Immune cells surveil aberrantly sialylated O-glycans on megakaryocytes to regulate platelet count.

Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti-Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1 adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK-/-). TF antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK-/- mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H-positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK-/- BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell-like signature and concomitant upregulation of the immunoglobulin rearrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.

Transfer of Functional Cargo in Exomeres.

Exomeres are a recently discovered type of extracellular nanoparticle with no known biological function. Herein, we describe a simple ultracentrifugation-based method for separation of exomeres from exosomes. Exomeres are enriched in Argonaute 1-3 and amyloid precursor protein. We identify distinct functions of exomeres mediated by two of their cargo, the ß-galactoside α2,6-sialyltransferase 1 (ST6Gal-I) that α2,6- sialylates N-glycans, and the EGFR ligand, amphiregulin (AREG). Functional ST6Gal-I in exomeres can be transferred to cells, resulting in hypersialylation of recipient cell-surface proteins including ß1-integrin. AREG-containing exomeres elicit prolonged EGFR and downstream signaling in recipient cells, modulate EGFR trafficking in normal intestinal organoids, and dramatically enhance the growth of colonic tumor organoids. This study provides a simplified method of exomere isolation and demonstrates that exomeres contain and can transfer functional cargo. These findings underscore the heterogeneity of nanoparticles and should accelerate advances in determining the composition and biological functions of exomeres.

An optimised assay for quantitative, high-throughput analysis of polysialyltransferase activity.

The polysialyltransferases are biologically important glycosyltransferase enzymes responsible for the biosynthesis of polysialic acid, a carbohydrate polymer that plays a critical role in the progression of several diseases, notably cancer. Having improved the chemical synthesis and purification of the fluorescently-labelled DMB-DP3 acceptor, we report optimisation and validation of a highly sensitive cell-free high-throughput HPLC-based assay for assessment of human polysialyltransferase activity.

Analyses of chicken sialyltransferases related to O-glycosylation.

The chicken ß-galactoside α2,3-sialyltransferase 1, 2, and 5 (ST3Gal1, 2, and 5) genes were cloned, and their enzymes were expressed in 293FT cells. ST3Gal1 and 2 exhibited enzymatic activities toward galactose-ß1,3-N-acetylgalactosamine and galactose-ß1,3-N-acetylglucosamine. ST3Gal5 only exhibited activity toward lactosylceramide. ST3Gal1 and 2 and previously cloned ST3Gal3 and 6 transferred CMP-sialic acid to asialofetuin. Reverse-transcription-quantitative PCR indicated that ST3Gal1 was expressed at higher levels in the trachea, lung, spleen, and magnum, and the strong expression of ST3Gal5 was observed in the spleen, magnum, and small and large intestines. ST3Gal1, 5, and 6 were also expressed in the tubular gland cells of the magnum, which secretes egg-white proteins. ST3Gal1, 5, and 6 were expressed in the egg chorioallantoic membrane, in which influenza viruses are propagated for the production of vaccines.

Glycolipid GD3 and GD3 synthase are key drivers for glioblastoma stem cells and tumorigenicity.

The cancer stem cells (CSCs) of glioblastoma multiforme (GBM), a grade IV astrocytoma, have been enriched by the expressed marker CD133. However, recent studies have shown that CD133(-) cells also possess tumor-initiating potential. By analysis of gangliosides on various cells, we show that ganglioside D3 (GD3) is overexpressed on eight neurospheres and tumor cells; in combination with CD133, the sorted cells exhibit a higher expression of stemness genes and self-renewal potential; and as few as six cells will form neurospheres and 20-30 cells will grow tumor in mice. Furthermore, GD3 synthase (GD3S) is increased in neurospheres and human GBM tissues, but not in normal brain tissues, and suppression of GD3S results in decreased GBM stem cell (GSC)-associated properties. In addition, a GD3 antibody is shown to induce complement-dependent cytotoxicity against cells expressing GD3 and inhibition of GBM tumor growth in vivo. Our results demonstrate that GD3 and GD3S are highly expressed in GSCs, play a key role in glioblastoma tumorigenicity, and are potential therapeutic targets against GBM.

Polysialic acid: biosynthesis, novel functions and applications.

