Pure Glycoforms Research Articles

Chemical and Enzymatic Synthesis of Sialylated Glycoforms of Human Erythropoietin

AbstractRecombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in‐vivo activity of EPO is carbohydrate‐dependent with the number of sialic acid residues regulating its circulatory half‐life. EPO carries threeN‐glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline‐assisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1 complexes with recombinant EPO receptor.

Chemical and Enzymatic Synthesis of Sialylated Glycoforms of Human Erythropoietin.

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in‐vivo activity of EPO is carbohydrate‐dependent with the number of sialic acid residues regulating its circulatory half‐life. EPO carries three N‐glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline‐assisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1 complexes with recombinant EPO receptor.

Glycosylation of Plant-Produced Immunoglobulins.

Many economically important protein-based therapeutics like monoclonal antibodies are glycosylated. Due to the recognized importance of this type of posttranslational modification, glycoengineering of expression systems to obtain highly active and homogenous therapeutics is an emerging field. Although most of the monoclonal antibodies on the market are still produced in mammalian expression platforms, plants are emerging as an alternative cost-effective and scalable production platform that allows precise engineering of glycosylation to produce targeted human glycoforms at large homogeneity. Apart from producing more effective antibodies, pure glycoforms are required in efforts to link biological functions to specific glycan structures. Much is already known about the role of IgG1 glycosylation and this antibody class is the dominant recombinant format that has been expressed in plants. By contrast, little attention has been paid to the glycoengineering of recombinant IgG subtypes and the other four classes of human immunoglobulins (IgA, IgD, IgE, and IgM). Except for IgD, all these antibody classes have been expressed in plants and the glycosylation has been analyzed in a site-specific manner. Here, we summarize the current data on glycosylation of plant-produced monoclonal antibodies and discuss the findings in the light of known functions for these glycans.

Recent Development in Ligation Methods for Glycopeptide and Glycoprotein Synthesis.

Glycoproteins are produced by the post-translational modification process of proteins and they play an important role in mediating various biological processes. Our understanding towards biochemical functions of individual glycoproteins has been seriously hampered due to the heterogeneous expression of carbohydrate parts in glycoproteins. Despite the advancement in recombinant expression and chromatographic techniques, the isolation of pure glycoforms remains nearly impossible. To obtain homogenous glycoproteins, tremendous efforts hves been spent in developing various ligation and glycosylation techniques. This minireview discusses selected methods for the preparation and ligation of glycopeptides. The importance of the development of new chemical synthesis method for glycoproteins has also been discussed, which would be one of the next directions in this field.

Glycoengineering of Antibodies for Modulating Functions.

Antibodies are immunoglobulins that play essential roles in immune systems. All antibodies are glycoproteins that carry at least one or more conserved N-linked oligosaccharides (N-glycans) at the Fc domain. Many studies have demonstrated that both the presence and fine structures of the attached glycans can exert a profound impact on the biological functions and therapeutic efficacy of antibodies. However, antibodies usually exist as mixtures of heterogeneous glycoforms that are difficult to separate in pure glycoforms. Recent progress in glycoengineering has provided useful methods that enable production of glycan-defined and site-selectively modified antibodies for functional studies and for improved therapeutic efficacy. This review highlights major approaches in glycoengineering of antibodies with a focus on recent advances in three areas: glycoengineering through glycan biosynthetic pathway manipulation, glycoengineering through in vitro chemoenzymatic glycan remodeling, and glycoengineering of antibodies for site-specific antibody-drug conjugation.

ChemInform Abstract: Design and Synthesis of Antifreeze Glycoproteins and Mimics

AbstractReview: [94 refs.]

Design and Synthesis of Antifreeze Glycoproteins and Mimics

Antifreeze glycoproteins are an important class of biological antifreezes that have potential applications in many areas of medicine, agriculture and industry in which ice crystal growth is damaging. While the synthesis of antifreeze glycoproteins as pure glycoforms has recently been achieved by using ligation and polymerisation strategies, the routine production of large quantities of pure glycoforms remains challenging. A range of C-linked analogues that are readily produced by solid-phase synthesis have delivered novel compounds that are not biological antifreezes, but are potent, non-cytotoxic, ice-recrystallisation inhibitors. Structure-activity studies, the identification of cyclic antifreeze glycoproteins and conformational studies have provided further insight into the requirements for antifreeze activity. These results, coupled with significant advances in approaches to the routine synthesis of different glycoproteins and mimics, present opportunities for the design and synthesis of novel ice-growth-inhibiting and antifreeze compounds.

Separation of α‐acid glycoprotein glycoforms using affinity‐based reversed micellar extraction and separation

A new method for the preparation of the glycoforms of bovine α1-acid glycoprotein (AGP) is described relying on affinity-reversed micellar extraction and separation (ARMES). This method has proven effective in separating structurally similar glycoproteins and separating glycoproteins from nonglycosylated proteins from natural sources. In this method, individual glycoforms complex with the lectin, concanavalin A (ConA) are extracted into an organic-phase reversed micellar solution formed by Aerosol OT (AOT). The purity of three AGP glycoforms isolated was assessed by hydroxyapatite high-performance liquid chromatography (HPLC), gel-permeation chromatography and SDS-PAGE. The glycan structure of the pure glycoforms was analyzed. Oligosaccharide mapping using capillary electrophoresis (CE) and PAGE showed the glycans obtained from each glycoform to be distinctly different. ARMES can be used for the semi-preparative scale resolution of the glycoforms of bovine AGP or other therapeutic glycoproteins. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 484–490, 2000.

Separation of α-acid glycoprotein glycoforms using affinity-based reversed micellar extraction and separation

A new method for the preparation of the glycoforms of bovine alpha(1)-acid glycoprotein (AGP) is described relying on affinity-reversed micellar extraction and separation (ARMES). This method has proven effective in separating structurally similar glycoproteins and separating glycoproteins from nonglycosylated proteins from natural sources. In this method, individual glycoforms complex with the lectin, concanavalin A (ConA) are extracted into an organic-phase reversed micellar solution formed by Aerosol OT (AOT). The purity of three AGP glycoforms isolated was assessed by hydroxyapatite high-performance liquid chromatography (HPLC), gel-permeation chromatography and SDS-PAGE. The glycan structure of the pure glycoforms was analyzed. Oligosaccharide mapping using capillary electrophoresis (CE) and PAGE showed the glycans obtained from each glycoform to be distinctly different. ARMES can be used for the semi-preparative scale resolution of the glycoforms of bovine AGP or other therapeutic glycoproteins.