A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation

Schwarz, Flavio; Huang, Wei; Li, Cishan; Schulz, Benjamin L.; Lizak, Christian; Palumbo, Alessandro; Numao, Shin; Neri, Dario; Aebi, Markus; Wang, Lai-Xi

doi:10.1038/nchembio.314

Cited by 179 publications

(119 citation statements)

References 25 publications

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“…Recently, an innovative glycoprotein production system using the Campylobacter N-glycosylation machinery was reported (32). The key component of the machinery is Campylobacter PglB.…”

Section: Discussionmentioning

confidence: 99%

Selective Control of Oligosaccharide Transfer Efficiency for the N-Glycosylation Sequon by a Point Mutation in Oligosaccharyltransferase

Igura

Kohda

2011

Journal of Biological Chemistry

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Asn-linked glycosylation is the most ubiquitous posttranslational protein modification in eukaryotes and archaea, and in some eubacteria. Oligosaccharyltransferase (OST) catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. Inefficient oligosaccharide transfer results in glycoprotein heterogeneity, which is particularly bothersome in pharmaceutical glycoprotein production. Amino acid variation at the X position of the Asn-XSer/Thr sequon is known to modulate the glycosylation efficiency. The best amino acid at X is valine, for an archaeal Pyrococcus furiosus OST. We performed a systematic alanine mutagenesis study of the archaeal OST to identify the essential and dispensable amino acid residues in the three catalytic motifs. We then investigated the effects of the dispensable mutations on the amino acid preference in the N-glycosylation sequon. One residue position was found to selectively affect the amino acid preference at the X position. This residue is located within the recently identified DXXKXXX(M/I) motif, suggesting the involvement of this motif in N-glycosylation sequon recognition. In applications, mutations at this position may facilitate the design of OST variants adapted to particular N-glycosylation sites to reduce the heterogeneity of glycan occupancy. In fact, a mutation at this position led to 9-fold higher activity relative to the wild-type enzyme, toward a peptide containing arginine at X in place of valine. This mutational approach is potentially applicable to eukaryotic and eubacterial OSTs for the production of homogenous glycoproteins in engineered mammalian and Escherichia coli cells.N-linked glycosylation is the most ubiquitous posttranslational protein modification in eukaryotes and archaea, and in some eubacteria (1-4). It plays an important role in determining the biological activities of glycoproteins as well as the medicinal efficacy of many pharmaceutical recombinant glycoproteins. The covalent attachment of an oligosaccharide occurs on the side chain of an asparagine residue in a polypeptide chain, within the Asn-X-Ser and Asn-X-Thr sequences, where X is any amino acid except for proline (5, 6). About one-third of the potential N-glycosylation sequons are not glycosylated in extracellular proteins (7-9). This fact indicates that the presence of a consensus sequence is essential but not sufficient for N-glycosylation. The percentage of proteins modified at a particular sequon is referred to as "site occupancy." A recent comprehensive proteome analysis of mouse tissues and blood plasma revealed that more than 98% of N-linked glycosylated peptides derived from glycoproteins were not found in their unmodified form, indicating the high occupancy of N-glycosylation sites (10). Thus, for most N-glycosylation sequons in vivo, the N-glycan attachment is robust, resulting in a homogeneously glycosylated (100% occupancy) or unglycosylated (0% occupancy) site.In contrast, for some recombinant glycoproteins produced in cultur...

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“…Recently, an innovative glycoprotein production system using the Campylobacter N-glycosylation machinery was reported (32). The key component of the machinery is Campylobacter PglB.…”

Section: Discussionmentioning

confidence: 99%

Selective Control of Oligosaccharide Transfer Efficiency for the N-Glycosylation Sequon by a Point Mutation in Oligosaccharyltransferase

Igura

Kohda

2011

Journal of Biological Chemistry

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“…Furthermore, as NGTs are encoded on the genome of relevant pathogens, it will be interesting to study how the immune system of the hosts reacts to this type of N-glycosylation. Finally, NGTs represent a promising tool that complements established platforms for production of glycoconjugates and for site-specific modification of proteins (35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Schwarz