As an anti-adhesive, a reservoir for key biological molecules, and a modulator of signaling, polysialic acid (polySia) is critical for nervous system development and maintenance, promotes cancer metastasis, tissue regeneration and repair, and is implicated in psychiatric diseases. In this review, we focus on the biosynthesis and functions of mammalian polySia, and the use of polySia in therapeutic applications. PolySia modifies a small subset of mammalian glycoproteins, with the neural cell adhesion molecule, NCAM, serving as its major carrier. Studies show that mammalian polysialyltransferases employ a unique recognition mechanism to limit the addition of polySia to a select group of proteins. PolySia has long been considered an anti-adhesive molecule, and its impact on cell adhesion and signaling attributed directly to this property. However, recent studies have shown that polySia specifically binds neurotrophins, growth factors, and neurotransmitters and that this binding depends on chain length. This work highlights the importance of considering polySia quality and quantity, and not simply its presence or absence, as its various roles are explored. The capsular polySia of neuroinvasive bacteria allows these organisms to evade the host immune response. While this "stealth" characteristic has made meningitis vaccine development difficult, it has also made polySia a worthy replacement for polyetheylene glycol in the generation of therapeutic proteins with low immunogenicity and improved circulating half-lives. Bacterial polysialyltransferases are more promiscuous than the protein-specific mammalian enzymes, and new studies suggest that these enzymes have tremendous therapeutic potential, especially for strategies aimed at neural regeneration and tissue repair.

A new assay for determining ganglioside sialyltransferase activities lactosylceramide-2,3-sialyltransferase (SAT I) and monosialylganglioside-2,3-sialyltransferase (SAT IV).

A new assay for the determination of lactosylceramide-2,3-sialyltransferase (SAT I, EC 2.4.99.9) and monosialoganglioside sialyltransferase (SAT IV, EC 2.4.99.2) is described. The assay utilised the commercially available fluorophore labelled sphingolipids, boron dipyrromethene difluoride (BODIPY) lactosylceramide (LacCer), and BODIPY-monosialotetrahexosylganglioside (GM1) as the acceptor substrates, for SAT I and SAT IV, respectively. HPLC coupled with fluorescence detection was used to analyse product formation. The analysis was performed in a quick and automated fashion. The assay showed good linearity for both BODIPY sphingolipids with a quantitative detection limit of 0.05 pmol. The high sensitivity enabled the detection of SAT I and SAT IV activities as low as 0.001 µU, at least 200 fold lower than that of most radiometric assays. This new assay was applied to the screening of SAT I and SAT IV activities in ovine and bovine organs (liver, heart, kidney, and spleen). The results provided evidence that young animals, such as calves, start to produce ganglioside sialyltransferases as early as 7 days after parturition and that levels change during maturation. Among the organs tested from a bovine source, spleen had the highest specific ganglioside sialyltransferase activity. Due to the organ size, the greatest total ganglioside sialyltransferase activities (SAT I and SAT IV) were detected in the liver of both bovine and ovine origin.

Influence of passage number on glycosylation of alkyl lactosides by Madin-Darby canine kidney (MDCK) cells.

The cell function on saccharide biosynthesis can be evaluated by employing the saccharide primer method. This study demonstrated that the characteristics of Madin-Darby canine kidney (MDCK) cells changed in relation with passage number when 12-azidododecyl ß-lactoside (Lac-12N(3) primer) was incorporated into MDCK cells and afforded GM3-, GD3-, sialylparagloboside (SPG), and NeuAc-Gal-GlcNAc-Gal-GlcNAc-Lac-type oligosaccharides. By measuring the amount of glycosylated products from relatively early to late passage numbers, results showed that there was an appropriate passage number that optimized oligosaccharide production and that the higher passage number resulted to a decrease in oligosaccharide production. Moreover, results suggested that aside from sialyltransferase, the activity of several kinds of enzymes that control the amount of saccharide production was presumably affected depending upon the biological senescence.

A high-throughput screen for polysialyltransferase activity.

Polysialic acid is common to humans and a few bacterial pathogens and it holds great potential for the development of new therapeutic reagents. Currently, the bacterial polysialyltransferases (polySTs) are the only source of polysialic acid for research and biotechnological purposes either directly, by enzymatic polysialylation of therapeutic proteins, or indirectly, by harvest of polysialic acid from bacterial fermentation. Further engineering and optimization of these enzymes is hindered by the lack of high-throughput screening methodologies for polysialyltransferase activity. Here we report the development of an efficient in vivo activity screen for bacterial polySTs. The screen exploits complementation of a dormant capsule export complex in the expression strain, Escherichia coli BL21-Gold(DE3). This strain was metabolically engineered to synthesize CMP-Neu5Ac, the donor sugar for the polysialylation reaction. Using the new strain, a colony blotting procedure that enables the routine testing of more than 10(4) polyST genes was developed. To test the usefulness of the methodology, we screened a library of N-terminally truncated polySTs derived from the Neisseria meningitidis serogroup B (NmB)-polyST. We identified truncations that remove a putative membrane interaction domain, resulting in soluble and active enzymes.

