Three missense variants of ST3GAL3 are known to be responsible for a congenital disorder of glycosylation determining a neurodevelopmental disorder (intellectual disability/epileptic encephalopathy). Here we report a novel nonsense variant, p.Y220*, in two dichorionic infant twins presenting a picture of epileptic encephalopathy with impaired neuromotor development. Upon expression in HEK-293T cells, the variant appears totally devoid of enzymatic activity in vitro, apparently accumulated with respect to the wild-type or the missense variants, as detected by western blot, and in large part properly localized in the Golgi apparatus, as assessed by confocal microscopy. Both patients were found to efficiently express the CA19.9 antigen in the serum despite the total loss of ST3GAL3 activity, which thus appears replaceable from other ST3GALs in the synthesis of the sialyl-Lewis a epitope. Kinetic studies of ST3GAL3 revealed a strong preference for lactotetraosylceramide as acceptor and gangliotetraosylceramide was also efficiently utilized in vitro. Moreover, the p.A13D missense variant, the one maintaining residual sialyltransferase activity, was found to have much lower affinity for all suitable substrates than the wild-type enzyme with an overall catalytic efficiency almost negligible. Altogether the present data suggest that the apparent redundancy of ST3GALs deduced from knock-out mouse models only partially exists in humans. In fact, our patients lacking ST3GAL3 activity synthesize the CA19.9 epitope sialyl-Lewis a, but not all glycans necessary for fine brain functions, where the role of minor gangliosides deserves further attention.

No abstract

ST3GAL5-CDG is a rare syndrome which is caused by variant GM3 synthases, the enzyme involved in the biosynthesis of a-b-c-series gangliosides. Here we report a novel homozygous ST3GAL5 variant, p.Gly342Ser, in a patient suffering from failure to thrive, severe hearing, visual, motor, and cognitive impairment, and respiratory chain dysfunction. A GM3 synthase assay towards the natural acceptor substrate lactosylceramide was performed upon transfection in HEK-293T cells of expression plasmids carrying wild type and mutated ST3GAL5 cDNAs. The assay revealed a complete loss of enzyme activity. Identical results were obtained with the other four ST3GAL5 variants which have been reported to be pathogenic. HEK-293T clones permanently expressing HaloTag-ST3GAL5 carrying each of the five variants were assessed by quantitative PCR, flow cytometry, western blotting, and confocal microscopy. The results indicated that transcription, translation, stability, and intracellular localization of the tagged protein were identical to those of the wild type construct. Compared with the very mild phenotype of st3gal5 KO mouse models, the results suggest that unknown mechanisms, in addition to the lack of a-b-c-series gangliosides, contribute to the syndrome. Direct enzyme assay upon transfection in model cells appears to be an effective tool for characterizing variants of glycosyltransferases involved in glycosphingolipid biosynthesis.

Sphingolipid metabolism starts with the biosynthesis of ceramide, a bioactive lipid and the backbone for the biosynthesis of complex sphingolipids such as sphingomyelin and glycosphingolipids. These are degraded back to ceramide and then to sphingosine, which enters the ceramide–sphingosine-1-phosphate signaling pathway or is further degraded. Several enzymes with multiple catalytic properties and subcellular localizations are thus involved in such metabolism. Hereditary defects of lysosomal hydrolases have been known for several years to be the cause of lysosomal storage diseases such as gangliosidoses, Gaucher disease, Niemann–Pick disease, Krabbe disease, Fabry disease, and Farber disease. More recently, many other inborn errors of sphingolipid metabolism have been recognized, involving enzymes responsible for the biosynthesis of ceramide, sphingomyelin, and glycosphingolipids. Concurrently, epidemiologic and biochemical evidence has established a link between Gaucher disease and Parkinson’s disease, showing that glucocerebrosidase variants predispose individuals to α-synuclein accumulation and neurodegeneration even in the heterozygous status. This appears to be due not only to lysosomal overload of non-degraded glucosylceramide, but to the derangement of vesicle traffic and autophagy, including mitochondrial autophagy, triggered by both sphingolipid intermediates and misfolded proteins. In this review, old and novel disorders of sphingolipid metabolism, in particular those of ganglioside biosynthesis, are evaluated in light of recent investigations of the link between Gaucher disease and Parkinson’s disease, with the aim of better understanding their pathogenic mechanisms and addressing new potential therapeutic strategies.

