Convenient and Sensitive Measurement of Lactosylceramide Synthase Activity Using Deuterated Glucosylceramide and Mass Spectrometry.

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.

N-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sda Synthase B4GALNT2.

The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

The story of the Sda antigen and of its cognate enzyme B4GALNT2: What is new?

The structure Siaα2,3(GalNAcß1,4)Gal- is the epitope of the Sda antigen, which is expressed on the erythrocytes and secretions of the vast majority of Caucasians, carried by N- and O-linked chains of glycoproteins, as well as by glycolipids. Sda is very similar, but not identical, to ganglioside GM2 [Siaα2,3(GalNAcß1,4)Galß1,4Glc-Cer]. The Sda synthase ß1,4 N-acetylgalactosaminyl transferase 2 (B4GALNT2) exists in a short and a long form, diverging in the aminoterminal domain. The latter has a very long cytoplasmic tail and displays a Golgi- as well as a post-Golgi localization. The biosynthesis of Sda is mutually exclusive with that of the cancer-associated sialyl Lewis antigens, whose structure is Siaα2,3Galß1,3/4(Fucα1,4/3)GlcNAc-. B4GALNT2 is down-regulated in colon cancer but patients with higher expression survive longer. In experimental systems, B4GALNT2 inhibits colon cancer progression,not only through inhibition of sialyl Lewis antigen biosynthesis. By contrast, in breast cancer B4GALNT2 is associated with malignancy. In colon cancer, the B4GALNT2 gene is regulated by multiple mechanisms, which include miRNA and transcription factor expression, as well as CpG methylation. In addition, Sda/B4GALNT2 regulates the susceptibility to infectious agents, the protection from muscle dystrophy, the activity of immune system in pregnancy and the immune rejection in xenotransplantation.

The Mutual Relationship between Glycosylation and Non-Coding RNAs in Cancer and Other Physio-Pathological Conditions.

Glycosylation, which consists of the enzymatic addition of sugars to proteins and lipids, is one of the most important post-co-synthetic modifications of these molecules, profoundly affecting their activity. Although the presence of carbohydrate chains is crucial for fine-tuning the interactions between cells and molecules, glycosylation is an intrinsically stochastic process regulated by the relative abundance of biosynthetic (glycosyltransferases) and catabolic (glycosidases) enzymes, as well as sugar carriers and other molecules. Non-coding RNAs, which include microRNAs, long non-coding RNAs and circRNAs, establish a complex network of reciprocally interacting molecules whose final goal is the regulation of mRNA expression. Likewise, these interactions are stochastically regulated by ncRNA abundance. Thus, while protein sequence is deterministically dictated by the DNA/RNA/protein axis, protein abundance and activity are regulated by two stochastic processes acting, respectively, before and after the biosynthesis of the protein axis. Consequently, the worlds of glycosylation and ncRNA are closely interconnected and mutually interacting. In this paper, we will extensively review the many faces of the ncRNA-glycosylation interplay in cancer and other physio-pathological conditions.

Glycosyltransferases in Cancer: Prognostic Biomarkers of Survival in Patient Cohorts and Impact on Malignancy in Experimental Models.

Background: Glycosylation changes are a main feature of cancer. Some carbohydrate epitopes and expression levels of glycosyltransferases have been used or proposed as prognostic markers, while many experimental works have investigated the role of glycosyltransferases in malignancy. Using the transcriptomic data of the 21 TCGA cohorts, we correlated the expression level of 114 glycosyltransferases with the overall survival of patients. Methods: Using the Oncolnc website, we determined the Kaplan−Meier survival curves for the patients falling in the 15% upper or lower percentile of mRNA expression of each glycosyltransferase. Results: Seventeen glycosyltransferases involved in initial steps of N- or O-glycosylation and of glycolipid biosynthesis, in chain extension and sialylation were unequivocally associated with bad prognosis in a majority of cohorts. Four glycosyltransferases were associated with good prognosis. Other glycosyltransferases displayed an extremely high predictive value in only one or a few cohorts. The top were GALNT3, ALG6 and B3GNT7, which displayed a p < 1 × 10−9 in the low-grade glioma (LGG) cohort. Comparison with published experimental data points to ALG3, GALNT2, B4GALNT1, POFUT1, B4GALT5, B3GNT5 and ST3GAL2 as the most consistently malignancy-associated enzymes. Conclusions: We identified several cancer-associated glycosyltransferases as potential prognostic markers and therapeutic targets.

