of recombinant vaccine antigens in plants, strategies such as ER retention of the glycoprotein or treatment with kifunensine, have been successfully employed (Roychowdhury et al., 2018; Izadi et al., 2023a). Similarly, inhibition of GMII using swainsonine has been used to generate Man5 glycans (Choi et al., 2018), which can also be produced by expressing glycoproteins in GNTI-deficient plants, as previously discussed. However, precise engineering of plant systems to produce defined mannosidic or hybrid glycans remains challenging, as current glycoengineering strategies still require further optimization. One possible strategy to control N-glycan processing could be the use of chimeric proteins carrying specific CTS regions that interfere with glycan maturation at a defined step. Mislocalization or interaction of exogenous glycosyltransferases with endogenous enzymes via their stem regions can modulate N-glycan processing by limiting downstream enzyme access. For instance, GNTI, a key enzyme in the cis/medial-Golgi, initiates hybrid N-glycan formation by transferring a GlcNAc residue to Man5 (Figure 1). This terminal GlcNAc is essential for subsequent processing by medial- and trans-Golgi glycosyltransferases to form complex N-glycans. Interestingly, overexpression of the CTS region from GNTI and MNS1 fused to mRFP was shown to disrupt normal glycan processing, resulting in the accumulation of Man₅ structures on recombinant glycoproteins that would otherwise carry complex glycans (GnGnXF) (Schoberer et al., 2023). This pioneering work demonstrated that transient expression of specific CTS domains can redirect glycosylation from complex to mannosidic forms. A similar approach could be employed to selectively block further processing in the medial-Golgi, facilitating the production of hybrid or mono-antennary N-glycans by preventing recognition by downstream glycosyltransferases (Figure 4). Supporting this concept, targeting of B4GALT1 to the medial-Golgi (^XYLTB4GALT1 and ^FUT11B4GALT1) resulted in the formation of hybrid (Man₄A and Man₅A) and mono-antennary (MA) β1,4-galactosylated N-glycans (Supplementary Table S1) (Dicker et al., 2015). Thus, the overexpression of specific CTS regions fused to an inactive catalytic domain could promote the synthesis of unprocessed glycans (M5Gn, Man4Gn and MGn, Figure 4).

Figure 4

Figure 4. Potential strategy to interfere with N-glycan maturation in plants. Overexpression of Golgi-targeted membrane anchors (CTS regions) lacking a functional catalytic domain (e.g. expressed as fluorescent fusion proteins or with an inactive catalytic domain) can disrupt endogenous glycosylation by forming non-productive complexes with native enzymes through their stem regions. This strategy, previously demonstrated using MNS1 and GNTI CTS domains to generate mannosidic glycans (Man5), could be extended to selectively block processing in the medial or medial/trans-Golgi, enabling the accumulation of hybrid or monoantennary N-glycans (Man5Gn, Man4Gn and MGn). (+/−): presence or absence of core epitopes, depending on the genetic background of the host (wild type or ΔXF lines). N-glycan structures were labelled according to the ProGlycAn nomenclature (Proglycan_nomenclature_2023.pdf).

4.3 Galactosylation and galactosidases

In plants, the only galactosylated native N-glycan structure is the Lewis A (Le^a) motif. These galactosylated glycans result from the activity of GALT1 that transfers galactose in β1,3-linkage to terminal GlcNAc residues, followed by the action of α1,4-fucosyltransferase (FUT13), which adds fucose in an α1,4-linkage to GlcNAc, thereby completing the synthesis of the Le^a epitope (Strasser et al., 2007a) (Figure 1, Supplementary Table S1). Le^a‐containing N‐glycans are present only on a limited number of, mostly unidentified, glycoproteins in plants, and their biological significance remains unclear (Wilson, 2001; Beihammer et al., 2021). Although rare on endogenous plant proteins, Le^a structures are frequently found on complex N-glycans of some plant-produced recombinant glycoproteins, such as human erythropoietin (EPO), SARS-CoV-2 spike protein, and hepatitis B virus (HBV) antigen, even when absent on the native versions of these proteins (Weise et al., 2007; Parsons et al., 2012; Jez et al., 2013; Balieu et al., 2022; Pantazica et al., 2023). Notably, overexpression of GALT1 significantly increases the abundance of Le^a-containing N-glycans on a monoclonal antibody, without the need to overexpress FUT13 (Nguyen et al., 2023).

Le^a determinants are sometimes referred to as human-type glycosylation, since both Le^a and sialyl-Le^a epitopes are significantly elevated in the sera of patients with colon cancer (Terracciano et al., 2005). However, they are rarely present on glycoproteins in healthy adult humans. To eliminate Le^a formation in plants, the synthesis pathway has been successfully disrupted in Physcomitrella patens by knocking out the α1,4-fucosyltransferase and β1,3-galactosyltransferase genes (Parsons et al., 2012).

In contrast to plant β1,3-galactosylated N-glycans, mammalian and helminth glycoproteins carry N-glycans extended with antennary β1,4-galactose residues. This widespread glycan modification can significantly influence the efficacy of therapeutic glycoproteins. For instance, Fc galactosylation has been shown to affect the conformation of IgG1 and its binding to Fc receptors (Houde et al., 2010; Raju and Jordan, 2012). Moreover, β1,4-galactosylated N-glycans serve as essential acceptor substrates for terminal sialylation, a modification known to enhance glycoprotein stability, prolong serum half-life, and improve therapeutic performance (Chia et al., 2023). However, the efficient synthesis of bi-antennary β1,4-galactosylated N-glycans (AA) varies depending on the target protein (Kriechbaum et al., 2020). In fact, except for monoclonal antibodies, achieving homogeneous human-like β1,4-galactosylation in recombinant glycoproteins remains challenging. Efforts to fine tuning the expression of B4GALT1 included CTS swapping to target the enzyme to the late Golgi (Table 3) and optimizing expression levels through selection of weaker promoters, as discussed above. These approaches have enabled improved synthesis of sialylated N-glycans as well as Lewis X (Le^X) and blood group structures on plant-produced glycoproteins.

The synthesis of Le^X ([AF], Supplementary Table S1) structures found on native helminth glycoproteins was achieved through co-expression of chimeric ^ST6B4GALT1 and ^ST6FUT9a (Figure 2A) (Wilbers et al., 2017). Additionally, two fucosyltransferases from the parasite Schistosoma mansoni, FucTD and FucTE, have also been shown to synthesize Le^X (van Noort et al., 2020). The predominance of monoantennary Le^X-carrying N-glycans suggests that overexpression of GNTII may be necessary to enhance glycan branching and support bi-antennary Le^X formation, as discussed above (Wilbers et al., 2017).

Apart from the common β1,4GalGlcNAc (LacNAc or LN) motif, many invertebrates, such as helminths, express glycans containing GalNAc, an amino sugar derivative of galactose, in the form of LDN motifs (Supplementary Table S1), which also occur in vertebrates and are present on several mammalian hormones.

Engineering GalNAc-carrying glycans in plants was first explored to produce mammalian mucin-type O-glycans (Daskalova et al., 2010; Yang et al., 2012; Castilho et al., 2012) by introducing the enzymatic machinery to transfer GalNAc residues to O-glycosylation sites of recombinant proteins. These studies revealed that, among the three proteins involved in the process (UDP-GlcNAc C4 epimerase, Golgi UDP-GalNAc transporter, and β1,4-N-acetylgalactosaminyltransferase), only GalNAcT was strictly necessary for GalNAc-glycan synthesis (Castilho et al., 2012; Dicker et al., 2016). This was later confirmed by the successful synthesis of LDN on the N-glycans of S. mansoni kappa-5 in N. benthamiana using only a C. elegans GalNAcT (CeGalNAcT) (Wilbers et al., 2017) (Figure 2A). This shows that, even though GalNAc is not native to plant N-glycans, UDP-GalNAc is present within the correct Golgi compartment and can be incorporated into the antennae of N-glycans of recombinant glycoproteins. Plant-derived LDN motifs can be further modified by adding an α1,3-fucose to the distal GlcNAc (via FucT9a, SmFucTD, or SmFucTE) to yield LDN-F and an additional α1,3-fucose to the terminal GalNAc (via SmFucTF) to produce F-LDN-F, resulting in mono- and bi-fucosylated LDN motifs (Figure 2A, Supplementary Table S1) (Wilbers et al., 2017; van Noort et al., 2020; van der Kaaij et al., 2022; Zwanenburg et al., 2023). Fucosylation of LDN motifs has been shown to enhance the accumulation of GalNAc-containing N-glycans, likely by increasing resistance to HEXO-mediated removal of unsubstituted GlcNAc and/or GalNAc residues (Strasser et al., 2007a).

The ABH(O) blood group antigens are specific glycan determinants on glycoproteins and glycolipids that also carry GalNAc residues, which are crucial for their antigenicity and the determination of blood types (Stowell and Stowell, 2019). In plants, synthesis of blood group A (type 1 and type 2) structures was recently achieved on the RBD of the SARS-CoV-2 spike protein expressed in N. benthamiana (Konig-Beihammer et al., 2021). These modifications were generated by co-expressing either the native A. thaliana GALT1 (type 1) or a chimeric ^ST6B4GALT1 (type 2) with a human α1,2-fucosyltransferase targeted to late Golgi (^ST6FUT1 or ^ST6FUT2) to produce H antigens (α1,2FucGalβ-R), followed by extension with α1,3-GalNAc residues via N-acetylgalactosaminyltransferase (ABO A). Interestingly, in this case co-expression of a Yersinia enterocolitica UDP-GlcNAc 4-epimerase (YeGNE), which converts UDP-GlcNAc to UDP-GalNAc, and a Caenorhabditis elegans UDP-GlcNAc/UDP-GalNAc transporter (Castilho et al., 2012) further improved the biosynthesis of blood group A type 2 trisaccharide structures (Beihammer et al., 2021) (Figure 2A, Supplementary Table S1). The efficiency of ABH antigen synthesis appears to depend on glycosite accessibility. Mono-antennary structures suggests partial removal of terminal galactose and GalNAc by endogenous apoplastic galactosidases (Kriechbaum et al., 2020), whereas terminal fucosylation protects these residues from enzymatic trimming, as also observed for fucosylated LDN motifs (Wilbers et al., 2017). Despite significant efforts to optimize terminal galactosylation, many plant-derived glycoproteins still display incompletely processed N-glycans with heterogeneous β1,4-galactosylation due to trimming of terminal galactose residues in the apoplast (Castilho et al., 2014; Wilbers et al., 2017; Bohlender et al., 2020; Kriechbaum et al., 2020). Consequently, studies assessing the impact of glycosylation on protein function or activity (other than monoclonal antibodies) rarely include β1,4-galactosylated variants (Jez et al., 2013; Schneider et al., 2014; Loos et al., 2014; Montero-Morales et al., 2019).

β-galactosidases (BGALs) exhibit specificity for β1,3-, β1,6-, or β1,4-galactosidic linkages (Ahn et al., 2007). In N. benthamiana, BGAL1 (NbBGAL1) is an active enzyme targeting both N- and O-glycans, capable of removing β1,4-linked galactose from N-glycans as well as terminal β1,3-galactose from Lewis-A epitopes and T-antigen O-glycans (Kriechbaum et al., 2020).

Recombinant proteins expressed in N. benthamiana exhibit varying sensitivity to β-galactosidases activity. While glycans on the IgG Fab fragment are fully exposed to the surrounding solvent, those in the Fc fragment are largely shielded by the opposing CH2 domain, making galactosylation at this site inherently more efficient. Interestingly, similar to HEXO3 sensitivity, the susceptibility of Fc glycans to β-galactosidase activity appears to be influenced by plant-specific core α1,3-fucosylation (Kriechbaum et al., 2020). This plant-specific modification seems to relax N-glycan or protein structural constraints, rendering terminal sugar residues more accessible to glycosidases, a phenomenon once again not reproduced by core α1,6-fucosylation (Shin et al., 2017; Kriechbaum et al., 2020).

