Expression Of Sialyltransferases Research Articles

Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in gram-negative bacteria.

Lipooligosaccharides (LOS) are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their LOS with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides (LOS) at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their LOS. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the LOS of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering (MOE) and Selective Exo-Enzymatic Labeling (SEEL), and that together they provide tools to modify, label, detect and visualize sialylation of bacterial LOS.

ST3GAL1 Promotes Malignant Phenotypes in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (iCCA) has a poor prognosis, and elucidation of the molecular mechanisms underlying iCCA malignancy is of great significance. Glycosylation, an important post-translational modification, is closely associated with tumor progression. Altered glycosylation, including aberrant sialylation resulting from abnormal expression of sialyltransferases (STs) and neuraminidases (NEUs), is a significant feature of cancer cells. However, there is limited information on the roles of STs and NEUs in iCCA malignancy. Here, utilizing our proteogenomic resources from a cohort of 262 patients with iCCA, we identified ST3GAL1 as a prognostically relevant molecule in iCCA. Moreover, overexpression of ST3GAL1 promoted proliferation, migration, and invasion and inhibited apoptosis of iCCA cells invitro. Through proteomic analyses, we identified the downstream pathway potentially regulated by ST3GAL1, which was the NF-κB signaling pathway, and further demonstrated that this pathway was positively correlated with malignancy in iCCA cells. Notably, glycoproteomics showed that O-glycosylation was changed in iCCA cells with high ST3GAL1 expression. Importantly, the altered O-glycopeptides underscored the potential utility of O-glycosylation profiling as a discriminatory marker for iCCA cells with ST3GAL1 overexpression. Additionally, miR-320b was identified as a post-transcriptional regulator of ST3GAL1, capable of suppressing ST3GAL1 expression and then reducing the proliferation, migration, and invasion abilities of iCCA cell lines. Taken together, these results suggest ST3GAL1 could serve as a promising therapeutic target for iCCA.

Altered O-linked glycosylation in benign and malignant meningiomas.

Changes in protein glycosylation have been reported in various diseases, including cancer; however, the consequences of altered glycosylation in meningiomas remains undefined. We established two benign meningioma cell lines-SUT-MG12 and SUT-MG14, WHO grade I-and demonstrated the glycan and glycosyltransferase profiles of the mucin-type O-linked glycosylation in the primary benign meningioma cells compared with two malignant meningioma cell lines-HKBMM and IOMM-Lee, WHO grade III. Changes in O-linked glycosylation profiles in malignant meningiomas were proposed. Primary culture technique, morphological analysis, and immunocytochemistry were used to establish and characterize two benign meningioma cell lines. The glycan profiles of the primary benign and malignant meningiomas cell lines were then analyzed using lectin cytochemistry. The gene expression of O-linked glycosyltransferases, mucins, sialyltransferases, and fucosyltransferases were analyzed in benign and malignant meningioma using the GEO database (GEO series GSE16581) and quantitative-PCR (qPCR). Lectin cytochemistry revealed that the terminal galactose (Gal) and N-acetyl galactosamine (GalNAc) were highly expressed in primary benign meningioma cells (WHO grade I) compared to malignant meningioma cell lines (WHO grade III). The expression profile of mucin types O-glycosyltransferases in meningiomas were observed through the GEO database and gene expression experiment in meningioma cell lines. In the GEO database, C1GALT1-specific chaperone (COSMC) and mucin 1 (MUC1) were significantly increased in malignant meningiomas (Grade II and III) compared with benign meningiomas (Grade I). Meanwhile, in the cell lines, Core 2 β1,6-N-acetylglucosaminyltransferase-2 (C2GNT2) was highly expressed in malignant meningiomas. We then investigated the complex mucin-type O-glycans structures by determination of sialyltransferases and fucosyltransferases. We found ST3 β-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) was significantly decreased in the GEO database, while ST3GAL1, ST3GAL3, α1,3 fucosyltransferases 1 and 8 (FUT1 and FUT8) were highly expressed in malignant meningioma cell lines-(HKBMM)-compared to primary benign meningioma cells-(SUT-MG12 and SUT-MG14). Our findings are the first to demonstrate the potential glycosylation changes in the O-linked glycans of malignant meningiomas compared with benign meningiomas, which may play an essential role in the progression, tumorigenesis, and malignancy of meningiomas.

