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All cells are decorated with complex carbohydrate structures called glycans that serve as ligands for glycan-binding proteins (GBPs) to mediate a wide range of biological processes. Understanding the specific functions of glycans is key to advancing an understanding of human health and disease. However, the lack of convenient and accessible tools to study glycan-based interactions has been a defining challenge in glycobiology. Thus, the development of chemical and biochemical strategies to address these limitations has been a rapidly growing area of research. In this review, we describe the use of glycosyltransferases (GTs) as versatile tools to facilitate a greater understanding of the biological roles of glycans. We highlight key examples of how GTs have streamlined the preparation of well-defined complex glycan structures through chemoenzymatic synthesis, with an emphasis on synthetic strategies allowing for site- and branch-specific display of glyco-epitopes. We also describe how GTs have facilitated expansion of glyco-engineering strategies, on both glycoproteins and cell surfaces. Coupled with advancements in bioorthogonal chemistry, GTs have enabled selective glyco-epitope editing of glycoproteins and cells, selective glycan subclass labeling, and the introduction of novel biomolecule functionalities onto cells, including defined oligosaccharides, antibodies, and other proteins. Collectively, these approaches have contributed great insight into the fundamental biological roles of glycans and are enabling their application in drug development and cellular therapies, leaving the field poised for rapid expansion. [ABSTRACT FROM AUTHOR]

High accumulation of a single high-mannose glycan structure is important to ensure the quality of therapeutic proteins. We developed a glyco-engineering strategy for ensuring high accumulation of the Man 5 GlcNAc 2 structure by combining N -acetylglucosaminyltransferase I (GnT I) gene suppression and mannosidase I (Man I) gene overexpression. Nicotiana tabacum SR1 was used as the glyco-engineered host owing to the lower risk of pathogenic contamination than that in mammalian cells. We generated three glyco-engineered plant strains (gnt, gnt-MANA1, and gnt-MANA2) with suppression of GnT I or the combined suppression of GnT I and overexpression of Man I A1 or A2. The quantitative reverse transcriptase-PCR analysis showed a higher level of upregulation of Man I expression in gnt-MANA1/A2 plants than in the wild-type plants. Man I activity assay showed that the gnt-MANA1 plants had a higher Man I activity than did the wild-type and gnt-MANA2 plants. N -glycan analysis independently performed on two plants of each plant strain showed that gnt-MANA1 plants had a low abundance of the Man 6-9 GlcNAc 2 structure (2.8%, 7.1%) and high abundance of the Man 5 GlcNAc 2 structure (80.0%, 82.8%) compared with those in the wild-type and gnt plants. These results indicated that GnT I knockdown suppressed further modification of the Man 5 GlcNAc 2 structure, and Man I overexpression enhanced the conversion of Man 6-9 GlcNAc 2 structures to the Man 5 GlcNAc 2 structure. The developed glyco-engineered plants have potential for serving as novel expression hosts for therapeutic proteins. [ABSTRACT FROM AUTHOR]

Influenza viruses bind to their target through a multivalent interaction of their hemagglutinins (HAs) with sialosides at the host cell surface. To fight the virus, one therapeutic approach consists in developing sialylated multivalent structures that can saturate the virus HAs and prevent the binding to host cells. We describe herein the biotechnological production of sialylated solid lipid microparticles (SSLMs) in 3 steps: (i) a microbiological step leading to the large-scale production of sialylated maltodextrins by metabolic engineering of an Escherichia coli strain, (ii) a new in vitro glycosylation process using the amylomaltase MalQ, based on the transglycosylation of the terminal sialoside ligand of the sialylated maltodextrin onto a long-chain alkyl glucoside, and (iii) the formulation of the final SSLMs presenting a multivalent sialic acid. We also describe the morphology and structure of the SSLMs and demonstrate their very promising properties as influenza virus inhibitors using hemagglutination inhibition and microneutralization assays on the human A/H1N1 pdm09 virus. [ABSTRACT FROM AUTHOR]

Heterologous glycoprotein production relies on host glycosylation‐dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco‐engineering to improve the production of a model HIV‐1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3‐fucosyltransferase and β1,2‐xylosyltransferase was used as an expression host to prevent plant‐specific complex N‐glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco‐engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell‐produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant‐produced and mammalian cell‐derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus‐vectored vaccines. This study demonstrates that engineering glycosylation‐directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants. [ABSTRACT FROM AUTHOR]