Transient downregulation via RNA interference (RNAi) or complete knockout of NbBGAL1 significantly reduces β-galactosidase activity, leading to higher levels of fully bi-galactosylated complex N-glycans on multiple plant-produced glycoproteins (Kriechbaum et al., 2020). More recently, NbBGAL3B was identified as a second major contributor to undesired β-galactosidase activity, displaying similar specificity for β1,4-linked galactose on N-glycans (van der Kaaij et al., 2025). BGALs hydrolyze terminal β-D-galactosyl residues from diverse substrates, including polysaccharides and glycoproteins, playing key roles in cell wall structure and function (Sampedro et al., 2012; Hoang et al., 2023). Consequently, knockout of β-galactosidase genes can markedly affect plant development due to their essential biological functions. In transient glycoengineering, approaches to achieve near-complete galactosylation on glycoproteins prone to galactosidase trimming have focused on exploiting the protective effect of capping sugars. This was demonstrated by producing recombinant proteins carrying terminal fucose or sialic acid residues, followed by selective removal of the capping sugars with specific fucosidases or sialidases to expose terminal β1,4-galactose (Castilho et al., 2014; van der Kaaij et al., 2025).

De novo β1,4-galactosylation of plant N-glycans provides a substrate for further elongation (e.g., blood group antigens and sialylation), but these newly introduced β1,4-linked galactoses can also serve as anchors for other sugars, leading to the formation of aberrant N-glycans. Several studies have shown that recombinant proteins co-expressed with B4GALT1 carry β1,4-galactosylated N-glycans decorated with additional pentoses and hexoses (Bohlender et al., 2020; Kittur et al., 2020). In N. tabacum and Physcomitrella patens, multiple pentosylations on β1,4-galactosylated N-glycans were identified as α-linked arabinoses (Bohlender et al., 2022). This modification likely arises because the β1,4-linked galactose creates a non-native acceptor site for endogenous arabinosyltransferases, which normally target arabinose to cell wall polysaccharides but can act promiscuously on engineered N-glycans. Arabinoses are absent in humans and therefore potentially immunogenic (Leonard et al., 2005). In addition, they may interfere with the full N-glycan humanization of plant-derived glycoproteins. Comprehensive characterization of this aberrant N-glycosylation and identification of the responsible glycosyltransferases remain essential future steps to enable their elimination and ensure the safety of biopharmaceuticals produced in plant-based systems.

4.4 Branched and bisected N-glycans

An essential step for generating complex human-type glycans is the formation of multi-antennary structures, common in human but absent in plant N-glycans. As stated above, transgenic Arabidopsis and N. benthamiana plants expressing the human GNTIV or/and GNTV enable the synthesis of proteins with tri- and tetra-antennary N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]) (Figure 2A, Supplementary Table S1), when the enzymes are targeted to the medial-Golgi via CTS swapping (^FUT11GNTIV, ^FUT11GNTV and ^XYLTGNTIV, ^XYLTGNTV, Table 3) (Nagels et al., 2011; Nagels et al., 2012). Low abundance of multi-antennary structures on endogenous proteins may reflect HEXO activity toward β1,2-, β1,4-, and β1,6-linked GlcNAc residues (Nagels et al., 2012). Transient expression of GNTIV and V confirmed that medial-Golgi targeting resulted in more homogeneous multi-antennary glycans on recombinant proteins such as Butyrylcholinesterase (BChE), A1AT, and EPO, though efficiency depended on protein and glycosylation site accessibility (Castilho et al., 2011b; Schneider et al., 2014; Castilho et al., 2014). For example, a monoclonal antibody with an additional glycosylation site in the Fab region (such as Cetuximab), and an EPO protein fused to the Fc domain of an IgG1 (EPO-Fc), showed differing levels of accessibility to ^FUT11GNTIV, ^XYLTGNTIV and ^FUT11GNTV. While glycans on the Fab region and on EPO were successfully branched with GlcNAc residues, the N-glycosylation site on the Fc fragment remained unmodified (Castilho et al., 2011b, Castilho et al., 2015).

Addition of a bisecting GlcNAc to the β-mannose of the tri-mannosyl core via a β1,4-linkage, catalyzed by GNTIII, represents another critical branching step (Figure 2A). Unlike other branches, bisecting GlcNAc is not elongated and often inhibits further processing by GnTIV/V and other glyco-processing enzymes (Schachter, 1986). This was demonstrated in early efforts to produce bisected N-glycans by expressing full-length human GNTIII in Nicotiana tabacum and BY2 cells, which primarily resulted in the formation of hybrid bisected glycans (Cox et al., 2006; Rouwendal et al., 2007; Karg et al., 2010). Similarly, the expression of chimeric GNTIII targeted to the medial Golgi (^GMIIGNTIII, ^XYLTGNTIII and ^FUT11GNTIII) in wild-type plants led to the production of incompletely processed N-glycans lacking core fucose and xylose (Man5Gnbi, Supplementary Table S1) (Castilho et al., 2011a; Castilho et al., 2011b). In contrast, fully processed bisected N-glycans were achieved when GNTIII was targeted to the trans-Golgi (^STGNTIII, Table 3) (Castilho et al., 2011a; Castilho et al., 2011b; Castilho et al., 2015). Finally, co-expression of the sequentially targeted ^XYLTGNTV, ^FUT11GNTIV, and ^STGNTIII enabled the synthesis of branched and bisected glycans on recombinant proteins with accessible glycosylation sites, such as human transferrin and EPO (Castilho et al., 2011b). As observed with other glycoengineering strategies, accessibility of Fc glycans to branching and bisecting enzymes significantly improved with α1,3-core fucosylation (FUT11), whereas α1,6-core fucosylation (FUT8) had a weaker effect (Castilho et al., 2011a; Castilho et al., 2015).

4.5 Bi-, multi- and poly-sialylated N-glycans

Sialylation, the attachment of sialic acid residues to glycoproteins, is a critical modification for therapeutic proteins, enhancing their stability, half-life, and biological activity (Zhu et al., 2024). It plays a vital role in various cellular processes and interactions, making it a key focus in the development and optimization of biopharmaceuticals. Ensuring proper sialylation can significantly improve the therapeutic efficacy and pharmacokinetics of glycoprotein drugs (Jeong et al., 2008; Bork et al., 2009). The broader applications of therapeutic N-glycan engineering are driven by the goal of optimizing α2,3- and α2,6-linked sialylation of glycoproteins (Zhong et al., 2022). Among these efforts, α2,6-sialylation has garnered particular attention in glycoengineering, especially in the context of antibodies. Research indicates that the anti-inflammatory activity of antibodies is largely attributed to Fc-sialylation, highlighting the significance of this modification in enhancing therapeutic efficacy (Anthony et al., 2008).

De novo sialic acid biosynthesis occurs in the cytosol through a four-step pathway. The key enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) initiates the process by converting UDP-GlcNAc into ManNAc-6P, which is subsequently condensed with phosphoenolpyruvate (PEP) by Neu5Ac-9-phosphate synthase (NANS) to form Neu5Ac-9P. This intermediate is then dephosphorylated by Neu5Ac-9-phosphate phosphatase (NANP) to yield free N-acetylneuraminic acid (Neu5Ac). Neu5Ac is activated in the nucleus by CMP-Neu5Ac synthetase (CMAS) to form CMP-Neu5Ac, which is then transported into the Golgi lumen via the CMP–sialic acid transporter (CSAT). In the final step, α2,3- or α2,6-sialyltransferases (ST3GAL4 and ST6GAL1) transfer Neu5Ac from CMP-Neu5Ac to β1,4-galactosylated acceptor substrates in the trans-Golgi, thereby completing the synthesis of complex-type mammalian N-glycans (Zhu et al., 2024).

Since plants naturally lack this pathway and its corresponding enzymes, engineering sialylation requires extensive metabolic and subcellular modifications to enable the formation of human-type sialylated glycans. Fine-tuning of the plant sialylation pathway commonly includes the use of a truncated CMAS variant lacking the 40 N-terminal amino acids, which destabilizes the enzyme, and a feedback-insensitive GNE mutant (GNE^R263L) to circumvent CMP-Neu5Ac–mediated negative feedback regulation (Castilho et al., 2010; Kallolimath et al., 2016; Bohlender et al., 2020). Interestingly, stable or transient co-expression of GNE, NANS, and CMAS in plants results in the accumulation of Neu5Ac rather than Neu5Ac-9-P, indicating that an endogenous plant phosphatase with NANP-like activity catalyzes the dephosphorylation step (Castilho et al., 2008; Castilho et al., 2010). However, co-expression of NANP can further increase Neu5Ac production (Bohlender et al., 2020).

In the past years several approaches have been performed to establish sialylation in plants. In N. benthamiana functional N-glycan sialylation could be achieved by MUTE of the sialylation pathway-genes (Izadi et al., 2023a). This resulted in an efficient sialylation (up to 90%) of several recombinant proteins produced in plants (Castilho et al., 2010; Castilho et al., 2014; Jez et al., 2013; Schneider et al., 2014; Kallolimath et al., 2016; Goritzer et al., 2019; Montero-Morales et al., 2019; Kallolimath et al., 2020; Izadi et al., 2025).

For proteins with exposed glycosylation sites, terminal sialic acid protects against trimming by BGAL1 and HEXO3. Accordingly, full galactosylation on these sites can be achieved by sialidase treatment (Kriechbaum et al., 2020). In contrast, the impact of core α1,3-fucosylation on sialylation of glycosylation sites with difference exposure has been demonstrated with cetuximab, which carries N-glycans in both the Fab and CH2 domains. While glycan processing in the Fab domain is unaffected by fucosylation, the presence of plant-specific α1,3-core fucose markedly increases the levels of bi-antennary sialylated Fc glycans (Castilho et al., 2015; Kallolimath et al., 2016; Kallolimath et al., 2020; Eidenberger et al., 2022), an effect not reproduced by the over-expression of mammalian α1,6-core fucosylation (FUT8) where mainly monosialylated N-glycans are detected (ANaF, Supplementary Table S1) (Castilho et al., 2015).

Among the genes required to establish sialylation in plants, the correct subcellular localization of B4GALT1 and of ST3GAL4 or ST6GAL1 represents a major bottleneck. In moss, co-expression of B4GALT1 and ST6GAL1 targeted to the late Golgi by CTS swapping with FUT13 (FT4) led to the synthesis of mono-antennary sialylated glycans (MNa, Supplementary Table S1) (Bohlender et al., 2020).

Specific glycosylation patterns, including the degree of branching and sialylation, can modulate the biological activity of glycoproteins and enhance their pharmacokinetic properties (Teare et al., 2019). Overexpression of GNTIV and GNTV, increases the glycan antennary structure and potentially the number of terminal GlcNAc residues available for sialylation. For example, the introduction of tri-antennary sialylated glycans on EPO significantly enhanced its hematopoietic activity in vivo (Yin et al., 2015). In plants, the combined expression of GNTIV and GNTV with genes required for protein α2,6-sialylation enabled the production of tri- and tetra-antennary sialylated N-glycans on several recombinant proteins (Figure 2A, Table 3) (Castilho et al., 2012; Castilho et al., 2013; Castilho et al., 2014; Schneider et al., 2014).

