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Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality

ABSTRACT: In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization. We generated four variants of a potentSARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Nicotiana Benthamiana

TISSUE(S): Plant Cell, Leaf

SUBMITTER: Richard Strasser

LAB HEAD: Richard Strasser

PROVIDER: PXD050139 | Pride | 2024-02-27

REPOSITORIES: Pride

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			Action	DRS
	H10KO_1434.raw	Raw
	H10KOplusO_1455.raw	Raw
	HEK_1432.raw	Raw
	Quantified.DAT	Other
	WT_1433.raw	Raw

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Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBD

Project description:Recombinant RBD variants from SARS-CoV-2 spike were produced by transient expression in Nicotiana benthamiana plants and purified. The purified proteins were digested with proteases and glycopeptides were analysed by MS to identify the N-glycan structures.

2024-01-26 | PXD044462 | Pride

Immunomolecular and reactivity landscapes of gut IgA subclasses in homeostasis and inflammatory bowel diseases.

Project description:The human gut includes plasma cells (PCs) expressing immunoglobulin A1 (IgA1) or IgA2, two structurally distinct IgA subclasses with elusive regulation, function and reactivity. We show here that intestinal IgA1+ and IgA2+ PCs co-emerged early in life, comparably accumulated somatic mutations, and were enriched within short-lived CD19+ and long-lived CD19− PC subsets, respectively. IgA2+ PCs were often clonally related to IgA1+ PCs and a subset of them presumably emerged from IgA1+ precursors. Of note, secretory IgA1 (SIgA1) and SIgA2 dually coated a large fraction of mucus-embedded bacteria, including Akkermansia muciniphila. Disruption of homeostasis by inflammatory bowel disease (IBD) increased newly formed and actively proliferating IgA1+ plasmablasts, depleted long-lived IgA2+ PCs, and increased SIgA1+SIgA2+ gut microbiota. Such increase featured enhanced IgA1 reactivity to pathobionts, including Escherichia coli, combined with depletion of beneficial Akkermansia muciniphila. Thus, gut IgA1 and IgA2 emerge from clonally related PCs and show unique changes of both frequency and reactivity in IBD.

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Role of IgG autoantibodies in the pathogenesis of IgA nephropathy assessed in a passive mouse model

Project description:IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, is characterized by IgA1-containing glomerular immunodeposits. These immunodeposits are thought to originate from the circulating immune complexes consisting of aberrantly glycosylated IgA1 (galactose-deficient IgA1; Gd-IgA1) bound by IgG autoantibodies. This hypothesis is supported by the findings that renal immunodeposits of IgA nephropathy patients are enriched for IgG autoantibodies specific for galactose-deficient IgA1 (Rizk et al., J. Am. Soc. Nephrol. 30(10), 2017-2026, 2019). However, experimental proof is needed. This study provides in vivo data in mice that support the pathogenic role of IgG autoantibodies in IgAN.

2021-01-31 | GSE156179 | GEO

microRNA-630 regulates underglycosylated IgA1 production in the tonsils by targeting TLR4 in IgA nephropathy

Project description:IgA nephropathy (IgAN) is the most common primary glomerular disease. The characteristic pathology involves immune complexes formed by the deposition of IgA1 and underglycosylated IgA1 aggregates in the mesangial area, which may be accompanied by the deposition of IgG and/or IgM and complement components. However, the molecular mechanisms of IgAN remain unclear. In the present study, microarray analysis showed that the expression of miR-630 was significantly reduced in palatal tonsils from IgAN patients compared with chronic tonsillitis. Additionally, bioinformatic analysis showed that Toll-like receptor 4 (TLR4) was the predicted target gene of miR-630 and was regulated by miR-630. When miR-630 was overexpressed in palatal tonsil mononuclear cells from IgAN patients, the expression of TLR4 was reduced and the content of IgA1 in the cell culture supernatant was decreased, and the level of galactosylation in the IgA1 hinge region was increased. Moreover, immunohistochemical analysis showed that the expression of TLR4 in IgAN patients was significantly increased and. After knocking down the expression of TLR4, both the concentration of IgA1 and the binding force of IgA1 with broad bean lectin were significantly reduced in IgAN. Furthermore, the mechanism study demonstrated that TLR4 might regulate the expression of IL-1β and IL-8 through NF-κB signaling pathway to modulate the concentration of IgA1 and the glycosylation level of IgA1. This interesting finding may offer new insight into the molecular mechanism of IgAN.

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Dual antagonism of the endothelin A and angiotensin II type 1 receptors protects from glomerular hypercellularity in a model of IgA nephropathy

Project description:IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, is characterized by glomerular deposition of immune complexes (IC) consisting of IgA1 with some O-glycans deficient in galactose (Gd-IgA1) and Gd-IgA1-specific IgG autoantibodies. These IC induce kidney injury, and in the absence of disease-specific therapy, up to 40% of IgAN patients progress to kidney failure. IgA1 with its clustered O-glycans is unique to humans, which hampered development of small-animal models of IgAN. To address this issue, we developed a model wherein engineered IC (EIC) formed from human Gd-IgA1 and recombinant IgG autoantibody are injected into nude mice to induce glomerular injury mimicking human IgAN. Here, we used this animal model to assess the protective effects of sparsentan, a single-molecule dual antagonist of endothelin A receptor (ETAR) and angiotensin II type 1 receptor (AT1R) (DEARA). Oral administration of sparsentan (60 or 120 mg/kg) to mice intravenously injected with EIC attenuated the EIC-induced glomerular hypercellularity.

