Comparative Conformational Analysis of Acyclic Sugar Alcohols Ribitol, Xylitol and d-Arabitol by Solution NMR and Molecular Dynamics Simulations.

Ribitol (C5H12O5) is an acyclic sugar alcohol that was recently identified in O-mannose glycan on mammalian α-dystroglycan. The conformation and dynamics of acyclic sugar alcohols such as ribitol are dependent on the stereochemistry of the hydroxyl groups; however, the dynamics are not fully understood. To gain insights into the conformation and dynamics of sugar alcohols, we carried out comparative analyses of ribitol, d-arabitol and xylitol by a crystal structure database search, solution NMR analysis and molecular dynamics (MD) simulations. The crystal structures of the sugar alcohols showed a limited number of conformations, suggesting that only certain stable conformations are prevalent among all possible conformations. The three-bond scholar coupling constants and exchange rates of hydroxyl protons were measured to obtain information on the backbone torsion angle and possible hydrogen bonding of each hydroxyl group. The 100 ns MD simulations indicate that the ribitol backbone has frequent conformational transitions with torsion angles between 180∘ and ±60∘, while d-arabitol and xylitol showed fewer conformational transitions. Taking our experimental and computational data together, it can be concluded that ribitol is more flexible than d-arabitol or xylitol, and the flexibility is at least in part defined by the configuration of the OH groups, which may form intramolecular hydrogen bonds.

Solution NMR Analysis of O-Glycopeptide-Antibody Interaction.

O-Linked glycans potentially play a functional role in cellular recognition events. Recent structural analyses suggest that O-glycosylation can be a specific signal for a lectin receptor which recognizes both the O-glycan and the adjacent polypeptide region. Further, certain antibodies specifically bind to the O-glycosylated peptide. There is growing interest in the mechanism by which O-glycans on proteins are specifically recognized by lectins and antibodies. The recognition system may be common to many O-glycosylated proteins; however, there is limited 3D structural information on the dual recognition of glycan and protein. This chapter describes a solution NMR analysis of the interaction between MUC1 O-glycopeptide and anti-MUC1 antibody MY.1E12.

Molecular Dynamics Simulation and Docking of MUC1 O-Glycopeptide.

Advances in computer performance and computational simulations allow increasing sophistication in applications in biological systems. Molecular dynamics (MD) simulations are especially suitable for studying conformation, dynamics, and interaction of flexible biomolecules such as free glycans and glycopeptides. Computer simulations are best performed when the scope and limitations in performance have been thoroughly assessed. Proper outputs are obtained only under suitable parameter settings, and results need to be properly validated. In this chapter, we will introduce an example of molecular dynamics simulations of MUC1 O-glycopeptide and its docking to anti-MUC1 antibody Fv fragment.

Prediction of protein structure and AI.

AlphaFold, an artificial intelligence (AI)-based tool for predicting the 3D structure of proteins, is now widely recognized for its high accuracy and versatility in the folding of human proteins. AlphaFold is useful for understanding structure-function relationships from protein 3D structure models and can serve as a template or a reference for experimental structural analysis including X-ray crystallography, NMR and cryo-EM analysis. Its use is expanding among researchers, not only in structural biology but also in other research fields. Researchers are currently exploring the full potential of AlphaFold-generated protein models. Predicting disease severity caused by missense mutations is one such application. This article provides an overview of the 3D structural modeling of AlphaFold based on deep learning techniques and highlights the challenges in predicting the pathogenicity of missense mutations.

A convenient assay for soluble Dectin-1 lectin domain binding to insoluble ß-glucans.

