#METABOLOMICS WORKBENCH maria_monticelli_20250107_065859 DATATRACK_ID:5508 STUDY_ID:ST003691 ANALYSIS_ID:AN006057 PROJECT_ID:PR002290
VERSION             	1
CREATED_ON             	January 27, 2025, 8:15 pm
#PROJECT
PR:PROJECT_TITLE                 	Beneficial effects of Glucose-1,6-bisphosphate modulation on mutant
PR:PROJECT_TITLE                 	phosphomannomutase-2
PR:PROJECT_TYPE                  	NMR analysis
PR:PROJECT_SUMMARY               	NMRbased metabolomic analysis performed on two cell lines to investigate
PR:PROJECT_SUMMARY               	glucose-1,6-bisphosphate modulation in PMM2-related congenital disorder of
PR:PROJECT_SUMMARY               	glycosylation for new drug discovery perspectives: 1. Arg141His/Phe119LeuPMM2
PR:PROJECT_SUMMARY               	patient-derived fibroblasts with a knock-out of the PMM1 gene 2.
PR:PROJECT_SUMMARY               	Arg141His/Phe119LeuPMM2 patient-derived fibroblasts scramble.
PR:INSTITUTE                     	Institute of Biomolecular Chemistry (ICB-CNR)
PR:LAST_NAME                     	Monticelli
PR:FIRST_NAME                    	Maria
PR:ADDRESS                       	via Campi Flegrei 34, 80078 Pozzuoli (NA)
PR:EMAIL                         	maria.monticelli@unina.it
PR:PHONE                         	+39 3663514733
PR:FUNDING_SOURCE                	PRIN 2022B2N2BY by MUR, Joint Bilateral Agreement CNR/Slovak Academy of
PR:FUNDING_SOURCE                	Sciences, EU NextGenerationEU through the Recovery and Resilience Plan for
PR:FUNDING_SOURCE                	Slovakia
PR:CONTRIBUTORS                  	Maria Monticelli, Debora Paris, Maria Chiara Monti, Elva Morretta, Zuzana
PR:CONTRIBUTORS                  	Pakanova, Marek Nemcovic, Rebeka Kodrikova, Maria Vittoria Cubellis, Giuseppina
PR:CONTRIBUTORS                  	Andreotti
#STUDY
ST:STUDY_TITLE                   	Beneficial effects of Glucose-1,6-bisphosphate modulation on mutant
ST:STUDY_TITLE                   	phosphomannomutase-2
ST:STUDY_SUMMARY                 	The metabolite Glucose-1,6-bisphosphate (Glc-1,6-P2) plays a vital role in human
ST:STUDY_SUMMARY                 	metabolism, and is a crucial activator and stabilizer for phosphomannomutase-2
ST:STUDY_SUMMARY                 	(PMM2) - mutations within this protein propagate the most common congenital
ST:STUDY_SUMMARY                 	disorder of glycosylation (PMM2-CDG). In vivo, Glc-1,6-P2 is hydrolysed by
ST:STUDY_SUMMARY                 	phosphomannomutase-1 (PMM1), predominantly in the brain, under the influence of
ST:STUDY_SUMMARY                 	inosine monophosphate. In the present study, we employed knockout PMM1 in
ST:STUDY_SUMMARY                 	Arg141His/Phe119LeuPMM2 patient-derived fibroblasts and investigated the
ST:STUDY_SUMMARY                 	phenotypic improvement. Increased Glc-1,6-P2 was associated with glycosylation
ST:STUDY_SUMMARY                 	enhancement, confirmed by glycan profiling. The prevalence of previously
ST:STUDY_SUMMARY                 	identified PMM2-CDG biomarkers, such as LAMP-1, PTX3 and lysosomal enzymes
ST:STUDY_SUMMARY                 	showed empirical increases - these findings were corroborated by metabolomic and
ST:STUDY_SUMMARY                 	proteomic analysis. Moreover, our results support the potential of Glc-1,6-P2
ST:STUDY_SUMMARY                 	modulation for PMM2-CDG, potentiating novel perspectives in drug discovery.
