Glycans are chains of sugars that attach to cells through proteins or lipids, changing their chemical characteristics. Glycans can be incredibly complex and branched in structure, contributing to the stability of the extracellular domain of cells and proteins. Exactly how and why proteins and lipids are glycosylated, or modified with glycans after synthesis, remains largely unknown.
The glycosylation of proteins to create glycoproteins is very common and has been linked to changes in cell adhesion, cell signaling, protein folding and receptor activation processes. Modifications to glycosylation have also been associated with disease: Disruption to the glycosylation of lipids and proteins has been linked to neurodegenerative disease, for instance.
To better understand the process of protein glycosylation, a team of researchers from the Institute for Glyco-Core Research (iGCORE) at Gifu University, the University of Mississippi, Hiroshima University and Osaka University investigated unique glycan-binding domains in N-acetylglucosaminyltransferase-IVa (GnT-IVa or MGAT4A) and GnT-IVb (MGAT4B), two glycosylation proteins responsible for attaching the glycan branch β1-4-GlcNAc to specific glycans attached to glycoproteins.
The team published their study in the September 27 issue of iScience.
"Most glycosylation enzymes, or glycosyltransferases, do not have an additional lectin, or sugar-recognition domain like GnT-IVa and GnT-IVb. Most glycosyltransferases only possess a catalytic domain that simply glycosylates their substrate protein. We aimed to elucidate the functions of the unique lectin domain of glycosylation enzymes GnT-IVa and GnT-IVb," said Yasuhiko Kizuka, professor at iGCORE at Gifu University and senior author of the research paper.
Another question addressed by the team's research is whether or not glycosylation enzymes recognize the protein part of the glycoproteins, or proteins bound to glycans, they modify. Many biochemical studies have demonstrated how glycosylation enzymes specifically recognize glycans attached to the glycoproteins they modify, but the protein-selection mechanisms used by glycosylation enzymes, if any, are unknown.
The research team created a series of GnT-IVa and GnT-IVb mutants that impaired only the lectin-binding activity of both glycosylation enzymes to determine whether or not lectin binding was required for enzyme activity. Without functional lectin domains, both enzymes demonstrated significantly lower glycosylation activity toward glycoproteins than normal GnT-IVa and GnT-IVb. Additional experiments suggested that the lectin domain of normal GnT-IVa and GnT-IVb had little to do with the actual formation of the β1-4-GlcNAc branch on substrate glycoproteins.
The team also performed molecular dynamics simulations to determine whether or not N-glycans, or multiple sugar molecules that are attached to asparagine (Asn) amino acids in proteins, attached to the GnT-IVa and GnT-IVb enzymes could interfere with the lectin domain of the enzymes. The simulation predicted this was a possibility, but that it depended on what type of N-glycan was attached to the GnT-IVa and GnT-IVb enzymes.
Additional experiments used GnT-IVa enzymes with different N-glycans capable of binding the GnT-IVa lectin domain: one with oligomannose (weak binding) and another with GlcNAc (strong binding). Importantly, activity assays determined that GnT-IVa enzyme with GlcNAc glycan branches (higher binding) near the lectin domain decreases the activity of modified GnT-IVa toward glycoprotein substrates. Put simply, GlcNAc bound to GnT-IVa decreased the addition of the GlcNAc glycan to proteins GnT-IVa would normally glycosylate.
"We discovered that GnT-IVa and GnT-IVb use their lectin domains to recognize their substrate proteins, showing a unique mechanism for recognition of a substrate glycoprotein by a glycosyltransferase. Another interesting finding is that recognition of a substrate glycoprotein can be inhibited by a glycosyltransferase's own glycans attaching to its lectin domains. Namely, their activity is self-regulated by their own glycans. This is, to my knowledge, is the first case of a specific glycan self-regulating its own biosynthesis," said Kizuka.
Despite the research team's findings, the field still has a lot to learn about the mechanisms of protein glycosylation. Ultimately, the authors would like to study other glycosyltransferases that possess unique non-catalytic domains. "Our study implied that some glycosyltransferases have unique mechanisms by which accessory domains regulate their activity. This could lead to further understanding of how glycosyltransferases choose their substrate proteins in cells," said Kizuka.
Other contributors include Naoko Osada from the Graduate School of Natural Science and Technology at Gifu University in Gifu, Japan; Sushil K. Mishra and Robert J. Doerksen from the Glycoscience Center of Research Excellence in the Department of BioMolecular Sciences at the University of Mississippi in Oxford, Mississippi; Miyako Nakano from the Graduate School of Integrated Sciences for Life at Hiroshima University in Higashihiroshima, Japan; Yuko Tokoro from the Institute for Glyco-core Research (iGCORE) at Gifu University; Masamichi Nagae from the Department of Molecular Immunology in the Research Institute for Microbial Diseases and the Laboratory of Molecular Immunology in the Immunology Frontier Research Center (IFReC) at Osaka University in Suita, Japan.
This work was supported by FOREST grant JPMJFR215Z from Japan Science and Technology Agency (JST); Core-to-Core Program JPJSCCA202000007; Grant-in-Aid for Scientific Research (B) [24K02222] from the Japan Society for the Promotion of Science (JSPS); AMED-CREST grant JP23gm1410011 from Japan Agency for Medical Research and Development (AMED); the Human Glycome Atlas Project (HGA) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT); and a grant from the Takeda Science Foundation. Computational work was supported by National Institutes of Health funding (P20GM130460) to the Glycoscience Center of Research Excellence.