ß-Glucan is a homopolymer with a backbone of ß-1,3-linked glucose residues. The solubility and biological activity of ß-glucan can be influenced by the length of the backbone and the length/interval of the ß-1,6 branches. Dectin-1 is crucial in innate immunity through its binding to exogenous ß-glucans. However, there are few quantitative binding affinities available and there is no comprehensive comparative analysis of the binding of Dectin-1 to insoluble ß-glucans. Here, we have developed a simple binding assay for the interaction between Dectin-1 lectin domain (Dectin-1 CTLD) and insoluble ß-glucans. We utilized the paramylon particle as a model of insoluble ß-glucans. Dectin-1 CTLD bound to paramylon (particle size 3.1 µm) was separated from unbound Dectin-1 CTLD by centrifugation using a membrane filter (pore size 0.2 µm). The protein in the filtrate was quantified by SDS-PAGE and densitometry. The amount decreased in proportion to the amount of paramylon in the mixture. A control experiment using the Dectin-1 CTLD inactive mutant W221A showed that the mutant passes through the filter without binding paramylon. These results are evidence of site-specific binding of Dectin-1 CTLD to paramylon and demonstrate that the separation of paramylon-bound/unbound Dectin-1 CTLD is achievable through centrifugation using a filter. The assay was extended to other insoluble ß-glucans including curdlan. Additionally, it can be utilized in competitive inhibition experiments with soluble short-chain ß-glucans such as laminarin. The assay system allows for quantitative comparison of the affinities between insoluble and soluble ß-glucans and Dectin-1 CTLD, and should be useful because of its low-tech convenience.

NMR characterization of uniformly 13C- and/or 15N-labeled, unsulfated chondroitins with high molecular weights.

Solution nuclear magnetic resonance (NMR) analysis of polysaccharides can provide valuable information not only on their primary structures but also on their conformation, dynamics, and interactions under physiological conditions. One of the main problems is that non-anomeric 1H signals typically overlap, and this often hinders detailed NMR analysis. Isotope enrichment, such as with 13C and 15N, will add a new dimension to the NMR spectra of polysaccharides, and spectral analysis can be performed with enhanced sensitivity using isolated peaks. For this purpose, here we have prepared uniformly 13C- and/or 15N-labeled chondroitin polysaccharides -4)-ß-D-glucuronopyranosyl-(1-3)-2-acetamido-2-deoxy-ß-D-galactopyranosyl-(1- with molecular weights in the range from 310 to 460 k by bacterial fermentation. The enrichment ratios for 13C and 15N were 98.9 and 99.8%, respectively, based on the mass spectrometric analysis of the constituent chondroitin disaccharides. 1H and 13C NMR signals were assigned mainly based on HSQC and 13C-detection experiments including INADEQUATE, HETCOR, and HETCOR-TOCSY. The carbonyl carbon signal of the N-acetyl-ß-D-galactosamine residue was unambiguously distinguished from the C6 carbon of the ß-D-glucuronic acid residue by the observation of 13C peak splitting due to 1JCN coupling in 13C- and 15N-labeled chondroitin. The T2* and T1 were measured and indicate that both rigid and mobile sites are present in the long sequence of chondroitin. The conformation, dynamics, and interactions of chondroitin and its derivatives will be further analyzed based on the results obtained in this study.

Exogenous l-fucose attenuates neuroinflammation induced by lipopolysaccharide.

α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of fucose to the innermost GlcNAc residue of N-glycan to form core fucosylation. Our previous studies showed that lipopolysaccharide (LPS) treatment highly induced neuroinflammation in Fut8 homozygous KO (Fut8-/-) or heterozygous KO (Fut8+/-) mice, compared with the WT (Fut8+/+) mice. To understand the underlying mechanism, we utilized a sensitive inflammation-monitoring mouse system that contains the human interleukin-6 (hIL6) bacterial artificial chromosome transgene modified with luciferase (Luc) reporter cassette. We successfully detected LPS-induced neuroinflammation in the central nervous system by exploiting this bacterial artificial chromosome transgenic monitoring system. Then we examined the effects of l-fucose on neuroinflammation in the Fut8+/- mice. The lectin blot and mass spectrometry analysis showed that l-fucose preadministration increased the core fucosylation levels in the Fut8+/- mice. Notably, exogenous l-fucose attenuated the LPS-induced IL-6 mRNA and Luc mRNA expression in the cerebral tissues, confirmed using the hIL6-Luc bioluminescence imaging system. The activation of microglial cells, which provoke neuroinflammatory responses upon LPS stimulation, was inhibited by l-fucose preadministration. l-Fucose also suppressed the downstream intracellular signaling of IL-6, such as the phosphorylation levels of JAK2 (Janus kinase 2), Akt (protein kinase B), and STAT3 (signal transducer and activator of transcription 3). l-Fucose administration increased gp130 core fucosylation levels and decreased the association of gp130 with the IL-6 receptor in Fut8+/- mice, which was further confirmed in BV-2 cells. These results indicate that l-fucose administration ameliorates the LPS-induced neuroinflammation in the Fut8+/- mice, suggesting that core fucosylation plays a vital role in anti-inflammation and that l-fucose is a potential prophylactic compound against neuroinflammation.