As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with β1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human β1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric β1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human β1,4-galactosyltransferase. Stable FTGT expression led to the desired β1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of β1,4-galactosylated N-glycans was reported earlier, e.g., on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

The glycoproteins of yeast contain a large outer chain on N-linked oligosaccharides; therefore, yeast is not suitable for producing therapeutic glycoproteins for human use. Using a deletion mutant strain of α1,6-mannosyltransferase (och1Δ), we previously produced humanized N-glycans in fission yeast; however, the Schizosaccharomyces pombe och1Δ cells displayed a growth delay even during vegetative growth, resulting in reduced productivity of heterologous proteins. To overcome this problem, here we performed a genome-wide screen for genes that would suppress the growth defect of temperature-sensitive och1Δ cells. Using a genomic library coupled with screening of 18,000 transformants, we identified two genes (pwp1+, SPBC1E8.05), both encoding GPI-anchored proteins, that increased the growth rate of och1Δ cells, lacking the outer chain. We further showed that a high copy number of the genes was needed to improve the growth rate. Mutational analysis of Pwp1p revealed that the GPI-anchored region of Pwp1p is important in attenuating the growth defect. Analysis of disruptants of pwp1+ and SPBC1E8.05 showed that neither gene was essential for cell viability; however, both mutants were sensitive β-glucanase, suggesting that Pwp1p and the protein encoded by SPBC1E8.05 non-enzymatically support β-glucan on the cell-surface of S. pombe. Collectively, our work not only sheds light on the functional relationships between GPI-anchored proteins and N-linked oligosaccharides of glycoproteins in S. pombe, but also supports the application of S. pombe to the production of human glycoprotein. Key points: • We screened for genes that suppress the growth defect of fission yeast och1Δ cells. • Appropriate expression of GPI-anchored proteins alleviates the growth delay of och1Δ cells. • The GPI-anchor domain of Pwp1p is important for suppressing the growth defect of och1Δ cells. [ABSTRACT FROM AUTHOR]

Glycans decorate all cells and are critical mediators of cellular processes through recognition by glycan-binding proteins (GBPs). While targeting glycan–protein interactions has great therapeutic potential, these interactions are challenging to study as they are generally transient and exhibit low binding affinities. Glycan-based photo-crosslinkable probes have enabled covalent capture and identification of unknown GBP receptors and glycoconjugate ligands. Here, we review recent progress in photo-crosslinking approaches targeting glycan-mediated interactions. We discuss two prominent emerging strategies: 1) development of photo-crosslinkable oligosaccharide ligands to identify GBP receptors; and 2) cell-surface glyco-engineering to identify glycoconjugate ligands of GBPs. Overall, photoaffinity labeling affords valuable insights into complex glycan–protein networks and is poised to help elucidate the glycan–protein interactome, providing novel targets for therapeutic intervention. [Display omitted] [ABSTRACT FROM AUTHOR]

Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient “helminthization” of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by β-N-acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcβ1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel β-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict β-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants.

Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient "helminthization" of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by β- N -acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcβ1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel β-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict β-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants. [ABSTRACT FROM AUTHOR]

Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells are among the most commonly used plant cell lines for producing biopharmaceutical glycoproteins. Recombinant glycoproteins are usually produced with a mix of high-mannose and complex N-glycans. However, N-glycan heterogeneity is a concern for the production of therapeutic or vaccine glycoproteins because it can alter protein activity and might lead to batch-to-batch variability. In this report, a BY-2 cell line producing glycoproteins devoid of complex N-glycans was obtained using CRISPR/Cas9 edition of two N-acetylglucosaminyltransferase I (GnTI) genes, whose activity is a prerequisite for the formation of all complex N-glycans. The suppression of complex N-glycans in the GnTI-knocked out (KO) cell lines was assessed by Western blotting. Lack of β1,2-xylose residues confirmed the abolition of GnTI activity. Unexpectedly, α1,3-fucose residues were still detected albeit dramatically reduced as compared with wild-type cells. To suppress the remaining α1,3-fucose residues, a second genome editing targeted both GnTI and α1,3-fucosyltransferase (FucT) genes. No β1,2-xylose nor α1,3-fucose residues were detected on the glycoproteins produced by the GnTI/FucT-KO cell lines. Absence of complex N-glycans on secreted glycoproteins of GnTI-KO and GnTI/FucT-KO cell lines was confirmed by mass spectrometry. Both cell lines produced high-mannose N-glycans, mainly Man5 (80 and 86%, respectively) and Man4 (16 and 11%, respectively). The high degree of N-glycan homogeneity and the high-mannose N-glycosylation profile of these BY-2 cell lines is an asset for their use as expression platforms.