2024-02-29 | GSE225447 | GEO

Metabolomics of immunoglobulin-producing cells in IgA nephropathy

Project description:IgA nephropathy (IgAN), the most common primary glomerulonephritis, is characterized by deposits of IgA-containing immune complexes in the kidney glomeruli, as first described by Berger and Hinglais in 1968. IgAN is a major cause of end-stage renal disease with its associated cardio-renal morbidity and mortality. Analyses of the IgA deposits revealed that the IgA is exclusively of the IgA1 subclass and that this IgA1 is aberrantly glycosylated, deficient in galactose in some O-glycans (Gd-IgA1). Patients with IgAN have elevated serum levels of Gd-IgA1 bound by anti-glycan autoantibodies in circulating immune complexes (CIC) that are fundamental in driving disease pathology in an autoimmune process. We have recently shown that elevated serum levels of Gd-IgA1 in patients with IgAN predict disease progression. Thus, understanding the mechanisms behind Gd-IgA1 production will improve future treatment options, as there is presently no disease-specific therapy. A total of 24 cell pellets (4 replicates from 6 cell lines) were analyzed by LCMS metabolomics. Immortalized immunoglobulin-producing cell lines were generated from peripheral-blood lymphocytes from patients with IgAN and healthy controls as described in Suzuki, H., Moldoveanu, Z., Hall, S., et al. IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1. J Clin Invest. 2008, 118, 629-639.

2017-02-17 | ST000560 | MetabolomicsWorkbench

The upregulation of miR-98-5p affects the glycosylation of IgA1 through cytokines in IgA nephropathy

Project description:Purpose: Increases in galactose-deficient IgA1 (Gd-IgA1) play a crucial role in the pathogenesis of IgA nephropathy (IgAN), and several recent experiments have shown that microRNAs (miRNAs) are involved in regulating the development and physiological function of the kidney. The aims of this study were to identify miRNAs that can affect the pathogenesis of IgAN and reveal the underlying regulatory mechanism of IgA1 glycosylation in peripheral blood Methods: The differentially expressed miRNAs in peripheral blood mononuclear cells (PBMCs) between IgAN patients and healthy controls were screened by high-throughput sequencing. The miRNA targets were predicted and verified by dual-luciferase reporter assays. We also explored the miRNA regulation of Gd-IgA1 through the transfection of miRNA mimics and related plasmids Results: The high-throughput sequencing results showed that miR-98-5p was highly expressed in the PBMCs of IgAN patients compared with healthy controls, and the luciferase reporter gene system confirmed that miR-98-5p might target chemokine ligand 3 (CCL3). The transfection of si-CCL3 confirmed that a decrease in CCL3 can affect the expression of interleukin-6 (IL-6) and C1GALT1. The overexpression of miR-98-5p in PBMCs via the transfection of miR-98-5p mimic reduced the CCL3 and C1GALT1 levels and increased the IL-6 levels. However, these changes in PBMCs were attenuated by cotransfection with the CCL3 plasmid. Conclusion: The results showed that miR-98-5p in PBMCs can target CCL3 to decrease its expression, which increased the IL-6 levels and the resulting increase in IL-6 can decrease C1GALT1 expression. Therefore, miR-98-5p might be involved in the development of IgAN.

2023-02-20 | GSE145652 | GEO

An oligosaccharyltransferase from Leishmania donovani increases the N-glycan occupancy on plant-produced IgG1

Project description:The monoclonal antibodies rituximab and cetuximab were transiently expressed in leaves of Nicotiana benthamiana plants and purified by affinity chromatography using protein A. Purified antibodies were analysed by intact MS. Purified antibodies were also subjected to trypsin digestion followed by LC-ESI-MS analysis of the glycopeptide carrying the N-glycsylation site Asn297 using the LC-OTMS, Orbitrap Exploris 480 from Thermo Scientific.

2023-07-31 | PXD043623 | Pride

In planta deglycosylation improves the SARS-CoV-2 neutralization activity of recombinant ACE2-Fc

Project description:The ectodomain of human ACE2-Fc was transiently expressed in leaves of Nicotiana benthamiana plants and protein A and SEC purified. SEC-purified ACE2-Fc was sequentially digested with chymotrypsin and trypsin and the glycopeptides were subjected to LC-ESI-MS analysis using the LC-OTMS, Orbitrap Exploris 480 from Thermo Scientific.

2023-04-18 | PXD041320 | Pride

miR-148b modulates the C1GALT1 expression explaining the abnormal glycosylation process in IgA Nephropathy

Project description:Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide characterized by aberrant O-glycosylation in the hinge region of IgA1. The basis for the abnormal glycosylation in IgAN is still unknown, but an important involvement of the enzyme core 1, beta 1,3-galactosyltransferase 1 (C1GALT1) is known. However, the role of microRNAs (miRNAs), a new family of key mRNA regulatory molecules, in the IgAN pathogenesis has not yet been reported. In this study, by high-throughput microRNA profiling, we identified 37 miRNAs differentially expressed in peripheral blood mononuclear cells (PBMCs) from IgAN patients compared to healthy subjects. Among them, upregulated miR-148b potentially targeted C1GALT1, INVS and PTEN, three genes notably downregulated in IgAN patients. C1GALT1 expression levels in IgAN patients were reduced and negatively correlated with the upregulated miR-148b expression. We demonstrated the biological relationship between miR-148b and C1GALT1 by transient transfection experiments ex vivo. When we reduced the upregulated miR-148b function in PBMCs of IgAN patients an increase of the C1GALT1 mRNA and protein levels was observed. We validated biologically also the miR-148b targeting of INVS , involved in the altered modulation of the WNT–β-catenin and PI3K/Akt pathways in IgAN patients. All together our data evidence an important role of miR-148b in the pathogenesis of IgAN, which could explain the aberrant glycosylation of IgA1 in the pathogenesis and should light on a potential target for the theraphy of the disease.

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