Pharmacological significance of glycosylation in therapeutic proteins

Li, Huijuan; d’Anjou, Marc

doi:10.1016/j.copbio.2009.10.009

Cited by 210 publications

(138 citation statements)

References 65 publications

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“…Although ER/Golgi-derived glycosylation is one of the most frequently occurring classes of PTMs (6), these extracellular modifications continue to be a major challenge because of technical difficulties both in their structural characterization and in deciphering their biological roles. However, there is a renewed interest in the characterization of glycosylation driven by the production of protein pharmaceuticals as well as by the search for biomarkers and by new findings about the function(s) of glycosylation (7)(8)(9)(10)(11)(12).…”

Section: Post-translational Modifications (Ptms)mentioning

confidence: 99%

N- and O-Glycosylation in the Murine Synaptosome

Trinidad

Schoepfer²,

Burlingame

et al. 2013

Molecular & Cellular Proteomics

162

230

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We present the first large scale study characterizing both N-and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N-and O-linked carbohydrate structures. Liquid chromatography-MS (LC/ MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N-and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues. Molecular & Cellular

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Section: Post-translational Modifications (Ptms)mentioning

confidence: 99%

N- and O-Glycosylation in the Murine Synaptosome

Trinidad

Schoepfer²,

Burlingame

et al. 2013

Molecular & Cellular Proteomics

162

230

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“…The high protease activity in the BY-2 medium (Robertson et al, 1997;Okushima et al, 2000) may require, for the production of some pharmaceutical proteins, tailored knockout of specific proteases. It is well established that N-and O-linked glycosylation may enhance protein stability, probably by acting as a physical barrier to protease-mediated degradation (Li and d'Anjou, 2009). The finding that O-linked GalNAc glycosylation protected the MUC1 protein offers an alternative approach to combating protease-mediated degradation.…”

Section: Discussionmentioning

confidence: 99%

“…N-and O-linked glycosylation are often essential for biological activity and pharmacokinetic properties (Li and d'Anjou, 2009). Recombinant production of glycoprotein therapeutics has so far generally aimed to mimic the glycosylation found on naturally produced human glycoproteins, primarily to avoid adverse immunological reactivity and improve circulatory half-life.…”

mentioning

confidence: 99%

Toward Stable Genetic Engineering of Human O-Glycosylation in Plants

et al. 2012

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Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating GalNAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O-glycoproteins was obtained, but a high degree of proline hydroxylation and hydroxyproline-linked arabinosides, on a mucin (MUC1)-derived substrate, was also observed. Addition of the prolyl 4-hydroxylase inhibitor 2,2-dipyridyl, however, effectively suppressed proline hydroxylation and arabinosylation of MUC1 in Bright Yellow-2 cells. In summary, stably engineered mammalian type O-glycosylation was established in transgenic plants, demonstrating that plants may serve as host cells for the production of recombinant O-glycoproteins. However, the present stable implementation further strengthens the notion that elimination of endogenous posttranslational modifications may be needed for the production of protein therapeutics.

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“…More than two-thirds of the marketed therapeutic proteins are glycoproteins (Li and d'Anjou 2009), produced predominantly in mammalian cell culture systems, with Chinese hamster ovary (CHO) cell systems being the most widely used production host (Durocher and Butler 2009). CHO cells have emerged as the preferred host due to their extensive characterization and human-like glycosylation, particularly in comparison with murine cell lines.…”

Section: Introductionmentioning

confidence: 99%

Effects of clonal variation on growth, metabolism, and productivity in response to trophic factor stimulation: a study of Chinese hamster ovary cells producing a recombinant monoclonal antibody

Dahodwala

Nowey

Mitina³

et al. 2011

Cytotechnology

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The growth, metabolism, and productivity of five Chinese hamster ovary (CHO) clones were explored in response to stimulation with insulin (5 mg/L) and LONG Ò R 3 IGF-I (20 lg/L or 100 lg/L). All five clones were derived from the same parental CHO cell line (DG44) and produced the same recombinant monoclonal antibody, with varying specific productivities. There was no uniform response among the clones to stimulation with the different trophic factors. One of the high productivity clones (clone D) exhibited significantly better growth in response to LONG Ò R 3 IGF-I; whereas the other clones showed equivalent or slightly better growth in the presence of insulin. Three out of the five clones had higher specific productivities in the presence of insulin (although not statistically significant); one was invariant, and the final clone exhibited slightly higher specific productivity in the presence of LONG Ò R 3 IGF-I. Total product titers exhibited moderate variation between culture conditions, again with neither trophic factor being clearly superior. Overall product titers were affected by variations in both integrated viable cell density and specific productivity. Nutrient uptake and metabolite generation patterns varied strongly between clones and much less with culture conditions. These results point to the need for careful clonal analysis when selecting clones, particularly for platform processes where media and culture conditions are predetermined.

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Pharmacological significance of glycosylation in therapeutic proteins

Cited by 210 publications

References 65 publications

N- and O-Glycosylation in the Murine Synaptosome

N- and O-Glycosylation in the Murine Synaptosome

Toward Stable Genetic Engineering of Human O-Glycosylation in Plants

Effects of clonal variation on growth, metabolism, and productivity in response to trophic factor stimulation: a study of Chinese hamster ovary cells producing a recombinant monoclonal antibody

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