Biology subjects

Nakai, H.

Publications and source records attributed to Nakai, H..

16 recordsLinked to original sources

bioRxiv2026

Megalin-mediated deterministic pseudotransduction in proximal tubules fundamentally challenges current understanding of AAV kidney transduction

Genetic kidney diseases are promising targets for gene therapy. Emerging AAV capsids that reportedly enable effective gene delivery to proximal tubules (PTs) following intravenous injection have raised hopes for treating diseases affecting renal tubules. Despite these promising developments, the scientific literature contains contradictory and non-reproducible findings, which prompted us to conduct a detailed and rigorous investigation of the mechanisms of AAV vector-mediated PT transduction. Here we provide compelling evidence that previously reported effective PT transduction following intravenous AAV-GFP or AAV-mCherry administration largely reflects megalin-mediated deterministic pseudotransduction rather than stochastic vector-mediated gene delivery. We demonstrate that this process is mediated by megalin-dependent uptake of circulating reporter proteins released from transduced organs including the liver and filtered through the glomeruli. These observations highlight the ongoing challenge of achieving true proximal tubule transduction via systemic AAV delivery and caution against relying on small reporters like GFP or mCherry, which can produce misleading signals.

bioengineering↗

bioRxiv2025

Discovery of a wide variety of α-1,6-cyclized β-1,2-glucan synthases: a new entrance for host-microbe interactions.

-1,6-Cyclized {beta}-1,2-glucans (C{beta}G) are established virulence factors in Xanthomonas, yet functional breadth and distribution of C{beta}Gs in nature have remained unclear. Here, we biochemically identified enzymes synthesizing C{beta}Gs with specific chain lengths, exhibiting potential natural occurrence of various C{beta}Gs. Structural analyses identify subtle variations in a loop, named Loop X, as a key determinant of product size, leading to understanding of the whole picture of the mechanism that controls the sizes. We further demonstrated that C{beta}G composed of 13 glucose units contributes to plant-virulence in Ralstonia pseudosolanacearum, expanding the functional scope of virulence-associated cyclic glucans. Overall, it provides the potential targets for regulating various plant-microbe interactions and also serves as a vital lead to discovering unknown host-microbe interactions with significant potential for agricultural applications.

biochemistry↗

bioRxiv2024

AAV Capsid Screening for Translational Pig Research Using a Mouse Xenograft Liver Model

In gene therapy, delivery vectors are a key component for successful gene delivery and safety, based on which adeno-associated viruses (AAVs) gained popularity in particular for the liver, but also for other organs. Traditionally, rodents have been used as animal models to develop and optimize treatments, but species and organ specific tropism of AAV desire large animal models more closely related to humans for preclinical in-depth studies. Relevant AAV variants with the potential for clinical translation in liver gene therapy were previously evolved in vivo in a xenogeneic mouse model transplanted with human hepatocytes. Here, we selected and evaluated efficient AAV capsids using chimeric mice with a >90% xenografted pig hepatocytes. The pig is a valuable preclinical model for therapy studies due to its anatomic and immunological similarities to humans. Using a DNA-barcoded recombinant AAV library containing 47 different capsids and subsequent Illumina sequencing of barcodes in the AAV vector genome DNA and transcripts in the porcine hepatocytes, we found the AAVLK03 and AAVrh20 capsid to be the most efficient delivery vectors regarding transgene expression in porcine hepatocytes. In attempting to validate these findings with primary porcine hepatocytes, we observed capsid-specific differences in cell entry and transgene expression efficiency where the AAV2, AAVAnc80, and AAVDJ capsids showed superior efficiency to AAVLK03 and AAVrh20. This work highlights intricacies of in vitro testing with primary hepatocytes and the requirements for suitable pre-clinical animal models but suggests the chimeric mouse to be a valuable model to predict AAV capsids to transduce porcine hepatocytes efficiently.

molecular biology↗

bioRxiv2024

Genomic loss of GPR108 disrupts AAV transduction in birds

The G protein-coupled receptor 108 (GPR108) gene encodes a protein factor identified as critical for adeno-associated virus (AAV) entry into mammalian cells, but whether it is universally involved in AAV transduction is unknown. Remarkably, we have discovered that GPR108 is absent in the genomes of birds and in most other sauropsids, providing a likely explanation for the overall lower AAV transduction efficacy of common AAV serotypes in birds compared to mammals. Importantly, transgenic expression of human GPR108 and manipulation of related glycan binding sites in the viral capsid significantly boost AAV transduction in zebra finch cells. These findings contribute to a more in depth understanding of the mechanisms and evolution of AAV transduction, with potential implications for the design of efficient tools for gene manipulation in experimental animal models, and a range of gene therapy applications in humans.