Fan

Schubert

et al. 2011

Journal of Biological Chemistry

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N-Linked glycosylation is a frequent protein modification that occurs in all three domains of life. This process involves the transfer of a preassembled oligosaccharide from a lipid donor to asparagine side chains of polypeptides and is catalyzed by the membrane-bound oligosaccharyltransferase (OST). We characterized an alternative bacterial pathway wherein a cytoplasmic N-glycosyltransferase uses nucleotide-activated monosaccharides as donors to modify asparagine residues of peptides and proteins. N-Glycosyltransferase is an inverting glycosyltransferase and recognizes the NX(S/T) consensus sequence. It therefore exhibits similar acceptor site specificity as eukaryotic OST, despite the unrelated predicted structural architecture and the apparently different catalytic mechanism. The identification of an enzyme that integrates some of the features of OST in a cytoplasmic pathway defines a novel class of N-linked protein glycosylation found in pathogenic bacteria.N-Linked glycosylation is characterized by an N-glycosidic linkage between the side chain amide of an asparagine residue of proteins and an oligosaccharide. This type of glycosylation occurs in both prokaryotes and eukaryotes and requires the assembly of an oligosaccharide on a polyisoprenoid lipid by sequential addition of monosaccharides, catalyzed by cytosolic glycosyltransferase. The resulting lipid-linked oligosaccharide is translocated to the luminal side of the endoplasmic reticulum membrane or the plasma membrane of prokaryotes, where it may be further elongated. The glycan is then transferred to the ␦-amino group of asparagine residues within the consensus sequence NX(S/T) of polypeptides. This reaction is catalyzed by oligosaccharyltransferase (OST), 4 a membrane-bound enzyme that can be composed of several different subunits (1). As oligosaccharide transfer takes place in the endoplasmic reticulum or in the periplasm, N-glycosylation affects proteins trafficking along the secretory pathway.N-Glycosylation exhibits important physiological functions. In the early secretory pathway of eukaryotes, N-glycans present on newly synthesized proteins orchestrate the folding of glycoproteins and act as a signal for directing misfolded polypeptides to degradation (2). After being processed in the Golgi organelle, N-linked glycans are relevant, among other processes, for the modulation of the immune system and for the control of immune cell homeostasis and inflammation (3, 4). The importance of this protein modification is supported by its incidence: more than half of all eukaryotic proteins are glycosylated (5).About 8 years ago, a study uncovered that the extracellular HMW1A adhesin of the Gram-negative bacterium Haemophilus influenzae is modified with hexose monosaccharides on asparagine residues (6). The pioneering work of St Geme and co-workers (7, 8) subsequently showed that the modified asparagine residues are found within the same consensus sequence recognized by OST (i.e. NX(S/T)) and that the enzyme responsible for this modification is the HMW1C p...

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“…However, variations in extravasation rates and disease-homing properties may often go unnoticed, unless quantitative biodistribution studies are performed. The findings of this study suggest that the engineering and development of biopharmaceuticals may favor either a complete absence of glycostructures or, alternatively, the engineering of welldefined sialylated carbohydrates (25)(26)(27). Pharmacokinetic and biodistribution studies combined with thorough glycan profiling appear mandatory for therapeutic glycoproteins, especially when the production process is modified or when biosimilar products are developed.…”

Section: Resultsmentioning

confidence: 99%

Glycosylation profiles determine extravasation and disease-targeting properties of armed antibodies

Venetz¹,

Hess²,

Lin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The ability of antibodies to extravasate out of blood vessels is critical for therapeutic activity, because molecular targets for most diseases are located outside of the endothelial lining. By performing detailed biodistribution studies with a novel IL9-armed cancer-specific antibody, we identified a clear correlation between N-linked glycan structures and tumor-targeting efficiencies. Site-specific glycan analysis provided a detailed view of the glycan microheterogeneity present on the IL9 portion of the recombinant protein. Nonsialylated glycan structures have a negative impact on disease-homing activity, highlighting the importance of glycosylation control and characterization during process development.

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A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation

Cited by 179 publications

References 25 publications

Selective Control of Oligosaccharide Transfer Efficiency for the N-Glycosylation Sequon by a Point Mutation in Oligosaccharyltransferase

Selective Control of Oligosaccharide Transfer Efficiency for the N-Glycosylation Sequon by a Point Mutation in Oligosaccharyltransferase

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Glycosylation profiles determine extravasation and disease-targeting properties of armed antibodies

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