Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism
The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To in...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2024-03-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227524000221 |
| _version_ | 1827304423077969920 |
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| author | Michele Dei Cas Linda Montavoci Claudia Pasini Anna Caretti Sara Penati Carla Martinelli Umberto Gianelli Sara Casati Francesca Nardecchia Annalaura Torella Nicola Brunetti-Pierri Marco Trinchera |
| author_facet | Michele Dei Cas Linda Montavoci Claudia Pasini Anna Caretti Sara Penati Carla Martinelli Umberto Gianelli Sara Casati Francesca Nardecchia Annalaura Torella Nicola Brunetti-Pierri Marco Trinchera |
| author_sort | Michele Dei Cas |
| collection | DOAJ |
| description | The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%–80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis. |
| first_indexed | 2024-04-24T17:30:51Z |
| format | Article |
| id | doaj.art-c066db706c1b439f81926ed07b97410d |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-04-24T17:30:51Z |
| publishDate | 2024-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-c066db706c1b439f81926ed07b97410d2024-03-28T06:36:40ZengElsevierJournal of Lipid Research0022-22752024-03-01653100517Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanismMichele Dei Cas0Linda Montavoci1Claudia Pasini2Anna Caretti3Sara Penati4Carla Martinelli5Umberto Gianelli6Sara Casati7Francesca Nardecchia8Annalaura Torella9Nicola Brunetti-Pierri10Marco Trinchera11Department of Health Sciences, Università degli Studi di Milano, Milan, ItalyDepartment of Health Sciences, Università degli Studi di Milano, Milan, ItalyDepartment of Health Sciences, Università degli Studi di Milano, Milan, ItalyDepartment of Health Sciences, Università degli Studi di Milano, Milan, ItalyDepartment of Health Sciences, Università degli Studi di Milano, Milan, ItalyDepartment of Health Sciences, Università degli Studi di Milano, Milan, ItalyDepartment of Health Sciences, Università degli Studi di Milano, Milan, Italy; S.C. di Anatomia Patologica, ASST- Santi Paolo e Carlo, Milan, ItalyDepartment of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, Milan, ItalyDepartment of Human Neuroscience, Unit of Child Neurology and Psychiatry, Sapienza University of Rome, ItalyDepartment of Precision Medicine, University of Campania ''Luigi Vanvitelli'', Naples, ItalyTelethon Institute of Genetics and Medicine, Pozzuoli, Italy; Department of Translational Medicine, Medical Genetics, University of Naples Federico II, Naples, Italy; Scuola Superiore Meridionale (SSM, School of Advanced Studies), Genomics and Experimental Medicine Program, University of Naples Federico II, Naples, ItalyDepartment of Medicine and Surgery (DMC), University of Insubria, Varese, Italy; For correspondence: Marco TrincheraThe last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%–80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis.http://www.sciencedirect.com/science/article/pii/S0022227524000221sphingolipidsceramideslipidomicsbrain lipidsglycolipidshypomyelinating leukodystrophy 18 |
| spellingShingle | Michele Dei Cas Linda Montavoci Claudia Pasini Anna Caretti Sara Penati Carla Martinelli Umberto Gianelli Sara Casati Francesca Nardecchia Annalaura Torella Nicola Brunetti-Pierri Marco Trinchera Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism Journal of Lipid Research sphingolipids ceramides lipidomics brain lipids glycolipids hypomyelinating leukodystrophy 18 |
| title | Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism |
| title_full | Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism |
| title_fullStr | Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism |
| title_full_unstemmed | Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism |
| title_short | Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism |
| title_sort | loss of function and reduced levels of sphingolipid desaturase degs1 variants are both relevant in disease mechanism |
| topic | sphingolipids ceramides lipidomics brain lipids glycolipids hypomyelinating leukodystrophy 18 |
| url | http://www.sciencedirect.com/science/article/pii/S0022227524000221 |
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