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N-glycan sialylation. Since sialic acids are a common terminal modification on human N-glycans, the property to perform N-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N-glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of β1,4-galactosylated acceptor N-glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.

While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery. In this study we developed an innovative stepwise methodology in which the CRISPR-Cas9 is used sequentially to target genome editing, followed by its own excision. The first step included a stable insertion of a CRISPR-Cas9 cassette, targeted to knockout the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. The second step included the excision of the inserted cassette of 14.3 kbp by induction of specific sgRNA designed to target the T-DNA boundaries. The genome editing step and the transgene removal step are achieved in one transformation run. This mechanism enables CRISPR genome editing and subsequently eliminating the introduced transgenes thus freeing the cells from foreign DNA no longer needed.

Production of monomeric IgA in mammalian cells and plant expression systems such as Nicotiana benthamiana is well-established and can be achieved by co-expression of the corresponding light and heavy chains. In contrast, the assembly of dimeric IgA requires the additional expression of the joining chain and remains challenging especially in plant-based systems. Here, we examined factors affecting the assembly and expression of HER2 binding dimeric IgA1 and IgA2m(2) variants transiently produced in N. benthamiana. While co-expression of the joining chain resulted in efficient formation of dimeric IgAs in HEK293F cells, a mixture of monomeric, dimeric and tetrameric variants was detected in plants. Mass-spectrometric analysis of site-specific glycosylation revealed that the N-glycan profile differed between monomeric and dimeric IgAs in the plant expression system. Co-expression of a single-subunit oligosaccharyltransferase from the protozoan Leishmania major in N. benthamiana increased the N-glycosylation occupancy at the C-terminal heavy chain tailpiece and changed the ratio of monomeric to dimeric IgAs. Our data demonstrate that N-glycosylation engineering is a suitable strategy to promote the formation of dimeric IgA variants in plants.

Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells are among the most commonly used plant cell lines for producing biopharmaceutical glycoproteins. Recombinant glycoproteins are usually produced with a mix of high-mannose and complex N -glycans. However, N -glycan heterogeneity is a concern for the production of therapeutic or vaccine glycoproteins because it can alter protein activity and might lead to batch-to-batch variability. In this report, a BY-2 cell line producing glycoproteins devoid of complex N -glycans was obtained using CRISPR/Cas9 edition of two N-acetylglucosaminyltransferase I (GnTI) genes, whose activity is a prerequisite for the formation of all complex N -glycans. The suppression of complex N -glycans in the GnTI -knocked out (KO) cell lines was assessed by Western blotting. Lack of β1,2-xylose residues confirmed the abolition of GnTI activity. Unexpectedly, α1,3-fucose residues were still detected albeit dramatically reduced as compared with wild-type cells. To suppress the remaining α1,3-fucose residues, a second genome editing targeted both GnTI and α 1,3-fucosyltransferase (FucT) genes. No β1,2-xylose nor α1,3-fucose residues were detected on the glycoproteins produced by the GnTI/FucT -KO cell lines. Absence of complex N -glycans on secreted glycoproteins of GnTI -KO and GnTI/FucT -KO cell lines was confirmed by mass spectrometry. Both cell lines produced high-mannose N -glycans, mainly Man5 (80 and 86%, respectively) and Man4 (16 and 11%, respectively). The high degree of N -glycan homogeneity and the high-mannose N -glycosylation profile of these BY-2 cell lines is an asset for their use as expression platforms. [ABSTRACT FROM AUTHOR]

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N -glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N -glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N -glycan sialylation. Since sialic acids are a common terminal modification on human N- glycans, the property to perform N -glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N -glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of β1,4-galactosylated acceptor N -glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-type N- glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein. [ABSTRACT FROM AUTHOR]

While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery. In this study we developed an innovative stepwise methodology in which the CRISPR-Cas9 is used sequentially to target genome editing, followed by its own excision. The first step included a stable insertion of a CRISPR-Cas9 cassette, targeted to knockout the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. The second step included the excision of the inserted cassette of 14.3 kbp by induction of specific sgRNA designed to target the T-DNA boundaries. The genome editing step and the transgene removal step are achieved in one transformation run. This mechanism enables CRISPR genome editing and subsequently eliminating the introduced transgenes thus freeing the cells from foreign DNA no longer needed. [ABSTRACT FROM AUTHOR]