Glycans are typically mono-sialylated, but sialic acids can also form homo-polymers of α2,8- or α2,9-linked residues known as polysialic acid (PSA) (Colley et al., 2014). Among the few naturally polysialylated proteins identified, the neural cell adhesion molecule (NCAM) is the best studied, carrying α2,8-linked NeuAc PSA on two of its N-glycans (Close et al., 2003). The addition of PSA to proteins, termed polysialylation (polySia), is a complex post-translational modification that can improve the pharmacokinetic behavior of therapeutic proteins while reducing their immunogenicity. Unlike synthetic polymers such as PEG, PSA is biodegradable and non-immunogenic, making it a safer alternative for therapeutic protein modification (Zhang et al., 2023; Chia et al., 2023).

Recent advances in biosynthetic engineering have enabled the polysialylation of recombinant proteins in both bacteria (Keys et al., 2017) and plants (Kallolimath et al., 2016). In bacterial systems, a biosynthetic pathway for site-specific polysialylation was established in Escherichia coli, in which a cytoplasmic polypeptide-glycosyltransferase installs a defined primer onto the target protein, followed by elongation with bacterial glycosyltransferases to generate long-chain polySia. This strategy has been successfully applied to modify green fluorescent proteins and therapeutic DARPins (designed ankyrin repeat proteins) (Keys et al., 2017).

In N. benthamiana, transient co-expression of two human polysialyltransferases (polySTs: ST8SiaII and ST8SiaIV) together with genes required for α2,6-sialylation demonstrated that the synthesis of biologically active α2,8-linked PSA is feasible (Figure 2A) (Kallolimath et al., 2016). Bacterial polysialyltransferases are generally more promiscuous than their mammalian counterparts (Willis et al., 2008; Lindhout et al., 2011), and engineered bacterial variants capable of generating oligo- and polysialic acids of defined length represent promising candidates to further enhance in planta synthesis of site-specific polySia on diverse recombinant glycoproteins.

Although substantial progress has been made in plants and other heterologous systems, several biochemical and engineering challenges remain. These include achieving precise control over polymer length, ensuring efficient transfer of defined polySia to target proteins, and optimizing downstream purification processes to handle highly hydrophilic polysialylated products. Continued development in biosynthetic pathway engineering, enzyme design, and analytical methods will be critical to fully realize the potential of polysialylation in plant-based expression platforms.

5 Final remarks

Advancements in glycoscience and the development of a comprehensive network in glycosylation pathways are creating new opportunities for designing next-generation therapeutic proteins with tailored glycosylation profiles (Zhong et al., 2022).

Glycans are intrinsically flexible molecules that can adopt many conformations (Yadav et al., 2025). In complex N-glycans, the core Man₃GlcNAc₂ structure has two arms: the α1,3-arm linked to the mannose at the 3-position and the α1,6-arm linked to the mannose at the 6-position. Molecular dynamics simulations have revealed that the α1,3-arm tends to be more extended and accessible to glycosyltransferases in the Golgi apparatus, whereas the α1,6-arm often folds closer to the protein surface or the core, reducing its accessibility (Andre et al., 2007; Andre et al., 2009). Kinetic factors further influence this pattern, as modification of the α1,3-arm can sterically hinder subsequent modification of the α1,6-arm. These structural differences are crucial for the efficiency of glycosylation processes.

The addition of specific sugars to the core glycan structure (e.g., sialic acids, fucose, GlcNAc) can induce conformational shifts that alter the glycan’s shape and exposure. For instance, addition of sialic acid in α2,6-linkage to galactose can cause the glycan arm to bend outward, increasing accessibility to lectins (Stadlmann et al., 2010). Also, core fucosylation and the introduction of bisecting GlcNAc residues have been shown to induce long-range effects on glycan conformation (Nishima et al., 2012; Thaysen-Andersen and Packer, 2012). Specifically, core fucosylation significantly alters the conformational equilibria and strongly influences glycan structure and flexibility. Structural and molecular dynamics studies show that these effects depend more on glycan accessibility than on the peptide backbone (Andre et al., 2009; Nishima et al., 2012) and that glycan modifications occur in a branch-specific manner (Barb et al., 2009). Crystal structures of human IgG1 Fc glycoforms further revealed that core fucosylation pushes glycan arms outward, altering their positioning and accessibility (Ferrara et al., 2011).

More recently, structural insights into complex human and plant N-glycans demonstrated that plant-specific α1,3-fucose induces a rotation of the chitobiose core (β1,4GlcNAc-GlcNAc), thereby promoting an open conformation of the α1,6-arm branch. In contrast, in α1,6-core fucosylated N-glycans, the α1,6-arm is predominantly in a folded conformation (Fogarty et al., 2020). These conformational effects suggest that plant-specific core α1,3-fucosylation can relieve N-glycans from structural constraints (e.g. Fc glycans), increasing the accessibility of terminal sugars to both glycosyltransferases (e.g., GNTs and STs) and glycosidases (e.g., HEXO3, BGALs), an effect not mimicked by human-like core α1,6-fucosylation (Castilho et al., 2015). Additionally, capping exposed N-glycans with α1,2-, α1,3- and α1,4-linked fucose can protect against trimming by glycosidases, which can be exploited to create tailored glycoforms via in planta fucosylation combined with in vitro enzymatic treatment (Leonard et al., 2009) (Figure 5).

Figure 5

Figure 5. Role of fucosylation in modulating accessibility and processing of exposed and buried N-glycosylation sites. (A) Exposed glycosylation sites are susceptible to hydrolytic trimming by endogenous glycosidases such as HEXO and BGAL1. Capping terminal sugars with terminal fucose residues (or with sialic acid in the case of terminal galactose) protects them from enzymatic removal. This strategy, combined with subsequent in vitro removal of fucose (or sialic acid) using specific glycosidases, enables the accumulation of fully processed complex N-glycans (e.g., GnGn or AA structures). (B) In contrast, buried glycosylation sites (such as those found in Fc domains of antibodies) are shielded from hydrolytic enzymes but may also be inaccessible to certain glycosyltransferases. In such cases, core fucosylation, particularly plant-specific α1,3-fucose, appears to induce a rotation of the chitobiose core that renders the α1,6-arm branch accessibility to glyco-processing enzymes (both glycosidases and glycosyltransferases). This approach has proven effective for modulating antibody glycosylation toward bisected, branched, and fully sialylated human-like structures. Combining this strategy with subsequent in vitro removal of core fucose can yield fully humanized glycans devoid of plant-specific epitopes.

Extending this strategy to remove core α1,3-fucose residues to achieve complete humanization of plant-derived N-glycans remains challenging, as core-specific α1,3-fucosidases have not yet been identified. Although a GH29 α-fucosidase from Omnitrophica bacterium, which shows a strong preference for hydrolyzing α1,6-linked core fucose, can also remove the core α1,3-fucose residue from plant complex-type N-glycans (Vainauskas et al., 2018), its biotechnological application for selective core fucose removal warrants further investigation.

Decorating plant-produced glycoproteins with non-human or pathogen-mimicking glycans provides a strategic means to enhance vaccine immunogenicity. For instance, incorporation of plant-specific β1,2-xylose and α1,3-fucose, as well as Lewis-A or helminth-like motifs such as LacdiNAc and fucosylated LDN, can act as self-adjuvants, boosting both humoral and cellular immune responses. This strategy allows the simultaneous optimization of antigenicity and immunostimulatory potential, making tailored glycosylation a powerful tool in recombinant vaccine design (Rosales-Mendoza et al., 2016; Zwanenburg et al., 2023; Vo and Trinh, 2025). Therefore, while the removal of plant-specific glyco-epitopes is essential for producing human-compatible biopharmaceuticals, it is important to note that such glycans (including core α1,3-fucose) are not inherently undesirable and can be leveraged as a glycoengineering tool.

The success of plant molecular farming, particularly using N. benthamiana as a production platform, greatly relies on its high tolerance for genetic manipulation and glycoengineering. However, major challenges remain, including the need to prevent unintended developmental phenotypes arising from the stable “humanization” of plant glycosylation pathways. Consequently, most major advances in producing helminth- and human-like glycans in plants have been achieved via transient expression of glycan-modifying genes, enabling controlled glycoengineering without long-term effects on plant development.

While plant glycoengineering still faces several limitations, ongoing research and innovative strategies that combine in planta and in vitro approaches are steadily overcoming these challenges, including leveraging plant-specific α1,3-fucose to design glycoproteins with optimized structure and immune-modulating properties.

6 Outstanding challenges and future directions in plant glycoengineering

Despite major advances, several challenges still limit the precision, predictability, and scalability of plant N-glycoengineering. One persistent issue is the batch-to-batch variability observed in transient expression systems, which raises concerns for regulatory compliance and good manufacturing practices. Variability can arise from fluctuating plant growth conditions, differences in agroinfiltration efficiency, and the performance of expression constructs (Akher et al., 2025). A design-of-experiments (DoE) approach has been proposed to systematically identify and mitigate these sources of variability by optimizing environmental parameters, infiltration conditions, and expression timing (Buyel and Fischer, 2014; Buyel, 2023). Establishing more standardized and tightly controlled transient expression workflows will be essential to reduce glycan heterogeneity and improve reproducibility (Akher et al., 2025; Annese et al., 2025).

Another major challenge lies in accurately predicting glycosite accessibility and the resulting glycan maturation. Glycosylation outcomes are strongly influenced by protein three-dimensional structure, domain flexibility, and steric constraints around individual glycosylation sites. Although computational tools, including molecular dynamics simulations, provide valuable insights, current models often lack the accuracy needed to reliably forecast in vivo glycan processing due to high parametric complexity and the intrinsic flexibility of glycans. Improving these predictive tools will require better integration of experimental structural data, refined algorithms that capture glycan dynamics, and the incorporation of machine learning approaches.

At the cellular level, mislocalization or suboptimal localization of native and heterologous glycosyltransferases in the Golgi apparatus remain a significant bottleneck. Even with engineered targeting motifs, differences in trafficking behavior and competitive interactions with endogenous enzymes can lead to incomplete or unintended glycan processing. Developing improved strategies for controlling enzyme residency times and Golgi compartment specificity will be critical for achieving more precise pathway engineering.

Looking ahead, progress will depend on the convergence of several technological advances. Synthetic biology tools and precision genome editing can enable more robust plant expression systems, while high-throughput analytical platforms will allow rapid screening and selection of optimal glycosylation conditions. Advances in computational modeling and artificial intelligence hold promise for enabling predictive control of glycosylation pathways, bridging the gap between theoretical design and experimental outcomes. By addressing these interconnected challenges, the field of plant glycoengineering is well positioned to deliver increasingly reliable and scalable production platforms for next-generation biopharmaceuticals.

Author contributions

KG: Writing – original draft, Writing – review & editing. VK: Data curation, Writing – review & editing, Writing – original draft. AC: Writing – original draft, Data curation, Funding acquisition, Conceptualization, Validation, Supervision, Writing – review & editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This research was funded by the Austrian Science Fund (FWF) 10.55776/PAT1041324.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2025.1734060/full#supplementary-material

Abbreviations

ABO-A, α1,3-N-acetylgalactosaminyltransferase; B4GALT1, β1,4-galactosyltransferase; CMAS, CMP-Neu5Ac synthetase; CSAT, CMP-Neu5Ac transporter; FUT1/2, α1,2-fucosyltransferases; FUT11/12, core α1,3-fucosyltransferases; FUT13, α1,4-fucosyltransferase; FUT8, α1,6 fucosyltransferase; FUT9a, α1,3-fucosyltransferase IXa; GalNAcT, α1,4-N-acetylgalactosaminyltransferase; GALT1, β1,3-galactosyltransferase 1; GMII, Golgi α-mannosidase II; GNE, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase; GnTI, α1,3-mannosyl-β1,2-N-acetylglucosaminyltransferase I; GnTII, α1,6-mannosyl-β1,2-N-acetylglucosaminyltransferase II; GNTIII, β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase III; GNTIV, α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase IVa; GNTV, α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase V; HEXO3, β-N-acetylhexosaminidase 3. MNS1/2, Golgi α-mannosidase I; MNS3, ER α-mannosidase I; NANS, N-acetylneuraminic acid phosphate-synthase; SmFucT D/E/F, Schistosoma mansoni α1,3-fucosyltransferases D, E, and F. ST3GAL4, α2,3-sialyltransferase; ST6GAL1, α2,6-sialyltransferase; ST8Sia II/IV, α2,8-sialyltransferases II and IV; XYLT, β1,2-xylosyltransferase.

