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.

Chemical and Enzymatic Synthesis of Sialylated Glycoforms of Human Erythropoietin.

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life. EPO carries three N-glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline-assisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1 complexes with recombinant EPO receptor.

Adenovirus Receptor Expression in Cancer and Its Multifaceted Role in Oncolytic Adenovirus Therapy.

Oncolytic adenovirus therapy is believed to be a promising way to treat cancer patients. To be able to target tumor cells with an oncolytic adenovirus, expression of the adenovirus receptor on the tumor cell is essential. Different adenovirus types bind to different receptors on the cell, of which the expression can vary between tumor types. Pre-existing neutralizing immunity to human adenovirus species C type 5 (HAdV-C5) has hampered its therapeutic efficacy in clinical trials, hence several adenoviral vectors from different species are currently being developed as a means to evade pre-existing immunity. Therefore, knowledge on the expression of appropriate adenovirus receptors on tumor cells is important. This could aid in determining which tumor types would benefit most from treatment with a certain oncolytic adenovirus type. This review provides an overview of the known receptors for human adenoviruses and how their expression on tumor cells might be differentially regulated compared to healthy tissue, before and after standardized anticancer treatments. Mechanisms behind the up- or downregulation of adenovirus receptor expression are discussed, which could be used to find new targets for combination therapy to enhance the efficacy of oncolytic adenovirus therapy. Additionally, the utility of the adenovirus receptors in oncolytic virotherapy is examined, including their role in viral spread, which might even surpass their function as primary entry receptors. Finally, future directions are offered regarding the selection of adenovirus types to be used in oncolytic adenovirus therapy in the fight against cancer.

Metabolic Reprogramming in Cancer Is Induced to Increase Proton Production.

Considerable metabolic reprogramming has been observed in a conserved manner across multiple cancer types, but their true causes remain elusive. We present an analysis of around 50 such reprogrammed metabolisms (RM) including the Warburg effect, nucleotide de novo synthesis, and sialic acid biosynthesis in cancer. Analyses of the biochemical reactions conducted by these RMs, coupled with gene expression data of their catalyzing enzymes, in 7,011 tissues of 14 cancer types, revealed that all RMs produce more H+ than their original metabolisms. These data strongly support a model that these RMs are induced or selected to neutralize a persistent intracellular alkaline stress due to chronic inflammation and local iron overload. To sustain these RMs for survival, cells must find metabolic exits for the nonproton products of these RMs in a continuous manner, some of which pose major challenges, such as nucleotides and sialic acids, because they are electrically charged. This analysis strongly suggests that continuous cell division and other cancerous behaviors are ways for the affected cells to remove such products in a timely and sustained manner. As supporting evidence, this model can offer simple and natural explanations to a range of long-standing open questions in cancer research including the cause of the Warburg effect. SIGNIFICANCE: Inhibiting acidifying metabolic reprogramming could be a novel strategy for treating cancer.

Sialic acid expression in human fetal skeletal muscle during limb early myogenesis.

Investigations on animal models demonstrated that changes of sialic acid (SA) expression, particularly the polymeric form, in the skeletal muscle during embryonic and post-natal development seem to be related to muscle differentiation and functionality onset. The aim of this study was to evaluate the monomeric and polymeric SA expression in human skeletal muscle during early stages of fetal development, when important morphofunctional events occur. Specimens of fetal skeletal muscle from limb, between 9 and 12 weeks of gestation (wg), were obtained from 19 pregnant women. To investigate some morphofunctional features occurring during this development period, haematoxylin-eosin staining, tunel assay and immunohistochemistry for connexin-43 (Cx43) and parvalbumin were performed. SA expression and characterization was evaluated using lectin histochemistry (MAA, SNA, PNA, SBA, DBA), associated with enzymatic and chemical treatments. Polysialic acid (PSA) expression was also evaluated using immunohistochemistry. The results showed apoptotic myotubes between 9 and 10.5 wg, disappearing from 11 wg; Cx43 was more abundant in myotubes/myoblasts between 9 and 9.5 wg, decreasing and/or disappearing from 10 wg and parvalbumin was present in myotubes between 10 and 10.5 wg. PSA was revealed in myotubes/myoblasts from 9 to 10.5 wg; from 11 wg it was reduced or disappeared. Monomeric SA appeared in myotubes/myoblasts from 10 wg, increasing successively; acetylated SA was present from 11 wg. These findings demonstrated that changes in expression of various types of SA, occurring in human fetal skeletal muscle during early development, seem to be related to some morphofunctional aspects distinctive of this organogenesis crucial period.

