Summary of Study ST003691
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002290. The data can be accessed directly via it's Project DOI: 10.21228/M8MP0C This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003691 |
| Study Title | Beneficial effects of Glucose-1,6-bisphosphate modulation on mutant phosphomannomutase-2 |
| Study Summary | The metabolite Glucose-1,6-bisphosphate (Glc-1,6-P2) plays a vital role in human metabolism, and is a crucial activator and stabilizer for phosphomannomutase-2 (PMM2) - mutations within this protein propagate the most common congenital disorder of glycosylation (PMM2-CDG). In vivo, Glc-1,6-P2 is hydrolysed by phosphomannomutase-1 (PMM1), predominantly in the brain, under the influence of inosine monophosphate. In the present study, we employed knockout PMM1 in Arg141His/Phe119LeuPMM2 patient-derived fibroblasts and investigated the phenotypic improvement. Increased Glc-1,6-P2 was associated with glycosylation enhancement, confirmed by glycan profiling. The prevalence of previously identified PMM2-CDG biomarkers, such as LAMP-1, PTX3 and lysosomal enzymes showed empirical increases - these findings were corroborated by metabolomic and proteomic analysis. Moreover, our results support the potential of Glc-1,6-P2 modulation for PMM2-CDG, potentiating novel perspectives in drug discovery. |
| Institute | Institute of Biomolecular Chemistry (ICB-CNR) |
| Last Name | Monticelli |
| First Name | Maria |
| Address | via Campi Flegrei 34, 80078 Pozzuoli (NA) |
| maria.monticelli@unina.it | |
| Phone | +39 3663514733 |
| Submit Date | 2025-01-07 |
| Num Groups | 2 |
| Total Subjects | 10 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | fid |
| Analysis Type Detail | NMR |
| Release Date | 2025-07-07 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002290 |
| Project DOI: | doi: 10.21228/M8MP0C |
| Project Title: | Beneficial effects of Glucose-1,6-bisphosphate modulation on mutant phosphomannomutase-2 |
| Project Type: | NMR analysis |
| Project Summary: | NMRbased metabolomic analysis performed on two cell lines to investigate glucose-1,6-bisphosphate modulation in PMM2-related congenital disorder of glycosylation for new drug discovery perspectives: 1. Arg141His/Phe119LeuPMM2 patient-derived fibroblasts with a knock-out of the PMM1 gene 2. Arg141His/Phe119LeuPMM2 patient-derived fibroblasts scramble. |
| Institute: | Institute of Biomolecular Chemistry (ICB-CNR) |
| Last Name: | Monticelli |
| First Name: | Maria |
| Address: | via Campi Flegrei 34, 80078 Pozzuoli (NA) |
| Email: | maria.monticelli@unina.it |
| Phone: | +39 3663514733 |
| Funding Source: | PRIN 2022B2N2BY by MUR, Joint Bilateral Agreement CNR/Slovak Academy of Sciences, EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia |
| Contributors: | Maria Monticelli, Debora Paris, Maria Chiara Monti, Elva Morretta, Zuzana Pakanova, Marek Nemcovic, Rebeka Kodrikova, Maria Vittoria Cubellis, Giuseppina Andreotti |
Subject:
| Subject ID: | SU003823 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Class |
|---|---|---|
| SA403645 | A1 | +/+PMM1 |
| SA403646 | A2 | +/+PMM1 |
| SA403647 | A3 | +/+PMM1 |
| SA403648 | A4 | +/+PMM1 |
| SA403649 | A5 | +/+PMM1 |
| SA403650 | B1 | -/-PMM1 |
| SA403651 | B2 | -/-PMM1 |
| SA403652 | B3 | -/-PMM1 |
| SA403653 | B4 | -/-PMM1 |
| SA403654 | B5 | -/-PMM1 |
| Showing results 1 to 10 of 10 |
Collection:
| Collection ID: | CO003816 |
| Collection Summary: | p.Arg141His/Phe119LeuPMM2 fibroblasts were a gift from Prof. Flemming Skovby (rigshospitalet.dk -informed consent obtained from patients in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). Cells were immortalised as described in Monticelli et al. 2022 [1] and grown in RPMI medium supplemented with 10% Fetal Bovine Serum, 2 mM glutamine, 0.5 mg/mL penicillin, 0.5 mg/mL streptomycin, and non-essential amino acids at 37 °C in 5% humidified CO2. -/-PMM1 was generated by the insertion of a puromycin resistance cassette into the first exon of PMM1, using the Origene kit KN202004. Following the manufacturer's instructions, puromycin -up to 0.3 ng/mL- was used for the selection, and resistant cells were analysed via PCR to verify the correct insertion. Positive cells were grown and further analysed via RT-qPCR and immunoblot to confirm the knock-out. For metabolomics analysis, cells from confluent 150 cm2 plates were washed with PBS and enzymatically detached using trypsin, then pelleted in PBS and washed again for 3 times. [1] Monticelli, L. Liguori, M. Allocca, A. Bosso, G. Andreotti, J. Lukas, M.C. Monti, E. Morretta, M.V. Cubellis, B. Hay Mele, Drug Repositioning for Fabry Disease: Acetylsalicylic Acid Potentiates the Stabilization of Lysosomal Alpha-Galactosidase by Pharmacological Chaperones, International Journal of Molecular Sciences 23 (2022) 5105. https://doi.org/10.3390/ijms23095105 |
| Sample Type: | Fibroblasts |
Treatment:
| Treatment ID: | TR003832 |
| Treatment Summary: | Arg141His/Phe119Leu-PMM2 patient-derived fibroblasts were knocked-out of the PMM1 gene and compared with the scramble fibroblasts (i.e., Arg141His/Phe119Leu-PMM2 patient-derived fibroblasts which were subjected to the CRISPR-Cas9 procedure but lacking the gRNA, to avoid gene knock-out). |
Sample Preparation:
| Sampleprep ID: | SP003830 |
| Sampleprep Summary: | Metabolites extraction was performed using methanol:chloroform:water 1:1:1 protocol, as suggested by Beckonert et al. 2007 [1]. Polar and nonpolar fractions were transferred into glass vials and the solvents were removed under a nitrogen stream at room temperature and stored at -80°C until they were analysed. For NMR analysis, polar fractions were resuspended in phosphate buffer saline (PBS, pH 7.4), containing 10% 2H2O (to provide a field frequency lock) and 1 mM sodium 3-trimethylsylyl [2,2,3,3-2H4] propionate as a chemical shift reference for 1H spectra. [1] O. Beckonert, H.C. Keun, T.M.D. Ebbels, J. Bundy, E. Holmes, J.C. Lindon, J.K. Nicholson, Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts, Nat Protoc 2 (2007) 2692–2703. https://doi.org/10.1038/nprot.2007.376. |
| Sampleprep Protocol ID: | NMR_sample_prep.pdf |
Analysis:
| Analysis ID: | AN006057 |
| Analysis Type: | NMR |
| Num Factors: | 2 |
| Num Metabolites: | 24 |
| Units: | Normalized bin area |
NMR:
| NMR ID: | NM000305 |
| Analysis ID: | AN006057 |
| Instrument Name: | Bruker Avance III–600 MHz spectrometer |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| NMR Comments: | For each metabolite, the corresponding bin intensity was normalized to the total area of the spectrum |
| Spectrometer Frequency: | 600 MHz |
