Discovery of a New Mechanism for Diabetes-Related Glycan-Synthesizing Enzymes

An international joint research team consisting of Professor Yasuhiko Kizuka from the Institute for Glyco-core Research (iGCORE), Gifu University, Assistant Professor Masamichi Nagae from the Research Institute for Microbial Diseases, Osaka University , Tohoku Medical and Pharmaceutical University , National Institute of Advanced Industrial Science and Technology , and the University of Mississippi in the USA, has discovered a new reaction mechanism for GnT-IVa, a glycan-synthesizing enzyme also involved in diabetes. GnT-IVa is an enzyme that makes branching structures on protein glycans (sugar chains). In addition to its glycan-producing region, GnT-IVa was discovered to have a region (lectin) that binds to glycans, which has not been found in previously known sugar chain glycan-synthesizing enzymes. Furthermore, by clarifying the steric structure of the region, researchers revealed that lectins are essential for enzymatic activity and promote efficient glycan synthesis by binding to specific glycans. The finding is expected to provide essential basic knowledge for elucidating the mechanisms of complex glycans on proteins, the pathogenesis of diabetes, and developing therapeutic methods. The team's findings were published in the journal Communications Biology.

Glycans are mainly found bound to proteins and lipids, and researchers know that the shape of glycans change when disease is present. Changes in glycans are used in medicine to diagnose cancer and other diseases. Since specific glycans play important roles in various diseases such as cancer, Alzheimer's disease, and diabetes, the team's findings are expected to lead to new therapeutic agents that target these conditions.

Glycans attached to proteins are produced by the action of glycosyltransferases (glycan-synthesizing enzymes) in cells. GnT-IVa, one of the 180 or so human glycosyltransferases, acts on N-glycans attached to proteins in the cell to create certain branching structures. This branching structure created by GnT-IVa has been found to be closely related to the onset and progression of diabetes. However, the steric structure of GnT-IVa itself and the details of the mechanism used by GnT-IVa to produce glycans on proteins were not fully understood.

This knowledge gap led Professor Kizuka and the group to first focus on GnT-IVa's amino acid sequence. The protein steric structure prediction tool Phyre2 revealed that GnT-IVa has a lectin domain on the C-terminal side in addition to the catalytic domain that produces glycans. Since lectins selectively bind to various glycans, this suggests that GnT-IVa binds to specific glycans in the C-terminal lectin domain. Many glycosyltransferases do not have such lectin domains, suggesting that GnT-IVa makes glycans using a unique mechanism.

To determine the role of the lectin domain in GnT-IVa's enzyme activity, the team expressed and purified GnT-IVa or a variant lacking the lectin domain in cells. The purified enzymes were mixed with a substrate glycan in a test tube, and the product of the enzymatic reaction that occurred was analyzed using high-performance chromatography. The results revealed that the GnT-IVa variant lacking the lectin domain did not exhibit enzymatic activity. This finding means that lectin domains are essential for enzyme activity.

Next, the team performed a detailed examination of the function and steric structure of the lectin domain. Since the lectin domains are highly likely to bind to glycans, they investigated which types they bind to by performing frontal affinity chromatography on 157 varieties, including N- and O-type glycans. These results revealed that lectin domains bind only to N-glycans with a branched structure created by GnT-IVa.

Furthermore, X-ray crystallography revealed the three-dimensional structure of the purified lectin domain. Based on the steric structure, molecular dynamics simulations showed that the lectin domain strongly interacts with N-glycans with the branching structure created by the catalytic domain. The team also found that aspartic acid (D445), GnT-IVa's 445th amino acid, is crucial for this interaction.

Finally, the team investigated the role of the lectin domain when GnT-IVa actually acts on proteins in the cell by replacing this amino acid (D445), which is essential for the lectin domain's glycan-binding capacity, with alanine. To accomplish this aim, it expressed a variant (D445A) with loss of function of the lectin domain in cells and examined its function. Results revealed that the binding of lectin domains to glycans is necessary for GnT-IVa to make glycans on proteins in cells efficiently.

Kizuka et al. have also recently revealed that GnT-V, an enzyme that creates another N-glycans branching structure, also recognizes substrate proteins via a domain separate from the catalytic domain. These findings suggest that each glycosyltransferase acts on the substrate protein using its own mechanism to create complex glycans. However, the details of how the binding of GnT-IVa's lectin domain to glycans contributes to efficient enzyme reactions have not yet been uncovered. In the future, understanding the steric structure of GnT-IVa as a whole, including the catalytic domain, is expected to provide further insight into its working mechanisms. This research also provides important insights into the mechanisms by which different glycans attach to different proteins, a topic that researchers in this field have yet to fully clarify.

Journal Information
Publication: Communications Biology
Title: Discovery of a lectin domain that regulates enzyme activity in mouse N-acetylglucosaminyltransferase-IVa (MGAT4A)
DOI: 10.1038/s42003-022-03661-w