Structure of O-glycan Core 1

The O-glycan core 1 structure is widely recognized as the most prevalent modification of the GalNAcα1-S/T core structure. A single core 1 β1-3 galactosyltransferase (T-synthase) is responsible for adding galactose in a β1-3 linkage to GalNAc.

Fig.1 Structure of O-glycan core 1. (CD BioGlyco)

The gene encoding this enzyme is widely expressed in various mammalian tissues, including the liver, kidney, heart, and placenta. Amazingly, the activity of the mammalian T-synthase depends on the presence of a molecular chaperone called Cosmc. Cosmc helps in folding and stabilizing the T-synthase, ensuring its proper localization to the Golgi apparatus. Mutations in the Cosmc gene lead to the loss of T-synthase from the Golgi apparatus and the loss of core 1 activity.

Fig.2 O-GalNAc-type O-glycosylation pathway. (Bagdonaite, et al., 2020)

O-glycan Core 1 Glycoengineering Service at CD BioGlyco

At CD BioGlyco, gene editing technology is employed to knock out the Cosmc gene related to the O-glycan core 1 structure into the surface of cells. This approach results in the inhibition of T-synthase activity, thereby impeding O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins.

Our glycoengineering can be applied to various cell surfaces based on client requirements. For example, we have generated genetically mosaic mouse embryo (ES) cells by deleting the Cosmc gene through targeted genetic modification.

• Firstly, we have designed a targeting vector that incorporates three LoxP sites into the murine Cosmc allele.
• After transfecting and selecting ES cells and screening with PCR, we obtain clones with the correct homologous recombination and confirm them by Southern blot.
• As a result, the Cosmc-null ES cells we produce lack T-synthase activity and express Tn antigen on their surfaces.
• Additionally, we use RT-PCR analysis further to confirm the absence of Cosmc transcripts in the Cosmc-null ES cells.

Besides, we also use our O-glycan core 1 glycoengineering to modify human therapeutic proteins.

• We have generated a glycoengineered strain of Escherichia coli for the production and transfer of an authentic core-1 O-glycan structure, to human therapeutic proteins.
• Human ppGalNAcT2, which is known to be the most versatile isoform of this enzyme family, and the bacterial β1,3-galactosyltransferase CgtB from Campylobacter jejuni are utilized to synthesize the core-1 O-glycan.
• Our approach has successfully realized the production of authentic core-1 O-glycan structure on the target recombinant proteins, hIFNα2b and hGH, with high yield using an E. coli system.

Applications

• Our technology offers a conceptual framework to investigate the biological effects of O-glycan core 1 expression in human diseases, including Tn syndrome and IgA nephropathy.
• This technique can be used to optimize the structure and composition of therapeutic glycoproteins to improve their biological properties shown in Fig.3. This rich field of glycoengineering offers tremendous potential for the development of more effective and efficient therapeutic interventions.

Fig.3 These biological properties are improved by glycoengineering. (CD BioGlyco)

Advantages

• Our method is precise for gene editing. The use of three LoxP sites allows for accurate manipulation of the Cosmc allele, enabling targeted gene knockout, knock-in, mutation, and editing with high precision.
• Our approach combines transfection, selection, and screening techniques such as PCR and Southern blot to allow for the identification and confirmation of clones with the desired homologous recombination.
• Our O-glycan glycoengineering is a versatile and powerful approach to significantly enhance the potency and efficacy of therapeutic proteins.

CD BioGlyco has earned a well-deserved reputation for innovation and expertise by making remarkable contributions to glycoengineering research. Our research team is composed of exceptional scientists and researchers. By using cutting-edge technologies and innovative methods, we strive to offer comprehensive Cell Surface Glycoengineering Services. If you have any inquiries or would like to know more about our services, please don't hesitate to contact us.