Hippo cell signaling and HS-proteoglycans regulate tissue form and function, age-dependent maturation, extracellular matrix remodeling, and repair.

This review examined how Hippo cell signaling and heparan sulfate (HS)-proteoglycans (HSPGs) regulate tissue form and function. Despite being a nonweight-bearing tissue, the brain is regulated by Hippo mechanoresponsive cell signaling pathways during embryonic development. HS-proteoglycans interact with growth factors, morphogens, and extracellular matrix components to regulate development and pathology. Pikachurin and Eyes shut (Eys) interact with dystroglycan to stabilize the photoreceptor axoneme primary cilium and ribbon synapse facilitating phototransduction and neurotransduction with bipolar retinal neuronal networks in ocular vision, the primary human sense. Another HSPG, Neurexin interacts with structural and adaptor proteins to stabilize synapses and ensure specificity of neural interactions, and aids in synaptic potentiation and plasticity in neurotransduction. HSPGs also stabilize the blood-brain barrier and motor neuron basal structures in the neuromuscular junction. Agrin and perlecan localize acetylcholinesterase and its receptors in the neuromuscular junction essential for neuromuscular control. The primary cilium is a mechanosensory hub on neurons, utilized by YES associated protein (YAP)-transcriptional coactivator with PDZ-binding motif (TAZ) Hippo, Hh, Wnt, transforming growth factor (TGF)-β/bone matrix protein (BMP) receptor tyrosine kinase cell signaling. Members of the glypican HSPG proteoglycan family interact with Smoothened and Patched G-protein coupled receptors on the cilium to regulate Hh and Wnt signaling during neuronal development. Control of glycosyl sulfotransferases and endogenous protease expression by Hippo TAZ YAP represents a mechanism whereby the fine structure of HS-proteoglycans can be potentially modulated spatiotemporally to regulate tissue morphogenesis in a similar manner to how Hippo signaling controls sialyltransferase expression and mediation of cell-cell recognition, dysfunctional sialic acid expression is a feature of many tumors.

Glycation Interferes with the Expression of Sialyltransferases and Leads to Increased Polysialylation in Glioblastoma Cells.

Glioblastoma (GBM) is a highly aggressive brain tumor that often utilizes aerobic glycolysis for energy production (Warburg effect), resulting in increased methylglyoxal (MGO) production. MGO, a reactive dicarbonyl compound, causes protein alterations and cellular dysfunction via glycation. In this study, we investigated the effect of glycation on sialylation, a common post-translational modification implicated in cancer. Our experiments using glioma cell lines, human astrocytes (hA), and primary glioma samples revealed different gene expressions of sialyltransferases among cells, highlighting the complexity of the system. Glycation has a differential effect on sialyltransferase expression, upregulating ST8SIA4 in the LN229 and U251 cell lines and decreasing the expression in normal hA. Subsequently, polysialylation increased in the LN229 and U251 cell lines and decreased in hA. This increase in polysialylation could lead to a more aggressive phenotype due to its involvement in cancer hallmark processes such as immune evasion, resistance to apoptosis, and enhancing invasion. Our findings provide insights into the mechanisms underlying GBM aggressiveness and suggest that targeting glycation and sialylation could be a potential therapeutic strategy.

RANK+TLR2+ myeloid subpopulation converts autoimmune to joint destruction in rheumatoid arthritis.

