Astragaloside IV Inhibits Galactose-Deficient IgA1 Secretion via miR-98-5p in Pediatric IgA Nephropathy

Purpose: The factor associated with IgA nephropathy (IgAN) is an abnormality of IgA known as galactose-deficient IgA1 (Gd-IgA1). The purpose of this study was to determine the molecular role played by miRNAs in the formation of Gd-IgA1 in IgAN and investigate the regulatory role of Astragaloside IV (AS-IV) in miRNAs.Patients and methods: Bioinformatics analysis, along with functional and mechanistic experiments, were used to investigate the relationship and function of miRNA, β-1, 3-galactosyltransferase (C1GALT1), Gd-IgA1, and AS-IV. Analyses involved a series of tools, including quantitative real-time polymerase chain reaction (qRT-qPCR), Western blot, enzyme-linked immunosorbent assay (ELISA), Vicia Villosa lectin-binding assay (VVA), Cell counting kit-8 assay (CCK-8), and the dual-luciferase reporter assay.Results: miRNA screening and validation showed that miR-98-5p was significantly upregulated in the peripheral blood mononuclear cells (PBMCs) of pediatric patients with IgAN compared with patients diagnosed with mesangial proliferative glomerulonephritis (MsPGN) and immunoglobulin A vasculitis nephritis (IgAV-N), and healthy controls (p < 0.05). Experiments with the dual-luciferase reporter confirmed that miR-98-5p might target C1GALT1. The overexpression of miR-98-5p in DAKIKI cells decreased both the mRNA and protein levels of C1GALT1 and increased the levels of Gd-IgA1 levels; these effects were reversed by co-transfection with the C1GALT1 plasmid, and vice versa. In addition, AS-IV downregulated the levels of Gd-IgA1 level in DAKIKI cells by inhibiting miR-98-5p.Conclusions: Our results revealed that AS-IV could inhibit Gd-IgA1 secretion via miR-98-5p. Increased levels of miR-98-5p in pediatric IgAN patients might affect the glycosylation of IgA1 by targeting C1GALT1. In addition, our analyses suggest that the pathogenesis of IgAN may differ from that of IgAV-N. Collectively, these results provide significant insight into the pathogenesis of IgAN and identify a potential therapeutic target.

A GlycoGene CRISPR-Cas9 lentiviral library to study lectin binding and human glycan biosynthesis pathways

Glycan biosynthesis on cell surface proteins and lipids is orchestrated by different classes of enzymes and proteins including the following: i. glycosyltransferases that add saccharides; ii. glycosidases that trim glycans; iii. conserved oligomeric golgi complex members that regulate intracellular transport; iv. enzymes aiding the biosynthesis of sugar–nucleotides; and v. sulfotransferases. This manuscript describes a pooled “glycoGene CRISPR” lentiviral library that targets 347 human genes involved in the above processes. Approximately 10 single-guide RNA (sgRNA) are included against each glycogene, with the putative editing site spanning the length of the target. A data analysis scheme is presented in order to determine glycosylation pathways regulating biological processes. As proof of principle, forward genetic screen results are presented to identify penetrating glycogenes that regulate the binding of P-/E-selectin, anti-sialyl Lewis-X monoclonal antibody HECA-452 and selected lectins (phaseolus vulgaris leucoagglutinin, vicia villosa lectin, peanut agglutinin) to HL-60 promyelocytic cells. Besides validating previously established biology, the study identifies three enzymes, PAPSS1, SLC35B2 and TPST2, as key molecules regulating sulfation of the major P-selectin glycoprotein ligand-1 in leukocytes. Approximately 80–90% of the sgRNA used in this study displayed high editing efficiency, and the CRISPR library picked up entire gene sets regulating specific biosynthetic pathways rather than only isolated genes. These data suggest that the glycoGene CRISPR library contains high-efficiency sgRNA. Further, this resource could be useful for the rapid screening of glycosylation-related genes and pathways that control lectin recognition in a variety of contexts.