genomics↗

bioRxiv2024

A beta-Galactosidase acting on unique galactosides: the structure and function of a beta-1,2-galactosidase from Bacteroides xylanisolvens, an intestinal bacterium

Galactosides are major carbohydrates that are found in plant cell walls and various prebiotic oligosaccharides. Studying the detailed biochemical functions of {beta}-galactosidases in degrading these carbohydrates is important. In particular, identifying {beta}-galactosidases with new substrate specificities could help in the production of potentially beneficial oligosaccharides. In this study, we identified a {beta}-galactosidase with novel substrate specificity from Bacteroides xylanisolvens, an intestinal bacterium. The enzyme did not show hydrolytic activity toward natural {beta}-galactosides during the first screening. However, when -D-galactosyl fluoride (-GalF) as a donor substrate and galactose or D-fucose as an acceptor substrate were incubated with a nucleophile mutant, reaction products were detected. The galactobiose produced from the -GalF and galactose was identified as {beta}-1,2-galactobiose using NMR. Kinetic analysis revealed that this enzyme effectively hydrolyzed {beta}-1,2-galactobiose and {beta}-1,2-galactotriose. In the complex structure with methyl {beta}-galactopyranose as a ligand, the ligand is only located at subsite +1. The 2-hydroxy group and the anomeric methyl group of methyl {beta}-galactopyranose faces in the direction of subsite -1 and the solvent, respectively. This observation is consistent with the substrate specificity of the enzyme regarding linkage position and chain length. Overall, we concluded that the enzyme is a {beta}-galactosidase acting on {beta}-1,2-galactooligosaccharides. SynopsisThe structural and functional analysis of {beta}-galactosidase from an intestinal bacterium led to the discovery of a new {beta}-galactosidase hydrolyzing unique {beta}-1,2-galactooligosaccharides.

biochemistry↗

bioRxiv2024

Beta-Glucanase superfamily identified by sequential, functional, and structural analyses

{beta}-1,2-Glucans are natural glucose polymers that play important physiological roles, including as symbiotic or pathogenic factors and in osmoregulation. Phylogenetically new glycoside hydrolase (GH) families have recently been identified from {beta}-1,2-glucanase (SGL) sequences from bacteria (GH144) and a fungus (GH162). In this study, we identified four phylogenetically new groups (Groups 1-4), and determined that these families, together with GH144, GH162, and GH189, a family of transglycosylase domains in cyclic {beta}-1,2-glucan synthases, form a superfamily. Biochemical analysis of six proteins in these groups revealed that the proteins in Groups 1-3 showed hydrolytic activity specific to {beta}-1,2-glucan. The kinetic parameters of the enzymes of Groups 1-3 were similar to GH144 and GH162 SGLs, indicating that these enzymes were SGLs. Optical rotation analysis revealed that the SGLs followed an anomer-inverting mechanism. Structural analysis and prediction of the proteins in Groups 1-4, GH144, GH162, and GH189 suggested that Groups 1-3 and GH144 had the same reaction mechanism. Nevertheless, Groups 1-3 were dispersed irregularly in the superfamily. Overall, we determined that Groups 1-3 were new GH families, GHxxx, GHyyy, and GHzzz, respectively, and proposed that this superfamily be called an SGL superfamily because of the phylogenetical, functional, and structural relationships within the superfamily. HighlightsWide variety of glycoside hydrolases is far beyond our understanding. Functional and structural analysis identified three new glycoside hydrolase families. Molecular evolution with irregular changes in reaction mechanism was revealed.