Joint destruction is the major clinic burden in patients with rheumatoid arthritis (RA). It is unclear, though, how this autoimmune disease progresses to the point of deterioration of the joint. Here, we report that in a mouse model of RA the upregulation of TLR2 expression and its α(2,3) sialylation in RANK+ myeloid monocytes mediate the transition from autoimmunity to osteoclast fusion and bone resorption, resulting in joint destruction. The expression of α(2,3) sialyltransferases was significantly increased in RANK+TLR2+ myeloid monocytes, and their inhibition or treatment with a TLR2 inhibitor blocked osteoclast fusion. Notably, analysis of our single-cell RNA-sequencing (scRNA-seq) libraries generated from RA mice revealed a novel RANK+TLR2- a subset that negatively regulated osteoclast fusion. Importantly, the RANK+TLR2+ subset was significantly diminished with the treatments, whereas the RANK+TLR2- subset was expanded. Moreover, the RANK+TLR2- subset could differentiate into a TRAP+ osteoclast lineage, but the resulting cells did not fuse to form osteoclasts. Our scRNA-seq data showed that Maf is highly expressed in the RANK+TLR2- subset, and the α(2,3) sialyltransferase inhibitor-induced Maf expression in the RANK+TLR2+ subset. The identification of a RANK+TLR2- subset provides a potential explanation for TRAP+ mononuclear cells in bone and their anabolic activity. Further, TLR2 expression and its α(2,3) sialylation in the RANK+ myeloid monocytes could be effective targets to prevent autoimmune-mediated joint destruction.

Targeting stromal cell sialylation reverses T cell-mediated immunosuppression in the tumor microenvironment

Immunosuppressive tumor microenvironments (TMEs) reduce the effectiveness of immune responses in cancer. Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote tumor progression by enhancing immune cell suppression in colorectal cancer (CRC). Hyper-sialylation of glycans promotes immune evasion in cancer through binding of sialic acids to their receptors, Siglecs, expressed on immune cells, which results in inhibition of effector functions. The role of sialylation in shaping MSC/CAF immunosuppression in the TME is not well characterized. In this study, we show that tumor-conditioned stromal cells have increased sialyltransferase expression, α2,3/6-linked sialic acid, and Siglec ligands. Tumor-conditioned stromal cells and CAFs induce exhausted immunomodulatory CD8+ PD1+ and CD8+ Siglec-7+/Siglec-9+ Tcell phenotypes. Invivo, targeting stromal cell sialylation reverses stromal cell-mediated immunosuppression, as shown by infiltration of CD25 and granzyme B-expressing CD8+ Tcells in the tumor and draining lymph node. Targeting stromal cell sialylation may overcome immunosuppression in the CRC TME.

A230 A MISSENSE MUTATION IN GOBLET CELL PROTEIN FCGBP ALTERS THE GLYCOMIC PROFILE AND FUNCTION OF THE COLONIC MUCUS BARRIER

Abstract Background MUC2 mucin, produced by colonic goblet cells, forms a mucus bilayer that provides a physical barrier between potential pathogens in the lumen and the underlying epithelial cells. Mucus is thus the first line of innate host defense in the gastrointestinal (GI) tract. Many GI diseases including inflammatory bowel disease and colon cancer affect the glycosylation of mucus. Goblet cells produce a variety of proteins that are associated with the mucus layer. Of these proteins, FCGBP is of significant interest due to its structural similarities to MUC2 mucin with unknown functions. In this study, we investigated how a missense mutation in FCGBP altered the glycosylation of goblet cell MUC2 and affected its barrier functions. Purpose Hypothesis: A missense mutation in FCGBP results an impaired mucus layer by the altering glycomic profiles of goblet cell mucins. Specific aims: 1) To determine mechanistically how FCGBP impeded the structural integrity of the mucus layer 2) To quantify MUC2 glycoprotein modifications in the altered mucus layer Method To investigate whether FCGBP impaired mucus barrier functions, two cell types were investigated: wildtype LS174T (WT) MUC2 mucus-producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS). To determine if FCGBP MS led to loss in barrier function in the mucus layer, the penetration of 0.2, 1, and 2 μm fluorescent beads (to mimic bacteria) through the mucus layer were quantified. To determine if the differences in penetrability were caused by differences in MUC2 glycosylation in the goblet cell lines, sensitive glycomic analyses were performed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Both intact cells and isolated MUC2 mucin granules were analyzed. To determine if differences in the glycomic profiles was caused by differences in glycotransferases, RT-PCR was performed on over 30 human glycosyltransferases. Result(s) FCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetration through the mucus layer in a temporal manner. FCGBP MS cells exhibited an altered glycomic profile with a notable increase in sialylated glycans as quantified by HPLC-MS. The increase in sialylated glycans was associated with a significant increase in sialyl-transferase expression in FCGBP MS cells. Conclusion(s) These data demonstrate that a single missense mutation in FCGBP altered the penetrability of the mucus layer associated with an increase in sialylated proteins, a signature hallmark of numerous colonic diseases. FCGBP was critical in providing structural integrity of the mucus layer and maintenance of goblet cell glycosylation profiles. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