The Cancer-Associated Antigens Sialyl Lewisa/x and Sda: Two Opposite Faces of Terminal Glycosylation.

Terminal carbohydrate structures are particularly relevant in oncology because they can serve as cancer markers and alter the phenotype of cancer cells. The Sda antigen and the sialyl Lewisx and sialyl Lewisa (sLex and sLea) antigens are terminal structures whose biosynthesis is mutually exclusive. In this review, we describe the main features of the Sda antigen in cancer and its relationship with sLex/a antigens. Information was obtained from an extensive literature search and from The Cancer Genome Atlas (TCGA) public database. The Sda biosynthetic enzyme B4GALNT2 undergoes downregulation in colorectal (CRC) and stomach cancer, while it is ectopically expressed by a minority of breast cancer (BRCA) patients. High expression of B4GALNT2 is associated with better prognosis and a less malignant gene expression profile in CRC, while the opposite occurs in BRCA. The regulation of B4GALNT2 expression in CRC is multifactorial, involving gene methylation and miRNA expression. Forced expression of B4GALNT2 inhibited sLea/sLex and reduced malignancy and stemness in cells constitutively expressing sLex/a antigens. However, consistent effects were observed upon B4GALNT2 forced expression and in cells not expressing sLex/a antigens. Thus, B4GALNT2 and the Sda antigen exert a tumor-restraining activity in CRC and probably other gastrointestinal cancers, independently of sLex/a antigens.

Immunoglobulin G Glycosylation Changes in Aging and Other Inflammatory Conditions.

Among the multiple roles played by protein glycosylation, the fine regulation of biological interactions is one of the most important. The asparagine 297 (Asn297) of IgG heavy chains is decorated by a diantennary glycan bearing a number of galactose and sialic acid residues on the branches ranging from 0 to 2. In addition, the structure can present core-linked fucose and/or a bisecting GlcNAc. In many inflammatory and autoimmune conditions, as well as in metabolic, cardiovascular, infectious, and neoplastic diseases, the IgG Asn297-linked glycan becomes less sialylated and less galactosylated, leading to increased expression of glycans terminating with GlcNAc. These conditions alter also the presence of core-fucose and bisecting GlcNAc. Importantly, similar glycomic alterations are observed in aging. The common condition, shared by the above-mentioned pathological conditions and aging, is a low-grade, chronic, asymptomatic inflammatory state which, in the case of aging, is known as inflammaging. Glycomic alterations associated with inflammatory diseases often precede disease onset and follow remission. The aberrantly glycosylated IgG glycans associated with inflammation and aging can sustain inflammation through different mechanisms, fueling a vicious loop. These include complement activation, Fcγ receptor binding, binding to lectin receptors on antigen-presenting cells, and autoantibody reactivity. The complex molecular bases of the glycomic changes associated with inflammation and aging are still poorly understood.

Glycobiology of the Epithelial to Mesenchymal Transition.

Glycosylation consists in the covalent, enzyme mediated, attachment of sugar chains to proteins and lipids. A large proportion of membrane and secreted proteins are indeed glycoproteins, while glycolipids are fundamental component of cell membranes. The biosynthesis of sugar chains is mediated by glycosyltransferases, whose level of expression represents a major factor of regulation of the glycosylation process. In cancer, glycosylation undergoes profound changes, which often contribute to invasion and metastasis. Epithelial to mesenchymal transition (EMT) is a key step in metastasis formation and is intimately associated with glycosylation changes. Numerous carbohydrate structures undergo up- or down-regulation during EMT and often regulate the process. In this review, we will discuss the relationship with EMT of the N-glycans, of the different types of O-glycans, including the classical mucin-type, O-GlcNAc, O-linked fucose, O-linked mannose and of glycolipids. Finally, we will discuss the role in EMT of galectins, a major class of mammalian galactoside-binding lectins. While the expression of specific carbohydrate structures can be used as a marker of EMT and of the propensity to migrate, the manipulation of the glycosylation machinery offers new perspectives for cancer treatment through inhibition of EMT.

Glycosyltransferase B4GALNT2 as a Predictor of Good Prognosis in Colon Cancer: Lessons from Databases.