Summary: Glyco‐design of proteins is a powerful tool in fundamental studies of structure–function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco‐engineering facilitate customized N‐glycosylation of plant‐derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human‐like β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐galactosyltransferase, many plant‐derived glycoproteins still exhibit incomplete processed N‐glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β‐galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4‐ and β1,3‐galactose residues on both N‐ and O‐glycans. Transient BGAL1 down‐regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N‐glycans on several plant‐produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N‐glycans of plant‐produced glycoproteins. [ABSTRACT FROM AUTHOR]

Sialic acid, a terminal monosaccharide present in N -glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production. Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells. Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2–44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode. • Sialidase accumulation in the culture supernatant reduced rhEPO sialyation. • Three sialidase genes were knocked out in CHO cells using the CRISPR/Cas9 system. • Sialidase KO clones maintained rhEPO sialyation throughout fed-batch cultures. • Additional KO of two pro-apoptotic genes further improved rhEPO production. • Multiple KO in rCHO cells is a useful tool for producing rhEPO in fed-batch mode. [ABSTRACT FROM AUTHOR]

Abstract Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rh A1AT, rh C1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest. Here, we present engineered CHO cell lines producing rh A1AT or rh C1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rh A1AT and rh C1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rh A1AT and rh C1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients. Highlights • Humanization of N-glycans on two recombinant therapeutic proteins. • N-glycosylation similar to human in engineered Chinese hamster ovary cells. • Knock out of ten target genes and expression of two human genes. [ABSTRACT FROM AUTHOR]

Intravenous immunoglobulins (IVIg) are used in the treatment of different autoimmune and inflammatory diseases, such as immune thrombocytopenia and hemolytic anemia. One of the modes of action of IVIg is preventing phagocytosis of autoantibody-opsonized blood cells by saturation of the Fc-gamma receptors of macrophages in spleen and liver. IgG contains a fixed glycan, which is in most cases biantennary, at the asparagine residue at position 297 in the Fc tail. This glycan consists of a core structure of N-acetyl glucosamine (GlcNAc) and mannose groups, variably extended with core fucose, bisecting GlcNAc as well as terminal galactose and sialic acid. This structural glycan influences the binding affinity of IgG to Fc-gamma receptors. By glyco-engineering, we generated monoclonal IgG antibodies with different Fc-tail glycans and tested both their opsonizing and blocking capacity in a phagocytosis assay of IgG-opsonized erythrocytes with human monocyte-derived macrophages. In contrast to a lack of effect in opsono-phagocytosis, these IgG glycovariants differentially inhibited the uptake of opsonized erythrocytes. The level of bisecting GlcNAc and galactosylation had unexpectedly larger impact than core fucosylation, and suggest that targeted modifications different from the core fucose may well improve the immunomodulating efficacy of IVIg treatment.

The programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis plays a central role in suppression of anti-tumor immunity. Blocking the axis by targeting PD-L1 with monoclonal antibodies is an effective and already clinically approved approach to treat cancer patients. Glyco-engineering technology can be used to optimize different properties of monoclonal antibodies, for example, binding to FcγRs. We generated two glycosylation variants of the same anti-PD-L1 antibody: one bearing core fucosylated N-glycans in its Fc part (92%) and its de-fucosylated counterpart (4%). The two glycosylation variants were compared to a non-glycosylated commercially available anti-PD-L1 antibody in various assays. No differences were observed regarding binding to PD-L1 and blocking of this interaction with its counter receptors PD-1 or CD80. The de-fucosylated anti-PD-L1 antibody showed increased FcγRIIIa binding resulting in enhanced antibody dependent cellular cytotoxicity (ADCC) activity against PD-L1+ cancer cells compared to the “normal”-glycosylated variant. Both glycosylation variants showed no antibody-mediated lysis of B cells and monocytes. The non-glycosylated reference antibody showed no FcγRIIIa engagement and no ADCC activity. Using mixed leukocyte reaction it was observed that the de-fucosylated anti-PD-L1 antibody induced the strongest CD8 T cell activation determined by expression of activation markers, proliferation, and cytotoxicity against cancer cells. The systematic comparison of anti-PD-L1 antibody glycosylation variants with different Fc-mediated potencies demonstrates that our glyco-optimization approach has the potential to enhance CD8 T cell-mediated anti-tumor activity which may improve the therapeutic benefit of anti-PD-L1 antibodies.

