Biology subjects

Kuno, A.

Publications and source records attributed to Kuno, A..

15 recordsLinked to original sources

bioRxiv2026

Spatial proteomics identifies altered thick ascending limb cells as drivers of kidney disease through uromodulin retention

Chronic kidney disease (CKD) is increasingly prevalent and is a leading cause of mortality. Recent transcriptomic studies have identified the persistence of injury-induced renal tubular epithelial cell states, yet the cell populations that drive CKD remain unresolved. Uromodulin (UMOD) is the most abundant urinary secreted protein expressed exclusively in thick ascending limb (TAL) epithelial cells in the kidney. UMOD is strongly associated with CKD risk in genome-wide association studies, suggesting a broader, yet unexplored role of UMOD and TAL cells in CKD pathogenesis. Here, using spatial proteomics, we identified an injury-induced TAL cell state marked by intracellular UMOD accumulation. Spatially resolved multimodal integration revealed that UMOD accumulation occurred without transcriptional upregulation. Impaired urinary UMOD secretion further supported pathological intracellular retention. These altered TAL cells with UMOD retention exhibited inflammatory signaling, formed profibrotic niches enriched with M2-like macrophages across renal compartments, and were associated with CKD progression in multiple experimental models. Reducing the burden of UMOD retention diminished altered TAL cells and their profibrotic microenvironments, and attenuated CKD development. A similar altered TAL state was identified in human kidney disease and was associated with worse kidney function. Together, we identify pathological UMOD retention and altered TAL cells as key drivers of CKD development and potential therapeutic targets. This study also highlights that protein-level alterations can redefine disease-associated cell states beyond transcript profiling.

cell biology↗

bioRxiv2026

Lectin-Assisted Imaging Mass Cytometry (Lectin-IMC) Enables Spatial Validation of Disease-Associated Glyco-Niches within a Multimodal Glycoprotein Analysis Framework

Understanding the pathological significance of protein glycosylation and translating disease-associated glycan alterations into diagnostic and therapeutic opportunities require integrated analysis of glycans, their carrier glycoproteins, and spatial context. Here, we present an expanded multimodal glycoprotein analysis framework incorporating lectin-assisted imaging mass cytometry (Lectin-IMC) for stepwise discovery and spatial validation of disease-associated glyco-niches. Disease-associated glycans identified by laser microdissection-assisted lectin microarray (LMD-LMA) tissue glycome mapping are spatially evaluated by Lectin-IMC in relation to cell types and tissue microenvironments, and candidate carrier glycoproteins identified by MS-based glycoproteomics are subsequently incorporated for higher-order evaluation of glycan-protein-cell type relationships. Multiplex panel design is guided by LMA-based assessment of lectin-lectin interactions and biologically informed probe selection. Using a dilated cardiomyopathy model, we constructed a five-lectin panel centered on Wisteria floribunda agglutinin (WFA), which recognizes fibrosis-associated asialo N-glycans identified previously. Glycan-dependent WFA detection was validated by competitive inhibition and PNGase F treatment, and WFA-reactive glycans were spatially associated with fibrotic regions containing ACTA2VIM myofibroblast-like cells. Among six extracellular matrix glycoprotein candidates identified in WFA-binding fractions, periostin showed the strongest spatial correspondence with WFA-positive regions by pixel-based quantitative analysis and was further associated with ACTA2VIMWFA fibrotic microenvironments. The optimized Lectin-IMC panel was also transferable to a human FFPE cardiomyopathy specimen. Collectively, Lectin-IMC provides intuitive spatial visualization and interpretability of glycan-protein-cell type relationships and serves as an on-tissue spatial validation and prioritization layer within an iterative multimodal framework in which spatially defined glyco-niches can guide subsequent proteomic or glycoproteomic discovery.

biochemistry↗

bioRxiv2026

Activation of the NAD⁺-Sirtuin Axis Protects Against Chronic Doxorubicin-Induced Subclinical Renal Tubular Injury Through Restoration of Mitochondrial Homeostasis and Suppression of Inflammation

