Structure and mechanism of cancer-associated N-acetylglucosaminyltransferase-V

Abstract

N-acetylglucosaminyltransferase-V (GnT-V) alters the structure of specific N-glycans by modifying α1-6-linked mannose with a β1-6-linked N-acetylglucosamine branch. β1-6 branch formation on cell surface receptors accelerates cancer metastasis, making GnT-V a promising target for drug development. However, the molecular basis of GnT-V's catalytic mechanism and substrate specificity are not fully understood. Here, we report crystal structures of human GnT-V luminal domain with a substrate analog. GnT-V luminal domain is composed of a GT-B fold and two accessary domains. Interestingly, two aromatic rings sandwich the α1-6 branch of the acceptor N-glycan and restrain the global conformation, partly explaining the fine branch specificity of GnT-V. In addition, interaction of the substrate N-glycoprotein with GnT-V likely contributes to protein-selective and site-specific glycan modification. In summary, the acceptor-GnT-V complex structure suggests a catalytic mechanism, explains the previously observed inhibition of GnT-V by branching enzyme GnT-III, and provides a basis for the rational design of drugs targeting N-glycan branching.

Fig. 1

GnT-V transfers β1-6 branch on N-glycan via C-terminal region. a The β1-6 branch formation of N-glycan catalyzed by GnT-V. The substrate of GnT-V is GlcNAc-terminated biantennary glycan termed “GnGnbi-Asn”. The β1-6 branch formation leads to further LacNAc extension, which is the target for galectin. The β1–4 branch at β-mannose catalyzed by GnT-III, named as bisecting GlcNAc, hampers β1-6 branch formation. Monosaccharide symbols follow the symbol nomenclature for glycans (SNFG) system. Nomenclature of sugar residues (red arrows) and glycosidic linkages (cyan arrows) are indicated in boxes. b Chemical structure of bisubstrate-type inhibitor of GnT-V used for co-crystallization. Donor and acceptor substrates are colored with blue and red, respectively. Three sugar residues (GlcNAcβ1-2Manα1-6Man) of acceptor moiety are labeled. In this study, the inhibitor of which the linker length is (n = 3) and K_i value is 18 μM was used. Acceptor trisaccharide corresponds to α1-6 branch of biantennary glycan (GnGnbi) as shown in the box. c Domain architecture of full length and truncated constructs of human GnT-V. Expression levels and catalytic activities of truncated constructs are indicated on the right of these constructs. d Enzymatic activity of GnT-V luminal domain and mini-GnT-V. The two constructs were expressed in COS-7 cells and purified through Ni²⁺-column, followed by SDS-PAGE with CBB staining (left) or activity assays (right). Lane 1, mock, lane 2, GnT-V luminal domain, and lane 3, mini-GnT-V. The eluates from Ni²⁺-column were incubated with acceptor substrate GnGnbi-PA and donor substrate UDP-GlcNAc, and the reaction mixtures were analyzed by HPLC

Fig. 2

Overall structure and catalytic residues of GnT-V luminal domain. a Overall structure of GnT-V luminal domain is shown in ribbon model. The N-terminal domain (A128-Y207 in blue and G337-P339 in cyan), middle domain 1 (H212-K329 and I345-L424 in green and E606-H627 in yellow), middle domain 2 (G425-Y605 in orange), and C-terminal domain (G628-L741 in pink) are shown in ribbon models. The disulfide bonds and N-glycan attached on N433 are indicated with rod models and labeled. Putative catalytic center of GnT-V is indicated with a red dotted box. Among three putative N-glycans (N334, N433, and N447), only GlcNAc residue of N433 is assigned in the final model. Three N-glycosylation sites are indicated. The three missing loops between N-terminal domain and middle domain 1 are also indicated. b Schematic representation of disulfide bond pattern of GnT-V luminal domain. Cysteine residues, which form disulfide bonds, are also indicated. c Structural positions of six glutamates (E280, E287, E297, E429, E520, and E526) in GnT-V in respect of mutational experiments are shown in rod model. E280 and E287 are located in the missing loop region (K279-P294) shown as green dotted line. Sequence of missing loop region is shown in lower panel. The side chain of R558, which forms a salt bridge with E526 is also shown in rod model. d Kinetic parameters of wild-type and mutated GnT-V luminal domains. V_max values are shown in bar graph representation. K_m values for UDP-GlcNAc (donor substrate) and GnGnbi-PA (acceptor substrate) are shown in lower panel. ^aND not detected

