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N‐acetylglucosaminyltransferase‐V (GnT‐V or MGAT5) is a glycosyltransferase involved in cancer metastasis that creates the β1,6‐branch on N‐glycans of target proteins such as cell adhesion molecules and cell surface receptors. The 3D structure of GnT‐V and its catalytic site, which are critical for the interaction with the N‐glycan terminal, have already been revealed. However, it remains unclear how GnT‐V recognizes the core part of N‐glycan or the polypeptide part of the acceptor. Using molecular dynamics simulations and biochemical experiments, we found that several residues outside the catalytic pocket are likely involved in the recognition of the core part of N‐glycan. Furthermore, our simulation suggested that UDP binding affects the orientation of the acceptor due to the conformational change at the Manα1,6‐Man linkage. These findings provide new insights into how GnT‐V recognizes its glycoprotein substrates. [ABSTRACT FROM AUTHOR]

The β1,6-GlcNAc branch in N -glycans, produced by a glycosyltransferase N -acetylglucosaminyltransferase V (GnT-V or MGAT5), is associated with cancer and autoimmune diseases. Here, we summarize the structure and activity regulation of GnT-V. We also describe the roles of the β1,6-GlcNAc branch on glycoproteins in cells and the phenotypes of Mgat5 -deficient mice, focusing on cancer and the immune system. GnT-V has a unique structure for substrate recognition, and its activity and function are regulated by shedding. The glycans produced by GnT-V play pivotal roles in the differentiation of neural cells, cancer malignancy and immunotherapy, and the development of autoimmune diseases by regulating the functions and cell surface residency of glycoproteins. Controlling the expression or activity of GnT-V could be a therapeutic option against cancer and autoimmune diseases. Future work should clarify how GnT-V selectively modifies the specific glycoproteins or N -glycosylation sites in vivo. • β1,6-GlcNAc branch produced by GnT-V is involved in various diseases. • GnT-V has a unique domain and amino acid residues for recognizing substrates. • β1,6-GlcNAc branches on glycoproteins regulate cell mobility and immune response. • Mechanism of how GnT-V selects substrate proteins should be clarified. [ABSTRACT FROM AUTHOR]

N -Glycosylation is crucial for protein folding, trafficking, and functions. N -Glycans have a different number of N -acetylglucosamine (GlcNAc) branches in a protein-selective manner, and the β1,6-linked GlcNAc branch on specific proteins produced by N -acetylglucosaminyltransferase-V (GnT-V or MGAT5) promotes cancer malignancy. However, little is known about how GnT-V acts on specific target proteins. Based on our structural model, we hypothesized that GnT-V interacts with the N -glycan core or polypeptide moiety as well as the accepter site of N -glycan. To explore this possibility, we selected four candidate residues involved in the interaction with the glycan core or surrounding amino acids, created point mutants of these residues, and examined the in vitro and in vivo activities of the mutants. Our in vitro enzyme assays using various types of substrates including oligosaccharides and glycoproteins revealed that the V354N mutant had dramatically reduced activity for all tested substrates with an altered substrate preference and that K361A had reduced activity for an oligosaccharide with asparagine (Asn), but not a shorter oligosaccharide without the reducing end of GlcNAc and Asn. These results suggest that V354 and K361 are involved in the recognition of N -glycan core and surrounding amino acids. We further performed rescue experiments using GnT-V knockout HeLa cells and confirmed the importance of these residues for modifications of glycoproteins in cells. We identified several residues involved in the action of GnT-V toward N -glycan cores and surrounding amino acids. Our data provide new insights into how GnT-V recognizes glycoproteins. • GnT-V is a glycosyltransferase that makes a GlcNAc branch in N -glycans. • The mechanism of the protein-selective action of GnT-V has not been elucidated. • Mutation of the residues outside the catalytic pocket altered the substrate preference. • GnT-V likely recognizes N -glycan core and/or surrounding amino acids. [ABSTRACT FROM AUTHOR]

Background : N -Glycan branching regulates various functions of glycoproteins. N -Acetylglucosaminyltransferase V (GnT-V) is a GlcNAc transferase that acts on N -glycans and the GnT-V-producing branch is highly related to cancer progression. This indicates that specific GnT-V inhibitors may be drug candidates for cancer treatment. To design novel GnT-V inhibitors, we focused on the unique and weak recognition of the donor substrate UDP-GlcNAc by GnT-V. On the basis of the catalytic pocket structure, we hypothesized that UDP-GlcNAc analogs with increasing hydrophobicity may be GnT-V inhibitors. Methods : We chemically synthesized 10 UDP-GlcNAc analogs in which one or two phosphate groups were replaced with hydrophobic groups. To test these compounds, we set up an HPLC-based enzyme assay system for all N -glycan-branching GlcNAc transferases in which GnT-I–V activity was measured using purified truncated enzymes. Using this system, we assessed the inhibitory effects of the synthesized compounds on GnT-V and their specificity. Results : Several UDP-GlcNAc analogs inhibited GnT-V activity, although the inhibition potency was modest. Compared with other GnTs, these compounds showed a preference for GnT-V, which suggested that GnT-V was relatively tolerant of hydrophobicity in the donor substrate. Docking models of the inhibitory compounds with GnT-V suggested the mechanisms of how these compounds interacted with GnT-V and inhibited its action. Conclusions : Chemical modification of the donor substrate may be a promising strategy to develop selective inhibitors of GnT-V. General significance : Our findings provide new insights into the design of GnT inhibitors and how GnTs recognize the donor substrate. • GnT-V is a cancer-related glycosyltransferase forming a GlcNAc branch in N -glycans. • Recognition of the donor substrate UDP-GlcNAc by GnT-V is unique and weak. • Ten UDP-GlcNAc analogs were chemically synthesized as potential GnT-V inhibitors. • Some of these compounds inhibited GnT-V activity. [ABSTRACT FROM AUTHOR]