Endoglycosidase-catalyzed Transglycosylation Research Articles

N-Glycan Engineering: Constructing the N-GlcNAc Stump.

N-Glycosylation is often essential for the structure and function of proteins. However, N-glycosylated proteins from natural sources exhibit considerable heterogeneity in the appended oligosaccharides, bringing daunting challenges to corresponding basic research and therapeutic applications. To address this issue, various synthetic, enzymatic, and chemoenzymatic approaches have been elegantly designed. Utilizing the endoglycosidase-catalyzed transglycosylation method, a single N-acetylglucosamine (N-GlcNAc, analogous to a tree stump) on proteins can be converted to various homogeneous N-glycosylated forms, thereby becoming the focus of research efforts. In this concept article, we briefly introduce the methods that allow the generation of N-GlcNAc and its close analogues on proteins and peptides and highlight the current challenges and opportunities the scientific community is facing.

Site-Specific Protein Modification with Reducing Carbohydrates.

Protein glycosylation plays critical roles in many biological processes. However, the fundamental study and application of glycobiology are hindered by the heterogeneousness of oligosaccharides in natural glycoproteins and the difficulty in constructing glycoproteins of human design. Herein, we describe a semisynthetic method to site-specifically modify proteins with reducing carbohydrates. The method involves the genetic incorporation of a side-chain-esterified aspartate, which was subsequently quantitatively converted into alanine-β-hydrazide (Aβz), and chemoselective conjugation of Aβz with a range of readily available reducing carbohydrates. The resulting Aβz-linked GlcNAc is a close mimic of native N-GlcNAc and could be installed on various proteins, including IL-17A and RNase A. Notably, Aβz-linked GlcNAc on proteins reacted with biantennary oligosaccharide oxazoline derivatives through endoglycosidase-catalyzed transglycosylation reactions to enable the assembly of homogeneous glycans on proteins.

Chemoenzymatic Glyco-engineering of Monoclonal Antibodies.

Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications.

Expeditious chemoenzymatic synthesis of CD52 glycopeptide antigens

CD52 is a glycosylphosphatidylinositol (GPI)-anchored glycopeptide antigen found on sperm cells and human lymphocytes. Recent structural studies indicate that sperm-associated CD52 antigen carries both a complex type N-glycan and an O-glycan on the polypeptide backbone. To facilitate functional and immunological studies of distinct CD52 glycoforms, we report in this paper the first chemoenzymatic synthesis of homogeneous CD52 glycoforms carrying both N- and O-glycans. The synthetic strategy consists of two key steps: monosaccharide primers GlcNAc and GalNAc were first installed at the pre-determined N- and O-glycosylation sites by a facile solid-phase peptide synthesis, and then the N- and O-glycans were extended by respective enzymatic glycosylations. It was found that the endoglycosidase-catalyzed transglycosylation allowed efficient attachment of an intact N-glycan in a single step at the N-glycosylation site, while the recombinant human T-synthase could independently extend the O-linked GalNAc to form the core 1 O-glycan. This chemoenzymatic approach is highly convergent and permits easy construction of various homogeneous CD52 glycoforms from a common polypeptide precursor. In addition, the introduction of a latent thiol group in the form of protected cysteamine at the C-terminus of the CD52 glycoforms will enable site-specific conjugation to a carrier protein to provide immunogens for generating CD52 glycoform-specific antibodies for functional studies.

Chemoenzymatic Synthesis and Lectin Array Characterization of a Class of N-Glycan Clusters

N-Glycans are major components of many glycoproteins. These sugar moieties are frequently involved in important physiological and disease processes via their interactions with a variety of glycan-binding proteins (GBP). Clustering effect is an important feature in many glycan-lectin interactions. We describe in this paper a chemoenzymatic synthesis of novel N-glycan clusters using a tandem endoglycosidase-catalyzed transglycosylation. It was found that the internal beta-1,2-linked GlcNAc moieties in the N-glycan core, once exposed in the nonreducing terminus, was able to serve as acceptors for transglycosylation catalyzed by Endo-A and EndoM-N175A. This efficient chemoenzymatic method allows a quick extension of the sugar chains to form a class of glycan clusters in which sugar residues are all connected by native glycosidic linkages found in natural N-glycans. In addition, a discriminative enzymatic reaction at the two GlcNAc residues could be fulfilled to afford novel hybrid clusters. Lectin microarray studies revealed unusual properties in glyco-epitope expression by this panel of structurally well-defined synthetic N-glycans. These new compounds are likely valuable for functional glycomics studies to unveil new functions of both glycans and carbohydrate-binding proteins.

