New Insights into the Mechanism of Glycan Branch Formation

Glycans are sugar chains that bind to cells through proteins or lipids, influencing their chemical properties. These complex, often branched structures stabilize the extracellular domains of cells and proteins. However, the precise reasons and mechanisms behind glycosylation—the post-synthesis modification of proteins and lipids with glycans—remain only partially understood.

Protein glycosylation, which creates glycoproteins, is common and influences cell adhesion, signaling, protein folding, and receptor activation. Changes in glycosylation are also linked to various diseases, such as neurodegenerative disorders, due to disruptions in the glycosylation of proteins and lipids.

To further understand protein glycosylation, researchers from Gifu University’s Institute for Glyco-Core Research (iGCORE), in collaboration with the University of Mississippi, Hiroshima University, and Osaka University, studied unique glycan-binding domains in the glycosylation proteins N-acetylglucosaminyltransferase-IVa (GnT-IVa or MGAT4A) and GnT-IVb (MGAT4B). These proteins attach a specific glycan branch, β1-4-GlcNAc, to glycoproteins. The team’s findings were published in iScience.

“Most glycosylation enzymes, or glycosyltransferases, only have a catalytic domain for glycosylating substrate proteins. Our study aimed to reveal the functions of the unique lectin, or sugar-recognition, domains in GnT-IVa and GnT-IVb,” explained Yasuhiko Kizuka, Professor at iGCORE, Gifu University.

The researchers also explored whether glycosylation enzymes recognize not just glycans but also the protein component of glycoproteins. While it is well-known that glycosylation enzymes specifically recognize glycans on glycoproteins, the way these enzymes select their protein substrates remains unclear.

To examine the lectin domain's role in glycosylation, the team engineered GnT-IVa and GnT-IVb mutants with disrupted lectin-binding abilities. Results showed that when these domains were non-functional, glycosylation activity on glycoproteins was significantly reduced compared to the unaltered GnT-IVa and GnT-IVb. Further experiments revealed that the lectin domains in GnT-IVa and GnT-IVb contribute minimally to forming the β1-4-GlcNAc branch directly on substrates.

To understand if N-glycans—sugar molecules attached to asparagine (Asn) residues—interact with the lectin domains of GnT-IVa and GnT-IVb, the team conducted molecular dynamics simulations. They found that N-glycans can interfere with these lectin domains, depending on the N-glycan type.

“We found that GnT-IVa and GnT-IVb use their lectin domains to recognize substrate proteins, showing a unique recognition mechanism. Surprisingly, we also discovered that the glycosyltransferases’ own glycans can attach to and inhibit their lectin domains, essentially self-regulating their activity—a novel example of a glycan regulating its own biosynthesis,” said Professor Kizuka.

Despite these findings, much remains unknown about protein glycosylation mechanisms. The researchers hope to investigate other glycosyltransferases with unique non-catalytic domains.

“Our study suggests that certain glycosyltransferases use accessory domains to regulate their activity, potentially shedding light on how these enzymes select their protein substrates within cells,” Kizuka concluded.