Fig. 3

Structure of mini-GnT-V E297A in complex with bisubstrate-type inhibitor. a Crystal structure of mini-GnT-V E297A in complex with bisubstrate-type inhibitor. Trisaccharide is shown in sphere model. Highly mobile region (G282-G296) and E297A are colored with red and black, respectively, and indicated. b Close-up views of domain boundary between N-terminal domain and middle domain 1 in GnT-V luminal domain (left panel) and mini-GnT-V E297A (right panel). Key residues of these two structures are labeled. Domain architectures are shown in bottom. c Omit map contoured at 3.0 σ level around acceptor binding site is shown in cyan mesh. Carbohydrate and amino-acid residues are shown in rod models. d Close-up view of interaction with acceptor trisaccharide. Direct and water-mediated interaction network is depicted with red dotted lines. Three water molecules, which link glycan and mini-GnT-V are conserved in the two complexes. Details of interaction network are also summarized in Supplementary Table 2. e Two aromatic residues, F380 and W401, restrict the conformation of trisaccharide and define the branch specificity. Two aromatic rings and trisaccharide residues are shown in rod and semi-transparent sphere models. The interaction of α1-6 branch observed in crystal structure (i) and docking model of α1-3 branch (ii) are shown in upper and lower panels, respectively. Steric clash is shown in asterisk

Fig. 4

Substrate specificity of GnT-V toward various types of N-glycans. a Docking model of N-glycan bearing α1-3 branch, chitobiose, core fucose, and asparagine. Sugar residues and GnT-V are shown in rod and surface models, respectively. Schematic drawing of interaction mode is also shown in right panel. Structure of N-glycan was built based on the atomic structures of biantennary glycans (PDB codes: 5XFI and 4BM7). b Schematic drawing of interaction modes in various types of N-glycans. (i) Galactose extension at two branches, (ii) core fucosylation, and (iii) addition of bisecting GlcNAc are shown. Structural details of these interactions are also shown in Supplementary Figures 7 and 8. c Superposition of bisecting GlcNAc onto trisaccharide structure shown in rod model. Bisecting GlcNAc and Man-2 are also shown in semi-transparent spheres. Steric conflict is indicated with asterisk. d Two differently labeled biantennary N-glycans applied to enzymatic assays. The oligosaccharide-type glycan (GnGnbi-PA) and asparagine-type glycan (GnGnbi-Asn-PNS) are shown in upper and lower panels, respectively. Schematic drawing of labeled glycans and close-up view of chemical structures around chitobiose unit of two substrates are also shown in left panel. e Enzymatic activity of GnT-V luminal domain toward the two types of substrates (100 pmol each per reaction) is shown. Activity was repeatedly measured (n = 3) using the same enzymes and substrates (n = 3). The graph shows means ± S.D

Fig. 5

Putative ternary complex structure of wild-type GnT-V and substrates. a Structural superposition of GnT-V luminal domain (apo form), mini-GnT-V in complex with acceptor, BaBshA-UDP complex (PDB code: 3MBO) and BaBshA-UMP-GlcNAc-malate complex (PDB code: 5D00). b Close-up view of ligand binding site. The acceptor trisaccharide (mini-GnT-V-acceptor complex), UDP, UMP, and GlcNAc-malate (BaBshA ligand complexes) are shown. The distance between E297 and S6 of Man-2 is also indicated. c Ternary complex model of wild-type GnT-V, acceptor trisaccharide and UDP-GlcNAc. The position of UDP-GlcNAc was inferred by superposing the structure of UDP-GlcNAc extracted from GnT-I-UDP-GlcNAc complex (PDB code: 1FOA) onto UDP moiety of BaBshA complex. The S6 of Man-2 is also rotated toward E297. The distances between S6 of Man-2 and E297 or C1 of UDP-GlcNAc are indicated. d Six glutamates selected for mutational experiments (Fig. 2c) are shown in the ternary complex

Fig. 6

Hypothetical interaction mode between GnT-V and E-cadherin. a Schematic representation of human E-cadherin. E-cadherin is a type I membrane protein and has five ectodomains (EC1–5). Two N-glycans at N554 and N633 are highlighted. EC5 connects to transmembrane helix via short linker region (698-709). b Crystal structure of murine E-cadherin EC4-EC5 ectodomains (PDB code: 3Q2V) is shown in ribbon model. Two N-glycosylation sites, N554 and N633, are shown in rod models. c Docking models of GnT-V and N-glycan at N554 of E-cadherin EC4-EC5 ectodomain. GnT-V, N-glycan and E-cadherin are shown in surface, sphere and ribbon models, respectively. d Docking models of GnT-V and N-glycan at N633 of E-cadherin EC4-EC5 ectodomains. GnT-V, N-glycan and E-cadherin are shown in surface, sphere and ribbon models, respectively. Steric clash is indicated with red dotted circle. The three figures, b–d, are depicted from the same view angle