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.

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.

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.

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.

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.

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.

Analysis of Selenoprotein F Binding to UDP-Glucose:Glycoprotein Glucosyltransferase (UGGT) by a Photoreactive Crosslinker.

In the endoplasmic reticulum glycoprotein quality control system, UDP-glucose : glycoprotein glucosyltransferase (UGGT) functions as a folding sensor. Although it is known to form a heterodimer with selenoprotein F (SelenoF), the details of the complex formation remain obscure. A pulldown assay using co-transfected SelenoF and truncated mutants of human UGGT1 (HUGT1) revealed that SelenoF binds to the TRXL2 domain of HUGT1. Additionally, a newly developed photoaffinity crosslinker was selectively introduced into cysteine residues of recombinant SelenoF to determine the spatial orientation of SelenoF to HUGT1. The crosslinking experiments showed that SelenoF formed a covalent bond with amino acids in the TRXL3 region and the interdomain between ßS2 and GT24 of HUGT1 via the synthetic crosslinker. SelenoF might play a role in assessing and refining the disulfide bonds of misfolded glycoproteins in the hydrophobic cavity of HUGT1 as it binds to the highly flexible region of HUGT1 to reach its long hydrophobic cavity. Clarification of the SelenoF-binding domain of UGGT and its relative position will help predict and reveal the function of SelenoF from a structural perspective.

O-Glycan-Dependent Interaction between MUC1 Glycopeptide and MY.1E12 Antibody by NMR, Molecular Dynamics and Docking Simulations.

Anti-mucin1 (MUC1) antibodies have been widely used for breast cancer diagnosis and treatment. This is based on the fact that MUC1 undergoes aberrant glycosylation upon cancer progression, and anti-MUC1 antibodies differentiate changes in glycan structure. MY.1E12 is a promising anti-MUC1 antibody with a distinct specificity toward MUC1 modified with an immature O-glycan (NeuAcα(2-3)Galß(1-3)GalNAc) on a specific Thr. However, the structural basis for the interaction between MY.1E12 and MUC1 remains unclear. The aim of this study is to elucidate the mode of interaction between MY.1E12 and MUC1 O-glycopeptide by NMR, molecular dynamics (MD) and docking simulations. NMR titration using MUC1 O-glycopeptides suggests that the epitope is located within the O-linked glycan and near the O-glycosylation site. MD simulations of MUC1 glycopeptide showed that the O-glycosylation significantly limits the flexibility of the peptide backbone and side chain of the O-glycosylated Thr. Docking simulations using modeled MY.1E12 Fv and MUC1 O-glycopeptide, suggest that VH mainly contributes to the recognition of the MUC1 peptide portion while VL mainly binds to the O-glycan part. The VH/VL-shared recognition mode of this antibody may be used as a template for the rational design and development of anti-glycopeptide antibodies.

3D Structures of IgA, IgM, and Components.

Immunoglobulin G (IgG) is currently the most studied immunoglobin class and is frequently used in antibody therapeutics in which its beneficial effector functions are exploited. IgG is composed of two heavy chains and two light chains, forming the basic antibody monomeric unit. In contrast, immunoglobulin A (IgA) and immunoglobulin M (IgM) are usually assembled into dimers or pentamers with the contribution of joining (J)-chains, which bind to the secretory component (SC) of the polymeric Ig receptor (pIgR) and are transported to the mucosal surface. IgA and IgM play a pivotal role in various immune responses, especially in mucosal immunity. Due to their structural complexity, 3D structural study of these molecules at atomic scale has been slow. With the emergence of cryo-EM and X-ray crystallographic techniques and the growing interest in the structure-function relationships of IgA and IgM, atomic-scale structural information on IgA-Fc and IgM-Fc has been accumulating. Here, we examine the 3D structures of IgA and IgM, including the J-chain and SC. Disulfide bridging and N-glycosylation on these molecules are also summarized. With the increasing information of structure-function relationships, IgA- and IgM-based monoclonal antibodies will be an effective option in the therapeutic field.

NMR assignments of the N-glycans of the Fc fragment of mouse immunoglobulin G2b glycoprotein.