Summary: Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N‐linked glycans, including the presence of β‐1,2‐xylose and core α‐1,3‐fucose residues in plants, can affect the activity, potency and immunogenicity of plant‐derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N‐glycosylation machinery to allow the synthesis of complex N‐glycans lacking β‐1,2‐xylose and core α‐1,3‐fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant‐specific α‐1,3‐fucosyltransferase and β‐1,2‐xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry‐based N‐glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64‐binding affinity of 2G12 glycovariants produced in wild‐type N. benthamiana, the newly generated FX‐KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco‐engineered antibody performed as well as its CHO‐produced counterpart. [ABSTRACT FROM AUTHOR]

Intravenous immunoglobulins (IVIg) are used in the treatment of different autoimmune and inflammatory diseases, such as immune thrombocytopenia and hemolytic anemia. One of the modes of action of IVIg is preventing phagocytosis of autoantibody-opsonized blood cells by saturation of the Fc-gamma receptors of macrophages in spleen and liver. IgG contains a fixed glycan, which is in most cases biantennary, at the asparagine residue at position 297 in the Fc tail. This glycan consists of a core structure of N-acetyl glucosamine (GlcNAc) and mannose groups, variably extended with core fucose, bisecting GlcNAc as well as terminal galactose and sialic acid. This structural glycan influences the binding affinity of IgG to Fc-gamma receptors. By glyco-engineering, we generated monoclonal IgG antibodies with different Fc-tail glycans and tested both their opsonizing and blocking capacity in a phagocytosis assay of IgG-opsonized erythrocytes with human monocyte-derived macrophages. In contrast to a lack of effect in opsono-phagocytosis, these IgG glycovariants differentially inhibited the uptake of opsonized erythrocytes. The level of bisecting GlcNAc and galactosylation had unexpectedly larger impact than core fucosylation, and suggest that targeted modifications different from the core fucose may well improve the immunomodulating efficacy of IVIg treatment. [ABSTRACT FROM AUTHOR]

Summary: N‐glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central‐protein complex facilitating the N‐glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N‐glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single‐subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well‐established production platform for recombinant proteins. A fluorescent protein‐tagged LmSTT3D variant was predominately found in the ER and co‐located with plant oligosaccharyltransferase subunits. Co‐expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N‐glycosylation site occupancy on all N‐glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N‐glycosylation efficiency in plants. [ABSTRACT FROM AUTHOR]

The programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis plays a central role in suppression of anti-tumor immunity. Blocking the axis by targeting PD-L1 with monoclonal antibodies is an effective and already clinically approved approach to treat cancer patients. Glyco-engineering technology can be used to optimize different properties of monoclonal antibodies, for example, binding to FcγRs. We generated two glycosylation variants of the same anti-PD-L1 antibody: one bearing core fucosylated N-glycans in its Fc part (92%) and its de-fucosylated counterpart (4%). The two glycosylation variants were compared to a non-glycosylated commercially available anti-PD-L1 antibody in various assays. No differences were observed regarding binding to PD-L1 and blocking of this interaction with its counter receptors PD-1 or CD80. The de-fucosylated anti-PD-L1 antibody showed increased FcγRIIIa binding resulting in enhanced antibody dependent cellular cytotoxicity (ADCC) activity against PD-L1+ cancer cells compared to the “normal”-glycosylated variant. Both glycosylation variants showed no antibody-mediated lysis of B cells and monocytes. The non-glycosylated reference antibody showed no FcγRIIIa engagement and no ADCC activity. Using mixed leukocyte reaction it was observed that the de-fucosylated anti-PD-L1 antibody induced the strongest CD8 T cell activation determined by expression of activation markers, proliferation, and cytotoxicity against cancer cells. The systematic comparison of anti-PD-L1 antibody glycosylation variants with different Fc-mediated potencies demonstrates that our glyco-optimization approach has the potential to enhance CD8 T cell-mediated anti-tumor activity which may improve the therapeutic benefit of anti-PD-L1 antibodies. [ABSTRACT FROM AUTHOR]

Plant-produced glycoproteins contain N-linked glycans with plant-specific residues of β(1,2)- xylose and core α(1,3)-fucose, which do not exist in mammalian-derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant-expressed proteins. We knocked out the β(1,2)- xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked-out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N-linked glycans lacking β(1,2)-xylose and/or α(1,3)-fucose. The knocked-out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild-type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco-engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.Plant-produced glycoproteins contain N-linked glycans with plant-specific residues of β(1,2)- xylose and core α(1,3)-fucose, which do not exist in mammalian-derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant-expressed proteins. We knocked out the β(1,2)- xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked-out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N-linked glycans lacking β(1,2)-xylose and/or α(1,3)-fucose. The knocked-out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild-type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco-engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells. [ABSTRACT FROM AUTHOR]

Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N-glycans with plant-typical residues [b(1,2)-xylose and core a(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: b(1,2)-xylosyltransferase (XylT) and a(1,3)- fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker (bar), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing b(1,2)-xylose and a(1,3)-fucose. Three lines showed a strong reduction of b(1,2)-xylose and a(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The absence of these carbohydrates was confirmed by mass spectrometry analysis of the extracellular proteins. PCR amplification and sequencing of the targeted region indicated small INDEL and/or deletions between the target sites. The KO lines did not show any particular morphology and grew as the wild-type. One KO line was transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been glycoengineered. The IgG glycosylation profile determined by mass spectrometry confirmed that no b(1,2)-xylose or a(1,3)-fucose were present on the glycosylation moiety and that the dominant glycoform was the GnGn structure. These data represent an important step toward humanizing the glycosylation of pharmacological proteins expressed in N. tabacum BY-2 cells. [ABSTRACT FROM AUTHOR]

Plants are attractive hosts for the production of recombinant glycoproteins for therapeutic use. Recent advances in glyco-engineering facilitate the elimination of nonmammalian-type glycosylation and introduction of missing pathways for customized N-glycan formation. However, some therapeutically relevant recombinant glycoproteins exhibit unwanted truncated (paucimannosidic) N-glycans that lack Glc NAc residues at the nonreducing terminal end. These paucimannosidic N-glycans increase product heterogeneity and may affect the biological function of the recombinant drugs. Here, we identified two enzymes, β-hexosaminidases ( HEXOs) that account for the formation of paucimannosidic N-glycans in Nicotiana benthamiana, a widely used expression host for recombinant proteins. Subcellular localization studies showed that HEXO1 is a vacuolar protein and HEXO3 is mainly located at the plasma membrane in N. benthamiana leaf epidermal cells. Both enzymes are functional and can complement the corresponding HEXO-deficient Arabidopsis thaliana mutants. In planta expression of HEXO3 demonstrated that core α1,3-fucose enhances the trimming of Glc NAc residues from the Fc domain of human IgG. Finally, using RNA interference, we show that suppression of HEXO3 expression can be applied to increase the amounts of complex N-glycans on plant-produced human α1-antitrypsin. [ABSTRACT FROM AUTHOR]

Bacterial polysialyltransferases (PSTs) are processive enzymes involved in the synthesis of polysialic capsular polysaccharides. They can also synthesize polysialic acid in vitro from disialylated and trisialylated lactoside acceptors, which are the carbohydrate moieties of GD3 and GT3 gangliosides, respectively. Here, we engineered a non-pathogenic Escherichia coli strain that overexpresses recombinant sialyltransferases and sialic acid synthesis genes and can convert an exogenous lactoside into polysialyl lactosides. Several PSTs were assayed for their ability to synthesize polysialyl lactosides in the recombinant strains. Fed-batch cultures produced α-2,8 polysialic acid or alternate α-2,8-2,9 polysialic acid in quantities reaching several grams per liter. Bacterial culture in the presence of propargyl-β-lactoside as the exogenous acceptor led to the production of conjugatable polysaccharides by means of copper-assisted click chemistry. [ABSTRACT FROM AUTHOR]

N-glycosylation is a process during which a glycan moiety attaches to the asparagine residue in the N-glycosylation consensus sequence (Asn-Xxx-Ser/Thr), where Xxx can be any amino acid except proline. Introduction of a new N-glycosylation site into a protein backbone leads to its hyper-glycosylation, and may improve the protein properties such as solubility, folding, stability, and secretion. Glyco-engineering is an approach to facilitate the hyper-glycosylation of recombinant proteins by application of the site-directed mutagenesis methods. In this regard, selection of a suitable location on the surface of a protein for introduction of a new N-glycosylation site is a main concern. In this work, a computational approach was conducted to select suitable location(s) for introducing new N-glycosylation sites into the human coagulation factor IX (hFIX). With this aim, the first 45 residues of mature hFIX were explored to find out suitable positions for introducing either Asn or Ser/Thr residues, to create new N-glycosylation site(s). Our exploration lead to detection of five potential positions, for hyper-glycosylation. For each suggested position, an analog was defined and subjected for N-glycosylation efficiency prediction. After generation of three-dimensional structures, by homology-based modeling, the five designed analogs were examined by molecular dynamic (MD) simulations, to predict their stability levels and probable structural distortions caused by amino acid substitutions, relative to the native counterpart. Three out of five suggested analogs, namely; E15T, K22N, and R37N, reached equilibration state with relatively constant Root Mean Square Deviation values. Additional analysis on the data obtained during MD simulations, lead us to conclude that, R37N is the only qualified analog with the most similar structure and dynamic behavior to that of the native counterpart, to be considered for further experimental investigations. [ABSTRACT FROM AUTHOR]