References

Ahn, Y. O., Zheng, M., Bevan, D. R., Esen, A., Shiu, S. H., Benson, J., et al. (2007). Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 35. Phytochemistry. 68, 1510–1520. doi: 10.1016/j.phytochem.2007.03.021

PubMed Abstract | Crossref Full Text | Google Scholar

Akher, S. A., Wang, K. Y., Hall, K., Hunpatin, O. S., Shan, M., Zhang, Z., et al. (2025). Harnessing transient expression systems with plant viral vectors for the production of biopharmaceuticals in nicotiana benthamiana. Int. J. Mol. Sci. 9, 5510. doi: 10.3390/ijms26125510

PubMed Abstract | Crossref Full Text | Google Scholar

Alvisi, N., van Noort, K., Dwiani, S., Geschiere, N., Sukarta, O., Varossieau, K., et al. (2021). β-hexosaminidases along the secretory pathway of have distinct specificities toward engineered helminth N-glycans on recombinant glycoproteins. Front. Plant Sci. 12. doi: 10.3389/fpls.2021.638454

PubMed Abstract | Crossref Full Text | Google Scholar

Andre, S., Kozár, T., Kojima, S., Unverzagt, C., and Gabius, H. J. (2009). From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans. Biol. Chem. 390, 557–565. doi: 10.1515/bc.2009.072

PubMed Abstract | Crossref Full Text | Google Scholar

Andre, S., Kozár, T., Schuberth, R., Unverzagt, C., Kojima, S., and Gabius, H. J. (2007). Substitutions in the N-glycan core as regulators of biorecognition: The case of core-fucose and bisecting GlcNAc moieties. Biochemistry-Us. 46, 6984–6995. doi: 10.1021/bi7000467

PubMed Abstract | Crossref Full Text | Google Scholar

Annese, D., Romani, F., Grandellis, C., Ives, L., Frangedakis, E., Buson, F. X., et al. (2025). Semi-automated workflow for high-throughput Agrobacterium-mediated plant transformation. Plant J. 122, e70118. doi: 10.1111/tpj.70118

PubMed Abstract | Crossref Full Text | Google Scholar

Anthony, R. M., Nimmerjahn, F., Ashline, D. J., Reinhold, V. N., Paulson, J. C., and Ravetch, J. V. (2008). Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science. 320, 373–376. doi: 10.1126/science.1154315

PubMed Abstract | Crossref Full Text | Google Scholar

Bakker, H., Bardor, M., Molthoff, J. W., Gomord, V., Elbers, I., Stevens, L. H., et al. (2001). Galactose-extended glycans of antibodies produced by transgenic plants. Proc. Natl. Acad. Sci. U S A. 98, 2899–2904. doi: 10.1073/pnas.031419998

PubMed Abstract | Crossref Full Text | Google Scholar

Bakker, H., Rouwendal, G. J. A., Karnoup, A. S., Florack, D. E. A., Stoopen, G. M., Helsper, J. P. F. G., et al. (2006). An antibody produced in tobacco expressing a hybrid β-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes. P Natl. Acad. Sci. U.S.A. 103, 7577–7582. doi: 10.1073/pnas.0600879103

PubMed Abstract | Crossref Full Text | Google Scholar

Balieu, J., Jung, J. W., Chan, P., Lomonossoff, G. P., Lerouge, P., and Bardor, M. (2022). Investigation of the N-glycosylation of the SARS-coV-2 S protein contained in VLPs produced in nicotiana benthamiana. Molecules 27, 5119. doi: 10.3390/molecules27165119

PubMed Abstract | Crossref Full Text | Google Scholar

Barb, A. W., Brady, E. K., and Prestegard, J. H. (2009). Branch-specific sialylation of igG-fc glycans by ST6Gal-I. Biochemistry-Us. 48, 9705–9707. doi: 10.1021/bi901430h

PubMed Abstract | Crossref Full Text | Google Scholar

Becerra-Arteaga, A. and Shuler, M. L. (2007). Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase. Biotechnol. Bioeng 97, 1585–1593. doi: 10.1002/bit.21344

PubMed Abstract | Crossref Full Text | Google Scholar

Beihammer, G., Maresch, D., Altmann, F., Van Damme, E. J. M., and Strasser, R. (2021). Lewis A glycans are present on proteins involved in cell wall biosynthesis and appear evolutionarily conserved among natural arabidopsis thaliana accessions. Front. Plant Sci. 12. doi: 10.3389/fpls.2021.630891

PubMed Abstract | Crossref Full Text | Google Scholar

Blumberg, L. C., Bakker, G. M., van der Kaaij, A., Gariepy, E., van de Geest, H., Slootweg, E. J., et al. (2025). Highly efficient transgene-free ErCas12a RNP-protoplast genome editing and single-cell regeneration in Nicotiana benthamiana for glyco-engineering. Plant Biotechnol. J. 16. doi: 10.1111/pbi.70141

PubMed Abstract | Crossref Full Text | Google Scholar

Bohlender, L. L., Parsons, J., Hoernstein, S. N. W., Bangert, N., Rodriguez-Jahnke, F., Reski, R., et al. (2022). Unexpected arabinosylation after humanization of plant protein N-glycosylation. Front. Bioeng Biotech. 10. doi: 10.3389/fbioe.2022.838365

PubMed Abstract | Crossref Full Text | Google Scholar

Bohlender, L. L., Parsons, J., Hoernstein, S. N. W., Rempfer, C., Ruiz-Molina, N., Lorenz, T., et al. (2020). Stable protein sialylation in physcomitrella. Front. Plant Sci. 11. doi: 10.3389/fpls.2020.610032

PubMed Abstract | Crossref Full Text | Google Scholar

Bork, K., Horstkorte, R., and Weidemann, W. (2009). Increasing the sialylation of therapeutic glycoproteins: the potential of the sialic acid biosynthetic pathway. J. Pharm. Sci-Us. 98, 3499–3508. doi: 10.1002/jps.21684

PubMed Abstract | Crossref Full Text | Google Scholar

Bunte, M. J. M., Schots, A., Kammenga, J. E., and Wilbers, R. H. P. (2022). Helminth glycans at the host-parasite interface and their potential for developing novel therapeutics. Front. Mol. Biosci. 8. doi: 10.3389/fmolb.2021.807821

PubMed Abstract | Crossref Full Text | Google Scholar

Buyel, J. F. (2023). Product safety aspects of plant molecular farming. Front. Bioeng Biotechnol. 11. doi: 10.3389/fbioe.2023.1238917

PubMed Abstract | Crossref Full Text | Google Scholar

Buyel, J. F. and Fischer, R. (2014). Characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study. Jove-J Vis. Exp. 31, e51216. doi: 10.3791/51216

PubMed Abstract | Crossref Full Text | Google Scholar

Castilho, A., Bohorova, N., Grass, J., Bohorov, O., Zeitlin, L., Whaley, K., et al. (2011a). Rapid high yield production of different glycoforms of Ebola virus monoclonal antibody. PloS One 6, e26040. doi: 10.1371/journal.pone.0026040

PubMed Abstract | Crossref Full Text | Google Scholar

Castilho, A., Gattinger, P., Grass, J., Jez, J., Pabst, M., Altmann, F., et al. (2011b). N-Glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans. Glycobiology. 21, 813–823. doi: 10.1093/glycob/cwr009

PubMed Abstract | Crossref Full Text | Google Scholar

Castilho, A., Gruber, C., Thader, A., Oostenbrink, C., Pechlaner, M., Steinkellner, H., et al. (2015). Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation. Mabs. 7, 863–870. doi: 10.1080/19420862.2015.1053683

PubMed Abstract | Crossref Full Text | Google Scholar

Castilho, A., Neumann, L., Daskalova, S., Mason, H. S., Steinkellner, H., Altmann, F., et al. (2012). Engineering of sialylated mucin-type O-glycosylation in plants. J. Biol. Chem. 287, 36518–36526. doi: 10.1074/jbc.m112.402685

PubMed Abstract | Crossref Full Text | Google Scholar

Castilho, A., Neumann, L., Gattinger, P., Strasser, R., Vorauer-Uhl, K., Sterovsky, T., et al. (2013). Generation of biologically active multi-sialylated recombinant human EPOFc in plants. PloS One 8, e54836. doi: 10.1371/journal.pone.0054836

PubMed Abstract | Crossref Full Text | Google Scholar

Castilho, A., Pabst, M., Leonard, R., Veit, C., Altmann, F., Mach, L., et al. (2008). Construction of a functional CMP-sialic acid biosynthesis pathway in Arabidopsis. Plant Physiol. 147, 331–339. doi: 10.1104/pp.108.117572

PubMed Abstract | Crossref Full Text | Google Scholar

Castilho, A. and Strasser, R. (2018). “Production of functionally active recombinant proteins in plants: manipulating N- and O-glycosylation,” in Molecular pharming: applications, challenges and emerging areas cp4 (John Wiley & Sons, Inc). doi: 10.1002/9781118801512.ch4

Crossref Full Text | Google Scholar

Castilho, A., Strasser, R., Stadlmann, J., Grass, J., Jez, J., Gattinger, P., et al. (2010). In planta Protein Sialylation through Overexpression of the Respective Mammalian Pathway. J. Biol. Chem. 285, 15923–15930. doi: 10.1074/jbc.m109.088401

PubMed Abstract | Crossref Full Text | Google Scholar

Castilho, A., Windwarder, M., Gattinger, P., Mach, L., Strasser, R., Altmann, F., et al. (2014). Proteolytic and N-glycan processing of human alpha1-antitrypsin expressed in Nicotiana benthamiana. Plant Physiol. 166, 1839–1851. doi: 10.1104/pp.114.250720

PubMed Abstract | Crossref Full Text | Google Scholar

Chia, S., Tay, S. J., Song, Z. W., Yang, Y. S., Walsh, I., and Pang, K. T. (2023). Enhancing pharmacokinetic and pharmacodynamic properties of recombinant therapeutic proteins by manipulation of sialic acid content. BioMed. Pharmacother. 163. doi: 10.1016/j.biopha.2023.114757

PubMed Abstract | Crossref Full Text | Google Scholar

Chincinska, I. A. (2021). Leaf infiltration in plant science: old method, new possibilities. Plant Methods 17, 83. doi: 10.1186/s13007-021-00782-x

PubMed Abstract | Crossref Full Text | Google Scholar

Choi, H. Y., Park, H., Hong, J. K., Kim, S. D., Kwon, J. Y., You, S., et al. (2018). N-glycan remodeling using mannosidase inhibitors to increase high-mannose glycans on acid alpha-glucosidase in transgenic rice cell cultures. Sci. Rep. 8, 16130. doi: 10.1038/s41598-018-34438-z

PubMed Abstract | Crossref Full Text | Google Scholar

Close, B. E., Mendiratta, S. S., Geiger, K. M., Broom, L. J., Ho, L. L., and Colley, K. J. (2003). The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II. J. Biol. Chem. 278, 30796–30805. doi: 10.1074/jbc.m305390200

PubMed Abstract | Crossref Full Text | Google Scholar

Colley, K. J., Kitajima, K., and Sato, C. (2014). Polysialic acid: Biosynthesis, novel functions and applications. Crit. Rev. Biochem. Mol. Biol. 49, 498–532. doi: 10.3109/10409238.2014.976606