Carbohydrate antigen 19.9 (CA19.9) is used as a tumor marker for clinical and research purposes assuming that it is abundantly produced by gastrointestinal cancer cells due to a cancer-associated aberrant glycosylation favoring its synthesis. Recent data has instead suggested a different picture, where immunodetection on tissue sections matches biochemical and molecular data. In addition to CA19.9, structurally related carbohydrate antigens Lewis a and Lewis b are, in fact, undetectable in colon cancer, due to the down-regulation of a galactosyltransferase necessary for their synthesis. In the pancreas, no differential expression of CA19.9 or cognate glycosyltransferases occurs in cancer. Ductal cells only express such Lewis antigens in a pattern affected by the relative levels of each glycosyltransferase, which are genetically and epigenetically determined. The elevation of circulating antigens seems to depend on the obstruction of neoplastic ducts and loss of polarity occurring in malignant ductal cells. Circulating Lewis a and Lewis b are indeed promising candidates for monitoring pancreatic cancer patients that are negative for CA19.9, but not for improving the low diagnostic performance of such an antigen. Insufficient biological data are available for gastric and bile duct cancer. Studying each patient in a personalized manner determining all Lewis antigens in the surgical specimens and in the blood, together with the status of the tissue-specific glycosylation machinery, promises fruitful advances in translational research and clinical practice.

These findings indicate that HMGB1 may be a useful prognostic biomarker in MPM when detected by immunohistochemistry. Conversely, as it is also expressed in normal and reactive mesothelial cells, HMGB1 cannot be considered a diagnostic biomarker in histological samples of mesothelioma.

Merkel cell polyomavirus (MCPyV) has been identified in samples of Merkel cell carcinoma (MCC), an aggressive skin cancer. Seroepidemiologic studies indicated a high frequency of MCPyV infection in humans, suggesting respiratory and faecal-oral routes, or transmission by skin contact. Since MCC is more frequent in immunocompromised patients, a reactivation of MCPyV latently infecting target cells has been proposed. However, neither definite ways of transmission nor specific target organs have been identified with certainty. Ten autopsies with an extensive organ sampling for a total of 121 specimens (tissue and blood samples) were collected. All tissue specimens were fixed in formalin and embedded in paraffin. Real-time PCR was performed to quantify the copy number of the large T antigen (LT) gene and the capsid VP1 gene of MCPyV. MCPyV LT and/or VP genes were detected in all of the collected specimens. A high prevalence of MCPyV was found in the blood (six cases) and lung (five cases); the brain was positive in three cases. The highest viral copy number was detected in blood from two autopsies (21 610 570.09 copies per 105 cells and 380 413.25 copies per 105 cells), whereas the viral copy number in the other organs was low. Our data confirm the high frequency of MCPyV infection in the general population, which seems to indicate that the respiratory tract is a possible route for viral transmission and viral persistence in the brain. The frequent detection of MCPyV DNA in blood suggests that circulating leukocytes could be one of the reservoirs of MCPyV, whereas the high viral copy number also seems to indicate the possibility of viral reactivation in immunocompetent adults.

In the last few decades, the newly emerging field of epigenetic regulation of glycosylation acquired more importance because it is unraveling physiological and pathological mechanisms related to glycan functions. Glycosylation is a complex process in which proteins and lipids are modified by the attachment of monosaccharides. The main actors in this kind of modification are the glycoenzymes, which are translated from glycosylation-related genes (or glycogenes). The expression of glycogenes is regulated by transcription factors and epigenetic mechanisms (mainly DNA methylation, histone acetylation and noncoding RNAs). This review focuses only on these last ones, in relation to cancer and other diseases, such as inflammatory bowel disease and IgA1 nephropathy. In fact, it is clear that a deeper knowledge in the fine-tuning of glycogenes is essential for acquiring new insights in the glycan field, especially if this could be useful for finding novel and personalized therapeutics.