#METABOLOMICS WORKBENCH silviaradenkovic_20230418_110829 DATATRACK_ID:3868 STUDY_ID:ST002564 ANALYSIS_ID:AN004225 PROJECT_ID:PR001653 VERSION 1 CREATED_ON April 18, 2023, 4:51 pm #PROJECT PR:PROJECT_TITLE Metabolomic profiling of PMM2-CDG after siRNA mediated KD of AKR1b1 and PR:PROJECT_TITLE neuraminidase treatment PR:PROJECT_SUMMARY Abnormal polyol metabolism has been predominantly associated with diabetes, PR:PROJECT_SUMMARY where excess glucose is converted to sorbitol by aldose reductase (AR). PR:PROJECT_SUMMARY Recently, abnormal polyol metabolism has also been implicated in PR:PROJECT_SUMMARY phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG), and PR:PROJECT_SUMMARY epalrestat, an AR inhibitor, proposed as a potential therapy for this disorder. PR:PROJECT_SUMMARY Given that the PMM enzyme is not closely connected to polyol metabolism, and, PR:PROJECT_SUMMARY unlike in diabetes, PMM2-CDG does not present with hyperglycemia in blood, the PR:PROJECT_SUMMARY increased polyol production, and the therapeutic mechanism of epalrestat in PR:PROJECT_SUMMARY PMM2-CDG remained largely elusive. PMM2-CDG is caused by deficiency of the PMM PR:PROJECT_SUMMARY enzyme and results in a depletion of mannose-1-P and guanosine diphosphate PR:PROJECT_SUMMARY mannose (GDP-mannose), which is essential for glycosylation. Here, we show that PR:PROJECT_SUMMARY apart from glycosylation abnormalities, PMM2 deficiency also leads to changes in PR:PROJECT_SUMMARY intracellular glucose flux, which results in an increase in intracellular PR:PROJECT_SUMMARY polyols. Ssing tracer glucose studies, we demonstrate that AR inhibition diverts PR:PROJECT_SUMMARY glucose flux away from polyol production towards the synthesis of sugar PR:PROJECT_SUMMARY nucleotides, which results in increase in glucose flux towards glycans. PR:INSTITUTE Mayo Clinic PR:LAST_NAME Radenkovic PR:FIRST_NAME Silvia PR:ADDRESS 200 2nd Ave SW Rochester MN PR:EMAIL radenkovic.silvia@mayo.edu PR:PHONE 507(77) 6-6107 PR:FUNDING_SOURCE NIH, KU Leuven #STUDY ST:STUDY_TITLE Metabolomic profiling of PMM2-CDG after siRNA mediated KD of AKR1b1 and ST:STUDY_TITLE neuraminidase treatment ST:STUDY_SUMMARY Abnormal polyol metabolism has been predominantly associated with diabetes, ST:STUDY_SUMMARY where excess glucose is converted to sorbitol by aldose reductase (AR). ST:STUDY_SUMMARY Recently, abnormal polyol metabolism has also been implicated in ST:STUDY_SUMMARY phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG), and ST:STUDY_SUMMARY epalrestat, an AR inhibitor, proposed as a potential therapy for this disorder. ST:STUDY_SUMMARY Given that the PMM enzyme is not closely connected to polyol metabolism, and, ST:STUDY_SUMMARY unlike in diabetes, PMM2-CDG does not present with hyperglycemia in blood, the ST:STUDY_SUMMARY increased polyol production, and the therapeutic mechanism of epalrestat in ST:STUDY_SUMMARY PMM2-CDG remained largely elusive. PMM2-CDG is caused by deficiency of the PMM ST:STUDY_SUMMARY enzyme and results in a depletion of mannose-1-P and guanosine diphosphate ST:STUDY_SUMMARY mannose (GDP-mannose), which is essential for glycosylation. Here, we show that ST:STUDY_SUMMARY apart from glycosylation abnormalities, PMM2 deficiency also leads to changes in ST:STUDY_SUMMARY intracellular glucose flux, which results in an increase in intracellular ST:STUDY_SUMMARY polyols. Ssing tracer glucose studies, we demonstrate that AR inhibition diverts ST:STUDY_SUMMARY