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Mannosidic glycans on misfolded Arabidopsis SUBEX glycopeptides

ABSTRACT: The mutated extracellular domain of Arabidopsis STRUBBELIG (SUBEX) was fused to GFP and either transiently expressed in Nicotiana benthamiana leaves or stably in Arabidopsis thaliana seedlings and purified. The purified SUBEX proteins were subjected to SDS-PAGE and CBB staining. Corresponding gel pieces were excised and the proteins digested with proteases and jack bean alpha-mannosidase. Glycopeptides were analysed by MS to identify the N-glycan structures. In addition, glycans were released from SUBEX peptides using PNGase A and analysed by PGC-LC-MS.

INSTRUMENT(S):

ORGANISM(S): Nicotiana Benthamiana Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf

SUBMITTER: Richard Strasser

LAB HEAD: Richard Strasser

PROVIDER: PXD066844 | Pride | 2025-10-13

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	1B_Peaks_annotated_all.tsv	Tabular
	1B_mns45_SUBEX.raw	Raw
	1B_os9_SUBEX.raw	Raw
	1C_mns45_FUNC001.DAT	Other
	1C_os9_FUNC001.DAT	Other

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1 - 5 of 14

Publications

Mannose trimming is the dominant signal for the release of misfolded glycoproteins from ER quality control.

Shin Yun-Ji YJ Vavra Ulrike U Maresch Daniel D Grünwald-Gruber Clemens C Strasser Richard R

The Journal of biological chemistry 20250828 10

N-glycosylation is essential for protein folding in the endoplasmic reticulum (ER). Glycan attachment facilitates the binding of newly synthesized polypeptides to calnexin and calreticulin, two ER-resident lectins that act as chaperones and promote folding. The regulatory mechanism underlying this process is dictated by the glycan composition, and this study has elucidated the function of mannose trimming in the release of misfolded glycoprotein from ER quality control and subsequent transfer to ...[more]

PMID: 40885387

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

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2024-02-27 | PXD050139 | 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

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.

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Pathogen protein modularity enables mimicry of a host phosphatase

Project description:Pathogens rely on a diverse complement of effector proteins to manipulate host cellular processes. The devastating plant pathogen Phytophthora produces numerous effectors that consist of tandem repeats of the “(L)WY” motif. Here, we discovered a specific (L)WY-LWY combination that enables efficient recruitment of the Serine/Threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Structural characterization of an effector-PP2A complex and biochemical analyses demonstrate multiple effectors adopt this module at the amino terminus to form functional PP2A holoenzymes but have divergent C-terminal LWY units. Our results discovered a virulence strategy widely employed by Phytophthora in which an essential phosphatase complex of the host is appropriated to promote disease and highlight how diversification in an effector repertoire is promoted by protein modularity.

2024-10-01 | PXD034179 | Pride

FIRE mimics a 14-3-3-binding motif to promote Phytophthora palmivora infection

Project description:The oomycete Phytophthora palmivora infects a wide range of tropical crops worldwide. Like other filamentous plant pathogens, it secretes effectors to colonise plant tissues. Here we characterise FIRE, an RXLR effector that contains a canonical mode I 14-3-3 phospho-sensor binding motif that is conserved in effectors of several Phytophthora species. FIRE is phosphorylated in planta and interacts with multiple 14-3-3 proteins. Binding is sensitive to the R18 14-3-3 inhibitor. FIRE promotes plant susceptibility and co-localises with its target around haustoria. This work uncovers a new type of oomycete effector target mechanism. It demonstrates that substrate mimicry for 14-3-3 proteins is a cross-kingdom effector strategy used by both prokaryotic and eukaryotic plant pathogens to suppress host immunity.

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How a tRNA modifying enzyme facilitates RNase P activity in Arabidopsis nuclei

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2023-10-20 | PXD040022 | Pride

Comparative proteomic analysis for revealing the advantage mechanisms of Salt-tolerant tomato (Solanum lycopersicum)

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2022-02-08 | PXD029130 | Pride

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TIHAMAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics Analysis

Project description:Protein post-translational modifications (PTMs) are pivotal molecular events in plant cells, encompassing processes such as protein folding and enzyme activation. Among these PTMs, N-glycosylation and phosphorylation hold particular importance, as they play essential roles in regulating plant growth and stress responses. The intricate PTM landscape is exemplified by the identification of multiple plant membrane-localized proteins subject to both phosphorylation and N-glycosylation, hinting at potential interplay influencing protein functionality. To investigate the crosstalk between phosphorylation and glycosylation in plant signaling, immobilized metal affinity chromatography (IMAC) and hydrophilic interaction chromatography (HILIC) have been primarily utilized for large-scale phosphopeptide and glycopeptide enrichment, respectively. In this work, we introduce a novel streamlined tandem S-Trap-IMAC-HILIC strategy, termed TIMAHAC, to enable serial enrichment of phosphopeptides and glycopeptides. This approach successfully integrates the two methods through tandem tip enrichment, eliminating the need for buffer exchange and sample transfer. As a result, it significantly minimizes sample loss during serial enrichment steps, and streamlines experimental procedures, ultimately saving time. We demonstrated the efficiency of the TIHAMAC strategy in the glycoproteomics and phosphoproteomics analyses using 200 μg of Arabidopsis digests. Up to 1,954 N-glycopeptides and 11,255 phosphopeptides were identified by timsTOF HT. Remarkably, 69% of N-glycopeptides were consistently quantified with a coefficient of variation less than 20%. Furthermore, more than one-third identified glycoproteins exhibited phosphorylation modifications. These results suggest that the applicability of the TIMAHAC method for studying the potential crosstalk of N-glycoproteome and phosphoproteome within complex plant signaling networks.

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