Each N-glycan on human IgA and J-chain uniquely affects oligomericity and stability.

BACKGROUND: Immunoglobulin A (IgA) plays a pivotal role in various immune responses, especially that of mucosal immunity. IgA is usually assembled into dimers with the contribution of J-chains. There are two N-glycosylation sites in human IgA1-Fc and one in the J-chain. There is no consensus as yet on the functional role of the N-glycosylation. METHODS: To gain a better understanding of their role, we designed a series of IgA1-Fc mutants, which were expressed in the absence or presence of the J-chain. RESULTS: IgA1-Fc without the J-chain, was predominantly expressed as a monomer, and in its presence dimers and some polymers appeared. N263 (Fc Cα2), N459 (Fc tailpiece) and N49 (J-chain) were shown to be site-specifically modified with N-glycans by mass spectrometry analysis. Mutant IgA1-Fc N459Q failed to form a proper dimer in the presence of the J-chain, instead higher-order aggregates appeared. Fluorescence experiments suggest that the N459-glycans cover a hydrophobic surface at the Fc tailpiece that prevents other Fc molecules from approaching the dimeric IgA. A thermofluor assay revealed that the N-glycans at N263 (Fc) and N49 (J-chain) both contribute in different ways to the thermal stability of the Fc-J-chain complex. NMR analysis of 13C-labeled Fc suggests that the N459-glycan is relatively flexible while the N263-glycan is more rigid. CONCLUSIONS: We conclude that the N459-glycan of IgA1-Fc is essential for dimer formation and prevention of higher-order aggregates while those at N263 (Fc) and N49 (J-chain) stabilize the Fc-J-chain complex. GENERAL SIGNIFICANCE: Site-specific role for N-glycan in molecular assembly is addressed.

Identification of a buried ß-strand as a novel disease-related motif in the human polysialyltransferases.

The polysialyltransferases ST8SIA2 and ST8SIA4 and their product, polysialic acid (polySia), are known to be related to cancers and mental disorders. ST8SIA2 and ST8SIA4 have conserved amino acid (AA) sequence motifs essential for the synthesis of the polySia structures on the neural cell adhesion molecule. To search for a new motif in the polysialyltransferases, we adopted the in silico Individual Meta Random Forest program that can predict disease-related AA substitutions. The Individual Meta Random Forest program predicted a new eight-amino-acids sequence motif consisting of highly pathogenic AA residues, thus designated as the pathogenic (P) motif. A series of alanine point mutation experiments in the pathogenic motif (P motif) showed that most P motif mutants lost the polysialylation activity without changing the proper enzyme expression levels or localization in the Golgi. In addition, we evaluated the enzyme stability of the P motif mutants using newly established calculations of mutation energy, demonstrating that the subtle change of the conformational energy regulates the activity. In the AlphaFold2 model, we found that the P motif was a buried ß-strand underneath the known surface motifs unique to ST8SIA2 and ST8SIA4. Taken together, the P motif is a novel buried ß-strand that regulates the full activity of polysialyltransferases from the inside of the molecule.

Predicting the pathogenicity of missense variants based on protein instability to support diagnosis of patients with novel variants of ARSL.

