Linda Montavoci

Glycosphingolipids (GSLs) are glycoconjugates in which a short and heterogeneous saccharide chain is attached to a lipid moiety called ceramide. Based on their sugar backbone, mammalian GSLs are primarily grouped into the ganglio-, lacto-/neolacto-, and globo-series. Sialic acid—containing GSLs are known as gangliosides. Complex ganglio-series gangliosides are particularly abundant in the brain, whereas simple ganglio-series gangliosides, as well as those belonging to other series or neutral GSLs, are less abundant and typical of non-neural tissues. Congenital disorders in the biosynthesis of the lipid moiety of sphingolipids (SLs) result from defects in enzymes and proteins involved in ceramide biosynthesis and transport. Congenital disorders in the biosynthesis of the sugar chain of GSLs specifically affect ganglio-series ganglioside biosynthesis and are caused by pathogenic variants in GM3 synthase (ST3GAL5) or GM2/GD2/asialo-GM2 synthase (B4GALNT1). Defective variants of the sialyltransferase ST3GAL3 and the galactosyltransferase B4GALT5 have been reported and proposed to impair GSL biosynthesis. The occurrence of these syndromes has provided new insights into the physiological and pathological roles of GSLs. Most of these disorders are associated with completely inactive enzyme variants, leading to severe neurological syndromes. Only a few cases highlighted variants that retained partial activity, resulting in milder phenotypes, which included non-syndromic intellectual disability. It is therefore conceivable that many undiagnosed patients, with mild neurological symptoms, may carry variants retaining residual enzyme activity, insufficient to ensure normal levels of brain GSLs. The purpose of this article is to encourage clinicians to look for additional GLS hereditary disorders associated with a milder phenotype. We also hope to boost future investigations by highlighting the most critical issues emerging from recent literature on SL and GSL biosynthesis and their related defects.

Background/Objectives: Retinitis pigmentosa (RP) is characterized by progressive degeneration of photoreceptors, with increasing evidence supporting the involvement of inflammation in disease progression. Aqueous humor (AH) reflects the intraocular microenvironment and represents an accessible source for biochemical analysis. This study aimed to characterize the profile of the main inflammatory proteins and bioactive lipids in the AH of RP patients and to compare it with healthy subjects. Methods: The AH was analyzed for cytokines using multiplex immunoassays and for lipid species using liquid chromatography–mass spectrometry. The concentrations of the analyzed molecules were compared between RP patients and the control group and then correlated with age and ellipsoid zone (EZ) width in RP patients. Results: A total of 26 RP patients and 13 controls were recruited. Significantly elevated levels of the pro-inflammatory IL-6 and a significant decrease in vascular endothelial growth factor (VEGF) were found in RP patients compared to controls. In RP patients, lipidomic analysis demonstrated significant increases in medium- and long-chain sphingomyelins (SMs) and very-long-chain unsaturated phosphatidylcholines (PCs). Higher levels of Cer 16:0, PC 32:0, and PC 34:0 were significantly associated with greater EZ preservation in RP patients. Additionally, in RP patients, VEGF and GM-CSF levels increased significantly with age, while IL-8 showed a non-significant decreasing trend. Conclusions: By integrating proteomic and, for the first time, lipidomic analyses of AH, we identified significant alterations in pro-inflammatory cytokines and bioactive lipid species in RP patients compared to controls, further highlighting a link between inflammatory activity, patient age, and disease stage. These preliminary findings need further validation in larger longitudinal cohorts to confirm the clinical utility of these bioactive mediators as potential disease biomarkers.

Background: Pathogenic ST3GAL3 variants cause neurological and cognitive impairment, defining a distinct congenital disorder of glycosylation (ST3GAL3-CDG). Nonetheless, limited enzyme characterization exists due to the lack of a non-radiochemical assay. Methods: Here, we developed an LC-MS/MS-based method using the artificial substrate para-nitrophenyl-lacto-N-biose (LNB-pNP; Galβ1,3GlcNAcβ1-O-C6H4NO2) to measure ST3GAL3 activity in vitro. Results: A peak corresponding to sialyl-LNB-pNP was detected in reactions with homogenate from HEK-293T cells transfected with pCDNA3 ST3GAL3 plasmid, but was virtually absent in mock-transfected cells. A substrate dependence curve provided an apparent Km value for the substrate (0.40 mM) and closely matched values from prior radiochemical methods. No activity was detected with homogenates from cells expressing pathogenic ST3GAL3 variants, except p.A13D, which is known to retain about 10% of residual activity. Compared to ST3GAL4 and ST3GAL6, ST3GAL3 showed markedly higher specificity toward LNB-pNP, lactotetraosylceramide (Lc4) and asialo-GM1, which are rather specific substrates. Instead, neo-lactotetraosylceramide (neoLc4) was processed by all three ST3GALs. Conclusions: These findings suggest that ST3GAL4 or ST3GAL6 cannot compensate for ST3GAL3 loss in the biosynthesis of gangliosides sialyl-Lc4 and GM1b, but may do so for sialyl-neoLc4. This non-radiochemical assay enables screening and diagnostic evaluation of novel ST3GAL3 variants potentially associated with ST3GAL3-CDG.