biochemistry↗

bioRxiv2023

Phytopathogenic cyclic glucohexadecaose from an inverting transglycosylase

Xanthomonas species contain numerous notoriously well-known plant pathogens. Among various pathogenic factors, the role of -1,6-cyclized {beta}-1,2-glucohexadecaose (C{beta}G16) produced by Xanthomonas campestris pv. campestris was shown previously to be vital for infecting model organisms Arabidopsis thaliana and Nicotiana benthamiana. However, enzymes responsible for biosynthesising C{beta}G16 are essentially unknown, which limits the generation of agrichemicals that inhibit C{beta}G16 synthesis. In this study, we discovered that OpgD from X. campestris pv. campestris converts linear {beta}-1,2-glucan to C{beta}G16. Structural and functional analyses revealed that OpgD from X. campestris pv. campestris possesses an anomer-inverting transglycosylation mechanism, which is unprecedented among carbohydrate-active enzymes. The discovery of this unprecedented glucan-generating mechanism reveals a new foundation for the enzymatic synthesis of carbohydrates. Furthermore, identifying C{beta}G16 synthase highly conserved in Xanthomonas provides a broadly adaptable drug target for new-genre agrichemicals that overcome antimicrobial-resistant bacterial issues.

biochemistry↗

bioRxiv2023

Functional and structural analysis of a cyclization domain in a cyclic beta-1,2-glucan synthase

Cyclic {beta}-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic {beta}-1,2-glucans (C{beta}Gs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that {beta}-glucosidase-resistant compounds are produced by TiCGSCy with linear {beta}-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are C{beta}Gs. Next, action pattern analyses using {beta}-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide as the minimum length of the substrate. Nevertheless, longer substrate {beta}-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce C{beta}Gs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of {beta}-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear difference in the reaction pathways between these enzymes. Therefore, we propose a novel reaction mechanism of TiCGSCy. Thus, we propose that the present group of CGSs should be defined as a new glycoside hydrolase family, GHxxx. Statements and DeclarationsCompeting Interests: The authors declare no competing interests. Key PointsIt was clearly evidenced that cyclization domain alone produces cyclic {beta}-1,2-glucans. The domain exclusively catalyzes transglycosylation without hydrolysis. The present catalytic domain should be defined as a new glycoside hydrolase family.

biochemistry↗

bioRxiv2023

Enhancing gene transfer to renal tubules and podocytes by context-dependent selection of AAV capsids

Despite recent remarkable advancements in adeno-associated virus (AAV) vector technologies, effective gene delivery to the kidney remains a significant challenge. Here we show that AAV vector transduction in proximal tubules and podocytes, the crucial targets for renal gene therapy, can be enhanced remarkably through a meticulous selection of both AAV capsids and route of administration, tailored to the condition of the kidney. In this study, we performed a side-by-side comparison of 47 AAV capsids using AAV Barcode-Seq and identified six AAV capsids, including AAV-KP1, that exhibit remarkable enhancement of renal transduction in mice when delivered locally via the renal vein or the renal pelvis. Individual capsid validation analyses revealed that local delivery of AAV-KP1, but not AAV9, enables remarkably enhanced proximal tubule transduction while minimizing off-target liver transduction. In a mouse model of chronic kidney disease, intravenous administration of AAV9, not AAV-KP1, showed efficient renal tubule and podocyte transduction, which was not observed in the control wild-type mice. We also provide evidence that these contrasting observations between AAV-KP1 and AAV9 are attributed to their distinct pharmacokinetic profiles. Thus, this study highlights the importance of context-dependent capsid selection and engineering for successful renal gene therapy.

cell biology↗

bioRxiv2023

Novel glycoside hydrolase family enzymes from Escherichia coli are associated with osmo-regulated periplasmic glucan synthesis

Most Gram-negative bacteria synthesize osmo-regulated periplasmic glucans (OPG) in the periplasm or extracellular space. Many pathogens lose their pathogenicity by knocking out opgG, an OPG-related gene indispensable for OPG synthesis. However, the biochemical functions of OpgG and OpgD, a paralog of OpgG, have not been elucidated. In this report, structural and functional analyses of OpgG and OpgD from Escherichia coli revealed that these proteins are {beta}-1,2-glucanases with remarkably different activity, establishing a new glycoside hydrolase family. Furthermore, a reaction mechanism with an unprecedentedly long proton transfer pathway is proposed for OpgD. The conformation of the region that forms the reaction pathway differs noticeably between OpgG and OpgD, which explains the observed low activity of OpgG. The findings enhance our understanding of OPG biosynthesis and provide insights into functional diversity for this novel enzyme family.