Perturbation of placental protein glycosylation by endoplasmic reticulum stress promotes maladaptation of maternal hepatic glucose metabolism

Placental hormones orchestrate maternal metabolic adaptations to support pregnancy. We hypothesized that placental ER stress, which characterizes early-onset pre-eclampsia (ePE), compromises glycosylation, reducing hormone bioactivity and these maladaptations predispose the mother to metabolic disease in later life. We demonstrate ER stress reduces the complexity and sialylation of trophoblast protein N-glycosylation, while aberrant glycosylation of vascular endothelial growth factor reduced its bioactivity. ER stress alters the expression of 66 of the 146 genes annotated with "protein glycosylation" and reduces the expression of sialyltransferases. Using mouse placental explants, we show ER stress promotes the secretion of mis-glycosylated glycoproteins. Pregnant mice carrying placentas with junctional zone-specific ER stress have reduced blood glucose, anomalous hepatic glucose metabolism, increased cellular stress and elevated DNA methyltransferase 3A. Using pregnancy-specific glycoproteins as a readout, we also demonstrate aberrant glycosylation of placental proteins in women with ePE, thus providing a mechanistic link between ePE and subsequent maternal metabolic disorders.

Synovial Fibroblast Sialylation Regulates Cell Migration and Activation of Inflammatory Pathways in Arthritogenesis.

Synovial fibroblasts have emerged as critical underlying factors to perpetuate chronic joint inflammation in Rheumatoid Arthritis. Like any other cell, synovial fibroblasts are covered with a complex layer of glycans that can change in response to extracellular signals, such as inflammation. We have previously shown that inflammatory synovial fibroblasts show decreased levels of sialic acid, but our understanding of sialic acid-dependent pathophysiological pathways in these stromal cells is still very limited. In this report, we used in vivo and in vitro studies with exogenous sialidases and RNA sequencing to investigate the responses of murine synovial fibroblasts upon desialylation. Our results show that hyposialylated fibroblasts present a dysregulated migratory ability and an activated phenotype characterized by the expression of inflammatory mediators, such as cytokines and chemokines, and anti-viral related mechanisms. Removal of surface sialic acid also affected the expression of sialyltransferases, revealing the existence of a positive feedback to sustain reduced sialylation. Moreover, we demonstrate that synovial fibroblasts subsets have distinct sialyltransferase expression profiles, both in healthy and arthritic mice. These findings underline the ability of sialic acid to modulate homeostatic and inflammatory responses in non-immune synovial fibroblasts, suggesting that sialylation plays a key role in perpetuating local inflammation in the arthritic joint.

Altered Rheological Properties of Saliva with Aging in Mouse Sublingual Gland

Mucin in saliva plays a critical role in the hydration and lubrication of the oral mucosa by retaining water molecules, and its impaired function may be associated with hyposalivation-independent xerostomia. Age-dependent effects on salivary gland function and rheological properties of secreted saliva are not fully understood as aging is a complex and multifactorial process. We aimed to evaluate age-related changes in the rheological properties of saliva and elucidate the underlying mechanism. We performed ex vivo submandibular gland (SMG) and sublingual gland (SLG) perfusion experiments to collect saliva from isolated glands of young (12 wk old) and aged (27 mo old) female C57BL/6J mice and investigate the rheological properties by determining the spinnbarkeit (viscoelasticity). While fluid secretion was comparable in SMG and SLG of both mice, spinnbarkeit showed a significant decrease in SLG saliva of aged mice than that of young mice. There were no significant differences in GalNAc concentration between young and aged SLG saliva. Liquid chromatography/tandem mass spectrometry analysis of SLG saliva revealed that (Hex)1 (HexNAc)1 (NeuAc)1 at m/z 793.31 was the most abundant O-glycan structure in SLG saliva commonly detected in both mice. Lectin staining of salivary gland tissue showed that SLG stained strongly with Maackia amurensis lectin II (MAL II) while Sambucus nigra agglutinin (SNA) stained little, if any, SLG. The messenger RNA expression of St3gal1 that encodes an α-2,3 sialic acid sialyltransferase SIAT4-A showed a decrease in SLG of aged mice, confirmed by a Western blot analysis. Lectin blot analysis in SLG saliva revealed that the relative signal intensity detected by MAL II was significantly lower in aged SLG. Our results suggest that spinnbarkeit decreases in SLG of aging mice due to downregulation of sialic acid linked to α-2,3 sialic acid sialyltransferase expression.