PubMed Abstract | Crossref Full Text | Google Scholar

Cox, K. M., Sterling, J. D., Regan, J. T., Gasdaska, J. R., Frantz, K. K., Peele, C. G., et al. (2006). Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat. Biotechnol. 24, 1591–1597. doi: 10.1038/nbt1260

PubMed Abstract | Crossref Full Text | Google Scholar

Daskalova, S. M., Radder, J. E., Cichacz, Z. A., Olsen, S. H., Tsaprailis, G., Mason, H., et al. (2010). Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins–towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation. BMC Biotechnol. 10, 62. doi: 10.1186/1472-6750-10-62

PubMed Abstract | Crossref Full Text | Google Scholar

Dianatkhah, S., Kogelmann, B., Melnik, S., Eminger, F., Kallolimath, S., Sun, L., et al. (2025). A plant cell-based platform for the expression of complex proteins with fucose-reduced sialylated N-glycans. Plant Biotechnol. J. 10. doi: 10.1111/pbi.70044

PubMed Abstract | Crossref Full Text | Google Scholar

Dicker, M., Schoberer, J., Vavra, U., and Strasser, R. (2015). Subcellular targeting of proteins involved in modification of plant N- and O-glycosylation. Methods Mol. Biol. 1321, 249–267. doi: 10.1007/978-1-4939-2760-9_18

PubMed Abstract | Crossref Full Text | Google Scholar

Dicker, M., Tschofen, M., Maresch, D., König, J., Juarez, P., Orzaez, D., et al. (2016). Transient glyco-engineering to produce recombinant igA1 with defined O- and N-glycans in plants. Front. Plant Sci. 7. doi: 10.3389/fpls.2016.00018

PubMed Abstract | Crossref Full Text | Google Scholar

Dirnberger, D., Steinkellner, H., Abdennebi, L., Remy, J. J., and van de Wiel, D. (2001). Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants. Eur. J. Biochem. 268, 4570–4579. doi: 10.1046/j.1432-1327.2001.02384.x

PubMed Abstract | Crossref Full Text | Google Scholar

Dünser, K. and Schoberer, J. (2025). Trafficking and localization of Golgi-resident N-glycan processing enzymes in plants. Front. Plant Sci. 16, e1624949. doi: 10.3389/fpls.2025.1624949

PubMed Abstract | Crossref Full Text | Google Scholar

Eidenberger, L., Eminger, F., Castilho, A., and Steinkellner, H. (2022). Comparative analysis of plant transient expression vectors for targeted N-glycosylation. Front. Bioeng Biotech. 10. doi: 10.3389/fbioe.2022.1073455

PubMed Abstract | Crossref Full Text | Google Scholar

Eidenberger, L., Kogelmann, B., and Steinkellner, H. (2023). Plant-based biopharmaceutical engineering. Nat. Rev. Bioeng. 1, 426–439. doi: 10.1038/s44222-023-00044-6

PubMed Abstract | Crossref Full Text | Google Scholar

Fanata, W. I. D., Lee, K. H., Son, B. H., Yoo, J. Y., Harmoko, R., Ko, K. S., et al. (2013). N-glycan maturation is crucial for cytokinin-mediated development and cellulose synthesis in Oryza sativa. Plant J. 73, 966–979. doi: 10.1111/tpj.12087

PubMed Abstract | Crossref Full Text | Google Scholar

Ferrara, C., Grau, S., Jäger, C., Sondermann, P., Brünker, P., Waldhauer, I., et al. (2011). Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose. P Natl. Acad. Sci. U.S.A. 108, 12669–12674. doi: 10.1073/pnas.1108455108

PubMed Abstract | Crossref Full Text | Google Scholar

Fischer, R. and Buyel, J. F. (2020). Molecular farming - The slope of enlightenment. Biotechnol. Adv. 40, 107519. doi: 10.1016/j.bioteChadv.2020.107519

PubMed Abstract | Crossref Full Text | Google Scholar

Fogarty, C. A., Harbison, A. M., Dugdale, A. R., and Fadda, E. (2020). How and why plants and human N-glycans are different: Insight from molecular dynamics into the "glycoblocks" architecture of complex carbohydrates. Beilstein J. Org Chem. 16, 2046–2056. doi: 10.3762/bjoc.16.171

PubMed Abstract | Crossref Full Text | Google Scholar

Forthal, D. N., Gach, J. S., Landucci, G., Jez, J., Strasser, R., Kunert, R., et al. (2010). Fc-glycosylation influences fcγ Receptor binding and cell-mediated anti-HIV activity of monoclonal antibody 2G12. J. Immunol. 185, 6876–6882. doi: 10.4049/jimmunol.1002600

PubMed Abstract | Crossref Full Text | Google Scholar

Fukuzawa, N., Matsuo, K., Atsumi, G., Tasaka, Y., and Mitsuda, N. (2024). Plant-made pharmaceuticals. Plant Biotechnol Tokyo. 41, 243–260. doi: 10.5511/plantbiotechnology.24.0716a

PubMed Abstract | Crossref Full Text | Google Scholar

Goritzer, K., Grandits, M., Grunwald-Gruber, C., Figl, R., Mercx, S., Navarre, C., et al. (2022). Engineering the N-glycosylation pathway of Nicotiana tabacum for molecular pharming using CRISPR/Cas9. Front. Plant Sci. 13. doi: 10.3389/fpls.2022.1003065

PubMed Abstract | Crossref Full Text | Google Scholar

Goritzer, K., Kallolimath, S., and Strasser, R. (2025). Posttranslational modifications of heterologous proteins expressed in Nicotiana benthamiana. Plant Biotechnol. J. 8. doi: 10.1111/pbi.70176

PubMed Abstract | Crossref Full Text | Google Scholar

Goritzer, K., Maresch, D., Altmann, F., Obinger, C., and Strasser, R. (2017). Exploring site-specific N-glycosylation of HEK293 and plant-produced human igA isotypes. J. Proteome Res. 16, 2560–2570. doi: 10.1021/acs.jproteome.7b00121

PubMed Abstract | Crossref Full Text | Google Scholar

Goritzer, K. and Strasser, R. (2021). Glycosylation of plant-produced immunoglobulins. Exp Suppl. 112, 519–543. doi: 10.1007/978-3-030-76912-3_16

PubMed Abstract | Crossref Full Text | Google Scholar

Goritzer, K., Turupcu, A., Maresch, D., Novak, J., Altmann, F., Oostenbrink, C., et al. (2019). Distinct Fcα receptor N-glycans modulate the binding affinity to immunoglobulin A (IgA) antibodies. J. Biol. Chem. 294, 13995–14008. doi: 10.1074/jbc.ra119.009954

PubMed Abstract | Crossref Full Text | Google Scholar

Hall, M. K., Weidner, D. A., Zhu, Y., Dayal, S., Whitman, A. A., and Schwalbe, R. A. (2016). Predominant expression of hybrid N-glycans has distinct cellular roles relative to complex and oligomannose N-glycans. Int. J. Mol. Sci. 17. doi: 10.3390/ijms17060925

PubMed Abstract | Crossref Full Text | Google Scholar

Hanania, U., Ariel, T., Tekoah, Y., Fux, L., Sheva, M., Gubbay, Y., et al. (2017). Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins. Plant Biotechnol. J. 15, 1120–1129. doi: 10.1111/pbi.12702

PubMed Abstract | Crossref Full Text | Google Scholar

Harmoko, R., Yoo, J. Y., Ko, K. S., Ramasamy, N. K., Hwang, B. Y., Lee, E. J., et al. (2016). N-glycan containing a core alpha1,3-fucose residue is required for basipetal auxin transport and gravitropic response in rice (Oryza sativa). New Phytol. 212, 108–122. doi: 10.1111/nph.14031

PubMed Abstract | Crossref Full Text | Google Scholar

He, M., Zhou, X., and Wang, X. (2024). Glycosylation: mechanisms, biological functions and clinical implications. Signal Transduct Target Ther. 9, 194. doi: 10.1038/s41392-024-01886-1

PubMed Abstract | Crossref Full Text | Google Scholar

Herman, X., Far, J., Courtoy, A., Bouhon, L., Quinton, L., De Pauw, E., et al. (2021). Inactivation of N-Acetylglucosaminyltransferase I and alpha1,3-Fucosyltransferase Genes in Nicotiana tabacum BY-2 Cells Results in Glycoproteins With Highly Homogeneous, High-Mannose N-Glycans. Front. Plant Sci. 12, e634023. doi: 10.3389/fpls.2021.634023

PubMed Abstract | Crossref Full Text | Google Scholar

Hesselink, T., Rouwendal, G. J., Henquet, M. G., Florack, D. E., Helsper, J. P., and Bosch, D. (2014). Expression of natural human beta1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans. Transgenic Res. 23, 717–728. doi: 10.1007/s11248-014-9806-z

PubMed Abstract | Crossref Full Text | Google Scholar

Hoang, T. V., Vo, K. T. X., Rahman, M. M., Zhong, R., Lee, C., Ketudat Cairns, J. R., et al. (2023). SPOTTED-LEAF7 targets the gene encoding beta-galactosidase9, which functions in rice growth and stress responses. Plant Physiol. 193, 1109–1125. doi: 10.1093/plphys/kiad359

PubMed Abstract | Crossref Full Text | Google Scholar

Houde, D., Peng, Y., Berkowitz, S. A., and Engen, J. R. (2010). Post-translational modifications differentially affect IgG1 conformation and receptor binding. Mol. Cell Proteomics. 9, 1716–1728. doi: 10.1074/mcp.m900540-mcp200

PubMed Abstract | Crossref Full Text | Google Scholar

Huebbers, J. W. and Buyel, J. F. (2021). On the verge of the market - Plant factories for the automated and standardized production of biopharmaceuticals. Biotechnol. Adv. 46. doi: 10.1016/j.bioteChadv.2020.107681

PubMed Abstract | Crossref Full Text | Google Scholar

Izadi, S., Abrantes, R., Gumpelmair, S., Kunnummel, V., Duarte, H. O., Steinberger, P., et al. (2025). An engineered PD1-Fc fusion produced in N. benthamiana plants efficiently blocks PD1/PDL1 interaction. Plant Cell Rep. 44, 80. doi: 10.1007/s00299-025-03475-0

PubMed Abstract | Crossref Full Text | Google Scholar

Izadi, S., Kunnummel, V., Steinkellner, H., Werner, S., and Castilho, A. (2023a). Assessment of transient expression strategies to sialylate recombinant proteins in N. benthamiana. J. Biotechnol. 365, 48–53. doi: 10.1016/j.jbiotec.2023.02.004

PubMed Abstract | Crossref Full Text | Google Scholar

Izadi, S., Vavra, U., Melnik, S., Grunwald-Gruber, C., Foderl-Hobenreich, E., Sack, M., et al. (2023b). In planta deglycosylation improves the SARS-CoV-2 neutralization activity of recombinant ACE2-Fc. Front. Bioeng Biotechnol. 11. doi: 10.3389/fbioe.2023.1180044

PubMed Abstract | Crossref Full Text | Google Scholar

Jansing, J., Sack, M., Augustine, S. M., Fischer, R., and Bortesi, L. (2019). CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking beta-1,2-xylose and core alpha-1,3-fucose. Plant Biotechnol. J. 17, 350–361. doi: 10.1111/pbi.12981

PubMed Abstract | Crossref Full Text | Google Scholar

Jeong, Y. T., Choi, O., Lim, H. R., Son, Y. D., Kim, H. J., and Kim, J. H. (2008). Enhanced sialylation of recombinant erythropoietin in CHO cells by human glycosyltransferase expression. J. Microbiol. Biotechnol. 18, 1945–1952. doi: 10.4014/jmb.0800.546