ST:INSTITUTE                     	Institute of Biomolecular Chemistry (ICB-CNR)
ST:LAST_NAME                     	Monticelli
ST:FIRST_NAME                    	Maria
ST:ADDRESS                       	via Campi Flegrei 34, 80078 Pozzuoli (NA)
ST:EMAIL                         	maria.monticelli@unina.it
ST:PHONE                         	+39 3663514733
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	10
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#FACTORS
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	A1	Class:+/+PMM1 | Sample source:Human-derived fibroblasts	RAW_FILE_NAME(Raw file name)=GA-KOPMM1-A1-H2O-1xii20
SUBJECT_SAMPLE_FACTORS           	-	A2	Class:+/+PMM1 | Sample source:Human-derived fibroblasts	RAW_FILE_NAME(Raw file name)=GA-KOPMM1-A2-H2O-1xii20
SUBJECT_SAMPLE_FACTORS           	-	A3	Class:+/+PMM1 | Sample source:Human-derived fibroblasts	RAW_FILE_NAME(Raw file name)=GA-KOPMM1-A3-H2O-1xii20
SUBJECT_SAMPLE_FACTORS           	-	A4	Class:+/+PMM1 | Sample source:Human-derived fibroblasts	RAW_FILE_NAME(Raw file name)=GA-KOPMM1-A4-H2O-1xii20
SUBJECT_SAMPLE_FACTORS           	-	A5	Class:+/+PMM1 | Sample source:Human-derived fibroblasts	RAW_FILE_NAME(Raw file name)=GA-KOPMM1-A5-H2O-1xii20
SUBJECT_SAMPLE_FACTORS           	-	B1	Class:-/-PMM1 | Sample source:Human-derived fibroblasts	RAW_FILE_NAME(Raw file name)=GA-KOPMM1-B1-H2O-1xii20
SUBJECT_SAMPLE_FACTORS           	-	B2	Class:-/-PMM1 | Sample source:Human-derived fibroblasts	RAW_FILE_NAME(Raw file name)=GA-KOPMM1-B2-H2O-1xii20
SUBJECT_SAMPLE_FACTORS           	-	B3	Class:-/-PMM1 | Sample source:Human-derived fibroblasts	RAW_FILE_NAME(Raw file name)=GA-KOPMM1-B3-H2O-1xii20
SUBJECT_SAMPLE_FACTORS           	-	B4	Class:-/-PMM1 | Sample source:Human-derived fibroblasts	RAW_FILE_NAME(Raw file name)=GA-KOPMM1-B4-H2O-1xii20
SUBJECT_SAMPLE_FACTORS           	-	B5	Class:-/-PMM1 | Sample source:Human-derived fibroblasts	RAW_FILE_NAME(Raw file name)=GA-KOPMM1-B5-H2O-1xii20
#COLLECTION
CO:COLLECTION_SUMMARY            	p.Arg141His/Phe119LeuPMM2 fibroblasts were a gift from Prof. Flemming Skovby
CO:COLLECTION_SUMMARY            	(rigshospitalet.dk -informed consent obtained from patients in accordance with
CO:COLLECTION_SUMMARY            	The Code of Ethics of the World Medical Association (Declaration of Helsinki).
CO:COLLECTION_SUMMARY            	Cells were immortalised as described in Monticelli et al. 2022 [1] and grown in
CO:COLLECTION_SUMMARY            	RPMI medium supplemented with 10% Fetal Bovine Serum, 2 mM glutamine,
CO:COLLECTION_SUMMARY            	0.5 mg/mL penicillin, 0.5 mg/mL streptomycin, and non-essential amino acids
CO:COLLECTION_SUMMARY            	at 37 °C in 5% humidified CO2. -/-PMM1 was generated by the insertion of a
CO:COLLECTION_SUMMARY            	puromycin resistance cassette into the first exon of PMM1, using the Origene kit
CO:COLLECTION_SUMMARY            	KN202004. Following the manufacturer's instructions, puromycin -up to 0.3 ng/mL-
CO:COLLECTION_SUMMARY            	was used for the selection, and resistant cells were analysed via PCR to verify
CO:COLLECTION_SUMMARY            	the correct insertion. Positive cells were grown and further analysed via
CO:COLLECTION_SUMMARY            	RT-qPCR and immunoblot to confirm the knock-out. For metabolomics analysis,
CO:COLLECTION_SUMMARY            	cells from confluent 150 cm2 plates were washed with PBS and enzymatically
CO:COLLECTION_SUMMARY            	detached using trypsin, then pelleted in PBS and washed again for 3 times. [1]
CO:COLLECTION_SUMMARY            	Monticelli, L. Liguori, M. Allocca, A. Bosso, G. Andreotti, J. Lukas, M.C.