Harnessing cancer cell metabolism for theranostic applications using metabolic glycoengineering of sialic acid in breast cancer as a pioneering example.

Abnormal cell surface display of sialic acids - a family of unusual 9-carbon sugars - is widely recognized as distinguishing feature of many types of cancer. Sialoglycans, however, typically cannot be identified with sufficiently high reproducibility and sensitivity to serve as clinically accepted biomarkers and similarly, almost all efforts to exploit cancer-specific differences in sialylation signatures for therapy remain in early stage development. In this report we provide an overview of important facets of glycosylation that contribute to cancer in general with a focus on breast cancer as an example of malignant disease characterized by aberrant sialylation. We then describe how cancer cells experience nutrient deprivation during oncogenesis and discuss how the resulting metabolic reprogramming, which endows breast cancer cells with the ability to obtain nutrients during scarcity, constitutes an "Achilles' heel" that we believe can be exploited by metabolic glycoengineering (MGE) strategies to develop new diagnostic methods and therapeutic approaches. In particular, we hypothesize that adaptations made by breast cancer cells that allow them to efficiently scavenge sialic acid during times of nutrient deprivation renders them vulnerable to MGE, which refers to the use of exogenously-supplied, non-natural monosaccharide analogues to modulate targeted aspects of glycosylation in living cells and animals. In specific, once non-natural sialosides are incorporated into the cancer "sialome" they can be exploited as epitopes for immunotherapy or as chemical tags for targeted delivery of imaging or therapeutic agents selectively to tumors.

New observation of sialuria prompts detection of liver tumor in previously reported patient.

UNLABELLED: Sialuria, a rare inborn error of metabolism, was diagnosed in a healthy 12-year-old boy through whole exome sequencing. The patient had experienced mild delays of speech and motor development, as well as persistent hepatomegaly. Identification of the 8th individual with this disorder, prompted follow-up of the mother-son pair of patients diagnosed over 15years ago. Hepatomegaly was confirmed in the now 19-year-old son, but in the 46-year-old mother a clinically silent liver tumor was detected by ultrasound and MRI. The tumor was characterized as an intrahepatic cholangiocarcinoma (IHCC) and DNA analysis of both tumor and normal liver tissue confirmed the original GNE mutation. As the maternal grandmother in the latter family died at age 49years of a liver tumor, a retrospective study of the remaining pathology slides was conducted and confirmed it to have been an IHCC as well. The overall observation generated the hypothesis that sialuria may predispose to development of this form of liver cancer. As proof of sialuria in the grandmother could not be obtained, an alternate cause of IHCC cannot be ruled out. In a series of 102 patients with IHCC, not a single instance was found with the allosteric site mutation in the GNE gene. This confirms that sialuria is rare even in a selected group of patients, but does not invalidate the concern that sialuria may be a risk factor for IHCC. SYNOPSIS: Sialuria is a rare inborn error of metabolism characterized by excessive synthesis and urinary excretion of free sialic acid with only minimal clinical morbidity in early childhood, but may be a risk factor for intrahepatic cholangiocarcinoma in adulthood.

Effect of the glycosyltransferases on the capsular polysaccharide synthesis of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (S. suis 2) is a serious zoonotic pathogen causing septicemia and meningitis in piglets and humans. The capsular polysaccharide (CPS) is an essential virulence factor for S. suis 2 to infect the host. The synthesis of CPS repeating units involves multiple glycosyltransferases. In this study, four genes (cps2E, cps2G, cps2J and cps2L) encoding different glycosyltransferases involved in CPS synthesis were researched in S. suis 2. Four deletion mutants (Δcps2E, Δcps2G, Δcps2J and Δcps2L) with their CPS incomplete and their sialic acid content significantly decreased were constructed in S. suis 2 SC19. All these four mutant strains showed enhanced adhesion to Hep-2 cells and increased sensitivity to phagocytosis. Flow cytometric analysis also revealed that these four mutants were more susceptible to the attack by the complement system. In a mouse model of infection, the mutant strains were rapidly cleared by the immune system, compared with the wild-type strain. In summary, this study characterized four genes (cps2E, cps2G, cps2J and cps2L) involved in CPS synthesis of S. suis 2 SC19 and it revealed that these genes were all crucial for SC19 to invade and survive in the host.