Rare diseases are estimated to affect 3.5%-5.9% of the population worldwide and are difficult to diagnose. Genome analysis is useful for diagnosis. However, since some variants, especially missense variants, are also difficult to interpret, tools to accurately predict the effect of missense variants are very important and needed. Here we developed a method, "VarMeter", to predict whether a missense variant is damaging based on Gibbs free energy and solvent-accessible surface area calculated from the AlphaFold 3D protein model. We applied this method to the whole-exome sequencing data of 900 individuals with rare or undiagnosed disease in our in-house database, and identified four who were hemizygous for missense variants of arylsulfatase L (ARSL; known as the genetic cause of chondrodysplasia punctata 1, CPDX1). Two individuals had a novel Ser89 to Asn (Ser89Asn) or Arg469 to Trp (Arg469Trp) substitution, respectively predicted as "damaging" or "benign"; the other two had an Arg111 to His (Arg111His) or Gly117 to Arg (Gly117Arg) substitution, respectively predicted as "damaging" or "possibly damaging" and previously reported in patients showing clinical manifestations of CDPX1. Expression and analysis of the missense variant proteins showed that the predicted pathogenic variants (Ser89Asn, Arg111His, and Gly117Arg) had complete loss of sulfatase activity and reduced protease resistance due to destabilization of protein structure, while the predicted benign variant (Arg469Trp) had activity and protease resistance comparable to those of wild-type ARSL. The individual with the novel pathogenic Ser89Asn variant exhibited characteristics of CDPX1, while the individual with the benign Arg469Trp variant exhibited no such characteristics. These findings demonstrate that VarMeter may be used to predict the deleteriousness of variants found in genome sequencing data and thereby support disease diagnosis.

The cancer-associated glycosyltransferase GnT-V (MGAT5) recognizes the N-glycan core via residues outside its catalytic pocket.

N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) is a glycosyltransferase involved in cancer metastasis that creates the ß1,6-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.

Correction: Hoshi et al. High Correlation among Brain-Derived Major Protein Levels in Cerebrospinal Fluid: Implication for Amyloid-Beta and Tau Protein Changes in Alzheimer's Disease. Metabolites 2022, 12, 355.

In the original publication [...].

Dual impacts of a glycan shield on the envelope glycoprotein B of HSV-1: evasion from human antibodies in vivo and neurovirulence.

Identification of the mechanisms of viral evasion from human antibodies is crucial both for understanding viral pathogenesis and for designing effective vaccines. Here we show in cell cultures that an N-glycan shield on the herpes simplex virus 1 (HSV-1) envelope glycoprotein B (gB) mediated evasion from neutralization and antibody-dependent cellular cytotoxicity due to pooled γ-globulins derived from human blood. We also demonstrated that the presence of human γ-globulins in mice and immunity to HSV-1 induced by viral infection in mice significantly reduced replication in their eyes of a mutant virus lacking the glycosylation site but had little effect on the replication of its repaired virus. These results suggest that an N-glycan shield on a specific site of HSV-1 envelope gB mediated evasion from human antibodies in vivo and from HSV-1 immunity induced by viral infection in vivo. Notably, we also found that an N-glycan shield on a specific site of HSV-1 gB was significant for HSV-1 neurovirulence and replication in the central nervous system of naïve mice. Thus, we have identified a critical N-glycan shield on HSV-1 gB that has dual impacts, namely evasion from human antibodies in vivo and viral neurovirulence. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latent and recurrent infections in humans. To produce recurrent infections that contribute to transmission of the virus to new human host(s), the virus must be able to evade the antibodies persisting in latently infected individuals. Here, we show that an N-glycan shield on the specific site of the envelope glycoprotein B (gB) of HSV-1 mediates evasion from pooled γ-globulins derived from human blood both in cell cultures and mice. Notably, the N-glycan shield on the specific site of gB was also significant for HSV-1 neurovirulence in naïve mice. Considering the clinical features of HSV-1 infection, these results suggest that the glycan shield not only facilitates recurrent HSV-1 infections in latently infected humans by evading antibodies but is also important for HSV-1 pathogenesis during the initial infection.

O-GalNAc glycosylation determines intracellular trafficking of APP and Aß production.

A primary pathology of Alzheimer's disease (AD) is amyloid ß (Aß) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aß precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aß production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Aß production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

High-precision plant height measurement by drone with RTK-GNSS and single camera for real-time processing.

