Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation.

Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Because the glycosylation of mAbs in the Fc region influences their interaction with effector cells that kill antibody-targeted cells, and the current method of antibody production is relatively expensive, efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAbs with desirable glycoforms. In this study, we demonstrate that the mAb trastuzumab expressed in glycoengineered P. pastoris can be remodeled through deglycosylation by endoglycosidases identified from the Carbohydrate Active Enzymes database and through transglycosylation using glycans with a stable leaving group to generate a homogeneous antibody designed to optimize the effector functions. The 10 newly identified recombinant bacterial endoglycosidases are complementary to existing endoglycosidases (EndoA, EndoH, EndoS), two of which can even accept sialylated tri- and tetraantennary glycans as substrates.

Influenza A surface glycosylation and vaccine design.

We have shown that glycosylation of influenza A virus (IAV) hemagglutinin (HA), especially at position N-27, is crucial for HA folding and virus survival. However, it is not known whether the glycosylation of HA and the other two major IAV surface glycoproteins, neuraminidase (NA) and M2 ion channel, is essential for the replication of IAV. Here, we show that glycosylation of HA at N-142 modulates virus infectivity and host immune response. Glycosylation of NA in the stalk region affects its structure, activity, and specificity, thereby modulating virus release and virulence, and glycosylation at the catalytic domain affects its thermostability; however, glycosylation of M2 had no effect on its function. In addition, using IAV without the stalk and catalytic domains of NA as a live attenuated vaccine was shown to confer a strong IAV-specific CD8+ T-cell response and a strong cross-strain as well as cross-subtype protection against various virus strains.

An Effective Bacterial Fucosidase for Glycoprotein Remodeling.

Fucose is an important component of many oligo- and polysaccharide structures as well as glycoproteins and glycolipids, which are often associated with a variety of physiological processes ranging from fertilization, embryogenesis, signal transduction, and disease progression, such as rheumatoid arthritis, inflammation, and cancer. The enzyme α-l-fucosidase is involved in the cleavage of the fucosidic bond in glycans and glycoconjugates, particularly the Fuc-α-1,2-Gal, Fuc-α-1,3/4-GlcNAc, and Fuc-α-1,6-GlcNAc linkages. Here, we report a highly efficient fucosidase, designated as BfFucH identified from a library of bacterial glycosidases expressed in E. coli from the CAZy database, which is capable of hydrolyzing the aforementioned fucosidic linkages, especially the α-1,6-linkage from the N-linked Fuc-α-1,6-GlcNAc residue on glycoproteins. Using BfFucH coupled with endoglycosidases and the emerging glycosynthases allows glycoengineering of IgG antibodies to provide homogeneous glycoforms with well-defined glycan structures and optimal effector functions.