Enzymatic Transfer of a Preassembled Trisaccharide Antigen to Cell Surfaces Using a Fucosyltransferase

Research Overview

The elucidation of the biological roles of specific cell-surface glycosylation remains a subject of intense investigation. In the recent reports, the potential significance of specific glycosylation is highlighted that the role of sialylated and fucosylated tetrasaccharide antigens is essential in the adhesion of neutrophils to endothelial cells that is an example of carbohydrate-mediated cell-to-cell recognition. Well characterized oligosaccharides are commonly used as inhibitors of the binding of proteins or cells with the natural glycoconjugate ligand. If inhibition is observed, then the carbohydrate sequence is regarded as a physiologically relevant recognition marker candidate. The Le-FucT enzyme is a potential tool to label animal cell surface with preassembled oligosaccharide and other complex markers.

In this research paper, Geeta Srivastava et al. investigated whether synthetic oligosaccharides could be used in alternative experiments, and whether it is feasible to add postulated complex carbohydrate recognition markers to proteins or cells without related structures.

Materials

• Fetuin
• Alkaline phosphatase conjugate of goat anti-mouse IgM
• Alkaline phosphatase substrate tablets containing 5 mg of p-nitrophenyl phosphate, Tween 20, and bovine serum albumin
• Anti-B mAbs
• Human milk
• Type I BSA conjugates
• Buffers: PBS, 7.8 mM Na2HPO4, 2.2 mM KH2PO4, etc.

Experimental procedures

• Chemical synthesis of GDP-Fuc derivatives
• Synthesis of GDP- "B"-Fuc (13)
• Synthesis of 6-O-Propyl-GDP-β-L-galactose (2)
• Comparison of transfer of GDP-Fuc with GDP-6-O-propyl Gal
• Detection of the transfer of fucose analogs to immobilized glycoconjugates by ELISA plate coating with type 1 BSA conjugates and fetuin
• ELISA incubations
• Transfer of the B-active trisaccharide to red cells detected by agglutination

Results

The first experiment demonstrated that Le-FucT can transfer a fucose residue chemically substituted at C-6. Much larger groups could also be attached to the C-6 of the Fuc residue of GDP-Fuc, which was confirmed by the team and a conclusion was drawn that covalent attachment of the blood group B-active trisaccharide to C-6 of the fucose residue in GDP-fucose does not destroy the activity of the donor. In addition, the transfer of blood group B trisaccharide antigen to the O Lea-b- red cells surface was detected by a monoclonal anti-B antibody.

Conclusion

The core of study, according to this research paper, is that large molecules can be attached to the six-position of the fucosyl residue of GDP-fucose, which is then transferred to the acceptor oligo-saccharides by means of Lewis α(1→3/4))fucosyltransferase. It should be possible to label glycoproteins that have FucT acceptor sequences in this way. Whether peptides or larger proteins attaching to the GDP-fucose residue could be transferred needs further experiments.

• Cell-Fluorescent Protein Conjugate (CFPC) Service
• Cell-Drug Conjugate (CDC) Service
• Cell-Adjuvant-loaded Nanoparticles (CALNP) Service
• Cell-Antibody Conjugate（CAC）Service
• Cell-Nucleic Acids Conjugate (CNAC) Service
• Cell-Fucose Conjugate (CFC) Service
• Cell-Peptide Conjugate (CPC) Service