Conventional crop height measurements performed using aerial drone images require 3D reconstruction results of several aerial images obtained through structure from motion. Therefore, they require extensive computation time and their measurement accuracy is not high; if the 3D reconstruction result fails, several aerial photos must be captured again. To overcome these challenges, this study proposes a high-precision measurement method that uses a drone equipped with a monocular camera and real-time kinematic global navigation satellite system (RTK-GNSS) for real-time processing. This method performs high-precision stereo matching based on long-baseline lengths (approximately 1 m) during the flight by linking the RTK-GNSS and aerial image capture points. As the baseline length of a typical stereo camera is fixed, once the camera is calibrated on the ground, it does not need to be calibrated again during the flight. However, the proposed system requires quick calibration in flight because the baseline length is not fixed. A new calibration method that is based on zero-mean normalized cross-correlation and two stages least square method, is proposed to further improve the accuracy and stereo matching speed. The proposed method was compared with two conventional methods in natural world environments. It was observed that error rates reduced by 62.2% and 69.4%, for flight altitudes between 10 and 20 m respectively. Moreover, a depth resolution of 1.6 mm and reduction of 44.4% and 63.0% in the error rates were achieved at an altitude of 4.1 m, and the execution time was 88 ms for images with a size of 5472 × 3468 pixels, which is sufficiently fast for real-time measurement.

Brain-Derived Major Glycoproteins Are Possible Biomarkers for Altered Metabolism of Cerebrospinal Fluid in Neurological Diseases.

Cerebrospinal fluid (CSF) plays an important role in the homeostasis of the brain. We previously reported that CSF major glycoproteins are biosynthesized in the brain, i.e., lipocalin-type prostaglandin D2 synthase (L-PGDS) and transferrin isoforms carrying unique glycans. Although these glycoproteins are secreted from distinct cell types, their CSF levels have been found to be highly correlated with each other in cases of neurodegenerative disorders. The aim of this study was to examine these marker levels and their correlations in other neurological diseases, such as depression and schizophrenia, and disorders featuring abnormal CSF metabolism, including spontaneous intracranial hypotension (SIH) and idiopathic normal pressure hydrocephalus (iNPH). Brain-derived marker levels were found to be highly correlated with each other in the CSF of depression and schizophrenia patients. SIH is caused by CSF leakage, which is suspected to induce hypovolemia and a compensatory increase in CSF production. In SIH, the brain-derived markers were 2-3-fold higher than in other diseases, and, regardless of their diverse levels, they were found to be correlated with each other. Another abnormality of the CSF metabolism, iNPH, is possibly caused by the reduced absorption of CSF, which secondarily induces CSF accumulation in the ventricle; the excess CSF compresses the brain's parenchyma to induce dementia. One potential treatment is a "shunt operation" to bypass excess CSF from the ventricles to the peritoneal cavity, leading to the attenuation of dementia. After the shunt operation, marker levels began to increase within a week and then further increased by 2-2.5-fold at three, six, and twelve months post-operation, at which point symptoms had gradually attenuated. Notably, the marker levels were found to be correlated with each other in the post-operative period. In conclusion, the brain-derived major glycoprotein markers were highly correlated in the CSF of patients with different neurological diseases, and their correlations were maintained even after surgical intervention. These results suggest that brain-derived proteins could be biomarkers of CSF production.

Core Fucosylation Is Required for the Secretion of and the Enzymatic Activity of SOD3 in Nonsmall-Cell Lung Cancer Cells.

