Reduced sialylation triggers homeostatic synapse and neuronal loss in middle-aged mice.

Sialic acid-binding Ig-like lectin (Siglec) receptors are linked to neurodegenerative processes, but the role of sialic acids in physiological aging is still not fully understood. We investigated the impact of reduced sialylation in the brain of mice heterozygous for the enzyme glucosamine-2-epimerase/N-acetylmannosamine kinase (GNE+/-) that is essential for sialic acid biosynthesis. We demonstrate that GNE+/- mice have hyposialylation in different brain regions, less synapses in the hippocampus and reduced microglial arborization already at 6 months followed by increased loss of neurons at 12 months. A transcriptomic analysis revealed no pro-inflammatory changes indicating an innate homeostatic immune process leading to the removal of synapses and neurons in GNE+/- mice during aging. Crossbreeding with complement C3-deficient mice rescued the earlier onset of neuronal and synaptic loss as well as the changes in microglial arborization. Thus, sialic acids of the glycocalyx contribute to brain homeostasis and act as a recognition system for the innate immune system in the brain.

Increased Expression of Immature Mannose-Containing Glycoproteins and Sialic Acid in Aged Mouse Brains.

Aging represents the accumulation of changes in an individual over time, encompassing physical, psychological, and social changes. Posttranslational modifications of proteins such as glycosylation, including sialylation or glycation, are proposed to be involved in this process, since they modulate a variety of molecular and cellular functions. In this study, we analyzed selected posttranslational modifications and the respective proteins on which they occur in young and old mouse brains. The expression of neural cell adhesion molecule (NCAM), receptor for advanced glycation endproducts (RAGE), as well as the carbohydrate-epitopes paucimannose and high-mannose, polysialic acid, and O-GlcNAc were examined. We demonstrated that mannose-containing glycans increased on glycoproteins in aged mouse brains and identified synapsin-1 as one major carrier of paucimannose in aged brains. In addition, we found an accumulation of so-called advanced glycation endproducts, which are generated by non-enzymatic reactions and interfere with protein function. Furthermore, we analyzed the expression of sialic acid and found also an increase during aging.

Ascorbic acid leads to glycation and interferes with neurite outgrowth.

Ascorbic acid better known as vitamin C, is a reducing carbohydrate needed for a variety of functions in the human body. The most important characteristic of ascorbic acid is the ability to donate two electrons, predestining it as a major player in balancing the physiological redox state and as a necessary cofactor in multiple enzymatic hydroxylation processes. Ascorbic acid can be reversibly oxidized in two steps, leading to semidehydroascorbic acid and dehydroascorbic acid, respectively. Further degradation is irreversible and generates highly reactive carbonyl-intermediates. These intermediates are able to induce glycation of proteins, a non-enzymatic and unspecific reaction of carbonyls with amino groups involved to several age-related diseases. In this study, we investigated the effect of ascorbic acid- and dehydroascorbic acid-induced glycation on PC12 cells, which represent a model for neuronal plasticity. We found that both applications of ascorbic acid or dehydroascorbic acid leads to glycation of cellular proteins, but that ascorbic acid interferes more with viability and neurite outgrowth compared with dehydroascorbic acid.

Structural Basis for Binding of Fluorescent CMP-Neu5Ac Mimetics to Enzymes of the Human ST8Sia Family.

Polysialyltransferases synthesize polysialic acid on cell surface-expressed glycoconjugates, which is crucial for developing processes and signaling pathways in eukaryotes. Recent advances in cancer research have rendered polysialyltransferases important drug targets because polysialic acid contributes to cancer cell progression, metastasis, and treatment of resistant tumors. To aid the development of high-throughput screening assays for polysialyltransferase inhibitors, we demonstrate that a previously developed class of fluorescent CMP-sialic acid mimetics for sialyltransferases has nanomolar affinities for oligo- and polysialyltransferases and can be used for the rapid screening of new polysialyltransferase inhibitors. We demonstrate that these CMP-Neu5Ac mimetics inhibit polysialylation in vitro and perform cell culture experiments, where we observe reduced polysialylation of NCAM. Furthermore, we describe the structural basis of CMP-Neu5Ac mimetics binding to the human oligosialyltransferase ST8SiaIII and extrapolate why their affinity is high for human polysialyltransferases. Our results show that this novel class of compounds is a promising tool for the development of potent and selective drugs against polysialyltransferase activity.

