Hideyuki Takeuchi

Some viruses, such as mumps virus (MuV), possess sialidases that cleave terminal sialic acids, primarily N -acetylneuraminic acid (Neu5Ac), from glycans. Previously, we developed a sialidase fluorescent imaging probe (BTP3-Neu5Ac) and visualized cells infected with sialidase–expressing viruses. However, BTP3-Neu5Ac could not specifically identify these viruses, as it reacted with a wide range of sialidases from viruses, bacteria, and mammals. Here, to confer specificity of BTP3-Neu5Ac to MuV sialidase, which features a hydrophobic cavity extending toward the C5 position of bound Neu5Ac, we evaluated BTP3-Neu5Ac derivatives containing hydrophobic groups at the C5 position of Neu5Ac moiety across various viral sialidases, including influenza A and B viruses. Linear N -acyl groups with 5–8 carbon atoms at this position improved specificity for MuV sialidase. These derivatives might enable precise identification and diagnosis of MuV infections. Our findings demonstrate that chemical modifications to Neu5Ac in BTP3-Neu5Ac can effectively modulate its specificity for viral sialidases.

Three types of O -linked glycosylation— O -glucose, O -fucose, and O - N -acetylglucosamine— are crucial for the function of Notch receptors, which regulate critical cell fate determination processes in a wide variety of contexts. O -Glucose glycans are transferred to serine residues located between the first and second conserved cysteines within the epidermal growth factor-like (EGF) repeats in the Notch extracellular domain. Previously, O -glucose glycans were shown to be extended to a trisaccharide structure with two xyloses via α1-3 linkages. Our recent studies, however, indicated that the O -glucose glycan on NOTCH1 EGF10 can be extended by hexose and Neu5Ac. Here, we demonstrated that this hexose- and Neu5Ac-extended glycan has a 3’-sialyllactose-like structure synthesized by specific members of two isoenzyme families, B4GALT1 and ST3GAL4. Using mass spectrometry, we identified this modification exclusively on NOTCH1 EGF10 and the analogous NOTCH3 EGF9 domain, with no detection in any other EGF domains in NOTCH1, NOTCH2, and NOTCH3. Sequence comparison and mutagenesis experiments identified one amino acid at position -2 of the fourth cysteine (C ⁴ -2) in the EGF domain as crucial for the galactose elongation of O -glucose glycans. We further demonstrated that this site-specific elongation of O -glucose on NOTCH1 EGF10 significantly impacts ligand binding and signal transduction of NOTCH1. In the context of early T cell development, the C ⁴ -2 mutants NOTCH1 A396Y and A396F enhance T cell differentiation through DLL1- and DLL4-dependent NOTCH1 signaling. Our findings contribute to the understanding of the intricate regulatory mechanisms of Notch receptor function mediated by distinct positions and structures of O -glycans.

Glycosylation, a key post-translational modification, regulates protein function in many contexts. Epidermal growth factor-like repeats undergo domain-specific O-glycosylation such as O-glucosylation, O-fucosylation, and O-GlcNAc'ylation. O-Glucose glycans are attached to specific serine residues by the action of protein O-glucosyltransferase 1 (POGLUT1) and can be elongated with two xylose residues by glucoside α1-3xylosyltransferase 1 (GXYLT1) or glucoside α1-3xylosyltransferase 2 (GXYLT2) and xyloside α1-3xylosyltransferase 1 (XXYLT1) in mammals. The xylosyl elongation of O-glucose as a negative regulator of Notch in Drosophila has recently been reported, but its role in mammalian Notch signaling remains elusive. Here, we investigated the impact of terminal xylosylation by XXYLT1 on NOTCH1 signaling in Jurkat cells, a T-cell acute lymphoblastic leukemia cell line with cell-autonomous NOTCH1 activation due to the juxtamembrane expansion mutation. Mass spectrometry analysis of NOTCH1 fragments overexpressed in Jurkat cells demonstrated that the O-glucose site on NOTCH1 EGF10 was modified with various elongating patterns of O-glucose. Genetic deletion of XXYLT1 in Jurkat cells led to enhanced activation of NOTCH1, suggesting that XXYLT1 inhibited NOTCH1 activation in Jurkat cells, whereas the cell surface expression of NOTCH1 was not altered. Unexpectedly, the proliferation of Jurkat cells was impaired in XXYLT1 knockout cells, with accompanying MYC downregulation, a Notch target gene. Our results revealed for the first time that mammalian Notch activation is fine-tuned by xylosylation in Jurkat cells, thus highlighting the potential of Notch agonism by the inhibition of xylosylation. Additionally, our findings regarding cell proliferation underscore the notion that there are possibly substrates other than NOTCH1 that XXYLT1 modifies, thereby regulating their functions in Jurkat cells.

