Hirokazu Yagi

B4GALNT1 is a glycosyltransferase essential for the synthesis of complex gangliosides and plays important roles in the nervous system. Although N-glycosylation is thought to regulate B4GALNT1, its functional significance has remained unclear. In this study, we generated human B4GALNT1 mutants carrying single or combined substitutions at three N-glycosylation sites (N1–N3) and analyzed their enzymatic activity, intracellular localization, and molecular assembly using transfected B78 mouse melanoma cells. Loss of N-glycosylation reduced GM2 expression and enzymatic activity in a site-dependent manner, with multiple-site mutants showing greater impairment. Golgi localization was also progressively disrupted as the number of mutated N-glycosylation sites increased. Non-reducing immunoblotting demonstrated that loss of the N1 glycosylation site markedly impaired B4GALNT1 dimer formation, with additional defects caused by the combined loss of the N3 and N2 sites. Predicted dimeric B4GALNT1 structures generated by AlphaFold2 revealed the spatial arrangement of N-glycans, suggesting a dominant role for the N1 site and a supportive role for the N3 site in stabilizing intermolecular contacts. These results indicated that N-glycosylation sites in the three-dimensional structure of B4GALNT1 are critical for the molecular integrity and enzymatic activity.

Protein N-glycosylation generates diverse glycoforms that influence protein structure and function, yet the molecular rules governing site-specific glycan maturation remain incompletely understood. Accumulating evidence indicates that glycosylation outcomes are shaped by determinants embedded within glycoproteins themselves, beyond enzyme availability alone. Here, we investigate how neighboring glycans modulate site-specific N-glycan maturation in human Fcγ receptor III (FcγRIII). Building on previous glycoproteomic studies showing restricted processing of the N45 glycan in FcγRIII molecules, we combined reciprocal glycosylation-site mutagenesis with molecular dynamics simulations. Introduction of an N64 glycosylation sequon into FcγRIIIa reduced N45 glycan maturation, whereas removal of the N64 sequon from FcγRIIIb produced the reciprocal effect. Simulations using glycan models, analyzed primarily by contact maps and contact frequencies, showed that the N45 glycan dynamically contacts neighboring N64 and N169 glycans. These findings support a model in which the isoform-specific N64 glycan, in addition to the adjacent N169 glycan shared by both FcγRIII isoform, contribute to reduced N45 glycan maturation by reshaping the local glycan environment around N45.

Glycosyltransferases often contain multiple structural modules that contribute to substrate recognition, catalytic coordination, and higher-order molecular organization. However, how multidomain glycosyltransferases dynamically organize their catalytic domains in solution remains poorly understood. Here, we investigated the assembly states and conformational dynamics of POMGNT2, LARGE1, K4CP, and L137 using high-speed atomic force microscopy (HS-AFM) integrated with complementary solution biophysical analyses. This multi-technique approach extends previous static structural studies by enabling a parallel comparison of solution-state dynamics among multiple glycosyltransferases. POMGNT2 formed a stable dimeric architecture with limited large-scale conformational fluctuation, consistent with its role in site-selective substrate recognition. In contrast, LARGE1 and K4CP exhibited concentration-dependent and heterogeneous assembly behavior. K4CP displayed pronounced open–closed interdomain motion and a tendency toward more compact conformations in the presence of substrate, suggesting dynamic catalytic-domain reorganization during glycan elongation. By comparison, the mimivirus glycosyltransferase candidate L137 predominantly behaved as a monomeric species under the tested conditions. These findings demonstrate that multidomain glycosyltransferases employ diverse dynamic organizational strategies ranging from rigid recognition architectures to highly flexible and reversible catalytic assemblies. Our results further suggest that glycosyltransferase function is governed not only by catalytic-domain structure, but also by dynamic conformational coordination adapted to distinct catalytic demands.