The expression of sialyltransferases is regulated by the bioavailability and biosynthesis of sialic acids.

Glycosylation is the most frequent and important post-translational modification of proteins. It occurs on specific consensus sequences but the final structure of a particular glycan is not coded on the DNA, rather it depends on the expression of the required enzymes and the availability of substrates (activated monosaccharides). Sialic acid (Sia) is the terminal monosaccharide of most glycoproteins or glycolipids (= glycoconjugates) and involved in a variety of function on molecular (e.g. determination of protein stability and half-life) and cellular level (e.g. influenza infection). Sia are synthesized in the cytosol from UDP-GlcNAc by the Roseman-Warren pathway. The key enzyme of this pathway is the UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Sia are transferred on glycoconjugates by a family of Golgi-located enzymes, so called sialyltransferases (ST). There are 20 (human) ST known, which all transfer CMP-activated Sia to specific acceptor-sites on glycoconjugates. The regulation of the expression of ST is still not understood. Using a GNE-deficient embryonic stem cell line, which cannot synthesize Sia endogenously and by supplementation of soluble Sia precursors, we present data that the cellular availability of Sia strongly regulates the expression of ST on the level of transcription. In summary, we suggest that the concentration of the donor substrate of sialyltransferases, which can be regarded as a sensor for the environmental conditions of a cell, regulates not only total sialylation, but also the quality of sialylation. This allows a cell to response to altered environmental conditions.

Mutation of a putative MAP kinase consensus site regulates NCAM endocytosis and NCAM-dependent neurite outgrowth.

The cytoplasmic domain of the neural cell adhesion molecule NCAM contains several putative serine/threonine phosphorylation sites whose functions are largely unknown. Human NCAM140 (NCAM140) possesses a potential MAP kinase phosphorylation site at threonine (T) 803. The aim of this study was to analyze a possible phosphorylation of NCAM140 by MAP kinases and to identify the functional role of T803. We found that NCAM140 is phosphorylated by the MAP kinase ERK2 in vitro. Exchange of T803 to aspartic acid (D) which mimics constitutive phosphorylation at the respective position resulted in increased endocytosis compared to NCAM140 in neuroblastoma cells and primary neurons. Consistently, NCAM140 endocytosis was inhibited by the MEK inhibitor U0126 in contrast to NCAM140-T803D or NCAM140-T803A endocytosis supporting a role of a potential ERK2 mediated phosphorylation at this site in endocytosis. Furthermore, cells expressing NCAM140-T803D developed significantly shorter neurites than NCAM140 expressing cells indicating that a potential phosphorylation of NCAM by ERK2 also regulates NCAM-dependent neurite outgrowth.

Novel insights in the dysfunction of human blood-brain barrier after glycation.

The blood-brain barrier (BBB) provides a dynamic and complex interface consisting of endothelial cells, pericytes and astrocytes, which are embedded in a collagen and fibronectin-rich basement membrane. This complex structure restricts the diffusion of small hydrophilic solutes and macromolecules as well as the transmigration of leukocytes into the brain. It has been shown that carbonyl stress followed by the formation of advanced glycation endproducts (AGE=glycation) interfere with the BBB integrity and function. Here, we present data that carbonyl stress induced by methylglyoxal leads to glycation of endothelial cells and the basement membrane, which interferes with the barrier-function and with the expression of RAGE, occludin and ZO-1. Furthermore, methylglyoxal induced carbonyl stress promotes the expression of the pro-inflammatory interleukins IL-6 and IL-8. In summary, this study provides new insights into the relationship between AGE formation by carbonyl stress and brain microvascular endothelial barrier dysfunction.

