Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

Yang, Qiang; An, Yanming; Zhu, Shilei; Zhang, Roushu; Loke, Chun Mun; Cipollo, John F.; Wang, Lai-Xi

doi:10.1021/acschembio.7b00282

Cited by 44 publications

(27 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore the high mannose type is an ideal substrate for Endo-H digestion, leaving a terminal GlcNac at the asparagine residue at each N-glycosylation site. An engineered glycosynthase based on Endo-M (EndoM-N175A) is known to be able to transfer N-glycans, including sialylated biantennary complex types based on an oxazoline derivate to a single GlcNAc residue 33 . For a further improvement of the glycan transfer to the GlcNAc residues a second acceptor substrate composed of a core focusylated EPO glycoform was investigated (Fucα1,6GlcNAc-EPO).…”

Section: Discussionmentioning

confidence: 99%

Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin

Zemella

Thoring

Hoffmeister

et al. 2018

Sci Rep

View full text Add to dashboard Cite

As one of the most complex post-translational modification, glycosylation is widely involved in cell adhesion, cell proliferation and immune response. Nevertheless glycoproteins with an identical polypeptide backbone mostly differ in their glycosylation patterns. Due to this heterogeneity, the mapping of different glycosylation patterns to their associated function is nearly impossible. In the last years, glycoengineering tools including cell line engineering, chemoenzymatic remodeling and site-specific glycosylation have attracted increasing interest. The therapeutic hormone erythropoietin (EPO) has been investigated in particular by various groups to establish a production process resulting in a defined glycosylation pattern. However commercially available recombinant human EPO shows batch-to-batch variations in its glycoforms. Therefore we present an alternative method for the synthesis of active glycosylated EPO with an engineered O-glycosylation site by combining eukaryotic cell-free protein synthesis and site-directed incorporation of non-canonical amino acids with subsequent chemoselective modifications.

show abstract

Section: Discussionmentioning

confidence: 99%

Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin

Zemella

Thoring

Hoffmeister

et al. 2018

Sci Rep

View full text Add to dashboard Cite

show abstract

“…The mutant was capable of synthesizing asialo biantennary and complex triantennary core-fucosylated glycoforms of rituximab (intact antibody) in yields over 95%. Further applications, indicating the high potential of the method for the synthesis of pharmaceutical relevant compounds were the chemoenzymatic production of vaccine candidates [ 72 ]; the site-selective glycosylation of HIV-1 polypeptide antigen bearing two different glycans (yields up to 95%) [ 73 ]; the glycan remodeling of human erythropoietin (EPO) [ 74 ]; and the synthesis of mannose-6-phosphate-containing glycoproteins [ 75 ]. Tang et al impressively demonstrated a one-pot N -glycan remodeling of IgG proteins by combining the wild type ( wt ) Endo-M glycosidase with the synthase variant Endo-S D322S [ 76 ].…”

Section: Glycoside Syntheses Using Glycosynthase Methodsmentioning

confidence: 99%

Synthesis of Glycosides by Glycosynthases

Hayes

Pietruszka

2017

Molecules

View full text Add to dashboard Cite

The many advances in glycoscience have more and more brought to light the crucial role of glycosides and glycoconjugates in biological processes. Their major influence on the functionality and stability of peptides, cell recognition, health and immunity and many other processes throughout biology has increased the demand for simple synthetic methods allowing the defined syntheses of target glycosides. Additional interest in glycoside synthesis has arisen with the prospect of producing sustainable materials from these abundant polymers. Enzymatic synthesis has proven itself to be a promising alternative to the laborious chemical synthesis of glycosides by avoiding the necessity of numerous protecting group strategies. Among the biocatalytic strategies, glycosynthases, genetically engineered glycosidases void of hydrolytic activity, have gained much interest in recent years, enabling not only the selective synthesis of small glycosides and glycoconjugates, but also the production of highly functionalized polysaccharides. This review provides a detailed overview over the glycosylation possibilities of the variety of glycosynthases produced until now, focusing on the transfer of the most common glucosyl-, galactosyl-, xylosyl-, mannosyl-, fucosyl-residues and of whole glycan blocks by the different glycosynthase enzyme variants.

show abstract

“…Consequently, in vitro glycan remodeling was developed to offer the advantage of adding customized oligosaccharides on recombinant proteins to produce a single uniform glycoform [ 26 , 38 , 39 ]. In particular, using the endoglycosidase-catalyzed glycan remodeling approach, N-linked glycoproteins are enzymatically deglycosylated such that the innermost GlcNAc or Fucα-1,6GlcNAc remains on the asparagine residue of the protein [ 40 , 41 , 42 , 43 ]. This approach allows for an N -glycan oxazoline substrate to be transferred en bloc onto that remaining GlcNAc of recombinant proteins to reconstitute homogeneous glycoproteins.…”

Section: Introductionmentioning

confidence: 99%

Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies

Bennett¹,

Yang

Berquist

et al. 2018

IJMS

Self Cite

View full text Add to dashboard Cite

N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or β-1,2-xylose residues was produced. When compared with the innovator product Rituxan®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics’ glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies.

show abstract

Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

Cited by 44 publications

References 45 publications

Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin

Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin

Synthesis of Glycosides by Glycosynthases

Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies

Contact Info

Product

Resources

About