Aims: The anticancer function of superoxide dismutases (SODs) is still controversial. SOD3 is an extracellular superoxide dismutase and contains a single N-glycan chain. The role played by the N-glycosylation of SOD3, as it relates to lung cancer, is poorly understood. For this, we performed the structural and functional analyses of the N-glycan of SOD3 in lung cancer. Results: We report herein that the fucose structure of the N-glycan in SOD3 was increased in the sera of patients with lung cancer. In cell lines of non-small lung cancer cell (NSCLC), we also found a high level of the core fucose structure in the N-glycan of SOD3, as determined by lectin blotting and mass spectrometry analysis. To address the roles of the core fucose structure of SOD3, we generated FUT8 (α1,6-fucosyltransferase) gene knockout A549 cells. Using these cells, we found that the core fucose structure of SOD3 was required for its secretion and enzymatic activity, which contributes to the suppression of cell growth of NSCLC cells. Innovation: The core fucosylation is required for the secretion and enzymatic activity of SOD3, which contributes to anti-tumor functions such as the suppression of cell growth of NSCLC. Conclusion: The N-glycans, especially those with core fucose structures, regulate the anti-tumor functions of SOD3 against NSCLC. Antioxid. Redox Signal. 38, 1201-1211.

Structural Analysis of Oligosaccharides and Glycoconjugates Using NMR.

Carbohydrate chains play critical roles in cellular recognition and subsequent signal transduction in the nervous system. Furthermore, gangliosides are targets for various amyloidogenic proteins associated with neurodegenerative disorders. To better understand the molecular mechanisms underlying these biological phenomena, atomic views are essential to delineate dynamic biomolecular interactions. Nuclear magnetic resonance (NMR) spectroscopy provides powerful tools for studying structures, dynamics, and interactions of biomolecules at the atomic level. This chapter describes the basics of solution NMR techniques and their applications to the analysis of 3D structures and interactions of glycoconjugates in the nervous system.

NMR Analysis of Mammalian Glycolipids.

Mammalian glycolipids play a variety of roles, often coupled with interactions with endogenous and exogenous molecules. The interactions can induce intracellular signaling and are the means by which glycolipids express biological phenotypes. Insights into the structure-function relationships of glycolipids (both glycan and lipid moieties) provide the basis for gaining an understanding of the mechanisms at play, an important area for further study. Solution nuclear magnetic resonance (NMR) spectroscopy is a unique and powerful method employed to provide, at the atomic level, structural information on glycolipids and other biomolecules in solutions. This chapter briefly describes how we measure NMR spectra of glycolipids and the information gained from NMR spectral analysis.

Characterization of novel antibodies that recognize sialylated keratan sulfate and lacto-N-fucopentaose I on human induced pluripotent cells: comparison with existing antibodies.

This report describes the isolation and characterization of two new antibodies, R-6C (IgM) and R-13E (IgM), which were generated in C57BL/6 mice (Mus musculus) using the Tic (JCRB1331) human induced pluripotent cell (hiPSC) line as an antigen, and their comparisons with two existing antibodies, R-10G (IgG1) and R-17F (IgG1). Their epitopes were studied by western blotting after various glycosidase digestions, binding analyses using enzyme-linked immunosorbent assays (ELISAs) and microarrays with various synthetic oligosaccharides. The minimum epitope structures identified were: Siaα2-3Galß1-3GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S)ß1 (R-6C), Fucα1-2Galß1-3GlcNAcß1-3Galß1 (R-13E), Galß1-4GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S)ß1 (R-10G), and Fucα1-2Galß1-3GlcNAß1-3Galß1-4Glc (lacto-N-fucopentaose I) (R-17F). Most glycoprotein epitopes are expressed as O-glycans. The common feature of these epitopes is the presence of an N-acetyllactosamine type 1 structure (Galß1-3GlcNAc) at their nonreducing termini, followed by a type 2 structure (Galß1-4GlcNAc); this arrangement comprises a type 1-type 2 motif. This motif is also shared by TRA-1-60, a traditional onco-fetal antigen. In contrast, the R-10G epitope has a type 2-type 2 motif. Among these antibodies, R-17F and R-13E exhibit cytotoxic activity toward hiPSCs. R-17F and R-13E exhibit extremely high similarity in the amino acid sequences in their complementarity-determining regions (CDRs), which is consistent with their highly similar glycan recognition. These antibodies are excellent tools for investigating the biological functions of glycoconjugates in hiPSCs/hESCs; they could be useful for the selection, isolation and selective killing of such undifferentiated pluripotent stem cells.