Background and Aims: Cholangiocarcinoma (CCA) is a globally rare, increasingly incident cancer. Metabolic reprogramming is common in cancer cells, and altered lipid homeostasis favors tumor development and progression. Previous studies have described lipid deregulation in HCC cells, while in CCA, the lipidome profile is still poorly characterized. Methods: We used liquid chromatography–tandem mass spectrometry to examine the lipid level profile of intrahepatic CCA (iCCA) and non-tumor surrounding tissue from patients, as well as in patients’ and healthy controls’ sera. Results: All lipid classes were upregulated in tumor specimens and iCCA-derived sera. Newly synthesized fatty acids (FAs) accumulated in iCCA and were only marginally directed to mitochondrial β-oxidation and scarcely folded in lipid droplets as neutral species. Metabolic flux assay showed that FAs were instead redirected toward plasma membrane formation and remodeling, being incorporated into phospholipids and sphingomyelin. A distinct lipid droplet and macrophage distribution was revealed by immunohistochemistry and Imaging Mass Cytometry. Lipid droplets were fewer in iCCA than in normal tissue and present mainly in the intratumoral fibrous septa and in M2 macrophages. Monocytes modified their lipid content and phenotype in the presence of iCCA cells, and the same effect could be recapitulated by FA supplementation. Conclusions: Our results reveal a profound alteration in the lipid content of iCCA tissues and demonstrate that FA accumulation prompts iCCA aggressiveness by supporting membrane biogenesis, generating bioactive lipids that boost proliferation, and by modifying macrophage phenotype.

Background/Objectives: Chronic graft-versus-host disease (cGVHD) and oral lichen planus (LPO) are chronic inflammatory conditions with similar oral manifestations. This study aimed to assess whether serum and salivary cytokines (IL-1α, IL-6, IL-17) could serve as reliable biomarkers for cGVHD. Methods: A prospective cohort study was conducted involving cGVHD patients, LPO patients, and healthy controls. Cytokine levels in serum and saliva were measured by ELISA and compared across the groups using the Kruskal–Wallis test. Results: IL-17 levels were significantly elevated in the serum of cGVHD patients compared to LPO patients and controls (p < 0.05). However, IL-6 and IL-1α did not show significant differences among the groups. A comparison of salivary samples between the three groups did not reach statistical significance (p > 0.05). Conclusions: This study suggests that IL-17 could be a potential biomarker for cGVHD-related inflammation, warranting further investigation. Salivary samples do not seem to be a reliable biological marker for the diagnosis of cGVHD. The findings underline the need for larger studies to validate these preliminary results.

Angiopoietin-like protein 3 (ANGPTL3) is a plasmatic protein that plays a crucial role in lipoprotein metabolism by inhibiting the lipoprotein lipase (LPL) and the endothelial lipase (EL) responsible for the hydrolysis of phospholipids on high-density lipoprotein (HDL). Interest in developing new pharmacological therapies aimed at inhibiting ANGPTL3 has been growing due to the hypolipidemic and antiatherogenic profile observed in its absence. The goal of this study was the in silico characterization of the interaction between ANGPTL3 and EL. Because of the lack of any structural information on both the trimeric coiled-coil N-terminal domain of ANGPTL3 and the EL homodimer as well as data regarding their interactions, the first step was to obtain the three-dimensional model of these two proteins. The models were then refined via molecular dynamics (MD) simulations and used to investigate the interaction mechanism. The analysis of interactions in different docking poses and their refinement via MD allowed the identification of three specific glutamates of ANGPTL3 that recognize a positively charged patch on the surface of EL. These ANGPTL3 key residues, i.e., Glu154, Glu157, and Glu160, could form a putative molecular recognition site for EL. This study paves the way for future investigations aimed at confirming the recognition site and at designing novel inhibitors of ANGPTL3.

Lactosylceramide is necessary for the biosynthesis of almost all classes of glycosphingolipids and plays a relevant role in pathways involved in neuroinflammation. It is synthesized by the action of galactosyltransferases B4GALT5 and B4GALT6, which transfer galactose from UDP-galactose to glucosylceramide. Lactosylceramide synthase activity was classically determined in vitro by a method based on the incorporation of radiolabeled galactose followed by the chromatographic separation and quantitation of the product by liquid scintillation counting. Here, we used deuterated glucosylceramide as the acceptor substrate and quantitated the deuterated lactosylceramide product by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We compared this method with the classical radiochemical method and found that the reactions have similar requirements and provide comparable results in the presence of high synthase activity. Conversely, when the biological source lacked lactosylceramide synthase activity, as in the case of a crude homogenate of human dermal fibroblasts, the radiochemical method failed, while the other provided a reliable measurement. In addition to being very accurate and sensitive, the proposed use of deuterated glucosylceramide and LC-MS/MS for the detection of lactosylceramide synthase in vitro has the relevant advantage of avoiding the costs and discomforts of managing radiochemicals.