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The interplay of glycosyltransferases determines the structures of glycans, their function and disease state

In solving the mystery of protein glycosylation, Dr. Chi-Huey Wong has led his team and opened up a new chapter. The team has unveiled how an enzyme called FUT8 selectively worked with certain glyco-molecules with a tri-antennary structure.

More than 50% of human proteins are glycosylated, but we do not quite understand why proteins are glycosylated. Typical examples include: why is blood type determined by the sugars on red blood cells? Why would influenza infection start with interaction of viral surface hemagglutinin and host cell surface sialic acid-containing glycan? Glycoproteins play important roles in all kinds of biological events. It has been demonstrated that glycosylation can modulate the folding, stability, function and half-life of glycoproteins. Therefore, understanding how glycans modulate the functions of proteins is an important yet challenging task. With the rapid development of biologics, the impact of glycans on glycoproteins has been highly investigated and received great attention; for example, antibodies with the lack of core fucosylation or with the presence of bi-antennary glycans having terminal alpha-2,6 sialylation could enhance the antibody-dependent cell-mediated cytotoxicity (ADCC). In addition, aberrant glycosylation of glycoproteins could potentially be a biomarker for cancers, such as alpha-fetoprotein (AFP)-L3, the AFP with core fucose, is a better biomarker than AFP itself for the early detection of hepatocellular carcinoma.

Though many structures of glycans on glycoproteins have been determined, it remains challenging to effectively manipulate the biosynthesis of glycoproteins or glycan structures on cell surface. During glycosylation, a variety of glycosyltransferases is involved in the assembly of glycans, but their interplay is not well understood and so how that affects the biosynthesis of glycoproteins is basically unknown. For example, in human, there are at least 13 fucosyltransferases and at least eight N-acetylglucosaminyltransferases (GnTs), each recognizing different substrates.

To understand the glycan substrate specificity of fucosyltransferase 8 (FUT8), which catalyzes the core fucosylation of glycoproteins, Dr. Chi-Huey Wong’s laboratory developed a combined chemical and enzymatic method (including the use of various glycosidases and GnT-III, IVa and V) to synthesize a panel of glycopeptides containing various glycans for testing as substrates for FUT8. This study reveals that FUT8 has the specificity toward the tri-antennary glycan A3(2,4,2) generated from GnT-IVa, but not the isomers A2B and A3(2,2,6) generated from GnT-III and GnT-V respectively (shown in the figure).

 

 

To observe the changes of glycoforms in cells, Dr. Wong’s and Dr. Jung-Lee Lin’s laboratories worked together to characterize the glycans on glycoproteins by high-temperature porous graphitic carbon liquid chromatography-mass spectrometry (HT-PGC LC-MS), and successfully distinguished three isomers (A2B, A3(2,4,2), A3(2,2,6)), in which A3(2,4,2) is the specific substrate for FUT8 (shown in the figure).

“This research helps us understand further how the glycans on glycoproteins are assembled. The application of the research can be extended to explore new glycan markers for diseases or cancers and elucidate the interplay of glycosyltransferases in cells (For example, GnT-IV was found to be highly expressed in breast cancer cells and thus affects the structures of glycans on proteins). Furthermore, this work could pave the way to design better glycoforms and lead to the development of better glycoprotein pharmaceuticals such as antibody drugs,” commented the team.

This work has been published in the Journal of the American Chemical Society in an article titled “Substrate Preference and Interplay of Fucosyltransferase 8 and N-Acetylglucosaminyltransferases”, online access is at http://pubs.acs.org/doi/pdf/10.1021/jacs.7b03729. The first author of the publication is Tzu-Hao Tseng, a Ph.D. candidate in the Institute of Microbiology and Immunology, National Yang-Ming University. Dr. Tsui-Ling Hsu helps direct the research project. This work was supported by the Summit Program of Academia Sinica.

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