biochemistry↗

bioRxiv2022

Cleavage of α-1,4-Glycosidic Linkages by the Glycosylphosphatidylinositol-Anchored α-Amylase AgtA Decreases the Molecular Weight of Cell Wall α-1,3-Glucan in Aspergillus oryzae

Aspergillus fungi contain -1,3-glucan with a low proportion of -1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored -amylases are conserved in Aspergillus fungi. The GPI-anchored -amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall -1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall -1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall -1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial -1,3-glucan. Furthermore, rAoAgtA cleaved 3--maltotetraosylglucose with a structure similar to the predicted boundary structure between the -1,3-glucan main chain and a short spacer composed of -1,4-linked glucose residues in cell wall -1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the -1,4-glycosidic bonds of 3--maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall -1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight (MW) of cell wall -1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored -amylases can degrade the spacer -1,4-glycosidic linkages in cell wall -1,3-glucan before its insolubilization, and this spacer cleavage decreases the MW of cell wall -1,3-glucan in vivo.

microbiology↗

bioRxiv2021

Tertiary lymphoid structures induced by CXCL13-producing CD4+ T cells increase tumor infiltrating CD8+ T cells and B cells in ovarian cancer

BackgroundTertiary lymphoid structures (TLSs) are transient ectopic lymphoid aggregates whose formation might be caused by chronic inflammation states, such as cancer. The presence of TLS is associated with a favorable prognosis in most solid malignancies. The recognition of the relevance of TLS to cancer has led to a growing interest in TLS as an immunomodulatory target to enhance tumor immunity, although how TLSs are induced in the tumor microenvironment (TME) and how they affect patient survival are not well understood. MethodsTLS distribution in relation to tumor infiltrating lymphocytes (TILs) and related gene expression were investigated in high grade serous ovarian cancer (HGSC) specimens. CXCL13 expression, which is strongly associated with TLS, and its localization in immune cells, were examined. We explored the tumor microenvironment for CXCL13 secretion by adding various inflammatory cytokines in vitro. The induction of TLS by CXCL13 was examined in a mouse model of ovarian cancer. ResultsCXCL13 gene expression correlated with TLS formation and the infiltration of T cells and B cells, and was a favorable prognostic factor for HGSC patients. The coexistence of CD8+ T cells and B-cell lineages in the TME was associated with a better prognosis of HGSC and was closely related to the presence of TLSs. CXCL13 expression was predominantly coincident with CD4+ T cells in TLSs and CD8+ T cells in TILs, and shifted from CD4+ T cells to CD21+ follicular dendritic cells as the TLS matured. Although TGF-{beta} was reported to stimulate CXCL13 production, our in vitro results revealed that CXCL13 secretion was promoted in CD4+ T cells under TGF-{beta} + IL-2-restricted conditions and in CD8+ T cells under TGF-{beta} + IL-12-rich conditions. In a mouse model of ovarian cancer, recombinant CXCL13 induced TLSs and enhanced survival by the infiltration of CD8+ T cells. ConclusionsTLS formation was promoted by CXCL13-producing CD4+ T cells and TLSs facilitated the coordinated antitumor responses of cellular and humoral immunity in ovarian cancer.

immunology↗

bioRxiv2021

EXPRESSION OF A SECRETABLE, CELL-PENETRATING CDKL5 PROTEIN ENHANCES THE EFFICACY OF AAV VECTOR-MEDIATED GENE THERAPY FOR CDKL5 DEFICIENCY DISORDER.

No therapy is currently available for CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD), a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although delivery of a wild-type copy of the mutated gene to cells represents the most curative approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for brain gene therapy. Herein, we used a secretable TATk-CDKL5 protein to enhance the efficiency of a gene therapy for CDD. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to a higher CDKL5 protein replacement due to secretion and transduction of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle-treated Cdkl5 KO mice or Cdkl5 KO mice treated with the AAVPHP.B_CDKL5 vector, indicating that a gene therapy based on a secretable recombinant TATk-CDKL5 protein is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5.

neuroscience↗

bioRxiv2021

Frequent PIK3CA mutations in eutopic endometrium of patients with ovarian clear cell carcinoma