A universal fluorescent acceptor for high-performance liquid chromatography analysis of pro- and eukaryotic polysialyltransferases.

Polysialyltransferases (polySTs) play critical roles in diverse biological processes, including neural development, tumorigenesis, and bacterial pathogenesis. Although the bacterial enzymes are presumed to have evolved to provide molecular mimics of the host-specific polysialic acid, no analytical technique is currently available to facilitate a direct comparison of the bacterial and vertebrate enzymes. Here we describe a new fluorescent acceptor, a 1,2-diamino-4,5-methylenedioxybenzene (DMB)-labeled trimer of α2,8-linked sialic acid (DMB-DP3), which primes both pro- and eukaryotic polySTs. High-performance liquid chromatography separation and fluorescence detection (HPLC-FD) of reaction products enabled the sensitive and quantitative detection of polyST activity, even using cell lysates as enzyme source, and revealed product profiles characteristic of each enzyme. Single product resolution afforded by this assay system revealed mechanistic insights into a kinetic lag phase exhibited by the polyST from Neisseria meningitidis serogroup B during chain elongation. DMB-DP3 is the first fluorescent acceptor shown to prime the mammalian polySTs. Moreover, product profiles obtained for the two murine polySTs provided direct biochemical evidence for enzymatic properties that had, until now, only been inferred from the analysis of biological samples. With DMB-DP3, we introduce a universal acceptor that provides an easy, fast, and reliable system for the comprehensive mechanistic and comparative analysis of polySTs.

Ganglioside GM3 levels are altered in a mouse model of HIBM: GM3 as a cellular marker of the disease.

OBJECTIVE: HIBM (Hereditary Inclusion Body Myopathy) is a recessive hereditary disease characterized by adult-onset, slowly progressive muscle weakness sparing the quadriceps. It is caused by a single missense mutation of each allele of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, a bifunctional enzyme catalyzing the first two steps of sialic acid synthesis in mammals. However, the mechanisms and cellular pathways affected by the GNE mutation and causing the muscle weakness could not be identified so far. Based on recent evidence in literature, we investigated a new hypothesis, i.e. the involvement in the disease of the GM3 ganglioside, a specific glycolipid implicated in muscle cell proliferation and differentiation. METHODS: qRT-PCR analysis of St3gal5 (GM3 synthase) gene expression and HPLC quantification of GM3 ganglioside were conducted on muscle tissue from a mouse model of HIBM harboring the M712T mutation of GNE (Gne(M712T/M712T) mouse) vs control mice (Gne(+/+) mouse). RESULTS: St3gal5 mRNA levels were significantly lower in Gne(M712T/M712T) mouse muscles vs Gne(+/+) mouse muscles (64.41%+/-10% of Gne(+/+) levels). GM3 ganglioside levels showed also a significant decrease in Gne(M712T/M712T) mouse muscle compared to Gne(+/+) mouse muscle (18.09%+/-5.33% of Gne(+/+) levels). Although these Gne(M712T/M712T) mice were described to suffer severe glomerular proteinuria, no GM3 alterations were noted in kidneys, highlighting a tissue specific alteration of gangliosides. CONCLUSION: The M712T mutation of GNE hampers the muscle ability to synthesize normal levels of GM3. This is the first time that a mutation of GNE can be related to the molecular pathological mechanism of HIBM.

Enhancing of GM3 synthase expression during differentiation of human blood monocytes into macrophages as in vitro model of GM3 accumulation in atherosclerotic lesion.

