Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural characterisation of the fungal Pmt4 homodimer - native MS and lipidomics

ABSTRACT: Protein O-mannosyltransferases (PMTs) are conserved endoplasmic reticulum membrane embedded enzymes responsible for the transfer of mannose from dolichol phosphate-mannose (Dol-P-Man) to serine/threonine-rich protein substrates or unfolded proteins. PMTs from three subfamilies form obligate dimers with different substrate specificities, and require the concerted action of their transmembrane domains (TMDs) and a luminal MIR domain for catalysis. Here, we present structures, native mass spectrometry and structure-based mutagenesis of the Chaetomium thermophilum and Saccharomyces cerevisiae Pmt4 homodimers. The core fold of the TMDs and MIR domain is conserved with the Pmt1-Pmt2 heterodimer, indicating a shared catalytic mechanism. Distinct to Pmt4, the MIR domain interacts in cis with the TMDs of the same subunit and has a beta-hairpin insertion required for O-mannosylation of substrates. We further identify a cytosolic binding site for substrate Dol33 P-Man within the Pmt4 TMDs, which is conserved amongst PMTs and important for in vivo activity. Thus, we provide a framework to understand the substrate specificity and regulation of the Pmt4 homodimer.

INSTRUMENT(S):

ORGANISM(S): Chaetomium Thermophilum (strain Dsm 1495 / Cbs 144.50 / Imi 039719) Saccharomyces Cerevisiae

SUBMITTER: Francesco Fiorentino

LAB HEAD: Carol V. Robinson

PROVIDER: PXD061837 | Pride | 2025-11-19

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	Lipidomics_Blank.raw	Raw
	Lipidomics_Pmt4.raw	Raw
	Molecular_masses_nativeMS.txt	Txt
	Molecular_masses_nativeMS.xlsx	Xlsx
	Pmt4_DDM-DM_ar_5_Fig_4A.raw	Raw

Items per page:

1 - 5 of 13

Similar Datasets

Global view of domain-specific O-linked mannose glycosylation in glycoengineered cells

Project description:Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved post-translational modification, whose biosynthesis is initiated by three non-redundant enzyme families, POMT1/POMT2, TMTC1-4 and TMEM260. In this study, we applied a targeted workflow for membrane glycoproteomics to five human cell lines and to a panel of genetically engineered cells with individual and combinatorial knock-out of O-Man glycosyltransferase genes to extensively map substrates, specificities, and crosstalk of O-Man enzymes. Our quantitative glycoproteomics results demonstrate new protein targets for the POMT1/POMT2 pathway and show that TMTC1-4 and TMEM260 widely target distinct Ig-like protein domains of plasma membrane proteins. This new evidence adds further knowledge on the emerging concept of domain-specific O-Man glycosylation and establishes a platform for functional studies of O-Man glycosylated adhesion molecules and receptors.

2024-06-10 | PXD045597 | Pride

Global Analysis of Carbohydrates Utilization of the Alkaline Hemicellulytic Bacteria Bacillus sp. N16-5

Project description:Alkaline hemicellulytic bacteria Bacillus sp. N16-5 has abroad substrate spectrum and exhibits great growth ability on complex carbohydrates. In order to get insight into its carbohydrate utilization mechanism, global transcriptional profiles were separately determined for growth on glucose, fructose, mannose, galactose, arabinose, xylose, galactomannan, xylan, pectin and carboxymethyl cellulose by using one-color microarrays. Substrate induced gene expression was measured when culture was grown on glucose, fructose, mannose, galactose, arabinose, xylose, galactomannan, xylan and CMC to mid-logarithmic phase.

2014-06-17 | E-GEOD-38276 | biostudies-arrayexpress

O-mannose glycosylations influence E-cadherin protein interactions

Project description:Cadherins are plasma membrane adhesion molecules that play critical roles in maintaining cell-cell adhesion and modulating cell signaling during development. Their functions are mediated by extracellular cadherin (EC) domains, which facilitate adhesive interactions and enable the formation of cis- and trans assemblies at adherens junctions and desmosomes. The EC domains adopt a characteristic immunoglobulin-like fold composed of seven beta-strands (A-G), and are further modified by N-linked and O-linked glycosylation, including alpha-linked mannose monosaccharides (O-Man) on conserved serine and threonine residues of B- and G-strands. EC domain O-Man glycosylation is catalyzed by TMTC enzymes: TMTC3 catalyzes modifications to the G-strands, whereas TMTC2 targets the B-strands. Given the site-specific deposition of O-Man glycans by dedicated enzymes and the central role of EC domains in cadherins functions, we hypothesized that these PTMs may fine-tune cellular adhesion strength and otherwise contribute to diverse physical interactions that involve cadherins. To test these hypotheses, we assayed for changes in protein-protein interactions formed with epithelial (E)-cadherin in model cells where O-Man PTMs were genetically ablated. Herein, we report O-Man-dependent, E-cadherin (CDH1) protein interactions, revealed by affinity proteomics; we orthogonally validate an altered association between CDH1 and CDH3 (P-cadherin), and demonstrate loss-of-function consequences of O-Man ablation on specific positions, highlighting the importance of these PTMs in cadherin-mediated adhesion. These findings provide new insights into how post-translational modifications regulate cadherin-dependent adhesion complexes.