Glycosyltransferases often contain multiple structural modules that contribute to substrate recognition, catalytic coordination, and higher-order molecular organization. However, how multidomain glycosyltransferases dynamically organize their catalytic domains in solution remains poorly understood. Here, we investigated the assembly states and conformational dynamics of POMGNT2, LARGE1, K4CP, and L137 using high-speed atomic force microscopy (HS-AFM) integrated with complementary solution biophysical analyses. POMGNT2 formed a stable dimeric architecture with limited large-scale conformational fluctuation, consistent with its role in site-selective substrate recognition. In contrast, LARGE1 and K4CP exhibited concentration-dependent and heterogeneous assembly behavior. K4CP displayed pronounced open–closed interdomain motion and substrate-dependent conformational compaction, indicating dynamic catalytic-domain reorganization during glycan elongation. By comparison, the mimivirus glycosyltransferase candidate L137 predominantly behaved as a monomeric species under the tested conditions. These findings demonstrate that multidomain glycosyltransferases employ diverse dynamic organizational strategies ranging from rigid recognition architectures to highly flexible and reversible catalytic assemblies. Our results further suggest that glycosyltransferase function is governed not only by catalytic-domain structure, but also by dynamic conformational coordination adapted to distinct catalytic demands.

Protein glycosylation, a fundamental modification critical for diverse biological functions, is well orchestrated within the Golgi apparatus, a central organelle in the secretory pathway. This review explores the mechanisms by which the Golgi governs protein-specific glycan modification, emphasizing the spatial organization of glycosyltransferases within the Golgi and the regulated transport of glycoproteins through its compartments. Specificity arises from the strategic localization of glycosyltransferases within the Golgi, enabling them to recognize both glycan structures and protein domains on their substrates. Advanced imaging reveals a complex and dynamic organization within the Golgi, challenging traditional models and highlighting specialized zones. Regulated transport of glycoproteins through the Golgi is critical for controlling their access to modifying enzymes. Specific sequence motifs within glycoproteins can act as 'passport sequence', directing them to distinct Golgi regions and influencing glycosylation by altering their proximity to specific enzymes and accessory proteins. These mechanisms support the hypothesis that sequence-dependent signals within substrate proteins influence their trafficking, enabling precise glycosylation as they transit the Golgi. Understanding these processes provides new insights into how glycan diversity is generated and regulated within the Golgi, highlighting the interplay between enzyme localization, protein trafficking, and the dynamic organization of this essential organelle.

Monoclonal antibodies are the cornerstone biopharmaceuticals whose safety and efficacy critically depend on their higher-order structure (HOS). Nuclear magnetic resonance (NMR) spectroscopy has emerged as a promising tool for HOS evaluation, yet its application has largely relied on fingerprinting approaches without residue-level interpretation. Here, we report site-specific assignments of methyl resonances in the Fc region of human IgG1, established through amino acid-selective labeling and correlation with backbone resonances using scalar coupling and NOE connectivities, further supported by mutagenesis. These assignments allowed us to identify glycoform-dependent spectral variations, including distinct signatures of core fucosylation and terminal galactosylation, as well as an Fc-specific amino acid substitution. Importantly, these spectral probes were detectable even in antibodies at natural isotopic abundance, enabling practical applications to therapeutic products without isotopic labeling. Furthermore, dynamic filtering highlighted methyl resonances from hinge and receptor-binding residues with elevated mobility, providing localized insights into functional sites. Collectively, our results establish unlabeled methyl NMR as a robust platform for sensitive and practical HOS assessment of therapeutic antibodies. This approach is broadly applicable to monitor glycosylation heterogeneity, chemical modifications, and batch-to-batch consistency, thereby offering a valuable framework for development and quality control of both innovative biopharmaceuticals and biosimilars.

