Dynamic movement of the Golgi unit and its glycosylation enzyme zones

Harada A., Kunii M., Kurokawa K., et al. Dynamic movement of the Golgi unit and its glycosylation enzyme zones. Nature Communications 15, 4514 (2024); https://doi.org/10.1038/S41467-024-48901-1.Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small ‘Golgi units’ that have 1-3 μm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call ‘zones’. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Structural Variation of Chondroitin Sulfate Chains Contributes to the Molecular Heterogeneity of Perineuronal Nets

Aggrecan, a chondroitin sulfate (CS) proteoglycan, forms lattice-like extracellular matrix structures called perineuronal nets (PNNs). Neocortical PNNs primarily ensheath parvalbumin-expressing inhibitory neurons (parvalbumin, PV cells) late in brain development. Emerging evidence indicates that PNNs promote the maturation of PV cells by enhancing the incorporation of homeobox protein Otx2 and regulating experience-dependent neural plasticity. Wisteria floribunda agglutinin (WFA), an N-acetylgalactosamine-specific plant lectin, binds to the CS chains of aggrecan and has been widely used to visualize PNNs. Although PNNs show substantial molecular heterogeneity, the importance of this heterogeneity in neural plasticity remains unknown. Here, in addition to WFA lectin, we used the two monoclonal antibodies Cat315 and Cat316, both of which recognize the glycan structures of aggrecan, to investigate the molecular heterogeneity of PNNs. WFA detected the highest number of PNNs in all cortical layers, whereas Cat315 and Cat316 labeled only a subset of PNNs. WFA+, Cat315+, and Cat316+ PNNs showed different laminar distributions in the adult visual cortex. WFA, Cat315 and Cat316 detected distinct, but partially overlapping, populations of PNNs. Based on the reactivities of these probes, we categorized PNNs into four groups. We found that two subpopulation of PNNs, one with higher and one with lower WFA-staining are differentially labeled by Cat316 and Cat315, respectively. CS chains recognized by Cat316 were diminished in mice deficient in an enzyme involved in the initiation of CS-biosynthesis. Furthermore, WFA+ and Cat316+ aggrecan were spatially segregated and formed microdomains in a single PNN. Otx2 co-localized with Cat316+ but not with WFA+ aggrecan in PNNs. Our results suggest that the heterogeneity of PNNs around PV cells may affect the functional maturation of these cells

Fundamental Studies on Electrokinetic Chromatography with PEGylated Phospholipid Micelles

In this study, micelles prepared from distearoylphosphatidylethanolamine with covalently attached poly(ethylene) glycol) (PEG) of molecular weight 2000 (DSPE-PEG-2000) were employed in micellar electrokinetic chromatography (MEKC) as pseudostationary phases. Since DSPE-PEG-2000 contains long hydrophobic alkyl chains, an anionic phosphate group, and hydrophilic PEG chains, the prepared micelles are expected to provide a characteristic retention behavior for both neutral and ionic compounds. As a typical example, a baseline separation of phenol and 2-naphthol was successfully achieved by using the DSPE-PEG-2000 micelles as a background electrolyte for MEKC; such success clearly shows that the micelles can retain electrically neutral compounds. The MEKC separations of anionic and cationic compounds with a DSPE-PEG-2000 micellar solution and the enantioseparation of binaphthyl compounds with mixed micelles containing bile salt are also discussed

Fundamental studies on electrokinetic chromatography with PEGylated phospholipid micelles

In this study, micelles prepared from distearoylphosphatidylethanolamine with covalently attached poly(ethylene) glycol) (PEG) of molecular weight 2000 (DSPE-PEG-2000) were employed in micellar electrokinetic chromatography (MEKC) as pseudostationary phases. Since DSPE-PEG-2000 contains long hydrophobic alkyl chains, an anionic phosphate group, and hydrophilic PEG chains, the prepared micelles are expected to provide a characteristic retention behavior for both neutral and ionic compounds. As a typical example, a baseline separation of phenol and 2-naphthol was successfully achieved by using the DSPE-PEG-2000 micelles as a background electrolyte for MEKC; such success clearly shows that the micelles can retain electrically neutral compounds. The MEKC separations of anionic and cationic compounds with a DSPE-PEG-2000 micellar solution and the enantioseparation of binaphthyl compounds with mixed micelles containing bile salt are also discussed