Background and purposeAnthracyclines, such as doxorubicin (DOX), are associated with late-onset kidney dysfunction; however, the mechanisms underlying chronic tubular injury remain poorly understood. We investigated whether chronic low-dose DOX exposure induces persistent mitochondrial dysfunction in renal tubules and evaluated the therapeutic potential of activating the NAD-Sirtuin axis. Experimental ApproachC57BL/6 mice were repeatedly administered low-dose DOX with or without resveratrol (RSV) or nicotinamide mononucleotide (NMN), a sirtuin activator. Renal injury was assessed using neutrophil gelatinase-associated lipocalin (NGAL) staining. Integrated proteomic and RNA sequencing analyses were performed to identify molecular alterations. Mitochondrial morphology and function were evaluated using structured illumination microscopy (SIM) of Massons trichrome-stained paraffin sections and ex vivo Seahorse analysis of freshly isolated renal tubules. Key ResultsChronic DOX administration induced tubular injury, despite preserving serum creatinine levels. Multi-omics analyses consistently demonstrated the suppression of mitochondrial pathways, including oxidative phosphorylation, fatty acid oxidation, and mitochondrial gene expression. SIM revealed mitochondrial fragmentation in tubular epithelial cells, whereas the Seahorse assay showed impaired mitochondrial respiratory capacity in isolated renal tubules. DOX also increased tubular acetylated superoxide dismutase 2 (SOD2) levels and activated inflammatory pathways. Importantly, both RSV and NMN attenuated tubular injury, restored mitochondrial metabolic pathways, reduced SOD2 acetylation, improved mitochondrial morphology, and suppressed inflammatory responses. Conclusions and ImplicationsChronic low-dose DOX exposure induces subclinical renal tubular injury characterized by mitochondrial dysfunction and inflammation. The pharmacological activation of sirtuins confers reno-protective effects by preserving mitochondrial homeostasis. These findings identify mitochondrial dysfunction as a central therapeutic target in DOX-induced nephrotoxicity and support sirtuin modulation as a potential strategy for preventing chemotherapy-related chronic kidney injury. Bullet point summaryO_ST_ABSWhat is already knownC_ST_ABSO_LIDoxorubicin causes cardiotoxicity through mitochondrial dysfunction and oxidative stress. C_LIO_LIDoxorubicin-induced tubular injury and the associated late-onset kidney dysfunction are clinically proven. C_LI What does this study addO_LIChronic low-dose doxorubicin induces tubular mitochondrial dysfunction, as identified by integrated multi-omics analyses. C_LIO_LIResveratrol and NMN preserve mitochondrial integrity and suppress inflammatory responses in renal tubules. C_LI Clinical significanceO_LIMitochondrial dysfunction may represent an early therapeutic target in doxorubicin-associated nephrotoxicity. C_LIO_LIActivation of the NAD-Sirtuin axis could prevent chronic kidney injury in cancer survivors. C_LI

pharmacology and toxicology↗

bioRxiv2026

Multi-omics definition of the sex-specific glycoproteome of murine tissues

Sex-specific differences in the glycoproteome remain poorly defined despite growing evidence that protein glycosylation is a key determinant of sex biology. Here we present a tissue-resolved glycoproteome atlas of adult male and female C57BL/6J mice, integrating transcriptomics, proteomics and glycoproteomics with sialic acid speciation and lectin microarray profiling across 19 tissues. Quantitative analysis of >26,800 protein- and site-specific N-glycoforms from 1,512 glycoproteins revealed highly distinct tissue glycoproteomes shaped by coordinated regulation of protein abundance and glyco-enzyme expression. Multi-omics integration identified strong glycophenotype-enzyme relationships, including control of tissue sialylation by Cmas and Cmah, suggesting rate-limiting roles in glycosylation. Pronounced sex-linked glycophenotypes were observed in salivary gland, liver and kidney, driven by differences in fucosylation, sialylation and protein abundance, whereas the brain glycome was largely conserved between sexes. An interactive online database (https://igcore.cloud/mta/atlas-viewer/) provides a resource for exploring sex-biased glycosylation across mouse tissues.