Mammalian sialyltransferases allow efficient Escherichia coli-based production of mucin-type O-glycoproteins but can also transfer Kdo.

The prospect of producing human-like glycoproteins in bacteria is becoming attractive as an alternative to already-established but costly mammalian cell expression systems. We previously described an Escherichia coli expression platform that uses a dual-plasmid approach to produce simple mucin type O-glycoproteins: one plasmid encoding the target protein and another O-glycosylation machinery. Here, we expand the capabilities of our platform to carry out sialylation and demonstrate the high-yielding production of human interferon α2b and human growth hormone bearing mono- and disialylated T-antigen glycans. This is achieved through engineering an E. coli strain to produce CMP-Neu5Ac and introducing various α-2,3- and α-2,6 mammalian or bacterial sialyltransferases into our O-glycosylation operons. We further demonstrate that mammalian sialyltransferases, including porcine ST3Gal1, human ST6GalNAc2 and human ST6GalNAc4, are very effective in vivo and outperform some of the bacterial sialyltransferases tested, including Campylobacter jejuni Cst-I and Cst-II. In the process, we came upon a way of modifying T-Antigen with Kdo, using a previously uncharacterised Kdo-transferase activity of porcine ST3Gal1. Ultimately, the heterologous expression of mammalian sialyltransferases in E. coli shows promise for the further development of bacterial systems in therapeutic glycoprotein production.

Glycation Interferes with the Expression of Sialyltransferases in Meningiomas.

Meningiomas are the most common non-malignant intracranial tumors and prefer, like most tumors, anaerobic glycolysis for energy production (Warburg effect). This anaerobic glycolysis leads to an increased synthesis of the metabolite methylglyoxal (MGO) or glyoxal (GO), which is known to react with amino groups of proteins. This reaction is called glycation, thereby building advanced glycation end products (AGEs). In this study, we investigated the influence of glycation on sialylation in two meningioma cell lines, representing the WHO grade I (BEN-MEN-1) and the WHO grade III (IOMM-Lee). In the benign meningioma cell line, glycation led to differences in expression of sialyltransferases (ST3GAL1/2/3/5/6, ST6GAL1/2, ST6GALNAC2/6, and ST8SIA1/2), which are known to play a role in tumor progression. We could show that glycation of BEN-MEN-1 cells led to decreased expression of ST3Gal5. This resulted in decreased synthesis of the ganglioside GM3, the product of ST3Gal5. In the malignant meningioma cell line, we observed changes in expression of sialyltransferases (ST3GAL1/2/3, ST6GALNAC5, and ST8SIA1) after glycation, which correlates with less aggressive behavior.

Recent advances in understanding the roles of sialyltransferases in tumor angiogenesis and metastasis.

Abnormal glycosylation is a common characteristic of cancer cells and there is a lot of evidence that glycans can regulate the biological behavior of tumor cells. Sialylation modification, a form of glycosylation modification, plays an important role in cell recognition, cell adhesion and cell signal transduction. Abnormal sialylation on the surface of tumor cells is related to tumor migration and invasion, with abnormal expression of sialyltransferases being one of the main causes of abnormal sialylation. Recent studies provide a better understanding of the importance of the sialyltransferases, and how they influences cancer cell angiogenesis, adhesion and Epithelial-Mesenchymal Transition (EMT). The present review will provide a direction for future studies in determining the roles of sialyltransferases in cancer metastasis, and abnormal sialyltransferases are likely to be potential biomarkers for cancer.

Regulation of ST6GAL1 sialyltransferase expression in cancer cells.