2025-04-04 | MSV000097524 | MassIVE

B_multivorans growth on mannitol

Project description:Burkholderia multivorans was grown on agar plates containing mannitol as substrate and compared to growth on control plates containing mannose

2014-03-02 | E-MEXP-3971 | biostudies-arrayexpress

Nucleocytoplasmic O-Man glycoproteome

Project description:Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine (Ser) and threonine (Thr) residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, e.g Saccharomyces cerevisiae. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation, and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway (HBP) to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-Mannose (O-Man) glycoproteome, and used this to identify a pleothora of O-linked mannose glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and doing so we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic and mitochondrial proteins in Saccharomyces cerevisiae. The type of nucleocytoplasmic proteins and the localization of identified O-Man residues mirrors that of the O-GlcNAc glycoproteome in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzyme machinery that is predicted to regulate the discovered nucleocytoplasmic O-Man glycosylation. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing.

2015-12-08 | PXD002924 | Pride

Locust bean gum derived galacto-manno-oligosaccharides utilization by Lactobacillus plantarum WCFS1

Project description:In order to understand LBG derived galacto-manno-oligosaccharides utilization by a probiotic bacterium, Lactobacillus plantarum WCFS1, we have grown Lactobacillus plantarum WCFS1 (in duplicates) till mid log phase (OD600nm ~0.5, 10 h) in carbon free MRS (de Man, Rogosa Sharpe ) media containing either galacto-manno-oligosaccharides, mannose, glucose or galactose (1% w/v) as the sole carbon source.

2019-07-30 | GSE117591 | GEO

The restricted N-glycome of neurons is programmed during differentiation

Project description:The protein glycome of individual cell types in the brain is unexplored, despite the critical function of these modifications in development and disease. In aggregate, the most abundant asparagine (N-) linked glycans in the adult brain are high mannose structures, and specifically Man5GlcNAc2 (Man-5), which normally exits the ER for further processing in the Golgi. Mannose structures are uncommon in other organs and often overlooked or excluded in most studies. To understand cell-specific contributions to the unique brain N-glycome and its abundance of Man-5, we performed RNA-seq and MALDI-MS TOF protein N-glycomics at several timepoints during differentiation of multiple cell types. To this end, homogeneous cultures of glutamatergic neurons, GABAergic neurons, and brain-specific endothelial cells were generated from monoclonal human inducible pluripotent stem cells (hiPSCs) through cellular reprogramming. Small molecule induction of stably integrated synthetic transcription units driving morphogen expression generated differentiated cells with distinct patterns mirroring intact tissue. Comparing uninduced hiPSCs for each cell type revealed identical transcriptomic and glycomic profiles before differentiation, with low quantities of Man-5. In differentiated glutamatergic and GABAergic neurons, the most abundant N-glycans became Man-5 and its immediate precursor Man-6, despite the presence of transcripts encoding enzymes for their subsequent modification. Differentiation to brain-specific endothelial cells showed an opposite effect, with the N-glycome displaying an abundance of complex N-glycans and terminal modifications of the late secretory pathway. These results confirm that the restricted N-glycome profile of brain is programmed into neuronal differentiation, with regulation independent of the transcriptome and under tight evolutionary constraint.

2025-10-23 | GSE280179 | GEO

NOT2 proteins promote Pol II-dependent transcription and interact with multiple miRNA biogenesis factors in Arabidopsis

Project description:MicroRNAs (miRNAs) play key regulatory roles in numerous developmental and physiological processes in animals and plants. The elaborate mechanism of miRNA biogenesis involves transcription and multiple processing steps. Here, we report the identification of a pair of evolutionarily conserved NOT2_3_5 domain containing proteins, NOT2a and NOT2b (previously known as At-NOT2 and VIP2, respectively), as components involved in Arabidopsis miRNA biogenesis. NOT2 was identified by its interaction with the Piwi/Ago/Zwille (PAZ) domain of Dicer-like 1 (DCL1), an interaction that is conserved between rice and Arabidopsis. Inactivation of both NOT2 genes in Arabidopsis caused severe defects in male gametophytes, and weak lines show pleiotropic defects reminiscent of miRNA pathway mutants. Impairment of NOT2s decreases the accumulation of pri-miRNAs and mature miRNAs and increases DCL1-containing nuclear speckle number in vivo. In addition, NOT2b protein interacts with Pol II and other miRNA processing factors including two cap-binding proteins, CBP80/ABH1, CBP20 and SERRATE (SE). Therefore, these results suggest that NOT2 proteins promote MIR transcription and facilitate efficient DCL1 recruitment in Arabidopsis. Examination of transcriptome of not2 and wild type by RNA-seq.

2013-01-30 | E-GEOD-42901 | biostudies-arrayexpress

CHO O-Mannosylation independent of POMT1/2

Project description:Protein O-mannosylation is found in yeast and metazoans and a family of conserved orthologous polypeptide O-mannosyltransferases is believed to initiate this important posttranslational modification. We recently discovered that the large families of cadherins and protocadherins carry highly conserved O-Man glycans in specific EC domains, and it was suggested that the function of E-Cadherin was dependent on the O-Man glycans. Deficiencies in the two human polypeptide O-mannosyltransferases, POMT1 and T2, underlie a subgroup of congenital muscular dystrophies (CMD) designated α-dystroglycanopathies, because deficient O-Man glycans on -dystroglycan impair laminin interaction with -dystroglycan and the dystrophin complex. In order to explore the functions of O-Man glycans on cadherins and protocadherins we used a combinatorial gene editing strategy in multiple cell lines to evaluate to the role of the two POMTs initiation O-Man glycosylation and the major enzyme elongating O-Man glycans, the POMGNT1 2GlcNAc-transferase. Surprisingly, we discovered that O-Man glycans on cadherins and protocadherins do not appear to require POMT1 and T2 and moreover that the O-Man glycans on these proteins are not elongated in contrast to those on dystroglycan.

2017-05-18 | PXD004341 | Pride

Human O-Mannosylation independent of POMT1/2

2017-05-18 | PXD004358 | Pride