CO:COLLECTION_SUMMARY            	Monti, E. Morretta, M.V. Cubellis, B. Hay Mele, Drug Repositioning for Fabry
CO:COLLECTION_SUMMARY            	Disease: Acetylsalicylic Acid Potentiates the Stabilization of Lysosomal
CO:COLLECTION_SUMMARY            	Alpha-Galactosidase by Pharmacological Chaperones, International Journal of
CO:COLLECTION_SUMMARY            	Molecular Sciences 23 (2022) 5105. https://doi.org/10.3390/ijms23095105
CO:SAMPLE_TYPE                   	Fibroblasts
#TREATMENT
TR:TREATMENT_SUMMARY             	Arg141His/Phe119Leu-PMM2 patient-derived fibroblasts were knocked-out of the
TR:TREATMENT_SUMMARY             	PMM1 gene and compared with the scramble fibroblasts (i.e.,
TR:TREATMENT_SUMMARY             	Arg141His/Phe119Leu-PMM2 patient-derived fibroblasts which were subjected to the
TR:TREATMENT_SUMMARY             	CRISPR-Cas9 procedure but lacking the gRNA, to avoid gene knock-out).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolites extraction was performed using methanol:chloroform:water 1:1:1
SP:SAMPLEPREP_SUMMARY            	protocol, as suggested by Beckonert et al. 2007 [1]. Polar and nonpolar
SP:SAMPLEPREP_SUMMARY            	fractions were transferred into glass vials and the solvents were removed under
SP:SAMPLEPREP_SUMMARY            	a nitrogen stream at room temperature and stored at -80°C until they were
SP:SAMPLEPREP_SUMMARY            	analysed. For NMR analysis, polar fractions were resuspended in phosphate buffer
SP:SAMPLEPREP_SUMMARY            	saline (PBS, pH 7.4), containing 10% 2H2O (to provide a field frequency lock)
SP:SAMPLEPREP_SUMMARY            	and 1 mM sodium 3-trimethylsylyl [2,2,3,3-2H4] propionate as a chemical shift
SP:SAMPLEPREP_SUMMARY            	reference for 1H spectra. [1] O. Beckonert, H.C. Keun, T.M.D. Ebbels, J. Bundy,
SP:SAMPLEPREP_SUMMARY            	E. Holmes, J.C. Lindon, J.K. Nicholson, Metabolic profiling, metabolomic and
SP:SAMPLEPREP_SUMMARY            	metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue
SP:SAMPLEPREP_SUMMARY            	extracts, Nat Protoc 2 (2007) 2692–2703.
SP:SAMPLEPREP_SUMMARY            	https://doi.org/10.1038/nprot.2007.376.
SP:SAMPLEPREP_PROTOCOL_ID        	NMR_sample_prep.pdf
#ANALYSIS
AN:ANALYSIS_TYPE                 	NMR
#NMR
NM:INSTRUMENT_NAME               	Bruker Avance III–600 MHz spectrometer
NM:INSTRUMENT_TYPE               	FT-NMR
NM:NMR_EXPERIMENT_TYPE           	1D-1H
NM:NMR_COMMENTS                  	For each metabolite, the corresponding bin intensity was normalized to the total
NM:NMR_COMMENTS                  	area of the spectrum
NM:SPECTROMETER_FREQUENCY        	600 MHz
#NMR_METABOLITE_DATA
NMR_METABOLITE_DATA:UNITS	Normalized bin area
NMR_METABOLITE_DATA_START
Samples	A1	A2	A3	A4	A5	B1	B2	B3	B4	B5
Factors	Class:+/+PMM1 | Sample source:Human-derived fibroblasts	Class:+/+PMM1 | Sample source:Human-derived fibroblasts	Class:+/+PMM1 | Sample source:Human-derived fibroblasts	Class:+/+PMM1 | Sample source:Human-derived fibroblasts	Class:+/+PMM1 | Sample source:Human-derived fibroblasts	Class:-/-PMM1 | Sample source:Human-derived fibroblasts	Class:-/-PMM1 | Sample source:Human-derived fibroblasts	Class:-/-PMM1 | Sample source:Human-derived fibroblasts	Class:-/-PMM1 | Sample source:Human-derived fibroblasts	Class:-/-PMM1 | Sample source:Human-derived fibroblasts