The O-GalNAcylating enzyme GALNT5 mediates carcinogenesis and progression of cholangiocarcinoma via activation of AKT/ERK signaling

Mucin type O-glycosylation is a posttranslational modification of membrane and secretory proteins. Transferring of N-acetylgalactosamine, the first sugar of O-glycosylation, is catalyzed by one of the 20 isoforms of polypeptide N-acetylgalactosaminyltransferases (GALNTs). In this study, Vicia villosa lectin (VVL), a lectin that recognizes O-GalNAcylated glycans, was used to detect VVL-binding glycans (VBGs) in cholangiocarcinoma (CCA). The elevation of VBGs in tumor tissues of the liver fluke associated with CCA from hamsters and patients was noted. VBGs were detected in hyperplastic/dysplastic bile ducts and CCA but not in normal biliary epithelia and hepatocytes, indicating the association of VBGs with CCA development and progression. GALNT5 was shown to be the major isoform found in human CCA cell lines with high VBG expression. Suppression of GALNT5 expression using siRNA significantly reduced VBG expression, signifying the connection of GALNT5 and VBGs observed. Knocked-down GALNT5 expression considerably inhibited proliferation, migration and invasion of CCA cells. Increased expression of GALNT5 using pcDNA3.1-GALNT5 expression vector induced invasive phenotypes in CCA cells with low GALNT5 expression. Increasing of claudin-1 and decreasing of slug and vimentin expression together with inactivation of Akt/Erk signaling were noted in GALNT5 knocked-down cells. These observations were reversed in GALNT5 over-expressing cells. GALNT5-modulated progression of CCA cells was shown to be, in part, via GALNT5-mediated autocrine/paracrine factors that stimulated activations of Akt/Erk signaling and the epithelial to mesenchymal transition process. GALNT5 and its O-GalNAcylated products may have important roles in promoting progression of CCA and could possibly be novel targets for treatment of metastatic CCA.

P6302GALNT4 enhances monocyte adherence and chemotaxis by regulating PSGL1 O-glycosylation in patients with acute coronary syndrome

Background GalNAc-T4 (GALNT4), a member of N-acetylgalactosaminyltransferases family, which transfers GalNAc to Serine and Threonine residues in the initial step of O-linked protein glycosylation. Genome wide association studies (GWAS) have predicted that GALNT4 gene play a causal role in the susceptibility to cardiovascular diseases, whereas the precise mechanisms of it in acute coronary disease (ACS) is unknown. Purpose In this study, we sought to determine the role of GALNT4 in regulating monocytes with ACS and the underlying mechanisms. Methods and results The expression of GALNT4 mRNA was significantly elevated in patience with STEMI (n=50) compare with stable coronary heart disease (SCHD) (n=50) and controls (n=50) (Clinicaltrial.gov: NCT 03335514). Moreover, GALNT4 siRNA knockdown suppressed monocytes adherence under static and flow condition with P-selectin stimulation, as well as chemotaxis triggered by monocyte chemotactic protein-1 (MCP-1). In contrast, adenovirus mediated GALNT4 overexpression enhanced monocytes adherence and chemotaxis. Furthermore, vicia villosa lectin (VVL) pull down and PSGL1 immunoprecipitation assays showed that GALNT4 overexpression increased VVL binding to PSGL-1 and P-selectin induce phosphorylation of Akt and mTOR. Conversely, knockdown of GALNT4 decreased VVL binding and attenuated the activity of Akt /mTOR. Additionally, mTOR inhibitor rapamycin blocked these effects of GALNT4 overexpression on monocytes. Conclusion Expression of GALNT4 was increased in patience with ACS, GALNT4 overexpression enhances the adherence and chemotaxis of monocytes via modifying O-glycosylation of PSGL-1 and activity Akt/mTOR pathway. These findings suggest that GALNT4 may be a potential therapeutic target for ACS. Acknowledgement/Funding The National Natural Science Foundation of China (No. 81100220 and No. 81770340)

Loss of the Golgi Matrix Protein 130 Cause Aberrant IgA1 Glycosylation in IgA Nephropathy

Background: Aberrant O-glycosylation IgA1 production is a major factor in the pathogenesis of IgA nephropathy, but the underlying mechanism is still unclear. IgA1 glycosylation modification is in Golgi, and downregulation of the Golgi peripheral membrane protein Golgi matrix protein 130 (GM130) could lead to glycosylation deficiency. In this study, we aimed to explore the role of GM130 in glycosylate deficiency IgA1 (Gd-IgA1) production. Methods: We enrolled 27 IgA nephropathy patients, 12 patients with chronic tonsillitis, 15 non-IgAN chronic kidney disease patients, and 15 healthy volunteers as healthy control. We explored GM130 expression in Tonsillar tissue by immunofluorescence staining and Western blotting and expression in peripheral blood mononuclear cells (PBMCs) by flow cytometry. The concentration of IgA1 and level of O-glycosylation were determined by ELISA and Vicia Villosa lectin-binding assay. Real-time PCR and Western blot were used to analyze the levels of β1,3-Gal transferase (C1GALT1) and ST6GalNAC2, respectively. To explore the contribution of GM130 in IgA1 O-glycosylation modification, cells were subjected to experiments for evaluation of GM130 silencing by GM130-siRNA transfection. Results: GM130 expression was significantly decreased in tonsil tissues and PBMC of IgAN patients; the expression of C1GALT1 decreased and Gd-IgA1 level increased significantly in patients with IgAN patients. The expression of GM130 was negatively related to Gd-IgA1 production. By siRNA transfection, our results clearly indicated that the downregulation of GM130 can increase IgA1 O-glycosylation deficiency, which is thought to reduce C1GALT1 expression but not affect the expression of ST6GalNAC2. Conclusion: We identified and demonstrated that GM130 plays an important role in IgA1 O-glycans deficiency in IgAN patients, by negatively regulating C1GALT1 expression. We believe that this finding will provide theoretical foundations for a new mechanism of Gd-IgA1 production in IgAN patients.