Plant glycans play a central role in vaccinology: they can serve as adjuvants and/or delivery vehicles or backbones for the synthesis of conjugated vaccines. In addition, genetic engineering is leading to the development of platforms for the production of novel polysaccharides in plant cells, an approach with relevant implications for the design of new types of vaccines. This review contains an updated outlook on this topic and provides key perspectives including a discussion on how the molecular pharming field can be linked to the production of innovative glycan-based and conjugate vaccines. [ABSTRACT FROM PUBLISHER]

Monoclonal antibody and Fc fusion protein drugs are complex heterogeneous mixtures of numerous different protein variants and modifications. N-glycosylation as one of the most complex post-translational modification influences the structural characteristics of the antibodies Fc part thereby potentially modulating effector function and pharmacokinetics. Several investigations on the relationship between N-glycosylation and pharmacokinetics have been published. However, this structure–function relationship is not fully understood. In this review potential alterations with focus on N-glycosylation of mAbs and Fc fusion proteins and the possible effects on the pharmacokinetics are reviewed and the current understandings of the underlying mechanisms are described. [ABSTRACT FROM AUTHOR]

Human interleukin-22 ( IL-22) is a member of the IL-10 cytokine family that has recently been shown to have major therapeutic potential. IL-22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL-22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL-22 in Nicotiana benthamiana plants and investigated the role of N-glycosylation on protein folding and biological activity. Expression levels of IL-22 were up to 5.4 μg/mg TSP, and N-glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human-like N-glycans on IL-22 by co-expressing mouse FUT8 in Δ XT/ FT plants a strong reduction in Lewis A was observed. Also, core α1,6-fucoylation did not improve the biological activity of IL-22. The combination of site-directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N-glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N-glycan on Asn54 contributes to the atypical N-glycan composition of plant-produced IL-22 and influences the N-glycan composition of N-glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N-glycosylation on folding and activity of recombinant glycoproteins, such as IL-22. [ABSTRACT FROM AUTHOR]

Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases ( TALENs) to knock out two α(1,3)-fucosyltransferase ( FucT) and the two β(1,2)-xylosyltransferase ( XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products. [ABSTRACT FROM AUTHOR]

The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins. [ABSTRACT FROM AUTHOR]

Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little is known about the overall impact of non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines ( Nicotiana benthamiana and Arabidopsis thaliana ) that stably express a modified version of human β1,4-galactosyltransferase ( ST GalT). While some transgenic plants grew normally, other lines exhibited a severe phenotype associated with stunted growth and developmental retardation. The severity of the phenotype correlated with both increased ST GalT mRNA and protein levels but no differences were observed between N-glycosylation profiles of plants with and without the phenotype. In contrast to non-transgenic plants, all ST GalT expressing plants synthesized significant amounts of incompletely processed (largely depleted of core fucose) N-glycans with up to 40% terminally galactosylated structures. While transgenic plants showed no differences in nucleotide sugar composition and cell wall monosaccharide content, alterations in the reactivity of cell wall carbohydrate epitopes associated with arabinogalactan-proteins and pectic homogalacturonan were detected in ST GalT expressing plants. Notably, plants with phenotypic alterations showed increased levels of hydrogen peroxide, most probably a consequence of hypersensitive reactions. Our data demonstrate that unfavorable phenotypical modifications may occur upon stable in planta expression of non-native glycosyltransferases. Such important issues need to be taken into consideration in respect to stable glycan engineering in plants. [ABSTRACT FROM AUTHOR]

When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library. [ABSTRACT FROM AUTHOR]