Glycosylation depends on luminal nucleotide sugars delivered by solute carrier 35 (SLC35) transporters. SLC35A3 is a uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter. In humans, biallelic mutations in SLC35A3 cause arthrogryposis, mental retardation, and seizures (AMRS). To define how loss of SLC35A3 function reshapes the neural glycome, we profiled N-, O-, and glycosaminoglycans (GAGs) in Slc35a3 knockout mouse brains. N- and O-glycans were analyzed by MALDI-TOF MS, and GAG disaccharides were quantified by anion-exchange HPLC. Knockout mouse brains exhibited attenuation of complex-type N-glycans with a reciprocal rise in high-mannose species, as revealed by MALDI-TOF MS profiling. In contrast, ConA lectin blotting showed no significant change, consistent with its preferential detection of mannose-rich glycans. Branching analysis revealed loss of tri- and tetra-antennary structures compared with biantennary species. O-glycan profiling showed core-2-type species (Hex₂HexNAc₂ backbone) decreased. The dominant disialyl core-1 remained stable. Total GAG output (chondroitin/dermatan sulfate, heparan sulfate, and hyaluronan) was preserved. These findings support a microdomain model in which SLC35A3 acts as a locally effective supplier of UDP-GlcNAc to MGAT4 (branching N-acetylglucosaminyltransferase that installs the β1,4-GlcNAc arm) in the brain, while alternative routes buffer UDP-GlcNAc delivery for GAG and mucin-type O-glycan biosynthesis. Accordingly, AMRS may be attributed to impaired higher-order N-glycan branching in the brain.

Giant viruses encode unusual glycosylation machinery distinct from their amoebal hosts, raising fundamental questions about how their glycans are synthesized and diversified. Here, we present a comparative glycomic analysis of mimivirus, tokyovirus and hokutovirus, together with their common host Acanthamoeba castellanii. The main objective of this study was to determine whether giant viruses rely on host-derived N-glycosylation, or alternatively employ virus-encoded pathways to generate lineage-specific O-glycans, and to assess how these processes differ across virus families. N-glycan profiling revealed that all three viruses lack canonical eukaryotic core structures, in contrast to amoebal high-mannose N-glycans carrying pentose and phosphate residues. This finding demonstrates that giant viruses do not exploit the host secretory pathway for N-glycosylation, but instead depend on alternative mechanisms. O-glycan analyses showed lineage-specific patterns: family Marseilleviridae members tokyovirus and hokutovirus, displayed highly similar profiles, with minor virus-specific differences, whereas mimivirus exhibited structurally distinct glycans. Genomic inspection revealed that tokyovirus encodes only five glycosyltransferase-like genes, while A. castellanii harbours candidate enzymes for unusual monosaccharides. These findings clarify the distinct contributions of host and viral pathways and highlight evolutionary diversification of glycosylation among giant viruses.

Spinal cord injury (SCI) damages neural circuits and triggers pro-inflammatory responses, resulting in locomotor impairment. The carbohydrate sulfotransferases GlcNAc6ST1 and GlcNAc6ST4 modulate the function of blood monocytes and microglia. However, their specific roles and enzymatic relationships in neuroinflammation and functional recovery after SCI remain unclear. In this study, we demonstrate that mice deficient in both GlcNAc6ST1 and GlcNAc6ST4 (DKO) exhibit improved locomotor recovery compared with mice with a single deficiency. DKO mice exhibit reduced levels of monocytes and activated macrophages/microglia at the injury site alongside increased serotonergic neural fibers, indicating enhanced neural plasticity. RNA sequencing reveals down-regulation of collagen I genes and up-regulation of genes encoding synaptic membrane components in the injured DKO spinal cord. In addition, GALAXY glycomic analysis shows an absence of GlcNAc-6-sulfated N -glycans in the DKO spinal cord. These results suggest that GlcNAc6ST1 and GlcNAc6ST4 play complementary roles in promoting detrimental inflammatory responses post-SCI. Targeting these sulfotransferases could offer a novel therapeutic strategy to improve locomotor recovery after SCI.