glucose flux away from polyol production towards the synthesis of sugar ST:STUDY_SUMMARY nucleotides, which results in increase in glucose flux towards glycans. ST:INSTITUTE Mayo Clinic ST:LAST_NAME Radenkovic ST:FIRST_NAME Silvia ST:ADDRESS 200 2nd Ave SW Rochester MN, USA ST:EMAIL radenkovic.silvia@mayo.edu ST:PHONE 507(77) 6-6107 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN WT/PMM2-CDG SU:AGE_OR_AGE_RANGE 5-45 SU:GENDER Male and female #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 P2 SR01 Genotype:PMM2-CDG | Treatment:5.5mM 13C GLU negative siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR01 SUBJECT_SAMPLE_FACTORS P2 SR02 Genotype:PMM2-CDG | Treatment:5.5mM 12C GLU negative siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR02 SUBJECT_SAMPLE_FACTORS P2 SR03 Genotype:PMM2-CDG | Treatment:5.5mM 13C GLU siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR03 SUBJECT_SAMPLE_FACTORS P2 SR04 Genotype:PMM2-CDG | Treatment:5.5mM 12C GLU siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR04 SUBJECT_SAMPLE_FACTORS P5 SR05 Genotype:PMM2-CDG | Treatment:5.5mM 13C GLU negative siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR05 SUBJECT_SAMPLE_FACTORS P5 SR06 Genotype:PMM2-CDG | Treatment:5.5mM 12C GLU negative siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR06 SUBJECT_SAMPLE_FACTORS P5 SR07 Genotype:PMM2-CDG | Treatment:5.5mM 13C GLU siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR07 SUBJECT_SAMPLE_FACTORS P5 SR08 Genotype:PMM2-CDG | Treatment:5.5mM 12C GLU siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR08 SUBJECT_SAMPLE_FACTORS C3 SR09 Genotype:WT | Treatment:5.5mM 13C GLU negative siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR09 SUBJECT_SAMPLE_FACTORS C3 SR10 Genotype:WT | Treatment:5.5mM 12C GLU negative siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR10 SUBJECT_SAMPLE_FACTORS C3 SR11 Genotype:WT | Treatment:5.5mM 13C GLU siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR11 SUBJECT_SAMPLE_FACTORS C3 SR12 Genotype:WT | Treatment:5.5mM 12C GLU siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR12 SUBJECT_SAMPLE_FACTORS C2 SR13 Genotype:WT | Treatment:5.5mM 13C GLU negative siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR13 SUBJECT_SAMPLE_FACTORS C2 SR14 Genotype:WT | Treatment:5.5mM 12C GLU negative siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR14 SUBJECT_SAMPLE_FACTORS C2 SR15 Genotype:WT | Treatment:5.5mM 13C GLU siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR15 SUBJECT_SAMPLE_FACTORS C2 SR16 Genotype:WT | Treatment:5.5mM 12C GLU siRNA Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR16 #COLLECTION CO:COLLECTION_SUMMARY Briefly, medium was removed from the cells and the cells were washed 3 times CO:COLLECTION_SUMMARY with 1 mL Dulbecco PBS containing 0.901 mM CaCl2 (Merck) and 0.492 mM MgCl2 CO:COLLECTION_SUMMARY (Merck). Next, cells were incubated with 1 mg/100 mL EZ-Link-Sulfo-NHS-LC-Biotin CO:COLLECTION_SUMMARY (Thermo) in Dulbecco for 30 min, RT, shaking. Cells were then washed twice with CO:COLLECTION_SUMMARY 1 mL Dulbecco, non-reacted biotin was blocked with 1 mL 20 mM glycine in CO:COLLECTION_SUMMARY Dulbecco for 15 min, and cells washed again with 1 mL Dulbecco. Dulbecco was CO:COLLECTION_SUMMARY then removed, cells scraped in 200 µL RIPA buffer (with protease inhibitors) CO:COLLECTION_SUMMARY and transferred to a fresh Eppendorf tube. Samples were lysed on ice with 3 CO:COLLECTION_SUMMARY consecutive freeze-thaw cycles. Further, 30 µL dynabeads streptavidin T1 CO:COLLECTION_SUMMARY (Invitrogen) was added to each sample and the samples were washed twice with 500 CO:COLLECTION_SUMMARY µL 10 mM ammonium bicarbonate and neuraminidase buffer (100 mM Sodium Acetate CO:COLLECTION_SUMMARY Buffer with 2 mM CaCl2 (Merck), pH 5.0). Neuraminidase buffer was removed and CO:COLLECTION_SUMMARY 500 µL PBS was added to the beads. 