ATP	0.00366	0.00416	0.00318	0.00445	0.00295	0.00318	0.00287	0.00294	0.00255	0.00286
UDP-glucose + UDP-glucuronic acid	0.00345	0.00362	0.00347	0.00384	0.00346	0.00458	0.00414	0.00419	0.00404	0.00433
UDP-glucuronic acid	0.00015	0.00022	0.00023	0.00005	0.00019	0.0011	0.00046	0.00041	0.00027	0.00026
UDP-N-acetylglucosamine	0.00003	0.00008	0.0002	0.00001	0.00009	0.00141	0.00043	0.00027	0.00012	0.00011
Serine	0.00413	0.00381	0.00417	0.004	0.00394	0.00585	0.00523	0.00509	0.00596	0.00627
Mannitol	0.00976	0.00801	0.0094	0.00911	0.00677	0.01013	0.00999	0.00889	0.01267	0.01412
Myo-inositol	0.00655	0.00701	0.00726	0.00678	0.00709	0.00541	0.00561	0.00602	0.00499	0.00448
Choline phosphate	0.01059	0.0104	0.01021	0.01134	0.01152	0.00652	0.00746	0.00776	0.0071	0.00707
Glycine	0.01083	0.01008	0.01098	0.01099	0.01002	0.01478	0.01395	0.01449	0.01567	0.01626
Proline	0.00204	0.00225	0.00286	0.00242	0.00224	0.00078	0.00194	0.0019	0.00167	0.00111
Arginine	0.00227	0.0025	0.00232	0.0023	0.00215	0.00274	0.00336	0.00319	0.00339	0.00306
sn-Glycero-3-phosphocholine + Choline phosphate	0.04565	0.04354	0.04467	0.04824	0.04649	0.01793	0.01775	0.01674	0.0147	0.01663
Choline	0.00669	0.00613	0.0067	0.00568	0.00714	0.00259	0.00392	0.0037	0.00301	0.00267
Citrulline	0.00163	0.00161	0.00191	0.00127	0.00203	0.00008	0.00095	0.00074	0.00093	0.00034
Creatine	0.0041	0.00409	0.00421	0.00398	0.00439	0.00508	0.00499	0.00498	0.00462	0.00504
Citric acid	0.00287	0.003	0.00346	0.003	0.00293	0.00123	0.00212	0.00193	0.00197	0.00163
Glutaric acid	0.00439	0.00438	0.00429	0.00424	0.00419	0.00297	0.00379	0.0036	0.00366	0.00335
Glutathione	0.013	0.01313	0.01244	0.01317	0.01286	0.01005	0.00946	0.01001	0.00891	0.00881
Lysine	0.00379	0.00393	0.00386	0.00333	0.00379	0.00423	0.00557	0.00544	0.00604	0.00501
Alanine	0.00504	0.00493	0.00529	0.00503	0.00591	0.00702	0.00671	0.00752	0.00694	0.00749
Lactic acid	0.09055	0.09311	0.09772	0.11244	0.1073	0.12316	0.11252	0.10833	0.12232	0.13703
Valine	0.00287	0.00289	0.00306	0.00267	0.00324	0.00368	0.00452	0.00421	0.00498	0.00508
Leucine	0.00595	0.00571	0.00604	0.00565	0.00647	0.00773	0.00888	0.0086	0.01	0.01066
Isoleucine	0.00547	0.005	0.00459	0.00472	0.00507	0.0057	0.00724	0.00708	0.00766	0.00808
NMR_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Kegg code
ATP	C00002
UDP-glucose + UDP-glucuronic acid	C00029
UDP-glucuronic acid	C00167
UDP-N-acetylglucosamine	C00043
Serine	C00065
Mannitol	C00392
Myo-inositol	C00137
Choline phosphate	C00588
Glycine	C00037
Proline	C00148
Arginine	C00062
sn-Glycero-3-phosphocholine + Choline phosphate	C00670
Choline	C00114
Citrulline	C00327
Creatine	C00300
Citric acid	C00158
Glutaric acid	C00489
Glutathione	C00051
Lysine	C00047
Alanine	C00041
Lactic acid	C00186
Valine	C00183
Leucine	C00123
Isoleucine	C00407
METABOLITES_END
#END