Enzymatic Glycosylation of Triazole‐Linked GlcNAc/Glc–Peptides: Synthesis, Stability and Anti‐HIV Activity of Triazole‐Linked HIV‐1 gp41 Glycopeptide C34 Analogues

Long-lasting sweet proteins: The chemoenzymatic synthesis of a triazole (T)-linked glycosylated C34 fragment from HIV-1 gp41 is described. The glycopeptide shows high solubility, excellent fusion inhibition, and as shown in the graph, promising protease resistance. Endoglycosidase-catalyzed transglycosylation of triazole-linked glucose (Glc) and N-acetylglucosamine (GlcNAc)-containing dipeptides and polypeptides was achieved by using synthetic sugar oxazoline as the donor substrate. It was found that both N- and C-linked Glc/GlcNAc-containing triazole derivatives were effective substrates for endo-beta-N-acetylglucosaminidase from Arthrobacter (Endo-A) for transglycosylation; this demonstrates a broad acceptor substrate specificity for Endo-A. This chemoenzymatic method was successfully used for the synthesis of a novel triazole-linked C34 glycopeptide derived from the HIV-1 envelope glycoprotein, gp41. We found that the synthetic C34 glycopeptide possesses potent anti-HIV activity with an IC(50) of 21 nM. The triazole-linked C34 glycopeptide demonstrated a much enhanced stability against protease- and glycoamidase-catalyzed digestion; this shows the protective effects of glycosylation and the stability of the triazole linkage. These favorable properties suggest that the triazole-linked C34 glycopeptide might be valuable for further development as an anti-HIV drug candidate.

Glycoengineering of Human IgG1-Fc through Combined Yeast Expression and in Vitro Chemoenzymatic Glycosylation

The presence and precise structures of the glycans attached at the Fc domain of monoclonal antibodies play an important role in determining antibodies' effector functions such as antibody-dependent cell cytotoxicity (ADCC), complement activation, and anti-inflammatory activity. This paper describes a novel approach for glycoengineering of human IgG1-Fc that combines high-yield expression of human IgG1-Fc in yeast and subsequent in vitro enzymatic glycosylation, using the endoglycosidase-catalyzed transglycosylation as the key reaction. Human IgG1-Fc was first overproduced in Pichia pastoris. Then the heterogeneous yeast glycans were removed by Endo-H treatment to give the GlcNAc-containing IgG1-Fc as a homodimer. Finally, selected homogeneous glycans were attached to the GlcNAc-primer in the IgG1-Fc through an endoglycosidase-catalyzed transglycosylation, using sugar oxazolines as the donor substrates. It was found that the enzymatic transglycosylation was efficient with native GlcNAc-containing IgG1-Fc homodimer without the need to denature the protein, and the reaction could proceed to completion to give homogeneous glycoforms of IgG1-Fc when an excess of oligosaccharide oxazolines was used as the donor substrates. The binding of the synthetic IgG1-Fc glycoforms to the FcgammaIIIa receptor was also investigated. This novel glycoengineering approach should be useful for providing various homogeneous, natural or synthetic glycoforms of IgG1-Fc for structure-function relationship studies, and for future clinical applications.

Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation

Homogeneous glycopeptides and glycoproteins are indispensable for detailed structural and functional studies of glycoproteins. It is also fundamentally important to correct glycosylation patterns for developing effective glycoprotein-based therapeutics. This review discusses a useful chemoenzymatic method that takes advantage of the endoglycosidase-catalyzed transglycosylation to attach an intact oligosaccharide to a polypeptide in a single step, without the need for any protecting groups. The exploration of sugar oxazolines (enzymatic reaction intermediates) as donor substrates has not only expanded substrate availability, but also has significantly enhanced the enzymatic transglycosylation efficiency. Moreover, the discovery of a novel mutant with glycosynthase-like activity has made it possible to synthesize homogeneous glycoproteins with full-size natural N-glycans. Recent advances in this highly convergent chemoenzymatic approach and its application for glycopeptide and glycoprotein synthesis are highlighted.

Glycopeptide Synthesis through endo‐Glycosidase‐Catalyzed Oligosaccharide Transfer of Sugar Oxazolines: Probing Substrate Structural Requirement

An array of sugar oxazolines was synthesized and tested as donor substrates for the Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A)-catalyzed glycopeptide synthesis. The experiments revealed that the minimum structure of the donor substrate required for Endo-A catalyzed transglycosylation is a Man beta1-->4-GlcNAc oxazoline moiety. Replacement of the beta-D-Man moiety with beta-D-Glc, beta-D-Gal, and beta-D-GlcNAc monosaccharides resulted in the loss of substrate activity for the disaccharide oxazoline. Despite this, the enzyme could tolerate modifications such as attachment of additional sugar residues or a functional group at the 3- and/or 6-positions of the beta-D-Man moiety, thus allowing a successful transfer of selectively modified oligosaccharides to the peptide acceptor. On the other hand, the enzyme has a great flexibility for the acceptor portion and could take both small and large GlcNAc-peptides as the acceptor. The studies implicate a great potential of the endoglycosidase-catalyzed transglycosylation for constructing both natural and selectively modified glycopeptides.

Chemoenzymatic Synthesis of HIV-1 V3 Glycopeptides Carrying Two N-Glycans and Effects of Glycosylation on the Peptide Domain

[structure: see text] A highly efficient chemoenzymatic synthesis of HIV-1 V3 domain glycopeptides carrying two N-linked core tri- and pentasaccharides was achieved. The synthesis consisted of two key steps: a solid-phase synthesis of the cyclic, 47-mer V3 domain peptide containing two GlcNAc residues and a novel endoglycosidase-catalyzed transglycosylation that simultaneously added two N-glycan moieties to the peptide precursor from the oligosaccharide oxazoline donor substrates. The availability of the synthetic glycopeptides allowed the probing of the effects of glycosylation on the HIV-1 V3 domain. It was demonstrated that glycosylation influenced the global conformations of the V3 domain and provided protection of the V3 domain against protease digestion.