Functions and Biosynthesis of O-Acetylated Sialic Acids.

Sialic acids have a pivotal functional impact in many biological interactions such as virus attachment, cellular adhesion, regulation of proliferation, and apoptosis. A common modification of sialic acids is O-acetylation. O-Acetylated sialic acids occur in bacteria and parasites and are also receptor determinants for a number of viruses. Moreover, they have important functions in embryogenesis, development, and immunological processes. O-Acetylated sialic acids represent cancer markers, as shown for acute lymphoblastic leukemia, and they are known to play significant roles in the regulation of ganglioside-mediated apoptosis. Expression of O-acetylated sialoglycans is regulated by sialic acid-specific O-acetyltransferases and O-acetylesterases. Recent developments in the identification of the enigmatic sialic acid-specific O-acetyltransferase are discussed.

The sialyltransferase ST3GAL6 influences homing and survival in multiple myeloma.

Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The role of altered sialylation in multiple myeloma (MM) cell trafficking has not been previously investigated. In the present study we identified high expression of ß-galactoside α-2,3-sialyltransferase, ST3GAL6, in MM cell lines and patients. This gene plays a key role in selectin ligand synthesis in humans through the generation of functional sialyl Lewis X. In MRC IX patients, high expression of this gene is associated with inferior overall survival. In this study we demonstrate that knockdown of ST3GAL6 results in a significant reduction in levels of α-2,3-linked sialic acid on the surface of MM cells with an associated significant reduction in adhesion to MM bone marrow stromal cells and fibronectin along with reduced transendothelial migration in vitro. In support of our in vitro findings, we demonstrate significantly reduced homing and engraftment of ST3GAL6 knockdown MM cells to the bone marrow niche in vivo, along with decreased tumor burden and prolonged survival. This study points to the importance of altered glycosylation, particularly sialylation, in MM cell adhesion and migration.

Expression and localization of MCsialec, a sialic acid-specific lectin in the marine bivalve Manila clam, Ruditapes philppinarum.

A novel sialic acid-specific lectin (MCsialec) was detected from an expressed sequenced tag (EST) sequence from Manila clam haemocytes infected with Perkinsus olseni. The cDNA of the lectin was cloned using gene-specific primers based on a previously determined EST and characterized. The full-length cDNA of MCsialec is 603 bp in length and encodes a polypeptide of 200 amino acids with a calculated molecular mass of 21.928 kDa. Sequence alignment and protein motif analyses showed that MCsialec shares identity with sialic acid-specific invertebrate lectins from Cepaea hortensis, Helix pomatia and Haliotis discus discus. The lectin was expressed in Escherichia coli M15 cells and purified using a Ni-NTA His-binding resin matrix for antibody production. The presence of the lectin in various tissues of Perkinsus-infected and uninfected Manila clams was analysed by both PCR and immunohistochemical localization assays. MCsialec was detected in each tissue of the clams; however, upon infection, the level of expression of the lectin increased in each tissue. Vibrio tapetis infection also induced high-level expression of MCsialec in the haemocytes. These data suggest that MCsialec plays a crucial role in the immune system of the Manila clam during pathogenic infection.

Regulation and pathophysiological implications of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) as the key enzyme of sialic acid biosynthesis.

The key enzyme for the biosynthesis of N-acetylneuraminic acid, from which all other sialic acids are formed, is the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). GNE is a highly conserved protein found throughout the animal kingdom. Its highest expression is seen in the liver and placenta. GNE is regulated by a variety of biochemical means, including tetramerization promoted by the substrate UDP-GlcNAc, phosphorylation by protein kinase C and feedback inhibition by CMP-Neu5Ac, which is defect in the human disease sialuria. GNE knock-out in mice leads to embryonic lethality, emphasizing the crucial role of this key enzyme for sialic acid biosynthesis. The metabolic capacity to synthesize sialic acid and CMP-sialic acid upon ManNAc loads is amazingly high. An additional characteristic of GNE is its interaction with proteins involved in the regulation of development, which might play a crucial role in the hereditary inclusion body myopathy. Due to the importance of increased concentrations of tumor-surface sialic acid, first attempts to find inhibitors of GNE have been successful.