The ST6GAL1 sialyltransferase, which adds α2-6 linked sialic acids to N-glycosylated proteins, is overexpressed in a wide range of human malignancies. Recent studies have established the importance of ST6GAL1 in promoting tumor cell behaviors such as invasion, resistance to cell stress and chemoresistance. Furthermore, ST6GAL1 activity has been implicated in imparting cancer stem cell characteristics. However, despite the burgeoning interest in the role of ST6GAL1 in the phenotypic features of tumor cells, insufficient attention has been paid to the molecular mechanisms responsible for ST6GAL1 upregulation during neoplastic transformation. Evidence suggests that these mechanisms are multifactorial, encompassing genetic, epigenetic, transcriptional and posttranslational regulation. The purpose of this review is to summarize current knowledge regarding the molecular events that drive enriched ST6GAL1 expression in cancer cells.

MiR-193b modulates osteoarthritis progression through targeting ST3GAL4 via sialylation of CD44 and NF-кB pathway

Osteoarthritis (OA) is a worldwide epidemic and debilitating disease. It is urgent to explore the potential molecular mechanisms of OA which has crucial roles in the treatment strategy. As a post-translational modification, sialylation mediates the progression of OA. In current study, differential expression of sialyltransferases (STs) in normal and OA cartilage tissues is detected. The ST3GAL4 expression is significantly increased and positively associated with modified Mankin's score in OA tissue. Alteration of ST3GAL4 respectively mediates the degradation of extracellular mechanisms (ECM), apoptosis and proliferation in chondrocytes. Additionally, miR-193b is identified as a direct regulatory target of ST3GAL4. Functional analysis shows that modulation of ST3GAL4 could be reversed by miR-193b. Over-expression ST3GAL4 modifies CD44 sialylation. Finally, sialylated CD44 reduces the binding capacity to lubricin and mediates the activity of the NF-кB pathway. Collectively, these researches indicate that miR-193b/ST3GAL4 axis impacts OA progression by regulating CD44 sialylation via NF-кB pathway. Our researches propose a precise molecular mechanism and provide a prospective therapeutic target in OA.

Sialylation of Human Natural Killer (NK) Cells is Regulated by IL-2.

Sialic acids are terminal sugars on the cell surface that are found on all cell types including immune cells like natural killer (NK) cells. The attachment of sialic acids to different glycan structures is catalyzed by sialyltransferases in the Golgi. However, the expression pattern of sialyltransferases in NK cells and their expression after activation has not yet been analyzed. Therefore, the present study determines which sialyltransferases are expressed in human NK cells and if activation with IL-2 changes the sialylation of NK cells. The expression of sialyltransferases was analyzed in the three human NK cell lines NK-92, NKL, KHYG-1 and primary NK cells. NK-92 cells were cultured in the absence or presence of IL-2, and changes in the sialyltransferase expression were measured by qPCR. Furthermore, specific sialylation was investigated by flow cytometry. In addition, polySia and NCAM were measured by Western blot analyses. IL-2 leads to a reduced expression of ST8SIA1, ST6GAL1 and ST3GAL1. α-2,3-Sialylation remained unchanged, while α-2,6-sialylation was increased after IL-2 stimulation. Moreover, an increase in the amount of NCAM and polySia was observed in IL-2-activated NK cells, whereas GD3 ganglioside was decreased. In this study, all sialyltransferases that were expressed in NK cells could be identified. IL-2 regulates the expression of some sialyltransferases and leads to changes in the sialylation of NK cells.

Cell Line-, Protein-, and Sialoglycosite-Specific Control of Flux-Based Sialylation in Human Breast Cells: Implications for Cancer Progression.