Seasonal influenza epidemics caused by influenza A viruses (IAV) and influenza B viruses (IBV) pose a substantial public health burden. Despite the significant impact of IBV, its restricted host range and the absence of documented pandemics have resulted in limited research attention relative to IAV. Understanding the viral infection mechanisms of both IAV and IBV is crucial for controlling seasonal epidemics. Previously, we demonstrated that 3′-O-sulfated galactosylceramide sulfatide binds to IAV and enhances viral replication, a finding with potential therapeutic implications. However, the role sulfatide plays in other influenza virus infections, including those caused by IBV, remains unknown. Accordingly, in this paper, we investigate the function of sulfatide during IBV infection. We demonstrate that sulfatide binds to IBV hemagglutinin (HA), and that sulfatide overexpression significantly enhances IBV replication, whereas treatment with sulfatase or an anti-sulfatide antibody markedly suppressed IBV replication. Moreover, further tests involving the inhibition of sulfatide biosynthesis resulted in the suppression of viral replication with impaired nuclear export of viral ribonucleoproteins (vRNPs). These findings establish that sulfatide is a critical regulator of IBV replication, which parallels its role in IAV infection, and suggest that targeting sulfatide-virus interactions can lead to broad-spectrum therapeutic strategies against influenza virus.

Three types of O -linked glycosylation - O -glucose, O -fucose, and O - N -acetylglucosamine - are crucial for the function of Notch receptors, which regulates critical cell fate determination processes in a wide variety of contexts. O -Glucose glycans are added to serine residues located between the first and second conserved cysteines within the epidermal growth factor (EGF) -like repeats in the Notch extracellular domain. Previously, O -glucose glycans were shown to be extended to a trisaccharide structure with two xyloses via an α1-3 linkage. Our recent studies, however, indicated that the O -glucose glycan on NOTCH1 EGF10 can be extended by hexose and Neu5Ac. Here, we demonstrated that this hexose- and Neu5Ac-extended glycan has a 3’-sialyllactose-like structure synthesized by specific members of two isoenzyme families, B4GALT1 and ST3GAL4. Using mass spectrometry, we identified this modification exclusively on NOTCH1 EGF10 and NOTCH3 EGF9 (equivalent to NOTCH1 EGF10), with no detection in any other EGF domains in NOTCH1-3. Sequence comparison and mutagenesis experiments identified one amino acid at position -2 of the fourth cysteine in the EGF domain as crucial for the galactose elongation of O -glucose glycans. We further demonstrated that this site-specific elongation of O -glucose on NOTCH1 EGF10 significantly impact ligand binding and signal transduction of NOTCH1. Our findings contribute to the understanding of the intricate regulatory mechanisms of Notch receptor function mediated by distinct positions and structures of O -glycans. Significance Statement: Notch, a signaling receptor critical for development, differentiation, and homeostasis in multicellular organisms, undergoes multiple post-translational glycan modifications to regulate its function. While O -glucose glycan elongation by xylose was previously known, we identified an O -glucose glycan extended by galactose and Neu5Ac on specific epidermal growth factor (EGF) -like repeats of certain Notch paralogs, NOTCH1 and NOTCH3. As far as we know, this structure has not been reported on any other proteins. We identify the enzymes involved in the biosynthesis of this glycan and demonstrate that modifications at a specific site on Notch direct receptor functions, including ligand binding and Notch signaling.

Background/Objectives: In Southeast Asia, the traditional use of young coconut (Cocos nucifera L.) juice (YCJ) by women to alleviate postmenopausal symptoms suggests potential estrogenic properties. However, few studies explore the impact of YCJ on pathologies associated with estrogen deficiency in postmenopausal animal models. This study examines the impact of YCJ supplementation on memory impairment and depression-like behavior in ovariectomized (Ovx) rats. Methods: Ten-week-old female rats underwent either a sham operation (Sham) or bilateral Ovx. The rats in the Ovx + YCJ group received 5×-concentrated YCJ by gavage at a dose of 15 mL/kg body weight. Twelve weeks later, the Morris water maze and forced swim tests were used to evaluate hippocampus-dependent spatial memory and depression-like behavior, respectively. Results: The Ovx rats displayed significant memory impairment (p < 0.05) and depression-like behaviors (p < 0.05), while the memory performance in the rats in the Ovx + YCJ group resembled that of the Sham rats. However, the administration of YCJ did not result in the improvement of depression-like behavior. Conclusions: These findings suggest that YCJ consumption may help ameliorate memory impairment in postmenopausal women.