Antibodies and antibody-based drugs are currently the fastest-growing class of therapeutics. Over the last three decades, more than 30 therapeutic monoclonal antibodies and derivatives thereof have been approved for and successfully applied in diverse indication areas including cancer, organ transplants, autoimmune/inflammatory disorders, and cardiovascular disease. The isotype of choice for antibody therapeutics is human IgG, whose Fc region contains a ubiquitous asparagine residue (N) that acts as an acceptor site for N-linked glycans. The nature of these glycans can decisively influence the therapeutic performance of a recombinant antibody, and their absence or modification can lead to the loss of Fc effector functions, greater immunogenicity, and unfavorable pharmacokinetic profiles. However, recent studies have shown that aglycosylated antibodies can be genetically engineered to display novel or enhanced effector functions and that favorable pharmacokinetic properties can be preserved. Furthermore, the ability to produce aglycosylated antibodies in lower eukaryotes and bacteria offers the potential to broaden and simplify the production platforms and avoid the problem of antibody heterogeneity, which occurs when mammalian cells are used for production. In this review, we discuss the importance of Fc glycosylation focusing on the use of aglycosylated and glyco-engineered antibodies as therapeutic proteins. [ABSTRACT FROM AUTHOR]

Background: Saccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies. Findings: In order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications. Conclusions: The developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications. [ABSTRACT FROM AUTHOR]

The highly glycosylated peptide hormone erythropoietin (EPO) plays a key role in the regulation of erythrocyte maturation. Currently, marketed EPO is produced by recombinant technology in mammalian cell cultures. The complementary DNA (cDNA) for human EPO (hEPO) was transiently and stably expressed in the moss Physcomitrella patens wild-type and Δ-fuc-t Δ-xyl-t mutant, the latter containing N-glycans lacking the plant-specific, core-bound α1,3-fucose and β1,2-xylose. New expression vectors were designed based on a Physcomitrella ubiquitin gene-derived promoter for the expression of hEPO cDNA. Transient expression in protoplasts was much stronger at 10 than at 20 °C. In Western blot analysis, the molecular size of moss-produced recombinant human EPO (rhEPO) was identified to be 30 kDa, and it accumulated in the medium of transiently transformed protoplasts to high levels around 0.5 µg/mL. Transgenic Physcomitrella Δ-fuc-t Δ-xyl-t mutant lines expressing EPO cDNA showed secretion of rhEPO through the cell wall to the culture medium. In 5- and 10-L photobioreactor cultures, secreted rhEPO accumulated to high levels above 250 µg/g dry weight of moss material after 6 days. Silver staining of rhEPO on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) taken from the bioreactor culture demonstrated a high purity of the over-expressed secreted rhEPO, with a very low background of endogenous moss proteins. Peptide mapping of rhEPO produced by the Physcomitrella Δ-fuc-t Δ-xyl-t mutant indicated correct processing of the plant-derived signal peptide. All three N-glycosylation sites of rhEPO were occupied by complex-type N-glycans completely devoid of the plant-specific core sugar residues fucose and xylose. [ABSTRACT FROM AUTHOR]

Plants are a low-cost and contamination-free factory for the production of recombinant pharmaceutical proteins. However, plant-made pharmaceuticals differ from their mammalian homologues by the structure of their N-linked glycans. For instance, most mammalian glycoproteins harbour terminal sialic acids that control their half-life in the bloodstream. The absence of the whole sialylation machinery in plants is of major concern as non-sialylated plant-made pharmaceuticals may not perform at their full potential in humans, because of their removal from the circulation through the involvement of hepatic cell receptors. In this context, we have investigated the synthesis of N-acetylneuraminic acid (Neu5Ac) in the cytosol of plants by either the re-routing of the endogenous 3-deoxy-d- manno-2-octulosonic acid (Kdo) biosynthetic pathway or the expression of microbial Neu5Ac-synthesizing enzymes. In this paper, we demonstrate that the plant Kdo-8P synthase is not able to use N-acetyld-mannosamine as a substrate, and thus re-routing of the Kdo pathway for the synthesis of Neu5Ac is not possible. Consequently, we expressed genes encoding Neu5Ac lyase from Escherichia coli and Neu5Ac synthase ( neuB2) from Campylobacter jejuni in plants. These resulted in the production of functional enzymes in the cytosol, which in turn can catalyse the synthesis of Neu5Ac in vitro. Experiments were carried out on two models, Bright Yellow 2 (BY2) tobacco cells and Medicago sativa (alfalfa), the perennial legume crop. [ABSTRACT FROM AUTHOR]

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core α(1,3)-fucose, as well as terminal Lewisa epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a α(1,3)-fucose and a β(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal β(1,4)-galactose residues as observed in the mammalian IgG. [ABSTRACT FROM AUTHOR]