The Fc portion of immunoglobulin G (IgG) promotes defensive effector functions in the immune system by interacting with Fcγ receptors and complement component C1q. These interactions critically depend on N-glycosylation at Asn297 of each CH2 domain, where biantennary complex-type oligosaccharides contain microheterogeneities resulting primarily from the presence or absence of non-reducing terminal galactose residues. Crystal structures of Fc have shown that a pair of N-glycans is located between the two CH2 domains. Here we applied our metabolic isotope labeling technique using mammalian cells for in-solution structural characterization of mouse IgG2b-Fc glycoforms with a molecular mass of 54 kDa. Based on spectral assignments of the N-glycans as well as polypeptide backbones of Fc, we probed conformational perturbations of Fc induced by N-glycan trimming, especially enzymatic degalactosylation. The results indicated that degalactosylation structurally perturbed the Fc region through rearrangement of glycan-protein interactions. The spectral assignments of IgG2b-Fc glycoprotein will provide the basis for NMR investigation of its dynamic conformations and interactions with effector molecules in solution.

Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly-identified brain sterol metabolite, galactosylated cholesterol.

ß-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form ß-cholesterylglucoside (ß-GlcChol) in vitro ß-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate ß-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (ß-GalChol), in addition to ß-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for ß-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for ß-GalChol formation. Liquid chromatography-tandem MS revealed that ß-GlcChol and ß-GalChol are present throughout development from embryo to adult in the mouse brain. We found that ß-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of ß-GalChol biosynthesis appeared to be during myelination. We also found that ß-GlcChol and ß-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form ß-GalChol. This is the first report of the existence of ß-GalChol in vertebrates and how ß-GlcChol and ß-GalChol are formed in the brain.

3D Structure and Function of Glycosyltransferases Involved in N-glycan Maturation.

Glycosylation is the most ubiquitous post-translational modification in eukaryotes. N-glycan is attached to nascent glycoproteins and is processed and matured by various glycosidases and glycosyltransferases during protein transport. Genetic and biochemical studies have demonstrated that alternations of the N-glycan structure play crucial roles in various physiological and pathological events including progression of cancer, diabetes, and Alzheimer's disease. In particular, the formation of N-glycan branches regulates the functions of target glycoprotein, which are catalyzed by specific N-acetylglucosaminyltransferases (GnTs) such as GnT-III, GnT-IVs, GnT-V, and GnT-IX, and a fucosyltransferase, FUT8s. Although the 3D structures of all enzymes have not been solved to date, recent progress in structural analysis of these glycosyltransferases has provided insights into substrate recognition and catalytic reaction mechanisms. In this review, we discuss the biological significance and structure-function relationships of these enzymes.

The Core Fucose on an IgG Antibody is an Endogenous Ligand of Dectin-1.

The core fucose, a major modification of N-glycans, is implicated in immune regulation, such as the attenuation of the antibody-dependent cell-mediated cytotoxicity of antibody drugs and the inhibition of anti-tumor responses via the promotion of PD-1 expression on T cells. Although the core fucose regulates many biological processes, no core fucose recognition molecule has been identified in mammals. Herein, we report that Dectin-1, a known anti-ß-glucan lectin, recognizes the core fucose on IgG antibodies. A combination of biophysical experiments further suggested that Dectin-1 recognizes aromatic amino acids adjacent to the N-terminal asparagine at the glycosylation site as well as the core fucose. Thus, Dectin-1 appears to be the first lectin-like molecule involved in the heterovalent and specific recognition of characteristic N-glycans on antibodies.

Characterization of Antibody Products Obtained through Enzymatic and Nonenzymatic Glycosylation Reactions with a Glycan Oxazoline and Preparation of a Homogeneous Antibody-Drug Conjugate via Fc N-Glycan.

Glycan engineering of antibodies has received considerable attention. Although various endo-ß- N-acetylglucosaminidase mutants have been developed for glycan remodeling, a side reaction has been reported between glycan oxazoline and amino groups. In this study, we performed a detailed characterization for antibody products obtained through enzymatic and nonenzymatic reactions with the aim of maximizing the efficiency of the glycosylation reaction with fewer side products. The reactions were monitored by an ultraperformance liquid chromatography system using an amide-based wide-pore column. The products were characterized by liquid chromatography coupled with tandem mass spectrometry. The side reactions were suppressed by adding glycan oxazoline in a stepwise manner under slightly acidic conditions. Through a combination of an azide-carrying glycan transfer reaction under optimized conditions and a bio-orthogonal reaction, a potent cytotoxic agent monomethyl auristatin E was site-specifically conjugated at N-glycosylated Asn297 with a drug-to-antibody ratio of 4. The prepared antibody-drug conjugate exhibited cytotoxicity against HER2-expressing cells.