In previous studies, we showed that ganglioside levels (GM3 being the main ganglioside) in human aortic intima isolated from atherosclerotic lesions were 5 times greater compared to intima from non-diseased vascular areas. Recently, we found that GM3 and GM3 synthase levels in differentiated in vitro macrophages were five and ten times higher, respectively, compared to freshly isolated human monocytes. In this article, we report that GM3 synthase mRNA levels were significantly higher in differentiated human monocyte-derived macrophages compared to monocytes and in atherosclerotic aorta compared to normal aorta. The depletion of GM3 synthesis in cultured monocyte-derived macrophages with DL-threo-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, an inhibitor of ganglioside synthesis, delayed the acquisition of CD206 antigen, prevented the loss of CD163 antigen and enhanced anti-inflammatory cytokine (CCL18) secretion. In the current study, we performed purification of CMP-N-acetylneuraminic acid:lactosylceramide alpha2,3-sialyltransferase (GM3 synthase) from Triton X-100 extract of human blood mononuclear cells by immunoaffinity chromatography on Sepharose coupled with anti-GM3 synthase antibody. Comparison with several glycolipid substrates showed high specificity of the purified enzyme for lactosylceramide. The apparent K(M) for lactosylceramide and CMP-NeuAc were 101 and 180 muM, respectively. Analysis of the purified enzyme by SDS-PAGE followed by the anti-GM3 synthase antibody probing detected two bands with apparent molecular masses of 60 and 64 kDa. There were no other protein bands as revealed by Coomassie Blue staining. Thus, ganglioside GM3 may be considered as a physiological modulator of macrophage differentiation in human atherosclerotic aorta. The presented data suggest that up-regulation of GM3 levels is an element of monocyte/macrophage differentiation that provides a tool for control of macrophage accumulation in inflammatory loci.

Identification of possible candidate biomarkers for local or whole body radiation exposure in C57BL/6 mice.

PURPOSE: Specific genes expressed as a result of whole body exposure to gamma-radiation have been previously identified. In this study, we examined the genes further as possible biomarkers for the blood lymphocytes of C57BL/6 mice after whole body or local irradiation of the thorax, abdomen, and left subphrenic area. METHODS AND MATERIALS: We performed reverse transcriptase-polymerase chain reaction and real-time reverse transcriptase-polymerase chain reaction analysis of genes encoding platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD in blood lymphocytes, lung tissue, spleen, and intestines. The protein expression in blood lymphocytes was confirmed by Western blot analysis. RESULTS: The expression of platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD was significantly greater after 3 days as a result of 1 Gy of whole body irradiation. Moreover, local irradiation to the thorax, abdomen, or left subphrenic area, which are frequently exposed to therapeutic radiation doses, showed a tendency toward radiation-induced increased expression of these genes in both the blood and the locally irradiated organs. Western blot analysis also corroborated these results. CONCLUSION: Platelet membrane glycoprotein IIb, protein tyrosine kinase, sialyltransferase, and Cu/ZnSOD might be candidates for biomarkers of radiation exposure. However, additional experiments are required to reveal the relationship between the expression levels and the prognostic effects after irradiation.

A beta-galactoside alpha2,6-sialyltransferase produced by a marine bacterium, Photobacterium leiognathi JT-SHIZ-145, is active at pH 8.

A gene encoding a sialyltransferase produced by Photobacterium leiognathi JT-SHIZ-145 was cloned, sequenced, and expressed in Escherichia coli. The sialyltransferase gene contained an open reading frame of 1494 base pairs (bp) encoding a predicted protein of 497 amino acid residues. The deduced amino acid sequence of the sialyltransferase had no significant similarity to mammalian sialyltransferases and did not contain sialyl motifs, but did show high homology to another marine bacterial sialyltransferase, a beta-galactoside alpha2,6-sialyltransferase produced by P. damselae JT0160. The acceptor substrate specificity of the new enzyme was similar to that of the alpha2,6-sialyltransferase from P. damselae JT0160, but its activity was maximal at pH 8. This property is quite different from the properties of all mammalian and bacterial sialyltransferases reported previously, which have maximal activity at acidic pH. In general, both sialosides and cytidine-5'-monophospho-N-acetylneuraminic acid, the common donor substrate of sialyltransferases, are more stable under basic conditions. Therefore, a sialyltransferase with an optimum pH in the basic range should be useful for the preparation of sialosides and the modification of glycoconjugates, such as asialo-glycoproteins and asialo-glycolipids. Thus, the sialyltransferase obtained from P. leiognathi JT-SHIZ-145 is a promising tool for the efficient production of sialosides.

Molecular basis for the presence of glycosylated onco-foetal fibronectin in oral carcinomas: the production of glycosylated onco-foetal fibronectin by carcinoma cells.

Glycosylated onco-foetal fibronectin (GOF) deposited in the stroma of oral squamous cell carcinomas correlates with survival. One of the two polypeptide GalNAc-transferases, GalNAc-T3 or GalNAc-T6, is required for the biosynthesis of GOF by the initiation of a unique O-glycan in the alternative spliced IIICS region. Using cell culture experiments, immunohistochemical staining of primary tissue, and RT-PCR of tumour cells isolated by laser capture techniques we have examined the molecular basis for the production of GOF in oral carcinomas. Immuno-histochemical investigation confirmed the stromal deposition of GOF in oral carcinomas. However, neither GalNAc-T3 nor GalNAc-T6 could be detected in stromal fibroblasts. In contrast both transferases were present in the oral squamous carcinoma cells, suggesting that GOF is produced by the oral cancer cells and not only the stromal cells. RT-PCR analysis of RNA isolated from carcinoma cells provided further support for this conclusion by demonstrating in-splicing of the alternative spliced IIICS domain in GOF.