Sialylation, a post-translational modification that impacts the structure, activity, and longevity of glycoproteins has been thought to be controlled primarily by the expression of sialyltransferases (STs). In this report we explore the complementary impact of metabolic flux on sialylation using a glycoengineering approach. Specifically, we treated three human breast cell lines (MCF10A, T-47D, and MDA-MB-231) with 1,3,4-O-Bu3ManNAc, a “high flux” metabolic precursor for the sialic acid biosynthetic pathway. We then analyzed N-glycan sialylation using solid phase extraction of glycopeptides (SPEG) mass spectrometry-based proteomics under conditions that selectively captured sialic acid-containing glycopeptides, referred to as “sialoglycosites.” Gene ontology (GO) analysis showed that flux-based changes to sialylation were broadly distributed across classes of proteins in 1,3,4-O-Bu3ManNAc-treated cells. Only three categories of proteins, however, were “highly responsive” to flux (defined as two or more sialylation changes of 10-fold or greater). Two of these categories were cell signaling and cell adhesion, which reflect well-known roles of sialic acid in oncogenesis. A third category—protein folding chaperones—was unexpected because little precedent exists for the role of glycosylation in the activity of these proteins. The highly flux-responsive proteins were all linked to cancer but sometimes as tumor suppressors, other times as proto-oncogenes, or sometimes both depending on sialylation status. A notable aspect of our analysis of metabolically glycoengineered breast cells was decreased sialylation of a subset of glycosites, which was unexpected because of the increased intracellular levels of sialometabolite “building blocks” in the 1,3,4-O-Bu3ManNAc-treated cells. Sites of decreased sialylation were minor in the MCF10A (<25% of all glycosites) and T-47D (<15%) cells but dominated in the MDA-MB-231 line (~60%) suggesting that excess sialic acid could be detrimental in advanced cancer and cancer cells can evolve mechanisms to guard against hypersialylation. In summary, flux-driven changes to sialylation offer an intriguing and novel mechanism to switch between context-dependent pro- or anti-cancer activities of the several oncoproteins identified in this study. These findings illustrate how metabolic glycoengineering can uncover novel roles of sialic acid in oncogenesis.

Comparison of α2,6-sialyltransferases for sialylation of therapeutic proteins.

The development of therapeutic proteins for the treatment of numerous diseases is one of the fastest growing areas of biotechnology. Therapeutic efficacy and serum half-life are particularly important, and these properties rely heavily on the glycosylation state of the protein. Expression systems to produce authentically fully glycosylated therapeutic proteins with appropriate terminal sialic acids are not yet perfected. The in vitro modification of therapeutic proteins by recombinant sialyltransferases offers a promising and elegant strategy to overcome this problem. Thus, the detailed expression and characterization of sialyltransferases for completion of the glycan chains is of great interest to the community. We identified a novel α2,6-sialyltransferase from Helicobacter cetorum and compared it to the human ST6Gal1 and a Photobacterium sp. sialyltransferase using glycoprotein substrates in a 96-well microtiter-plate-based assay. We demonstrated that the recombinant α2,6-sialyltransferase from H. cetorum is an excellent catalyst for modification of N-linked glycans of different therapeutic proteins.

Sialyltransferase inhibition leads to inhibition of tumor cell interactions with E-selectin, VCAM1, and MADCAM1, and improves survival in a human multiple myeloma mouse model.

Aberrant glycosylation resulting from altered expression of sialyltransferases, such as ST3 β-galactoside α2-3-sialyltransferase 6, plays an important role in disease progression in multiple myeloma (MM). Hypersialylation can lead to increased immune evasion, drug resistance, tumor invasiveness, and disseminated disease. In this study, we explore the in vitro and in vivo effects of global sialyltransferase inhibition on myeloma cells using the pan-sialyltransferase inhibitor 3Fax-Neu5Ac delivered as a per-acetylated methyl ester pro-drug. Specifically, we show in vivo that 3Fax-Neu5Ac improves survival by enhancing bortezomib sensitivity in an aggressive mouse model of MM. However, 3Fax-Neu5Ac treatment of MM cells in vitro did not reverse bortezomib resistance conferred by bone marrow (BM) stromal cells. Instead, 3Fax-Neu5Ac significantly reduced interactions of myeloma cells with E-selectin, MADCAM1 and VCAM1, suggesting that reduced sialylation impairs extravasation and retention of myeloma cells in the BM. Finally, we showed that 3Fax-Neu5Ac alters the post-translational modification of the α4 integrin, which may explain the reduced affinity of α4β1/α4β7 integrins for their counter-receptors. We propose that inhibiting sialylation may represent a valuable strategy to restrict myeloma cells from entering the protective BM microenvironment, a niche in which they are normally protected from chemotherapeutic agents such as bortezomib. Thus, our work demonstrates that targeting sialylation to increase the ratio of circulating to BM-resident MM cells represents a new avenue that could increase the efficacy of other anti-myeloma therapies and holds great promise for future clinical applications.