Beyond glycosylation: sialic acid precursors act as signaling molecules and are involved in cellular control of differentiation of PC12 cells.

Sialic acids represent a family of 9-carbon acidic amino sugars expressed mainly as terminal monosaccharides on most mammalian glycoconjugates. Sialic acids play an outstanding role during cellular processes, such as development and regeneration, as they are involved in a variety of molecular interactions. Sialic acids are synthesized in the cytosol starting from UDP-N-acetylglucosamine by the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE), which is the key enzyme in the biosynthesis of sialic acid that catalyzes the generation of N-acetylmannosamine, which in turn is an intermediate of the sialic acid pathway that represents the natural molecular precursor of all sialic acids. Of increasing interest are the influence of the sialic acid precursor N-acetylmannosamine (or related N-acylmannosamines), GNE, and sialic acids themselves on cellular processes such as proliferation, gene expression, or cell differentiation. Here, we present recent data and review indications that N-acylmannosamines (the direct precursors of all sialic acids) may act as signaling molecules, and that the key enzyme of the sialic acid metabolism is directly involved in the regulation of cell proliferation and cell differentiation.

Hexosamine template. A platform for modulating gene expression and for sugar-based drug discovery.

This study investigates the breadth of cellular responses engendered by short chain fatty acid (SCFA)-hexosamine hybrid molecules, a class of compounds long used in "metabolic glycoengineering" that are now emerging as drug candidates. First, a "mix and match" strategy showed that different SCFA (n-butyrate and acetate) appended to the same core sugar altered biological activity, complementing previous results [Campbell et al. J. Med. Chem. 2008, 51, 8135-8147] where a single type of SCFA elicited distinct responses. Microarray profiling then compared transcriptional responses engendered by regioisomerically modified ManNAc, GlcNAc, and GalNAc analogues in MDA-MB-231 cells. These data, which were validated by qRT-PCR or Western analysis for ID1, TP53, HPSE, NQO1, EGR1, and VEGFA, showed a two-pronged response where a core set of genes was coordinately regulated by all analogues while each analogue simultaneously uniquely regulated a larger number of genes. Finally, AutoDock modeling supported a mechanism where the analogues directly interact with elements of the NF-kappaB pathway. Together, these results establish the SCFA-hexosamine template as a versatile platform for modulating biological activity and developing new therapeutics.

The key enzyme of sialic acid biosynthesis (GNE) promotes neurite outgrowth of PC12 cells.

Axonal outgrowth is a prerequisite for the development of the most complex organ, the brain. It depends partially on the attachment of sialic acid on glycans of (sialo)-glycoproteins expressed on the plasma membrane. In our study, we showed that nerve growth factor-induced neurite outgrowth of PC12-cells enhances the expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE), the key enzyme for the biosynthesis of sialic acid. Furthermore, we could show that overexpression of GNE induces neurite outgrowth in PC12 cells. The neurite-outgrowth promoting activity of overexpressed GNE, however, does not lead to an increased biosynthesis of sialic acid. These data suggest a novel role of GNE during neurite outgrowth, which is independent to its specific enzymatic activity.

Sialic acid and N-acyl sialic acid analog production by fermentation of metabolically and genetically engineered Escherichia coli.

Sialic acid is the terminal sugar found on most glycoproteins and is crucial in determining serum half-life and immunogenicity of glycoproteins. Sialic acid analogs are antiviral therapeutics as well as crucial tools in bacterial pathogenesis research, immunobiology and development of cancer diagnostic imaging. The scarce supply of sialic acid hinders production of these materials. We have developed an efficient, rapid and cost effective fermentation route to access sialic acid. Our approach uses low cost feedstock, produces an industrially relevant amount of sialic acid and is scalable to manufacturing levels. We have also shown that precursor directed biosynthesis can be used to produce a N-acyl sialic acid analog. This work demonstrates the feasibility of engineering manufacturing-friendly bacteria to produce complex, unavailable small molecules.

Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment.

The inherent promiscuity of the polysialic acid (PSA) biosynthetic pathway has been exploited by the use of exogenous unnatural sialic acid precursor molecules to introduce unnatural modifications into cellular PSA, and has found applications in nervous system development and tumor vaccine studies. The sialic acid precursor molecules N-propionyl- and N-butanoyl-mannosamine (ManPr, ManBu) have been variably reported to affect PSA biosynthesis ranging from complete inhibition to de novo production of modified PSA, thus illustrating the need for further investigation into their effects. In this study, we have used a monoclonal antibody (mAb) 13D9, specific to both N-propionyl-PSA and N-butanoyl-PSA (NPrPSA and NBuPSA), together with flow cytometry, to study precursor-treated tumor cells and NT2 neurons at different stages of their maturation. We report that both ManPr and ManBu sialic acid precursors are metabolized and the resultant unnatural sialic acids are incorporated into de novo surface sialylglycoconjugates in murine and human tumor cells and, for the first time, in human NT2 neurons. Furthermore, neither precursor treatment deleteriously affected endogenous PSA expression; however, with NT2 cells, PSA levels were naturally downregulated as a function of their maturation into polarized neurons independent of sialic acid precursor treatment.

Engineering the sialic acid in organs of mice using N-propanoylmannosamine.

Sialic acids play an important role during development, regeneration and pathogenesis. The precursor of most physiological sialic acids, such as N-acetylneuraminic acid is N-acetyl-D-mannosamine. Application of the novel N-propanoylmannosamine leads to the incorporation of the new sialic acid N-propanoylneuraminic acid into cell surface glycoconjugates. Here we analyzed the modified sialylation of several organs with N-propanoylneuraminic acid in mice. By using peracetylated N-propanoylmannosamine, we were able to replace in vivo between 1% (brain) and 68% (heart) of physiological sialic acids by N-propanoylneuraminic acid. The possibility to modify cell surfaces with engineered sialic acids in vivo offers the opportunity to target therapeutic agents to sites of high sialic acid concentration in a variety of tumors. Furthermore, we demonstrated that application of N-propanoylmannosamine leads to a decrease in the polysialylation of the neural cell adhesion molecule in vivo, which is a marker of poor prognosis for some tumors with high metastatic potential.

The collapsin response mediator protein 1 (CRMP-1) and the promyelocytic leukemia zinc finger protein (PLZF) bind to UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis.

Sialic acids (Sia) are expressed as terminal sugars in many glycoconjugates. They are involved in a variety of cell-cell interactions and therefore play an important role during development and regeneration. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme in the de novo synthesis of Sia and it is a regulator of cell surface sialylation. Inactivation of GNE in mice results in early embryonic lethality. Mutations in the GNE gene are of clinical relevance in hereditary inclusion body myopathy, but these mutations do not necessarily decrease the enzymatic activity of GNE. In this study, we searched for novel function of the GNE protein beside its enzymatic function in the Sia biosynthesis. We here report the identification of novel GNE-interacting proteins. Using a human prey matrix we identified four proteins interacting with GNE in a yeast two-hybrid assay. For two of them, the collapsin response mediator protein 1 and the promyelocytic leukemia zinc finger protein, we could verify protein-protein interaction with GNE.

Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell.

Plant cells have no beta1,4-galactosylated and sialylated glycan, which plays important roles in biological functions in animal cells. Previously, we generated transgenic tobacco BY2 suspension-cultured cells that produced human beta1,4-galactosyltransferase [N.Q. Palacpac, S. Yoshida, H. Sakai, Y. Kimura, K. Fujiyama, T. Yoshida, T. Seki, Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation pattern, Proc. Natl. Acad. Sci. USA 96 (1999) 4692-4697]. In this study, we introduced two critical genes encoding human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter into tobacco suspension-cultured cell to pave a route for sialic biosynthetic pathway. The recombinant human proteins showed their biological activities. These results show that the plant cell can be a useful bioreactor for the production of mammalian glycoproteins.