BACKGROUND: glycosyltransferase B4GALNT2 and its cognate carbohydrate antigen Sda are highly expressed in normal colon but strongly downregulated in colorectal carcinoma (CRC). We previously showed that CRC patients expressing higher B4GALNT2 mRNA levels displayed longer survival. Forced B4GALNT2 expression reduced the malignancy and stemness of colon cancer cells. METHODS: Kaplan-Meier survival curves were determined in "The Cancer Genome Atlas" (TCGA) COAD cohort for several glycosyltransferases, oncogenes, and tumor suppressor genes. Whole expression data of coding genes as well as miRNA and methylation data for B4GALNT2 were downloaded from TCGA. RESULTS: the prognostic potential of B4GALNT2 was the best among the glycosyltransferases tested and better than that of many oncogenes and tumor suppressor genes; high B4GALNT2 expression was associated with a lower malignancy gene expression profile; differential methylation of an intronic B4GALNT2 gene position and miR-204-5p expression play major roles in B4GALNT2 regulation. CONCLUSIONS: high B4GALNT2 expression is a strong predictor of good prognosis in CRC as a part of a wider molecular signature that includes ZG16, ITLN1, BEST2, and GUCA2B. Differential DNA methylation and miRNA expression contribute to regulating B4GALNT2 expression during colorectal carcinogenesis.

L1CAM as an E-selectin Ligand in Colon Cancer.

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.

The Sda Synthase B4GALNT2 Reduces Malignancy and Stemness in Colon Cancer Cell Lines Independently of Sialyl Lewis X Inhibition.

BACKGROUND: The Sda antigen and its biosynthetic enzyme B4GALNT2 are highly expressed in healthy colon but undergo a variable down-regulation in colon cancer. The biosynthesis of the malignancy-associated sialyl Lewis x (sLex) antigen in normal and cancerous colon is mediated by fucosyltransferase 6 (FUT6) and is mutually exclusive from that of Sda. It is thought that the reduced malignancy associated with high B4GALNT2 was due to sLex inhibition. METHODS: We transfected the cell lines SW480 and SW620, derived respectively from a primary tumor and a metastasis of the same patient, with the cDNAs of FUT6 or B4GALNT2, generating cell variants expressing either the sLex or the Sda antigens. Transfectants were analyzed for growth in poor adherence, wound healing, stemness and gene expression profile. RESULTS: B4GALNT2/Sda expression down-regulated all malignancy-associated phenotypes in SW620 but only those associated with stemness in SW480. FUT6/sLex enhanced some malignancy-associated phenotypes in SW620, but had little effect in SW480. The impact on the transcriptome was stronger for FUT6 than for B4GALNT2 and only partially overlapping between SW480 and SW620. CONCLUSIONS: B4GALNT2/Sda inhibits the stemness-associated malignant phenotype, independently of sLex inhibition. The impact of glycosyltransferases on the phenotype and the transcriptome is highly cell-line specific.

High Expression of the Sda Synthase B4GALNT2 Associates with Good Prognosis and Attenuates Stemness in Colon Cancer.

BACKGROUND: The carbohydrate antigen Sda and its biosynthetic enzyme B4GALNT2 are highly expressed in normal colonic mucosa but are down-regulated to a variable degree in colon cancer tissues. Here, we investigated the clinical and biological importance of B4GALNT2 in colon cancer. METHODS: Correlations of B4GALNT2 mRNA with clinical data were obtained from The Cancer Genome Atlas (TCGA) database; the phenotypic and transcriptomic changes induced by B4GALNT2 were studied in LS174T cells transfected with B4GALNT2 cDNA. RESULTS: TCGA data indicate that patients with high B4GALNT2 expression in cancer tissues display longer survival than non-expressers. In LS174T cells, expression of B4GALNT2 did not affect the ability to heal a scratch wound or to form colonies in standard growth conditions but markedly reduced the growth in soft agar, the tridimensional (3D) growth as spheroids, and the number of cancer stem cells, indicating a specific effect of B4GALNT2 on the growth in poor adherence and stemness. On the transcriptome, B4GALNT2 induced the down-regulation of the stemness-associated gene SOX2 and modulated gene expression towards an attenuation of the cancer phenotype. CONCLUSIONS: The level of B4GALNT2 can be proposed as a marker to identify higher- and lower-risk colorectal cancer patients.

A novel nonsense and inactivating variant of ST3GAL3 in two infant siblings suffering severe epilepsy and expressing circulating CA19.9.

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.

Carcinoembryonic antigen is a sialyl Lewis x/a carrier and an E­selectin ligand in non­small cell lung cancer.