PubMed Abstract | Crossref Full Text | Google Scholar

Jez, J., Castilho, A., Grass, J., Vorauer-Uhl, K., Sterovsky, T., Altmann, F., et al. (2013). Expression of functionally active sialylated human erythropoietin in plants. Biotechnol. J. 8, 371–382. doi: 10.1002/biot.201200363

PubMed Abstract | Crossref Full Text | Google Scholar

Joly, C., Maftah, A., and Riou-Khamlichi, C. (2004). Alteration of gibberellin response in transgenic tobacco plants which express a human Lewis fucosyltransferase. Plant Physiol. Biochem. 42, 629–637. doi: 10.1016/j.plaphy.2004.03.011

PubMed Abstract | Crossref Full Text | Google Scholar

Jugler, C., Grill, F. J., Eidenberger, L., Karr, T. L., Grys, T. E., Steinkellner, H., et al. (2022). Humanization and expression of IgG and IgM antibodies in plants as potential diagnostic reagents for Valley Fever. Front. Plant Sci. 13, e925008. doi: 10.3389/fpls.2022.925008

PubMed Abstract | Crossref Full Text | Google Scholar

Jung, J. W., Shin, J. H., Lee, W. K., Begum, H., Min, C. H., Jang, M. H., et al. (2021). Inactivation of the β (1, 2)-xylosyltransferase and the α (1, 3)-fucosyltransferase gene in rice (Oryza sativa) by multiplex CRISPR/Cas9 strategy. Plant Cell Rep. 40, 1025–1035. doi: 10.1007/s00299-021-02667-8

PubMed Abstract | Crossref Full Text | Google Scholar

Kallolimath, S., Castilho, A., Strasser, R., Grunwald-Gruber, C., Altmann, F., Strubl, S., et al. (2016). Engineering of complex protein sialylation in plants. Proc. Natl. Acad. Sci. U S A. 113, 9498–9503. doi: 10.1073/pnas.1604371113

PubMed Abstract | Crossref Full Text | Google Scholar

Kallolimath, S., Gruber, C., Steinkellner, H., and Castilho, A. (2018). Promoter choice impacts the efficiency of plant glyco-engineering. Biotechnol. J. 13. doi: 10.1002/biot.201700380

PubMed Abstract | Crossref Full Text | Google Scholar

Kallolimath, S., Hackl, T., Gahn, R., Grunwald-Gruber, C., Zich, W., Kogelmann, B., et al. (2020). Expression profiling and glycan engineering of igG subclass 1–4 in nicotiana benthamiana. Front. Bioeng Biotechnol. 8. doi: 10.3389/fbioe.2020.00825

PubMed Abstract | Crossref Full Text | Google Scholar

Kang, J. S., Frank, J., Kang, C. H., Kajiura, H., Vikram, M., Ueda, A., et al. (2008). Salt tolerance of Arabidopsis thaliana requires maturation of N-glycosylated proteins in the Golgi apparatus. Proc. Natl. Acad. Sci. U S A. 105, 5933–5938. doi: 10.1073/pnas.0800237105

PubMed Abstract | Crossref Full Text | Google Scholar

Kao, M. R., Karmarkar Saldivar, R., and Hsieh, Y. S. Y. (2024). Production of therapeutic glycoproteins in glycoengineered plant: old farm for new crops. Curr. Opin. Biotechnol. 87, 103145. doi: 10.1016/j.copbio.2024.103145

PubMed Abstract | Crossref Full Text | Google Scholar

Karg, S. R., Frey, A. D., and Kallio, P. T. (2010). Reduction of N-linked xylose and fucose by expression of rat β1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression. J. Biotechnol. 146, 54–65. doi: 10.1016/j.jbiotec.2010.01.005

PubMed Abstract | Crossref Full Text | Google Scholar

Katoh, T. and Yamamoto, K. (2021). Innovative preparation of biopharmaceuticals using transglycosylation activity of microbial endoglycosidases. J. Appl. Glycosci 68, 1–9. doi: 10.5458/jag.jag.jag-2020_0013

PubMed Abstract | Crossref Full Text | Google Scholar

Keys, T. G., Wetter, M., Hang, I., Rutschmann, C., Russo, S., Mally, M., et al. (2017). A biosynthetic route for polysialylating proteins in Escherichia coli. Metab. Eng. 44, 293–301. doi: 10.1016/j.ymben.2017.10.012

PubMed Abstract | Crossref Full Text | Google Scholar

Kittur, F. S., Hung, C. Y., Zhu, C., Shajahan, A., Azadi, P., Thomas, M. D., et al. (2020). Glycoengineering tobacco plants to stably express recombinant human erythropoietin with different N-glycan profiles. Int. J. Biol. Macromol. 157, 158–169. doi: 10.1016/j.ijbiomac.2020.04.199

PubMed Abstract | Crossref Full Text | Google Scholar

Kogelmann, B., Melnik, S., Bogner, M., Kallolimath, S., Stoger, E., Sun, L., et al. (2024). A genome-edited N. benthamiana line for industrial-scale production of recombinant glycoproteins with targeted N-glycosylation. Biotechnol. J. 19, e2300323. doi: 10.1002/biot.202300323

PubMed Abstract | Crossref Full Text | Google Scholar

Komarova, T. V., Sheshukova, E. V., and Dorokhov, Y. L. (2019). Plant-made antibodies: properties and therapeutic applications. Curr. Med. Chem. 26, 381–395. doi: 10.2174/0929867325666171212093257

PubMed Abstract | Crossref Full Text | Google Scholar

Beihammer, J., Vavra, U., Shin, Y. J., Veit, C., Grunwald-Gruber, C., Gillitschka, Y., et al. (2021). In planta production of the receptor-binding domain from SARS-coV-2 with human blood group A glycan structures. Front. Chem. 9. doi: 10.3389/fchem.2021.816544

PubMed Abstract | Crossref Full Text | Google Scholar

Kriechbaum, R., Ziaee, E., Grunwald-Gruber, C., Buscaill, P., van der Hoorn, R. A. L., and Castilho, A. (2020). BGAL1 depletion boosts the level of beta-galactosylation of N- and O-glycans in N. benthamiana. Plant Biotechnol. J. 18, 1537–1549. doi: 10.1111/pbi.13316

PubMed Abstract | Crossref Full Text | Google Scholar

Leonard, R., Petersen, B. O., Himly, M., Kaar, W., Wopfner, N., Kolarich, D., et al. (2005). Two novel types of O-glycans on the mugwort pollen allergen Art v 1 and their role in antibody binding. J. Biol. Chem. 280, 7932–7940. doi: 10.1074/jbc.m410407200

PubMed Abstract | Crossref Full Text | Google Scholar

Leonard, R., Strasser, R., and Altmann, F. (2009). Plant glycosidases acting on protein-linked oligosaccharides. Phytochemistry. 70, 318–324. doi: 10.1016/j.phytochem.2009.01.006

PubMed Abstract | Crossref Full Text | Google Scholar

Li, J., Stoddard, T. J., Demorest, Z. L., Lavoie, P. O., Luo, S., Clasen, B. M., et al. (2016). Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production. Plant Biotechnol. J. 14, 533–542. doi: 10.1111/pbi.12403

PubMed Abstract | Crossref Full Text | Google Scholar

Liebminger, E., Veit, C., Pabst, M., Batoux, M., Zipfel, C., Altmann, F., et al. (2011). Beta-N-acetylhexosaminidases HEXO1 and HEXO3 are responsible for the formation of paucimannosidic N-glycans in Arabidopsis thaliana. J. Biol. Chem. 286, 10793–10802. doi: 10.1074/jbc.m110.178020

PubMed Abstract | Crossref Full Text | Google Scholar

Limkul, J., Iizuka, S., Sato, Y., Misaki, R., Ohashi, T., Ohashi, T., et al. (2016). The production of human glucocerebrosidase in glyco-engineered Nicotiana benthamiana plants. Plant Biotechnol. J. 14, 1682–1694. doi: 10.1111/pbi.12529

PubMed Abstract | Crossref Full Text | Google Scholar

Lindhout, T., Iqbal, U., Willis, L. M., Reid, A. N., Li, J., Liu, X., et al. (2011). Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes. Proc Natl Acad Sci. U.S.A. 108, 7397–7402. doi: 10.1073/pnas.1019266108

PubMed Abstract | Crossref Full Text | Google Scholar

Liu, C. F., Niu, G. T., Zhang, H. C., Sun, Y. F., Sun, S. B., Yu, F. G., et al. (2018). Trimming of N-glycans by the golgi-localized α-1,2-mannosidases, MNS1 and MNS2, is crucial for maintaining RSW2 protein abundance during salt stress in arabidopsis. Mol. Plant 11, 678–690. doi: 10.1016/j.molp.2018.01.006

PubMed Abstract | Crossref Full Text | Google Scholar

Loos, A. and Castilho, A. (2015). Transient glyco-engineering of N. benthamiana aiming at the synthesis of multi-antennary sialylated proteins. Methods Mol. Biol. 1321, 233–248. doi: 10.1007/978-1-4939-2760-9_17

PubMed Abstract | Crossref Full Text | Google Scholar

Loos, A., Gruber, C., Altmann, F., Mehofer, U., Hensel, F., Grandits, M., et al. (2014). Expression and glycoengineering of functionally active heteromultimeric IgM in plants. Proc. Natl. Acad. Sci. U S A. 111, 6263–6268. doi: 10.1073/pnas.1320544111

PubMed Abstract | Crossref Full Text | Google Scholar

Loos, A. and Steinkellner, H. (2014). Plant glyco-biotechnology on the way to synthetic biology. Front. Plant Sci. 5. doi: 10.3389/fpls.2014.00523

PubMed Abstract | Crossref Full Text | Google Scholar

Margolin, E. A., Strasser, R., Chapman, R., Williamson, A. L., Rybicki, E. P., and Meyers, A. E. (2020). Engineering the plant secretory pathway for the production of next-generation pharmaceuticals. Trends Biotechnol. 38, 1034–1044. doi: 10.1016/j.tibtech.2020.03.004

PubMed Abstract | Crossref Full Text | Google Scholar

McGinness, A. J., Brooks, S. A., Strasser, R., Schoberer, J., and Kriechbaumer, V. (2025). Suborganellar resolution imaging for the localisation of human glycosylation enzymes in tobacco Golgi bodies. J. Microsc. 297, 258–273. doi: 10.1111/jmi.13311

PubMed Abstract | Crossref Full Text | Google Scholar

Mercx, S., Smargiasso, N., Chaumont, F., De Pauw, E., Boutry, M., and Navarre, C. (2017). Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 Cells by a Multiplex CRISPR/Cas9 Strategy Results in Glycoproteins without Plant-Specific Glycans. Front. Plant Sci. 8. doi: 10.3389/fpls.2017.00403

PubMed Abstract | Crossref Full Text | Google Scholar

Montero-Morales, L., Maresch, D., Crescioli, S., Castilho, A., Ilieva, K. M., Mele, S., et al. (2019). In planta glycan engineering and functional activities of igE antibodies. Front. Bioeng Biotechnol. 7. doi: 10.3389/fbioe.2019.00242

PubMed Abstract | Crossref Full Text | Google Scholar

Montero-Morales, L. and Steinkellner, H. (2018). Advanced plant-based glycan engineering. Front. Bioeng Biotechnol. 6. doi: 10.3389/fbioe.2018.00081

PubMed Abstract | Crossref Full Text | Google Scholar

Mukherjee, M., Caroll, E., and Wang, Z. Q. (2021). Rapid assembly of multi-gene constructs using modular golden gate cloning. J. Vis. Exp. 5. doi: 10.3791/61993

PubMed Abstract | Crossref Full Text | Google Scholar

Nagels, B., Van Damme, E. J., Callewaert, N., and Weterings, K. (2012). Introduction of tri-antennary N-glycans in Arabidopsis thaliana plants. Plant Sci. 185-186, 161–168. doi: 10.1016/j.plantsci.2011.10.002