cell biology↗

bioRxiv2026

TSUMUGI: a platform for phenotype-driven gene network identification from comprehensive knockout mouse phenotyping data

SummaryDeciphering complex organismal phenotypes requires elucidation of coordinated functions among multiple genes, yet this remains a fundamental challenge in functional genetics. The International Mouse Phenotyping Consortium (IMPC) has recently established a comprehensive phenotypic atlas based on systematic single-gene knockout mouse lines, providing an unprecedented resource for gene-phenotype associations in mammals. However, extracting phenotype-associated multiple gene relationships remains challenging. Here, we present TSUMUGI, a platform that identifies a phenotype-driven gene network by leveraging gene-phenotype associations from the IMPC. TSUMUGI enables users to initiate analyses from a user-specified phenotype or gene list and interactively explore a gene network. In addition, TSUMUGI highlights human disease-associated genes, supports flexible network filtering, and allows seamless export of a gene network for downstream analyses. By linking shared phenotypic signatures to putative functional gene modules, TSUMUGI provides a framework for systematic interpretation and hypothesis generation of complex organismal phenotypes. Availability and implementationThe web application is available online at https://larc-tsukuba.github.io/tsumugi/. The command-line tools are distributed via PyPI (https://pypi.org/project/TSUMUGI) and Bioconda (https://bioconda.github.io/recipes/tsumugi/README.html). Source code and documentation are hosted at GitHub (https://github.com/akikuno/TSUMUGI-dev) and archived on Zenodo (https://doi.org/10.5281/zenodo.18464478) under the MIT license.

bioinformatics↗

bioRxiv2025

Comparative Evaluation of Glycoproteomics Software for Rare Glycopeptide Identification

Advancements in glycoproteomics software have improved glycopeptide identification; however, algorithm differences cause discrepancies in identified glycopeptide, even when identical datasets. We compared five state-of-the-art glycoproteomics software programs (Byonic, MSFragger-Glyco, pGlyco3, Glyco-Decipher, and GRable), investigating their unique capabilities, and examined their ability to identify rare sialic acid-containing glycopeptides (NeuGc and KDN) derived from BJAB-K20 cells, which lack UDP-N-acetylglucosamine 2-epimerase, the rate-limiting enzyme for sialic acid synthesis. Approximately half of the identified glycopeptides were unique to individual tools. Byonic identified the highest number of glycopeptides, whereas Glyco-Decipher and GRable identified complex highly branched glycan structures. NeuGc- and KDN-containing glycopeptides were identified by specific programs, highlighting their capability to handle rare glycan structures. To assess the reliability of these identifications, we reanalyzed the MS/MS spectra for the presence of diagnostic ions corresponding to each identified glycopeptide. Some software programs identified glycopeptides without detecting the corresponding diagnostic ions, raising concerns regarding result reliability. However, leveraging the distinct capabilities of each software enabled us to achieve a comprehensive and reliable analysis of glycopeptides, including those with rare glycan structures. Combining multiple glycoproteomics software programs with complementary strengths and incorporating post- verification steps, such as diagnostic ion analysis, enhances the accuracy and depth of glycopeptide identification.

biochemistry↗

bioRxiv2025

LM-GlycoRepo Version 1.0: A novel repository system for mouse tissue glycome mapping data