The formation of distant metastasis resulting from vascular dissemination is one of the leading causes of mortality in non­small cell lung cancer (NSCLC). This metastatic dissemination initiates with the adhesion of circulating cancer cells to the endothelium. The minimal requirement for the binding of leukocytes to endothelial E­selectins and subsequent transmigration is the epitope of the fucosylated glycan, sialyl Lewis x (sLex), attached to specific cell surface glycoproteins. sLex and its isomer sialyl Lewis a (sLea) have been described in NSCLC, but their functional role in cancer cell adhesion to endothelium is still poorly understood. In this study, it was hypothesised that, similarly to leukocytes, sLe glycans play a role in NSCLC cell adhesion to E­selectins. To assess this, paired tumour and normal lung tissue samples from 18 NSCLC patients were analyzed. Immunoblotting and immunohistochemistry assays demonstrated that tumour tissues exhibited significantly stronger reactivity with anti­sLex/sLea antibody and E­selectin chimera than normal tissues (2.2­ and 1.8­fold higher, respectively), as well as a higher immunoreactive score. High sLex/sLea expression was associated with bone metastasis. The overall α1,3­fucosyltransferase (FUT) activity was increased in tumour tissues, along with the mRNA levels of FUT3, FUT6 and FUT7, whereas FUT4 mRNA expression was decreased. The expression of E­selectin ligands exhibited a weak but significant correlation with the FUT3/FUT4 and FUT7/FUT4 ratios. Additionally, carcinoembryonic antigen (CEA) was identified in only 8 of the 18 tumour tissues; CEA­positive tissues exhibited significantly increased sLex/sLea expression. Tumour tissue areas expressing CEA also expressed sLex/sLea and showed reactivity to E­selectin. Blot rolling assays further demonstrated that CEA immunoprecipitates exhibited sustained adhesive interactions with E­selectin­expressing cells, suggesting CEA acts as a functional protein scaffold for E­selectin ligands in NSCLC. In conclusion, this work provides the first demonstration that sLex/sLea are increased in primary NSCLC due to increased α1,3­FUT activity. sLex/sLea is carried by CEA and confers the ability for NSCLC cells to bind E­selectins, and is potentially associated with bone metastasis. This study contributes to identifying potential future diagnostic/prognostic biomarkers and therapeutic targets for lung cancer.

Impact of sialyltransferase ST6GAL1 overexpression on different colon cancer cell types.

Cancer-associated glycan structures can be both tumor markers and engines of disease progression. The structure Siaα2,6Galß1,4GlcNAc (Sia6LacNAc), synthesized by sialyltransferase ST6GAL1, is a cancer-associated glycan. Although ST6GAL1/Sia6LacNAc are often overexpressed in colorectal cancer (CRC), their biological and clinical significance remains unclear. To get insights into the clinical relevance of ST6GAL1 expression in CRC, we interrogated The Cancer Genome Atlas with mRNA expression data of hundreds of clinically characterized CRC and normal samples. We found an association of low ST6GAL1 expression with microsatellite instability (MSI), BRAF mutations and mucinous phenotype but not with stage, response to therapy and survival. To investigate the impact of ST6GAL1 expression in experimental systems, we analyzed the transcriptome and the phenotype of the CRC cell lines SW948 and SW48 after retroviral transduction with ST6GAL1 cDNA. The two cell lines display the two main pathways of CRC transformation: chromosomal instability and MSI, respectively. Constitutive ST6GAL1 expression induced much deeper transcriptomic changes in SW948 than in SW48 and affected different genes in the two cell lines. ST6GAL1 expression affected differentially the tyrosine phosphorylation induced by hepatocyte growth factor, the ability to grow in soft agar, to heal a scratch wound and to invade Matrigel in the two cell lines. These results indicate that the altered expression of a cancer-associated glycosyltransferase impacts the gene expression profile, as well as the phenotype, although in a cancer subtype-specific manner.

Total loss of GM3 synthase activity by a normally processed enzyme in a novel variant and in all ST3GAL5 variants reported to cause a distinct congenital disorder of glycosylation.

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.

The extended cytoplasmic tail of the human B4GALNT2 is critical for its Golgi targeting and post-Golgi sorting.