30 µL of prepared mix was added to each CO:COLLECTION_SUMMARY sample, and samples were incubated, shaking overnight at 4 °C. Samples were put CO:COLLECTION_SUMMARY on a dynabead rack (Invitrogen) and the supernatant was transferred to a new CO:COLLECTION_SUMMARY Eppendorf tube and used for protein concentration assay. Beads containing CO:COLLECTION_SUMMARY membrane fractions were washed 2 times with 500 µL lysis buffer (2 % IGEPAL CO:COLLECTION_SUMMARY (Sigma), 1% Triton X-100 (Sigma), and 10 % glycerol inPBS) and then washed with CO:COLLECTION_SUMMARY 1 mL PBS. Samples are centrifuged at 500 rcf, 5 min, 4 °C and PBS was removed. CO:COLLECTION_SUMMARY 100 µL neuraminidase buffer containing 0.05 U neuraminidase was added to each CO:COLLECTION_SUMMARY sample and samples were incubated overnight at 37 °C, shaking. Next, CO:COLLECTION_SUMMARY supernatant was transferred to a new Eppendorf tube and lyophilized at 4 °C. CO:COLLECTION_SUMMARY Finally, pellets were resuspended in 100 µL of extraction buffer (80 % MeOH, CO:COLLECTION_SUMMARY IS). Sialic acid was measured by LC/MS as described below. El Maven Polly CO:COLLECTION_SUMMARY software was used to annotate sialic acid based on m/z ratio and elution time, CO:COLLECTION_SUMMARY and determine fractional contribution of glucose in sialic acid. CO:SAMPLE_TYPE Fibroblasts CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Cells were treated with vehicle or 5nM siRNA targeting AKR1B1, Before TR:TREATMENT_SUMMARY collection, medium was removed from the cells and the cells were washed 3 times TR:TREATMENT_SUMMARY with 1 mL Dulbecco PBS containing 0.901 mM CaCl2 (Merck) and 0.492 mM MgCl2 TR:TREATMENT_SUMMARY (Merck). Next, cells were incubated with 1 mg/100 mL EZ-Link-Sulfo-NHS-LC-Biotin TR:TREATMENT_SUMMARY (Thermo) in Dulbecco for 30 min, RT, shaking. Cells were then washed twice with TR:TREATMENT_SUMMARY 1 mL Dulbecco, non-reacted biotin was blocked with 1 mL 20 mM glycine in TR:TREATMENT_SUMMARY Dulbecco for 15 min, and cells washed again with 1 mL Dulbecco. Dulbecco was TR:TREATMENT_SUMMARY then removed, cells scraped in 200 µL RIPA buffer (with protease inhibitors) TR:TREATMENT_SUMMARY and transferred to a fresh Eppendorf tube. Samples were lysed on ice with 3 TR:TREATMENT_SUMMARY consecutive freeze-thaw cycles. Further, 30 µL dynabeads streptavidin T1 TR:TREATMENT_SUMMARY (Invitrogen) was added to each sample and the samples were washed twice with 500 TR:TREATMENT_SUMMARY µL 10 mM ammonium bicarbonate and neuraminidase buffer (100 mM Sodium Acetate TR:TREATMENT_SUMMARY Buffer with 2 mM CaCl2 (Merck), pH 5.0). Neuraminidase buffer was removed and TR:TREATMENT_SUMMARY 500 µL PBS was added to the beads. 