Lectin microarray (LMA) is a high-sensitive profiling method of protein glycosylation. The increasing use of this method in many studies has led to a growing demand for a repository system that meets the FAIR data principles (Findable, Accessible, Interoperable, and Reusable). Herein, we present a novel repository system, "LM-GlycoRepo," for lectin-based multimodal (LM) data, including LMA data, in accordance with the international guideline MIRAGE (Minimum Information Required for a Glycomics Experiment). As a first step in our efforts to provide a general repository for storing various types of LM data, LM-GlycoRepo Version 1.0 is specialized for mouse tissue glycome mapping data obtained using standardized laser microdissection (LMD)-assisted LMA procedures. This system allows users to deposit datasets containing LMD images, LMA data, and high-resolution lectin staining images as LM data. In addition, this repository adopted an "embargo" system that allows users to specify the release date of datasets, allowing compatibility with an article peer review system. Notably, after the release date, the deposited data were visualized using an existing web tool called LM-GlycomeAtlas. LM-GlycoRepo will evolve into a comprehensive tool for lectin-based multimodal data for various biospecimens, including human samples. LM-GlycoRepo is freely available at the GlyCosmos portal (https://lm-glycorepo.glycosmos.org/lm_glycorepo/).

bioinformatics↗

bioRxiv2024

KOnezumi-AID: A Software to Automate the Design of Multiplex Knockouts Using Target-AID

With the groundbreaking advancements in genome editing technologies, particularly CRISPR-Cas9, creating knockout mutants has become highly efficient. However, the CRISPR-Cas9 system introduces DNA double-strand breaks, which can increase the risk of chromosomal rear-rangements, presenting a major obstacle when attempting to knockout multiple genes simultaneously. Base editing systems like Target-AID provide a safer alternative by enabling precise base modifications without requiring DNA double-strand breaks, making them a promising solution for addressing this challenge. Nevertheless, the absence of adequate tools to support Target-AID-based gene knockouts highlights the need for a comprehensive system to design guide RNA (gRNA) for the simultaneous knockout of multiple genes. Here, we present KOnezumi-AID, a command-line tool designed to facilitate gRNA design for Target-AID-mediated genome editing. KOnezumi-AID supports gene knockout strategies by inducing premature termination codons or promoting exon skipping. It generates experiment-ready gRNA designs for both mouse and human genomes. Additionally, KOnezumi-AID offers batch processing capabilities, enabling rapid and precise gRNA design for large-scale genome editing projects such as CRISPR screening. In summary, KOnezumi-AID provides an efficient and user-friendly tool for gRNA design, streamlining genome editing workflows and advancing gene knockout research.

bioinformatics↗

bioRxiv2023

GRable version 1.0: A software tool for site-specific glycoform analysis using the improved Glyco-RIDGE method with parallel clustering and MS2 information

High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we have developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographic properties of glycopeptides and then searches for each combination of core peptides and glycan compositions by matching their mass and retention time differences. Here we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorithms, including "parallel clustering." This unique function improved the comprehensiveness of glycopeptide detection and allowed the analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the "confidence level" of the GRable results, especially using MS2 information. This function facilitated reduced misassignment of the core peptide and glycan composition and improved the interpretation of the results. Additional improved points are: "correction function" for accurate monoisotopic peak picking; one-to-one correspondence of clusters and core peptides even for multiply sialylated glycopeptides; and "inter-cluster analysis" function for understanding the reason for detected but unmatched clusters. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large scale and in depth. Therefore, this software will help to analyze the status and changes in glycans to obtain biological and clinical insights into protein glycosylation by complementing the comprehensiveness of MS2-based glycoproteomics. GRable can run freely online using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/564073v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@12ae798org.highwire.dtl.DTLVardef@1cac352org.highwire.dtl.DTLVardef@dd6b92org.highwire.dtl.DTLVardef@c16ddf_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions

Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. In contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea (ENU) and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR-& Transposase-based RegionaL Mutagenesis (CTRL-Mutagenesis). We demonstrated that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/557308v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@f70563org.highwire.dtl.DTLVardef@182dc6borg.highwire.dtl.DTLVardef@550e32org.highwire.dtl.DTLVardef@16f7dd_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2023

Universal method for generating knockout mice in multiple genetic backgrounds using zygote electroporation