PubMed Abstract | Crossref Full Text | Google Scholar

Nagels, B., Van Damme, E. J. M., Pabst, M., Callewaert, N., and Weterings, K. (2011). Production of complex multiantennary N-glycans in nicotiana benthamiana plants. Plant Physiol. 155, 1103–1112. doi: 10.1104/pp.110.168773

PubMed Abstract | Crossref Full Text | Google Scholar

Navarre, C., Bailly, N., Balieu, J., Perruchon, O., Herman, X., Mercier, A., et al. (2025). Nicotiana tabacum contains two alpha1,3-fucosyltransferase types, one of which is able to catalyze core fucosylation of high-mannose N-glycans. Glycobiology 35. doi: 10.1093/glycob/cwaf024

PubMed Abstract | Crossref Full Text | Google Scholar

Nguyen, K. D., Kajiura, H., Kamiya, R., Yoshida, T., Misaki, R., and Fujiyama, K. (2023). Production and N-glycan engineering of Varlilumab in Nicotiana benthamiana. Front. Plant Sci. 14. doi: 10.3389/fpls.2023.1215580

PubMed Abstract | Crossref Full Text | Google Scholar

Nishima, W., Miyashita, N., Yamaguchi, Y., Sugita, Y., and Re, S. (2012). Effect of bisecting glcNAc and core fucosylation on conformational properties of biantennary complex-type N-glycans in solution. J. Phys. Chem. B. 116, 8504–8512. doi: 10.1021/jp212550z

PubMed Abstract | Crossref Full Text | Google Scholar

Nomura, K., Liu, Y., and Kajihara, Y. (2022). Synthesis of homogeneous glycoproteins with diverse N-glycans. Adv. Carbohydr Chem. Biochem. 81, 57–93. doi: 10.1016/bs.accb.2022.09.004

PubMed Abstract | Crossref Full Text | Google Scholar

Pantazica, A. M., van Eerde, A., Dobrica, M. O., Caras, I., Ionescu, I., Costache, A., et al. (2023). The "humanized" N-glycosylation pathway in CRISPR/Cas9-edited Nicotiana benthamiana significantly enhances the immunogenicity of a S/preS1 Hepatitis B Virus antigen and the virus-neutralizing antibody response in vaccinated mice. Plant Biotechnol. J. 21, 1176–1190. doi: 10.1111/pbi.14028

PubMed Abstract | Crossref Full Text | Google Scholar

Parsons, J., Altmann, F., Arrenberg, C. K., Koprivova, A., Beike, A. K., Stemmer, C., et al. (2012). Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants. Plant Biotechnol. J. 10, 851–861. doi: 10.1111/j.1467-7652.2012.00704.x

PubMed Abstract | Crossref Full Text | Google Scholar

Pedersen, C. T., Loke, I., Lorentzen, A., Wolf, S., Kamble, M., Kristensen, S. K., et al. (2017). N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research. Plant J. 91, 394–407. doi: 10.1111/tpj.13570

PubMed Abstract | Crossref Full Text | Google Scholar

Raju, T. S. and Jordan, R. E. (2012). Galactosylation variations in marketed therapeutic antibodies. Mabs. 4, 385–391. doi: 10.4161/mabs.19868

PubMed Abstract | Crossref Full Text | Google Scholar

Rosales-Mendoza, S., Salazar-González, J. A., Decker, E. L., and Reski, R. (2016). Implications of plant glycans in the development of innovative vaccines. Expert Rev. Vaccines 15, 915–925. doi: 10.1586/14760584.2016.1155987

PubMed Abstract | Crossref Full Text | Google Scholar

Rouwendal, G. J., Florack, D. E., Hesselink, T., Cordewener, J. H., Helsper, J. P., and Bosch, D. (2009). Synthesis of Lewis X epitopes on plant N-glycans. Carbohydr Res. 344, 1487–1493. doi: 10.1016/j.carres.2009.05.003

PubMed Abstract | Crossref Full Text | Google Scholar

Rouwendal, G. J., Wuhrer, M., Florack, D. E., Koeleman, C. A., Deelder, A. M., Bakker, H., et al. (2007). Efficient introduction of a bisecting GlcNAc residue in tobacco N-glycans by expression of the gene encoding human N-acetylglucosaminyltransferase III. Glycobiology. 17, 334–344. doi: 10.1093/glycob/cwl078

PubMed Abstract | Crossref Full Text | Google Scholar

Roychowdhury, S., Oh, Y. J., Kajiura, H., Hamorsky, K. T., Fujiyama, K., and Matoba, N. (2018). Hydroponic Treatment of Nicotiana benthamiana with Kifunensine Modifies the N-glycans of Recombinant Glycoprotein Antigens to Predominantly Man9 High-Mannose Type upon Transient Overexpression. Front. Plant Sci. 9. doi: 10.3389/fpls.2018.00062

PubMed Abstract | Crossref Full Text | Google Scholar

Rup, B., Alon, S., Amit-Cohen, B. C., Brill Almon, E., Chertkoff, R., Tekoah, Y., et al. (2017). Immunogenicity of glycans on biotherapeutic drugs produced in plant expression systems-The taliglucerase alfa story. PloS One 12, e0186211. doi: 10.1371/journal.pone.0186211

PubMed Abstract | Crossref Full Text | Google Scholar

Saint-Jore-Dupas, C., Nebenführ, A., Boulaflous, A., Follet-Gueye, M. L., Plasson, C., Hawes, C., et al. (2006). Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway. Plant Cell. 18, 3182–3200. doi: 10.1105/tpc.105.036400

PubMed Abstract | Crossref Full Text | Google Scholar

Sampedro, J., Gianzo, C., Iglesias, N., Guitián, E., Revilla, G., and Zarra, I. (2012). AtBGAL10 is the main xyloglucan β-galactosidase in arabidopsis, and its absence results in unusual xyloglucan subunits and growth defects. Plant Physiol. 158, 1146–1157. doi: 10.1104/pp.111.192195

PubMed Abstract | Crossref Full Text | Google Scholar

Sariyatun, R., Florence, Kajiura, H., Ohashi, T., Misaki, R., and Fujiyama, K. (2021). Production of human acid-alpha glucosidase with a paucimannose structure by glycoengineered arabidopsis cell culture. Front. Plant Sci. 12. doi: 10.3389/fpls.2021.703020

PubMed Abstract | Crossref Full Text | Google Scholar

Sasai, K., Ikeda, Y., Honke, K., and Taniguchi, N. (2001). The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V. The involvement of a domain homophilic interaction. J. Biol. Chem. 276, 759–765. doi: 10.1074/jbc.m004972200

PubMed Abstract | Crossref Full Text | Google Scholar

Schachter, H. (1986). Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. Biochem. Cell Biol. 64, 163–181. doi: 10.1139/o86-026

PubMed Abstract | Crossref Full Text | Google Scholar

Schneider, J. D., Castilho, A., Neumann, L., Altmann, F., Loos, A., Kannan, L., et al. (2014). Expression of human butyrylcholinesterase with an engineered glycosylation profile resembling the plasma-derived orthologue. Biotechnol. J. 9, 501–510. doi: 10.1002/biot.201300229

PubMed Abstract | Crossref Full Text | Google Scholar

Schneider, J., Castilho, A., Pabst, M., Altmann, F., Gruber, C., Strasser, R., et al. (2015). Characterization of plants expressing the human beta1,4-galactosyltrasferase gene. Plant Physiol. Biochem. 92, 39–47. doi: 10.1016/j.plaphy.2015.04.010

PubMed Abstract | Crossref Full Text | Google Scholar

Schoberer, J., Izadi, S., Kierein, C., Vavra, U., Konig-Beihammer, J., Ruocco, V., et al. (2023). The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation. Front. Plant Sci. 14. doi: 10.3389/fpls.2023.1320051

PubMed Abstract | Crossref Full Text | Google Scholar

Schoberer, J. and Strasser, R. (2011). Sub-compartmental organization of Golgi-resident N-glycan processing enzymes in plants. Mol. Plant 4, 220–228. doi: 10.1093/mp/ssq082

PubMed Abstract | Crossref Full Text | Google Scholar

Shaaltiel, Y., Bartfeld, D., Hashmueli, S., Baum, G., Brill-Almon, E., Galili, G., et al. (2007). Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system. Plant Biotechnol. J. 5, 579–590. doi: 10.1111/j.1467-7652.2007.00263.x

PubMed Abstract | Crossref Full Text | Google Scholar

Shaaltiel, Y. and Tekoah, Y. (2016). Plant specific N-glycans do not have proven adverse effects in humans. Nat. Biotechnol. 34, 706–708. doi: 10.1038/nbt.3556

PubMed Abstract | Crossref Full Text | Google Scholar

Shanmugaraj, B., CJ, I. B., and Phoolcharoen, W. (2020). Plant molecular farming: A viable platform for recombinant biopharmaceutical production. Plants(Basel) 9. doi: 10.3390/plants9070842

PubMed Abstract | Crossref Full Text | Google Scholar

Shin, Y. J., Castilho, A., Dicker, M., Sadio, F., Vavra, U., Grunwald-Gruber, C., et al. (2017). Reduced paucimannosidic N-glycan formation by suppression of a specific beta-hexosaminidase from Nicotiana benthamiana. Plant Biotechnol. J. 15, 197–206. doi: 10.1111/pbi.12602

PubMed Abstract | Crossref Full Text | Google Scholar

Sourrouille, C., Marquet-Blouin, E., D'Aoust, M. A., Kiefer-Meyer, M. C., Seveno, M., Pagny-Salehabadi, S., et al. (2008). Down-regulated expression of plant-specific glycoepitopes in alfalfa. Plant Biotechnol. J. 6, 702–721. doi: 10.1111/j.1467-7652.2008.00353.x

PubMed Abstract | Crossref Full Text | Google Scholar

Stadlmann, J., Pabst, M., and Altmann, F. (2010). Analytical and functional aspects of antibody sialylation. J. Clin. Immunol. 30, S15–S19. doi: 10.1007/s10875-010-9409-2

PubMed Abstract | Crossref Full Text | Google Scholar

Stowell, C. P. and Stowell, S. R. (2019). Biologic roles of the ABH and Lewis histo-blood group antigens Part I: infection and immunity. Vox Sang. 114, 426–442. doi: 10.1111/vox.12787

PubMed Abstract | Crossref Full Text | Google Scholar

Strasser, R. (2014). Biological significance of complex N-glycans in plants and their impact on plant physiology. Front. Plant Sci. 5. doi: 10.3389/fpls.2014.00363

PubMed Abstract | Crossref Full Text | Google Scholar

Strasser, R. (2016). Plant protein glycosylation. Glycobiology. 26, 926–939. doi: 10.1093/glycob/cww023

PubMed Abstract | Crossref Full Text | Google Scholar

Strasser, R. (2022). Recent developments in deciphering the biological role of plant complex N-glycans. Front. Plant Sci. 13. doi: 10.3389/fpls.2022.897549

PubMed Abstract | Crossref Full Text | Google Scholar

Strasser, R., Altmann, F., Mach, L., Glossl, J., and Steinkellner, H. (2004). Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose. FEBS Lett. 561, 132–136. doi: 10.1016/s0014-5793(04)00150-4

PubMed Abstract | Crossref Full Text | Google Scholar

Strasser, R., Altmann, F., and Steinkellner, H. (2014). Controlled glycosylation of plant-produced recombinant proteins. Curr. Opin. Biotechnol. 30, 95–100. doi: 10.1016/j.copbio.2014.06.008