Beta-galactoside alpha2,6-sialyltransferase and the sialyl alpha2,6-galactosyl-linkage in tissues and cell lines.

beta-Galactoside alpha2,6-sialyltransferase (ST6Gal.I) is the principal sialyltransferase responsible for the biosynthesis of the sialyl alpha2,6-galactosyl linkage. This enzyme and its cognate glycosidic structure are overexpressed in several malignancies and are related to cancer progression. The expression of the enzyme is regulated primarily through the expression of three principal mRNA species differing in the 5'-untranslated exons. The form known as YZ is considered associated with the basal expression of the gene, while forms H and X are specific to the liver and B-lymphocytes, respectively. The authors have studied the expression of ST6Gal.I activity by two different methods using a panel of human cancer cell lines: the expression of alpha2,6-sialylated sugar chains by the lectin from Sambucus nigra (SNA), and the expression of the different mRNA species by RT-PCR using oligonucleotide primers complementary to the isoform-specific regions. Very high levels of ST6Gal.I activity result in high levels of SNA reactivity and are associated with the expression of the H transcript in colon and liver cell lines, and of the X transcript in B cells.

Sialyltransferase STX (ST8SiaII): a novel molecular marker of metastatic neuroblastoma.

Polysialic acid (PSA) is highly expressed in many human cancers, including neuroblastoma (NB), and is critical for cellular adhesion, neuronal migration and tumor metastasis. The key enzyme responsible for PSA synthesis is sialyltransferase STX (ST8SiaII). Using quantitative RT-PCR we (i) studied STX expression in 39 NB tumors and 8 cell lines and (ii) examined its potential clinical utility as an early response marker in the bone marrows of the entire cohort of 136 high-risk NB patients treated with an immunotherapy protocol utilizing anti-GD2 antibody 3F8 and GM-CSF. Based on the quantitation of 24 normal marrow and peripheral blood samples, a normalized STX transcript value below the mean + 2SD was defined as negative. Sensitivity of this assay was 1 NB cell in 10(6) normal mononuclear cells. STX expression was high among NB tumors of all stages, as well as NB cell lines of different phenotypes. Evaluation for early (2.5 months from protocol entry) marrow response by univariate Cox model indicated that STX marker status (positive versus negative) was strongly associated with both progression-free and overall survival (p < 0.0005 for both). Similarly, the STX transcript level of posttreatment marrows was also highly prognostic of outcome (PFS, p = 0.001; OS, p < 0.0005). We conclude that STX mRNA has potential clinical utility as a molecular marker of metastatic NB.

Correlation analysis between tumor-associated antigen sialyl-Tn expression and ST6GalNAc I activity in human colon adenocarcinoma.

OBJECTIVES: Sialyl-Tn (sTn) is a mucin carbohydrate-associated antigen that is strongly expressed in a large number of colorectal carcinomas. In this study, we combined immunohistochemical and enzymatic techniques in order to find the correlation between sTn tissue expression and the sialyltransferase activity (ST6GalNAc I) responsible for its synthesis in colorectal cancer (CRC) patients. METHODS: We compared sTn expression in healthy (n = 46), tumorous (n = 60) and transitional tissue (n = 46) from CRC patients, and correlated sTn altered expression with clinicopathologic variables of the patient. Furthermore, we determined ST6GalNAc I tissue activity employing asialo-ovine submaxillary mucin (asialo-OSM) as glycoprotein acceptor (n = 27). RESULTS: The rates of sTn positive expression obtained for healthy, tumorous and transitional tissues were 15, 67 and 63%, respectively. These rates led to statistically significant differences between healthy and tumorous or transitional tissue (p = 0.001); sTn expression was related to the first stages of the tumor invasion in transitional tissue. As regards ST6GalNAc I activity, we found an enhancement in transitional tissue. Statistical correlation analysis did not reveal association between sTn expression and ST6GalNAc I activity. CONCLUSIONS: Our findings indicated that sTn antigen tissue expression and ST6GalNAc I activity levels were not correlated in CRC, in spite of the overexpression of the antigen in tumorous and transitional tissue.