Emerging Technologies for Making Glycan-Defined Glycoproteins

Wang, Lai-Xi; Lomino, Joseph V.

doi:10.1021/cb200429n

Cited by 138 publications

(125 citation statements)

References 123 publications

(241 reference statements)

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“…Most of the presently available glycoengineering techniques address and alter the processing machinery by knocking down, inhibiting, or overexpressing glycosylation enzymes (22,25,30,32,55,56) and thus are designed to modify simultaneously all the glycans carried by a given protein. Combining site-directed glycoprotein mutagenesis and classic glycoengineering techniques toward the limiting enzymes, as determined by flux analysis, can yield specific and targeted distributions of glycan on a single site.…”

Section: Discussionmentioning

confidence: 99%

“…Prime examples are the N-glycan structure of the Fc-domain glycan in IgG molecules (18) or the high mannose structure on gp120 of HIV as part of the epitope of neutralizing antibodies (19). Recently, glycan microheterogeneity has become a major hindrance in the production of glycoproteins (20)(21)(22). Chinese hamster ovary (CHO) cells are a mammalian cell line frequently used to produce recombinant glycoprotein (23)(24)(25).…”

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“…Altering glycostructures in production processes is mostly done by variation of production conditions or by changing the activity level of specific glycosyltransferases and glycosyl hydrolases and has primarily been studied on glycoproteins with a single N-glycosylation site (monoclonal antibody) (22,(29)(30)(31)(32)(33). The analysis of site-specific microheterogeneity on a protein with multiple N-glycosylation sites in cell or tissue-derived extracts requires more elaborate analytical methods that have become available only recently (16,17,(34)(35)(36).…”

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Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/ protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity. FASEB J. 31, 4623-4635 (2017). www.fasebj.org KEY WORDS: glycoengineering • glycobiology • glycoprotein maturation • Golgi processing • enzymatic flux N-glycosylation is a major posttranslational modification of proteins that modulates folding, maturation, trafficking, and secretion, as well as specific protein functions (1-3). Contrary to many other posttranslational modifications, N-glycans are composed of many monosaccharide units, creating a large diversity of glycan structures (4, 5). In eukaryotic cells, the N-glycosylation occurs in the endoplasmic reticulum (ER) after a conserved pathway. The oligosaccharide GlcNAc 2 Man 9 Glc 3 is assembled on the lipid dolicholpyrophosphate at the membrane of the ER and then is covalently linked to the side-chain amide of the asparagine residue in the sequon N-X-S/T by oligosaccharyltransferase (OST) (1, 6, 7). Multiple sites on one glycoprotein can be modified by the OST complex and with the increasing complexity of multicellular organism, an increasing number of N-glycosylation sites in the glycoproteome are observed. It is estimated that up to 6000 different sites are modified by OST in mammalian cells (8). In the second phase of the N-glycosylation pathway, the protein-linked glycan is trimmed by glucosidases and mannosidases (1, 9) and is subsequently modified by different glycosyltransferases spatially distributed along the secretory pathway. N-glycan processing enzymes have defined substrate specificities and locations in the secretory pathway (9, 10), and their expression level can be regulated upon internal and external signals.Glycan maturation in the secretory pathway is not a template-driven process. The...

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Section: Discussionmentioning

confidence: 99%

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Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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“…Many elegant chemical and biochemical strategies have been explored for making homogeneous glycoproteins and mimics, including total chemical synthesis with native chemical ligation (13-17), chemoselective ligation (18, 19), chemoenzymatic synthesis (20 -24), and glycosylation pathway engineering in host expression systems (25-28). As part of these efforts, we have attempted to develop a chemoenzymatic method for construction of complex N-glycopeptides and glycoproteins that is based on the transglycosylation activity of a class of endo-␤-N-acetylglucosaminidases (the endoglycosidases that hydrolyze N-glycans of glycoproteins) for convergent native ligation of preassembled glycans and GlcNAc-peptide/ protein (20,23,24). The success of this approach relies on the availability of novel endoglycosidase-derived glycosynthase mutants that are devoid of hydrolysis activity but can use the highly activated glycan oxazolines as substrates for transglycosylation.…”

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Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

Giddens

Lomino

Amin

et al. 2016

Journal of Biological Chemistry

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115

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Chemoenzymatic synthesis is emerging as a promising approach to the synthesis of homogeneous glycopeptides and glycoproteins highly demanded for functional glycomics studies, but its generality relies on the availability of a range of enzymes with high catalytic efficiency and well defined substrate specificity. We describe in this paper the discovery of glycosynthase mutants derived from Elizabethkingia meningoseptica endoglycosidase F3 (Endo-F3) of the GH18 family, which are devoid of the inherent hydrolytic activity but are able to take glycan oxazolines for transglycosylation. Notably, the Endo-F3 D165A and D165Q mutants demonstrated high acceptor substrate specificity toward ␣1,6-fucosyl-GlcNAc-Asn or ␣1, 6-fucosyl-GlcNAc-polypeptide in transglycosylation, enabling a highly convergent synthesis of core-fucosylated, complex CD52 glycopeptide antigen. The Endo-F3 mutants were able to use both bi-and triantennary glycan oxazolines as substrates for transglycosylation, in contrast to previously reported endoglycosidases derived from Endo-S, Endo-M, Endo-D, and Endo-A mutants that could not recognize triantennary N-glycans. Using rituximab as a model system, we have further demonstrated that the Endo-F3 mutants are highly efficient for glycosylation remodeling of monoclonal antibodies to produce homogeneous intact antibody glycoforms. Interestingly, the new triantennary glycan glycoform of antibody showed much higher affinity for galectin-3 than that of the commercial antibody. The Endo-F3 mutants represent the first endoglycosidase-based glycosynthases capable of transferring triantennary complex N-glycans, which would be very useful for glycoprotein synthesis and glycosylation remodeling of antibodies.Protein glycosylation is one of the most prevalent posttranslational modifications, found in almost all living organisms ranging from bacteria to eukaryotes (1). Glycosylation can profoundly affect a protein's intrinsic properties, such as folding, stability, intracellular trafficking, and immunogenicity. In addition, the oligosaccharide components of glycoproteins can participate directly in a number of important biological recognition processes, including cell adhesion, signaling, hostpathogen interactions, and immune responses (2-8). It is well documented that subtle changes in glycosylation can lead to a significant impact on the biological functions and, in the case of therapeutic glycoproteins, such as monoclonal antibodies, the in vivo stability and therapeutic efficacy (7, 9 -12). Natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ only in the structures of pendant glycans, from which pure glycoforms are extremely difficult to isolate by current chromatographic techniques. As a result, synthetic homogeneous glycopeptides and glycoproteins emerge as indispensable tools for functional studies and for drug/vaccine discoveries. Many elegant chemical and biochemical strategies have been explored for making homogeneous glycoproteins and mimics, including total chemic...

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“…Glycocluster‐based dendrimers, liposomes, and nanoparticles have been examined using in vivo kinetics and/or biodistribution studies 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. In an effort to design multivalency into certain proteins of interest, several studies have attempted to immobilize monosaccharides or glycans onto proteins using bioorthogonal click reactions,30, 31, 32, 33 reactions around the amino group in lysine residues25, 30, 34, 35 or the thiol group in cysteine,30, 36, 37, 38 or using enzymatic glycosylation 30, 39, 40, 41…”

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confidence: 99%