PubMed Abstract | Crossref Full Text | Google Scholar

Strasser, R., Bondili, J. S., Schoberer, J., Svoboda, B., Liebminger, E., Glossl, J., et al. (2007a). Enzymatic properties and subcellular localization of Arabidopsis beta-N-acetylhexosaminidases. Plant Physiol. 145, 5–16. doi: 10.1104/pp.107.101162

PubMed Abstract | Crossref Full Text | Google Scholar

Strasser, R., Bondili, J. S., Vavra, U., Schoberer, J., Svoboda, B., Glossl, J., et al. (2007b). A unique beta1,3-galactosyltransferase is indispensable for the biosynthesis of N-glycans containing Lewis a structures in Arabidopsis thaliana. Plant Cell. 19, 2278–2292. doi: 10.1105/tpc.107.052985

PubMed Abstract | Crossref Full Text | Google Scholar

Strasser, R., Castilho, A., Stadlmann, J., Kunert, R., Quendler, H., Gattinger, P., et al. (2009). Improved virus neutralization by plant-produced anti-HIV antibodies with a homogeneous beta1,4-galactosylated N-glycan profile. J. Biol. Chem. 284, 20479–20485. doi: 10.1074/jbc.m109.014126

PubMed Abstract | Crossref Full Text | Google Scholar

Strasser, R., Seifert, G., Doblin, M. S., Johnson, K. L., Ruprecht, C., Pfrengle, F., et al. (2021). Cracking the "Sugar code": A snapshot of N- and O-glycosylation pathways and functions in plants cells. Front. Plant Sci. 12. doi: 10.3389/fpls.2021.640919

PubMed Abstract | Crossref Full Text | Google Scholar

Strasser, R., Stadlmann, J., Schahs, M., Stiegler, G., Quendler, H., Mach, L., et al. (2008). Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol. J. 6, 392–402. doi: 10.1111/j.1467-7652.2008.00330.x

PubMed Abstract | Crossref Full Text | Google Scholar

Teare, J. M., Kates, D. S., Shah, A., and Garger, S. (2019). Increased branching and sialylation of N-linked glycans correlate with an improved pharmacokinetic profile for BAY 81–8973 compared with other full-length rFVIII products. Drug Des. Devel Ther. 13, 941–948. doi: 10.2147/dddt.s188171

PubMed Abstract | Crossref Full Text | Google Scholar

Tekoah, Y., Tzaban, S., Kizhner, T., Hainrichson, M., Gantman, A., Golembo, M., et al. (2013). Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems. Biosci. Rep. 33, e00071. doi: 10.1042/bsr20130081

PubMed Abstract | Crossref Full Text | Google Scholar

Terracciano, D., Mariano, A., Macchia, V., and Di Carlo, A. (2005). Analysis of glycoproteins in human colon cancers, normal tissues and in human colon carcinoma cells reactive with monoclonal antibody NCL-19-9. Oncol. Rep. 14, 719–722. doi: 10.3892/or.14.3.719

PubMed Abstract | Crossref Full Text | Google Scholar

Thaysen-Andersen, M. and Packer, N. H. (2012). Site-specific glycoproteomics confirms that protein structure dictates formation of N-glycan type, core fucosylation and branching. Glycobiology. 22, 1440–1452. doi: 10.1093/glycob/cws110

PubMed Abstract | Crossref Full Text | Google Scholar

Tomida, S., Nagae, M., and Kizuka, Y. (2022). The stem region of α1,6-fucosyltransferase FUT8 is required for multimer formation but not catalytic activity. J. Biol. Chem. 298, 102676. doi: 10.1016/j.jbc.2022.102676

PubMed Abstract | Crossref Full Text | Google Scholar

Tu, L. and Banfield, D. K. (2010). Localization of golgi-resident glycosyltransferases. Cell Mol. Life Sci. 67, 29–41. doi: 10.1007/s00018-009-0126-z

PubMed Abstract | Crossref Full Text | Google Scholar

Vainauskas, S., Kirk, C. H., Petralia, L., Guthrie, E. P., McLeod, E., Bielik, A., et al. (2018). A novel broad specificity fucosidase capable of core α1–6 fucose release from N-glycans labeled with urea-linked fluorescent dyes. Sci. Rep. 22, 9504. doi: 10.1038/s41598-018-27797-0

PubMed Abstract | Crossref Full Text | Google Scholar

van der Kaaij, A., Bunte, M. J. M., Nijhof, L., Mokhtari, S., Overmars, H., Schots, A., et al. (2025). Identification of beta-galactosidases along the secretory pathway of Nicotiana benthamiana that collectively hamper engineering of galactose-extended glycans on recombinant glycoproteins. Plant Biotechnol. J. 7. doi: 10.1111/pbi.70126

PubMed Abstract | Crossref Full Text | Google Scholar

van der Kaaij, A., van Noort, K., Nibbering, P., Wilbers, R. H. P., and Schots, A. (2022). Glyco-engineering plants to produce helminth glycoproteins as prospective biopharmaceuticals: recent advances, challenges and future prospects. Front. Plant Sci. 13. doi: 10.3389/fpls.2022.882835

PubMed Abstract | Crossref Full Text | Google Scholar

van Noort, K., Nguyen, D. L., Kriechbaumer, V., Hawes, C., Hokke, C. H., Schots, A., et al. (2020). Functional characterization of Schistosoma mansoni fucosyltransferases in Nicotiana benthamiana plants. Sci. Rep. 10, 18528. doi: 10.1038/s41598-020-74485-z

PubMed Abstract | Crossref Full Text | Google Scholar

Vo, D. K. and Trinh, K. T. L. (2025). Molecular farming for immunization: current advances and future prospects in plant-produced vaccines. Vaccines(Basel). 13, 191. doi: 10.3390/vaccines13020191

PubMed Abstract | Crossref Full Text | Google Scholar

Vonschaewen, A., Sturm, A., Oneill, J., and Chrispeels, M. J. (1993). Isolation of a mutant arabidopsis plant that lacks N-acetyl glucosaminyl transferase-I and is unable to synthesize golgi-modified complex N-linked glycans. Plant Physiol. 102, 1109–1118. doi: 10.1104/pp.102.4.1109

PubMed Abstract | Crossref Full Text | Google Scholar

Weise, A., Altmann, F., Rodriguez-Franco, M., Sjoberg, E. R., Bäumer, W., Launhardt, H., et al. (2007). High-level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Delta-fuc-t Delta-xyl-t mutant. Plant Biotechnol. J. 5, 389–401. doi: 10.1111/j.1467-7652.2007.00248.x

PubMed Abstract | Crossref Full Text | Google Scholar

Welch, L. G. and Munro, S. (2019). A tale of short tails, through thick and thin: investigating the sorting mechanisms of Golgi enzymes. FEBS Lett. 593, 2452–2465. doi: 10.1002/1873-3468.13553

PubMed Abstract | Crossref Full Text | Google Scholar

Wilbers, R. H., Westerhof, L. B., Reuter, L. J., Castilho, A., van Raaij, D. R., Nguyen, D. L., et al. (2016). 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. 14, 670–681. doi: 10.1111/pbi.12414

PubMed Abstract | Crossref Full Text | Google Scholar

Wilbers, R. H., Westerhof, L. B., van Noort, K., Obieglo, K., Driessen, N. N., Everts, B., et al. (2017). Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants. Sci. Rep. 7, 45910. doi: 10.1038/srep45910

PubMed Abstract | Crossref Full Text | Google Scholar

Willis, L. M., Gilbert, M., Karwaski, M. F., Blanchard, M. C., and Wakarchuk, W. W. (2008). Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme. Glycobiology 18, 177–186. doi: 10.1093/glycob/cwm126

PubMed Abstract | Crossref Full Text | Google Scholar

Wilson, I. B. (2001). Identification of a cDNA encoding a plant Lewis-type alpha1,4-fucosyltransferase. Glycoconj J. 18, 439–447. doi: 10.1023/a:1016030000527

PubMed Abstract | Crossref Full Text | Google Scholar

Yadav, N., Poveda, A., Mena, Y. V., Rosenthal, M., Ogawa, Y., Jiménez-Barbero, J., et al. (2025). Controlling glycan folding with ionic functional groups. J. Am. Chem. Soc 147, 15126–15135. doi: 10.1021/jacs.4c17992

PubMed Abstract | Crossref Full Text | Google Scholar

Yagi, H., Tateo, S., Saito, T., Ohta, Y., Nishi, E., Obitsu, S., et al. (2024). Deciphering the sub-Golgi localization of glycosyltransferases via 3D super-resolution imaging. Cell Struct. Funct. 49, 47–55. doi: 10.1247/csf.24008

PubMed Abstract | Crossref Full Text | Google Scholar

Yang, W. H., Aziz, P. V., Heithoff, D. M., Mahan, M. J., Smith, J. W., and Marth, J. D. (2015). An intrinsic mechanism of secreted protein aging and turnover. Proc. Natl. Acad. Sci. U S A. 112, 13657–13662. doi: 10.1073/pnas.1515464112

PubMed Abstract | Crossref Full Text | Google Scholar

Yang, Z., Drew, D. P., Jorgensen, B., Mandel, U., Bach, S. S., Ulvskov, P., et al. (2012). Engineering mammalian mucin-type O-glycosylation in plants. J. Biol. Chem. 287, 11911–11923. doi: 10.1074/jbc.m111.312918

PubMed Abstract | Crossref Full Text | Google Scholar

Yang, Z., Wang, S., Halim, A., Schulz, M. A., Frodin, M., Rahman, S. H., et al. (2015). Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nat. Biotechnol. 33, 842–844. doi: 10.1038/nbt.3280

PubMed Abstract | Crossref Full Text | Google Scholar

Yin, B. J., Gao, Y., Chung, C. Y., Yang, S., Blake, E., Stuczynski, M. C., et al. (2015). Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation. Biotechnol. Bioeng. 112, 2343–2351. doi: 10.1002/bit.25650

PubMed Abstract | Crossref Full Text | Google Scholar

Yuen, W. M., Song, S., Leung, S. K. P., Leung, M. F., Lo, S. W., Hwang, I., et al. (2024). Utilizing plant protein secretion system for pharmaceutical protein production. Methods Mol. Biol. 2841, 101–109. doi: 10.1007/978-1-0716-4059-3_9

PubMed Abstract | Crossref Full Text | Google Scholar

Zhang, Q. X., Li, S. S., He, L., and Feng, X. T. (2023). A brief review of polysialic acid-based drug delivery systems. Int. J. Biol. Macromol. 230, 123151. doi: 10.1016/j.ijbiomac.2023.123151

PubMed Abstract | Crossref Full Text | Google Scholar

Zhong, X., D'Antona, A. M., Scarcelli, J. J., and Rouse, J. C. (2022). New opportunities in glycan engineering for therapeutic proteins. Antibodies (Basel) 11. doi: 10.3390/antib11010005

PubMed Abstract | Crossref Full Text | Google Scholar

Zhou, Q. and Qiu, H. (2019). The mechanistic impact of N-glycosylation on stability, pharmacokinetics, and immunogenicity of therapeutic proteins. J. Pharm. Sci. 108, 1366–1377. doi: 10.1016/j.xphs.2018.11.029

PubMed Abstract | Crossref Full Text | Google Scholar

Zhu, W., Zhou, Y., Guo, L., and Feng, S. (2024). Biological function of sialic acid and sialylation in human health and disease. Cell Death Discov. 10, 415. doi: 10.1038/s41420-024-02180-3

PubMed Abstract | Crossref Full Text | Google Scholar

Zwanenburg, L., Borloo, J., Decorte, B., Bunte, M. J. M., Mokhtari, S., Serna, S., et al. (2023). Plant-based production of a protective vaccine antigen against the bovine parasitic nematode Ostertagia ostertagi. Sci. Rep. 13, 20488. doi: 10.1038/s41598-023-47480-3

PubMed Abstract | Crossref Full Text | Google Scholar