The Sda /Cad antigen reported on glycoconjugates of human tissues has an increasingly recognized wide impact on the physio-pathology of different biological systems. The last step of its biosynthesis relies on the enzymatic activity of the ß1,4-N-acetylgalactosaminyltransferase-II (B4GALNT2), which shows the highest expression level in healthy colon. Previous studies reported the occurrence in human colonic cells of two B4GALNT2 protein isoforms that differ in the length of their cytoplasmic tail, the long isoform showing an extended 66-amino acid tail. We examined here, the subcellular distribution of the two B4GALNT2 protein isoforms in stably transfected colonic LS174T cells and in transiently transfected HeLa cells using fluorescence microscopy. While a similar subcellular distribution at the trans-Golgi cisternae level was observed for the two isoforms, our study pointed to an atypical subcellular localization of the long B4GALNT2 isoform into dynamic vesicles. We demonstrated a critical role of its extended cytoplasmic tail for its Golgi targeting and post-Golgi sorting and highlighted the existence of a newly described post-Golgi sorting signal as well as a previously undescribed fate of a Golgi glycosyltransferase. DATABASE: The proteins ß1,4GalNAcT II, ß1,4-GalT1, FucT I, FucT VI and ST3Gal IV are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4, whereas the corresponding human genes are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4 according to the HUGO nomenclature.

Diseases of ganglioside biosynthesis: An expanding group of congenital disorders of glycosylation.

Among the numerous congenital disorders of glycosylation concerning glycoproteins, only a single mutation in ganglioside biosynthesis had been reported until a few years ago: one in the ST3GAL5 gene, encoding GM3 synthase. More recently, additional mutations in the same gene were reported, together with several distinct mutations in the B4GALNT1 gene, encoding GM2/GD2/GA2 synthase. Patients suffering from ST3GAL5 deficiency present a devastating syndrome characterized by early onset and dramatic neurological and cognitive impairment, sometimes associated with dyspigmentation and an increased blood lactate concentration. On the other hand, B4GALNT1 mutations give rise to a form of complicated hereditary spastic paraplegia (HSP), previously referred to as HSP26. It is characterized by the late onset of lower limb weakness and mild to moderate intellectual impairment, which is usually not progressive. In addition to the most typical signs, some patients present ocular and endocrine signs, pes cavus, and psychiatric illness. Since the nineties, mice lacking genes for single glycosyltransferases involved in ganglioside biosynthesis, including ST3GAL5 and B4GALNT1, were created and studied. The resulting phenotypes were frequently mild or very mild, so double knock-out animals were created to effectively study the function of gangliosides. The main clinical and biochemical features of patients suffering from GM3 synthase or GM2/GD2/GA2 synthase deficiency, compared with the phenotypes described in mice that are null for single or multiple glycosyltransferase genes, provide suggestions to improve the recognition of novel mutations and potentially related disorders.

Glycosylation as a Main Regulator of Growth and Death Factor Receptors Signaling.

Glycosylation is a very frequent and functionally important post-translational protein modification that undergoes profound changes in cancer. Growth and death factor receptors and plasma membrane glycoproteins, which upon activation by extracellular ligands trigger a signal transduction cascade, are targets of several molecular anti-cancer drugs. In this review, we provide a thorough picture of the mechanisms bywhich glycosylation affects the activity of growth and death factor receptors in normal and pathological conditions. Glycosylation affects receptor activity through three non-mutually exclusive basic mechanisms: (1) by directly regulating intracellular transport, ligand binding, oligomerization and signaling of receptors; (2) through the binding of receptor carbohydrate structures to galectins, forming a lattice thatregulates receptor turnover on the plasma membrane; and (3) by receptor interaction with gangliosides inside membrane microdomains. Some carbohydrate chains, for example core fucose and ß1,6-branching, exert a stimulatory effect on all receptors, while other structures exert opposite effects on different receptors or in different cellular contexts. In light of the crucial role played by glycosylation in the regulation of receptor activity, the development of next-generation drugs targeting glyco-epitopes of growth factor receptors should be considered a therapeutically interesting goal.

Oxidative damage and response to Bacillus Calmette-Guérin in bladder cancer cells expressing sialyltransferase ST3GAL1.

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 (Galß1,3GalNAc) Thomsen-Friedenreich (T) antigen, and its sialylated counterpart (Siaα2,3Galß1,3GalNAc) 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 HT1376sT and 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 HT1376sT cells induced a stronger macrophage secretion of IL-6, IL-1ß, TNFα and IL-10 than that of HT1376T cells. Transcriptomic analysis revealed that ST3GAL1 overexpression and T/sT replacement modulated hundreds of genes. Several genes preserving genomic stability were down-regulated in HT1376sT cells which, as a consequence, displayed increased sensitivity to oxidative damage. After BCG challenge, the transcriptome of HT1376sT cells showed higher susceptibility to BCG modulation than that of HT1376T cells. 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.