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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.

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.

This work was supported by: Portuguese Foundation for Science and Technology (FCT) PhD grants SFRH/BD/45120/2008 (Paulo F. Severino), SFRH/BD/81860/2011 (Mariana Silva) and SFRH/BD/100970/2014 (Mylene Carrascal), Liga Portuguesa Contra o Cancro 2011 (Mylene A. Carrascal); Premio Bluepharma Inovacao Universidade de Coimbra and the Santander Totta/Universidade NOVA de Lisboa prizes (Paula Videira). Grants from the University of Bologna (Fabio Dall'Olio). Mariangela Catera and Giulia Venturi are PhD students supported by grants from the University of Bologna. BACKGROUND: Treatment with Bacillus Calmette-Guérin (BCG) is the gold standard adjuvant immunotherapy of non-muscle invasive bladder cancer (NMIBC), although it fails in one third of the patients. NMIBC expresses two tumor-associated O-linked carbohydrates: the disaccharide (Galβ1,3GalNAc) Thomsen-Friedenreich (T) antigen, and its sialylated counterpart (Siaα2,3Galβ1,3GalNAc) sialyl-T (sT), synthesized by sialyltransferase ST3GAL1, whose roles in BCG response are unknown. METHODS: The human bladder cancer (BC) cell line HT1376 strongly expressing the T antigen, was retrovirally transduced with the ST3GAL1 cDNA or with an empty vector, yielding the cell lines HT1376sTand HT1376T, that express, respectively, either the sT or the T antigens. Cells were in vitro challenged with BCG. Whole gene expression was studied by microarray technology, cytokine secretion was measured by multiplex immune-beads assay. Human macrophages derived from blood monocytes were challenged with the secretome of BCG-challenged BC cells. RESULTS: The secretome from BCG-challenged HT1376sTcells induced a stronger macrophage secretion of IL-6, IL-1β, TNFα and IL-10 than that of HT1376Tcells. Transcriptomic analysis revealed that ST3GAL1 overexpression and T/sT replacement modulated hundreds of genes. Several genes preserving genomic stability were down-regulated in HT1376sTcells which, as a consequence, displayed increased sensitivity to oxidative damage. After BCG challenge, the transcriptome of HT1376sTcells showed higher susceptibility to BCG modulation than that of HT1376Tcells. CONCLUSIONS: High ST3GAL1 expression and T/sT replacement in BCG challenged-BC cancer cells induce a stronger macrophage response and alter the gene expression towards genomic instability, indicating a potential impact on BC biology and patient's response to BCG. publishersversion published

// Paulo F. Severino 1, 3, * , Mariana Silva 1, * , Mylene Carrascal 1 , Nadia Malagolini 3 , Mariella Chiricolo 3 , Giulia Venturi 3 , Annalisa Astolfi 4 , Mariangela Catera 3 , Paula A. Videira 1, 2 and Fabio Dall’Olio 3 1 Centro de Estudos de Doencas Cronicas, CEDOC, NOVA Medical School, Faculdade de Ciencias Medicas, Universidade NOVA de Lisboa, Lisboa, Portugal 2 UCIBIO, Departamento Ciencias da Vida, Faculdade de Ciencias e Tecnologia, Universidade NOVA de Lisboa, Lisboa, Portugal 3 Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Sede di Patologia Generale, Universita di Bologna, Bologna, Italy 4 Centro Interdipartimentale Ricerche sul Cancro “Giorgio Prodi”, Universita di Bologna, Bologna, Italy * Co-first authors Correspondence to: Fabio Dall’Olio, email: fabio.dallolio@unibo.it Paula A. Videira, email: p.videira@fct.unl.pt Keywords: Bacillus Calmette-Guerin, bladder cancer, glycosylation, sialyl-Tn antigen, ST6GALNAC1 sialyltransferase Received: May 30, 2016 Accepted: March 30, 2017 Published: April 17, 2017 ABSTRACT The sialyl-Tn (sTn) antigen is an O -linked carbohydrate chain aberrantly expressed in bladder cancer (BC), whose biosynthesis is mainly controlled by the sialyltransferase ST6GALNAC1. Treatment with Bacillus Calmette-Guerin (BCG) is the most effective adjuvant immunotherapy for superficial BC but one third of the patients fail to respond. A poorly understood correlation between the expression of sTn and BC patient’s response to BCG was previously observed. By analyzing tumor tissues, we showed that patients with high ST6GALNAC1 and IL-6 mRNA expression were BCG responders. To investigate the role of sTn in BC cell biology and BCG response, we established the cell lines MCR sTn and MCR Nc by retroviral transduction of the BC cell line MCR with the ST6GALNAC1 cDNA or with an empty vector, respectively. Compared with MCR Nc , BCG-stimulated MCR sTn secreted higher levels of IL-6 and IL-8 and their secretome induced a stronger IL-6, IL-1β, and TNFα secretion by macrophages, suggesting the induction of a stronger inflammatory response. Transcriptomic analysis of MCR Nc and MCR sTn revealed that ST6GALNAC1 /sTn expression modulates hundreds of genes towards a putative more malignant phenotype and down-regulates several genes maintaining genomic stability. Consistently, MCR sTn cells displayed higher H 2 O 2 sensitivity. In MCR sTn, , BCG challenge induced an increased expression of several regulatory non coding RNA genes. These results indicate that the expression of ST6GALNAC1 /sTn improves the response to BCG therapy by inducing a stronger macrophage response and alters gene expression towards malignancy and genomic instability, increasing the sensitivity of BC cells to the oxidizing agents released by BCG.