SLC35A2-CDG is an X-linked congenital disorder of glycosylation (CDG), characterized by defective UDP-galactose transport into the Golgi and endoplasmic reticulum and consequent insufficient galactosylation of glycans. Clinically, this translates into a range of predominantly neurological symptoms. Although the pathomechanism of this disorder is not fully understood, oral galactose supplementation has led to clinical and biochemical improvement in some patients. Here, we show that protein glycosylation (N- and O-linked) was only minimally disturbed in SLC35A2-CDG patient-derived fibroblasts. However, lipid glycosylation was significantly impaired, with accumulation of glucosylceramide and deficiency of digalactosylated glycosphingolipids (GSLs) and complex gangliosides. Galactose supplementation increased UDP-galactose, its transport into the Golgi, and improved deficient GSL synthesis through direct incorporation of the provided galactose. This improved GSL homeostasis in all patient-derived fibroblasts and in another SLC35A2 deficient cell model (CHO-Lec8). Additionally, SLC35A2-CDG serum analysis identified hydroxylated GSLs, particularly GM3, as potential disease biomarkers. Given the essential role of gangliosides in central nervous system function, their deficiency is likely a key factor in the neurological involvement of this disorder. These findings pave the way for new nutritional therapies with GSL supplements and highlight the importance of studying lipid glycosylation to better understand the complex pathophysiology of CDG.

Center for Metabolic Diseases Department of Paediatrics University Hospitals Leuven Leuven 3000 Belgium

Center for Pediatric and Adolescent Medicine Department Pediatrics 1 Heidelberg University 69120 Heidelberg Germany

Child Neurology and Psychiatry Unit Department of Clinical and Experimental Medicine University of Catania Catania 95131 Italy

Clinical Department of Laboratory Medicine University Hospitals Leuven Leuven 3000 Belgium

Department of Cardiovascular Sciences KU Leuven Leuven 3000 Belgium

Department of Chronic Diseases Metabolism and Ageing KU Leuven Leuven 3000 Belgium

Department of Development and Regeneration KU Leuven Leuven 3000 Belgium

Department of Genetics and Genomic Sciences Icahn School of Medicine at Mount Sinai New York NY 10029 USA

Department of Hepatology University Hospitals Leuven Leuven 3000 Belgium

Department of Paediatrics and Inherited Metabolic Disorders 1st Medical Faculty Charles University and General University Hospital Prague Prague 2 CZ 121 08 Czech Republic

Laboratory of Applied Mass Spectrometry Department of Cellular and Molecular Medicine KU Leuven Leuven 3000 Belgium

Laboratory of Lipid Metabolism and Cancer Department of Oncology Leuven Cancer Institute KU Leuven Leuven 3000 Belgium

Metabolic Center University Hospitals Leuven Leuven 3000 Belgium

Metabolomics Expertise Center Center for Cancer Biology VIB Leuven 3000 Belgium

Pediatric Neurology Department Hospital Sant Joan de Déu Esplugues de Llobregat Barcelona 08950 Spain

Research Unit of Rare Diseases and Neurodevelopmental Disorders Oasi Research Institute IRCCS Troina 94018 Italy

UMR 8576 UGSF Unité de Glycobiologie Structurale et Fonctionnelle CNRS University of Lille Lille F 59000 France

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