A cell culture platform forcryptosporidiumthat enables long-term cultivation and new tools for the systematic investigation of its biology

Cryptosporidiumparasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking. Currently,Cryptosporidiumoocysts for research must be freshly produced in animals and cannot be long-term stored. Here, we show that COLO-680N cells infected with two differentCryptosporidium parvumstrains (Moredun, Iowa) produce sufficient infectious oocysts to infect subsequent cultures. Oocyst identity was confirmed by specific staining (Crypt-a-glo, Vicia Villosa lectin, Sporo-glo), PCR-based amplification ofCryptosporidium-specific genes, lipidomics fingerprinting, and atomic force microscopy (AFM). Antibody-stained oocysts produced unstained oocysts confirming production of novel oocysts. Infected cultures could be cryoconserved and continued to produce infectious oocysts after resuscitation. Transmission electron microscopy identified all keyCryptosporidiumlife cycle stages. Infected cultures produced thick-walled (primarily involved inCryptosporidiumtransmission between organisms) and thin-walled oocysts (important forCryptosporidiumpropagation within a host/tissue) as indicated by DAPI staining (only thin-walled oocysts are permeable to DAPI staining, thus allowing visualisation of sporozoites) and AFM. In conclusion, we present a novel, easy-to-handle cell culture system that enables the propagation, cryopreservation and detailed investigation ofCryptosporidiumat a laboratory scale. Its availability will accelerate research onCryptosporidiumand the development of anti-Cryptosporidiumdrugs.

The Number of Chandelier and Basket Cells Are Differentially Decreased in Prefrontal Cortex in Autism

An interneuron alteration has been proposed as a source for the modified balance of excitation / inhibition in the cerebral cortex in autism. We previously demonstrated a decreased number of parvalbumin (PV)-expressing interneurons in prefrontal cortex in autism. PV-expressing interneurons include chandelier (Ch) and basket (Bsk) cells. We asked whether the decreased PV+ interneurons affected both Ch cells and Bsk cells in autism. The lack of single markers to specifically label Ch cells or Bsk cells presented an obstacle for addressing this question. We devised a method to discern between PV-Ch and PV-Bsk cells based on the differential expression of Vicia villosa lectin (VVA). VVA binds to N-acetylgalactosamine, that is present in the perineuronal net surrounding some cell types where it plays a role in intercellular communication. N-acetylgalactosamine is present in the perineuronal net surrounding Bsk but not Ch cells. We found that the number of Ch cells is consistently decreased in the prefrontal cortex of autistic (n = 10) when compared with control (n = 10) cases, while the number of Bsk cells is not as severely affected. This finding expand our understanding of GABAergic system functioning in the human cerebral cortex in autism, which will impact translational research directed towards providing better treatment paradigms for individuals with autism.

Detection of O-glycosylated proteins from different Trichinella species muscle larvae total extracts

The aim of the work was to analyze oligosaccharide composition with the focus on O-linked glycoproteins presence in the total extract obtained from different Trichinella species muscle larvae by means of lectin affino-blot with lectins selected for their sugar specificity. The absence of reactivity with two lectins, Tritrichomonas mobilensis lectin and Maackia amurensis agglutinin, indicated that the species of the Trichinella genus do not synthesize sialic acid. Reactivity with Helix pomatia lectin, Vicia villosa lectin-B4, peanut agglutinin and Ulex europeus lectin-I identified the presence of O-linked glycans identical to carcinoma-associated Tn-antigen (GalNAc-α-Ser/Thr) and T-antigen (Gal-β1,3-GalNAc-α-Ser/Thr) and also structures analogous to ABH-blood group antigens. The results obtained may contribute to a better understanding of the glycobiology of this parasitic nematode in relation to occupation of its intracellular niches.