Recent studies have reported cancer-associated mutations in normal endometrium. Mutations in eutopic endometrium may lead to endometriosis and endometriosis-associated ovarian cancer. We investigated PIK3CA mutations (PIK3CAm) for three hotspots (E542K, E545K, H1047R) in eutopic endometrium in patients with ovarian cancer and endometriosis from formalin-fixed paraffin-embedded specimens by laser-capture microdissection and droplet digital PCR. The presence of PIK3CAm in eutopic endometrial glands with mutant allele frequency [≥]15% were as follows: ovarian clear cell carcinoma (OCCC) with PIK3CAm in tumors, 20/300 hotspots in 11/14 cases; OCCC without PIK3CAm, 42/78 hotspots in 11/12 cases; high-grade serous ovarian carcinoma, 8/45 hotspots in 3/5 cases; and endometriotic cysts, 5/63 hotspots in 5/6 cases. These rates were more frequent than in non-cancer non-endometriosis controls (7/309 hotspots in 5/17 cases). In OCCC without PIK3CAm, 7/12 (58%) cases showed multiple hotspot mutations in the same eutopic endometrial glands. In 3/54 (5.6%) cases, PIK3CAm was found in eutopic endometrial stroma. Multi-sampling of the OCCC tumors with PIK3CAm showed intratumor heterogeneity in three of eight cases. In two cases, PIK3CAm was detected in the stromal component of the tumor. Homogenous PIK3CAm in the epithelial component of the tumor matched the mutation in eutopic endometrial glands in only one case. Eutopic endometrial glands in ovarian cancer and endometriosis show high frequency of PIK3CAm that is not consistent with tumors, and multiple hotspot mutations are often found in the same glands. Our results suggest that most PIK3CAm in eutopic endometrial glands are passenger rather than driver mutations.

cancer biology↗

bioRxiv2020

In vivo repair of a protein underlying a neurological disorder by programmable RNA editing

RNA base editing is gaining momentum as an approach to repair mutations, but its application to neurological disease has not been established. We have succeeded in directed transcript editing of a pathological mutation in a mouse model of the neurodevelopmental disease, Rett syndrome. Specifically, we directed editing of a guanosine to adenosine mutation in RNA encoding Methyl CpG Binding Protein 2 (MECP2). Repair was mediated by injecting the hippocampus of juvenile Rett mice with an adeno-associated virus expressing both an engineered enzyme containing the catalytic domain of Adenosine Deaminase Acting on RNA 2 and a Mecp2 targeting guide. After one month, 50% of Mecp2 RNA was recoded in three different hippocampal neuronal subtypes, and the ability of MeCP2 protein to associate with heterochromatin was similarly restored to 50% of wild-type levels. This study represents the first in vivo programmable RNA editing applied to a model of neurological disease.

neuroscience↗

bioRxiv2019

Hydroxide Ion Carrier for Proton Pump in Bacteriorhodopsin: Primary Proton Transfer

Bacteriorhodopsin (BR) is a model protein for light-driven proton pumps, where the vectorial active proton transport results in light-energy conversion. To clarify the microscopic mechanism of primary proton transfer from retinal Schiff base (SB) to Asp85 in BR, herein we performed quantum-mechanical metadynamics simulations of the whole BR system ([~]3800 atoms). The simulations showed a novel proton transfer mechanism, viz. hydroxide ion mechanism, in which the deprotonation of specific internal water (Wat452) yields the protonation of Asp85 via Thr89, after which the resulting hydroxide ion accepts the remaining proton from retinal SB. Furthermore, systematic investigations adopting four sequential snapshots obtained by the time-resolved serial femtosecond crystallography revealed that proton transfer took 2-5.25 s on the photocycle. The presence of Wat401, which is the main difference between snapshots at 2 and 5.25 s, is found to be essential in assisting the primary proton transfer. SIGNIFICANCEBacteriorhodopsin (BR), the benchmark of light-driven proton pumps, has attracted much attention from diverse areas in terms of energy conversion. Despite the significant experimental and theoretical efforts, the microscopic mechanism of the proton transfers in BR is not completely unveiled. In this study, quantum-mechanical molecular dynamics simulations of whole BR system were performed to elucidate the primary proton transfer in the L intermediate state with the latest snapshots obtained from X-ray free electron laser. As a result, it is found that the hydroxide ion originating from the specific internal water, which appears at the active site only in the L state, acts as a carrier for the primary proton transfer, demonstrating the importance of hydroxide ions in proton pumps.

biophysics↗