Genetically engineered mouse models are essential tools for understanding mammalian gene functions and disease pathogenesis. Genome editing allows for the generation of these models in multiple inbred strains of mice without backcrossing. Zygote electroporation dramatically removed the barrier for introducing the CRISPR-Cas9 complex in terms of cost and labour. However, the editing conditions and protocols to produce knockout lines have been optimised for a limited number of strains or stocks. Here, we demonstrate a novel and universal approach for generating knockout mice in multiple inbred strains. By combining in vitro fertilisation and electroporation, we obtained founders for knockout alleles in 8 common inbred strains. Long-read sequencing analysis detected not only intended mutant alleles but also differences in read frequency of intended and unintended alleles among strains. Successful germline transmission of knockout alleles demonstrated that our novel approach can establish mutant mice targeting the same locus in multiple inbred strains for phenotyping analysis, contributing to reverse genetics and human disease research. Summary statementUniversal method for zygote genome editing in multiple inbred mouse strains allows for generation of novel mutant mice for understanding mammalian gene function and human disease pathogenesis.

genetics↗

bioRxiv2021

Combinatorial approach with mass spectrometry and lectin microarray dissected glycoproteomic features of virion-derived spike protein of SARS-CoV-2

The COVID-19 pandemic caused by the novel coronavirus, SARS-CoV-2, has a global impact on public health. Since glycosylation of the viral envelope glycoproteins is known to be deeply associated with their immunogenicity, intensive studies on the glycans of its major glycoprotein, S protein, have been conducted. Nevertheless, the detailed site-specific glycan compositions of virion-associated S protein have not yet been clarified. Here, we conducted intensive glycoproteomic analyses of SARS-CoV-2 S protein using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarray. Using our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, we identified 1,759 site-specific glycan compositions. The most frequent was HexNAc:Hex:Fuc:NeuAc:NeuGc = 6:6:1:0:0, suggesting a tri-antennary N-glycan terminating with LacNAc and having bisecting GlcNAc and a core fucose, which was found in 20 of 22 glycosylated sites. The subsequent lectin microarray analysis emphasized intensive outer arm fucosylation of glycans, which efficiently complemented the glycoproteomic features. The present results illustrate the high-resolution glycoproteomic features of SARS-CoV-2 S protein and significantly contribute to vaccine design, as well as the understanding of viral protein synthesis.

molecular biology↗

bioRxiv2021

Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure

The trimeric spike protein (S) mediates host-cell entry and membrane fusion of SARS-CoV-2. S protein is highly glycosylated, whereas its O-glycosylation is still poorly understood. Herein, we site-specifically examine the O-glycosylation of S protein through a mass spectrometric approach with HCD-triggered-ETD model. We identify 15 high-confidence O-glycosites and at least 10 distinct O-glycan structures on S protein. Peptide microarray assays prove that human ppGalNAc-T6 actively participates in O-glycosylation of S protein. Importantly, the upregulation of ppGalNAc-T6 expression can profoundly enhance the O-glycosylation level by generating new O-glycosites and increasing both O-glycan heterogeneity and intensities. Further molecular dynamics simulations reveal that the O-glycosylation on the protomer-interface regions, which are mainly modified by ppGalNAc-T6, can potentially stabilize the trimeric S protein structure. Our work provides deep molecular insights of how viral infection harnesses the host O-glycosyltransferases to dynamically regulate the O-glycosylation level of the viral envelope protein responsible for membrane fusion.

biochemistry↗

bioRxiv2020

DAJIN-assisted multiplex genotyping to validate the outcomes of CRISPR-Cas-based genome editing

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

genomics↗

bioRxiv2020

EXOC1 regulates cell morphology of spermatogonia and spermatocytes in mice

Spermatogenesis requires high regulation of germ cell morphology. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest of spermatogenesis at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1 is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. We found that while EXOC1 contributes to the inactivation of Rac1 in the pseudopod formation of spermatogonia, in spermatocyte syncytium formation, EXOC1 and SNAP23 cooperate with STX2. Our results showed that EXOC1 functions in concert with various cell morphology regulators in spermatogenesis. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

developmental biology↗