30 µL of prepared mix was added to each TR:TREATMENT_SUMMARY sample, and samples were incubated, shaking overnight at 4 °C. Samples were put TR:TREATMENT_SUMMARY on a dynabead rack (Invitrogen) and the supernatant was transferred to a new TR:TREATMENT_SUMMARY Eppendorf tube and used for protein concentration assay. Beads containing TR:TREATMENT_SUMMARY membrane fractions were washed 2 times with 500 µL lysis buffer (2 % IGEPAL TR:TREATMENT_SUMMARY (Sigma), 1% Triton X-100 (Sigma), and 10 % glycerol inPBS) and then washed with TR:TREATMENT_SUMMARY 1 mL PBS. Samples are centrifuged at 500 rcf, 5 min, 4 °C and PBS was removed. TR:TREATMENT_SUMMARY 100 µL neuraminidase buffer containing 0.05 U neuraminidase was added to each TR:TREATMENT_SUMMARY sample and samples were incubated overnight at 37 °C, shaking. Next, TR:TREATMENT_SUMMARY supernatant was transferred to a new Eppendorf tube and lyophilized at 4 °C. TR:TREATMENT_SUMMARY Finally, pellets were resuspended in 100 µL of extraction buffer (80 % MeOH, TR:TREATMENT_SUMMARY IS). Sialic acid was measured by LC/MS as described below. El Maven Polly TR:TREATMENT_SUMMARY software was used to annotate sialic acid based on m/z ratio and elution time, TR:TREATMENT_SUMMARY and determine fractional contribution of glucose in sialic acid. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After treatment with neuraminidase, supernatant was transferred to a new SP:SAMPLEPREP_SUMMARY Eppendorf tube and lyophilized at 4 °C. Finally, lyophilized pellets were SP:SAMPLEPREP_SUMMARY resuspended in 100 µL of extraction buffer (80 % MeOH, IS). SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY C18 iP REVERSE PHASE CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 10mM tributylamine; 15mM acetic acid CH:SOLVENT_B 100% methanol CH:FLOW_GRADIENT The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B CH:FLOW_GRADIENT until 2 min post injection. A linear gradient to 37% B was carried out until 7 CH:FLOW_GRADIENT min and increased to 41% until 14 min. Between 14 and 26 minutes the gradient CH:FLOW_GRADIENT increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the CH:FLOW_GRADIENT gradient returned to 5% B. The chromatography was stopped at 40 min. CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS El-Maven polly, ThermoFisher Xcalibur; Sialic acid was annotated using the MS:MS_COMMENTS inhouse standard metabolite library- elution time and m/z values. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AUC MS_METABOLITE_DATA_START Samples SR01 SR02 SR03 SR04 SR05 SR06 SR07 SR08 SR09 SR10 SR11 SR12 SR13 SR14 SR15 SR16 Factors Genotype:PMM2-CDG | Treatment:5.5mM 13C GLU negative siRNA Genotype:PMM2-CDG | Treatment:5.5mM 12C GLU negative siRNA Genotype:PMM2-CDG | Treatment:5.5mM 13C GLU siRNA Genotype:PMM2-CDG | Treatment:5.5mM 12C GLU siRNA Genotype:PMM2-CDG | Treatment:5.5mM 13C GLU negative siRNA Genotype:PMM2-CDG | Treatment:5.5mM 12C GLU negative siRNA Genotype:PMM2-CDG | Treatment:5.5mM 13C GLU siRNA Genotype:PMM2-CDG | Treatment:5.5mM 12C GLU siRNA Genotype:WT | Treatment:5.5mM 13C GLU negative siRNA Genotype:WT | Treatment:5.5mM 12C GLU negative siRNA Genotype:WT | Treatment:5.5mM 13C GLU siRNA Genotype:WT | Treatment:5.5mM 12C GLU siRNA Genotype:WT | Treatment:5.5mM 13C GLU negative siRNA Genotype:WT | Treatment:5.5mM 12C GLU negative siRNA Genotype:WT | Treatment:5.5mM 13C GLU siRNA Genotype:WT | Treatment:5.5mM 12C GLU siRNA Sialic acid 1.68E+08 1.57E+08 1.40E+08 1.38E+08 1.65E+08 1.94E+08 2.42E+08 2.88E+08 2.28E+08 1.68E+08 2.65E+08 3.21E+08 1.48E+08 1.73E+08 1.24E+08 1.71E+08 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name parent formula M/Z HMDB ID isotopeLabel isotope count Sialic acid C11H19NO9 308.09845 HMDB00230 C12 PARENT m0-m11 METABOLITES_END #END