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

N-acetylglucosaminyltransferase-V (GnT-V) alters the structure of specific N-glycans by modifying α1-6-linked mannose with a ß1-6-linked N-acetylglucosamine branch. ß1-6 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.

Implication of C-type lectin receptor langerin and keratan sulfate disaccharide in emphysema.

Glycosylation is profoundly involved in various diseases, and interactions between glycan binding proteins and their sugar ligands are plausible drug targets. Keratan sulfate (KS), a glycosaminoglycan, is downregulated in lungs by cigarette smoking, suggesting that KS is involved in smoking-related diseases, such as chronic obstructive pulmonary disease (COPD). We found that a highly sulfated KS disaccharide, L4, suppresses lung inflammation and is effective against COPD and its exacerbation in mouse models. Its anti-inflammatory activity was comparable to that of a steroid. As a possible mechanism, langerin, a C-type lectin receptor (CLR) expressed in dendritic cells, was suggested to function as an L4 receptor. Oligomeric L4 derivatives were chemically designed to create new ligands with higher affinity and activity. The synthetic L4 oligomers bound to langerin with over 1000-fold higher affinity than the L4 monomer, suggesting that these compounds are effective drug candidates against COPD and inflammatory diseases.

Rapid increase of 'brain-type' transferrin in cerebrospinal fluid after shunt surgery for idiopathic normal pressure hydrocephalus: a prognosis marker for cognitive recovery.

Idiopathic normal pressure hydrocephalus (iNPH) is a dementia-inducing disorder. Primary cause of iNPH is speculated to be a reduction of cerebrospinal fluid (CSF) absorption, which secondarily induces hydrocephalus, compression of brain, and reduction of CSF production. Patients are treated by surgically inserting a shunt to deliver excess CSF to the abdominal cavity. The prognosis for cognitive improvement after shunt surgery has been difficult to predict. We therefore investigated various CSF proteins, hoping to find a biomarker predictive of cognitive performance one to two years after shunt surgery. CSF proteins of 34 iNPH and 15 non-iNPH patients were analysed by Western blotting, revealing two glycan isoforms of transferrin (Tf); 'brain-type' Tf with N-acetylglucosaminylated glycans and 'serum-type' Tf with α2, 6-sialylated glycans. Brain-type Tf levels decreased in iNPH but rapidly returned to normal levels within 1-3 months after shunt surgery. This change was positively correlated with recovery from dementia, per Mini-Mental State Examination and Frontal Assessment Battery scores at 11.8 ± 7.7 months post-operation, suggesting that brain-type Tf is a prognostic marker for recovery from dementia after shunt surgery for iNPH. Histochemical staining with anti-Tf antibody and an N-acetylglucosamine-binding lectin suggests that brain-type Tf is secreted from choroid plexus, CSF-producing tissue.

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr134/Thr138/Thr143/Thr147/Thr152) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr134/Thr138 or Thr143/Thr147/Thr152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvß3 and ß1 integrins, as well as αvß3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation.

Characterization of Conformational Ensembles of Protonated N-glycans in the Gas-Phase.

Ion mobility mass spectrometry (IM-MS) is a technique capable of investigating structural changes of biomolecules based on their collision cross section (CCS). Recent advances in IM-MS allow us to separate carbohydrate isomers with subtle conformational differences, but the relationship between CCS and atomic structure remains elusive. Here, we characterize conformational ensembles of gas-phase N-glycans under the electrospray ionization condition using molecular dynamics simulations with enhanced sampling. We show that the separation of CCSs between isomers reflects folding features of N-glycans, which are determined both by chemical compositions and protonation states. Providing a physicochemical basis of CCS for N-glycans helps not only to interpret IM-MS measurements but also to estimate CCSs of complex glycans.