Pathobiological implications of mucin glycans in cancer: Sweet poison and novel targets.

Mucins are large glycoproteins expressed on the epithelia that provide a protective barrier against harsh insults from toxins and pathogenic microbes. These glycoproteins are classified primarily as being secreted and membrane-bound; both forms are involved in pathophysiological functions including inflammation and cancer. The high molecular weight of mucins is attributed to their large polypeptide backbone that is extensively covered by glycan moieties that modulate the function of mucins and, hence, play an important role in physiological functions. Deregulation of glycosylation machinery during malignant transformation results in altered mucin glycosylation. This review describes the functional implications and pathobiological significance of altered mucin glycosylation in cancer. Further, this review delineates various factors such as glycosyltransferases and tumor microenvironment that contribute to dysregulation of mucin glycosylation during cancer. Finally, this review discusses the scope of mucin glycan epitopes as potential diagnostic and therapeutic targets.

Neuroprotective and neuroregenerative effects of nimodipine in a model system of neuronal differentiation and neurite outgrowth.

Nimodipine is a Ca2+-channel antagonist mainly used for the management of aneurysmal subarachnoid hemorrhage (aSAH) to prevent cerebral vasospasms. However, it is not clear if the better outcome of nimodipine-treated patients is mainly due to vasodilatation or whether other cellular neuroprotective or neuregenerative effects of nimodipine are involved. We analysed PC12 cells after different stress stimuli with or without nimodipine pretreatment. Cytotoxicity of 200 mM EtOH and osmotic stress (450 mosmol/L) was significantly reduced with nimodipine pretreatment, while nimodipine has no influence on the hypoxia-induced cytotoxicity in PC12 cells. The presence of nimodipine also increased the NGF-induced neurite outgrowth in PC12 cells. However, nimodipine alone was not able to induce neurite outgrowth in PC12 cells. These results support the idea that nimodipine has general neuroprotective or neuregenerative effect beside its role in vasodilatation and is maybe useful also in other clinical applications beside aSAH.

Advanced glycation endproducts interfere with adhesion and neurite outgrowth.

Advanced glycation endproducts (AGEs) represent a non-enzymatic posttranslational protein modification. AGEs are generated by a series of chemical reactions of free reducing monosaccharides, such as glucose, fructose or metabolites of the monosaccharide metabolism with amino groups of proteins. After oxidation, dehydration and condensation, stable AGE-modifications are formed. AGE-modified proteins accumulate in all cells and tissues as a normal feature of ageing and correlate with the glucose concentration in the blood. AGEs are increased in diabetic patients and play a significant role in the pathogenesis of most age-related neural disorders, such as Alzheimer's disease. We examined the role of AGEs on neurite outgrowth of PC12 cells. We induced the formation of AGEs using the reactive carbonyl compound methylglyoxal (MGO) as a physiological metabolite of glucose. We found that AGE-modification of laminin or collagen interfered with adhesion but not with neurite outgrowth of PC12 cells. Furthermore, the AGE-modification of PC12 cell proteins reduced NGF-induced neurite outgrowth. In conclusion, our data show that AGEs negatively influence neural plasticity.

Sialic acid metabolic engineering: a potential strategy for the neuroblastoma therapy.

BACKGROUND: Sialic acids (Sia) represent negative-charged terminal sugars on most glycoproteins and glycolipids on the cell surface of vertebrates. Aberrant expression of tumor associated sialylated carbohydrate epitopes significantly increases during onset of cancer. Since Sia contribute towards cell migration ( =  metastasis) and to chemo- and radiation resistance. Modulation of cellular Sia concentration and composition poses a challenge especially for neuroblastoma therapy, due to the high heterogeneity and therapeutic resistance of these cells. Here we propose that Metabolic Sia Engineering (MSE) is an effective strategy to reduce neuroblastoma progression and metastasis. METHODS: Human neuroblastoma SH-SY5Y cells were treated with synthetic Sia precursors N-propanoyl mannosamine (ManNProp) or N-pentanoyl mannosamine (ManNPent). Total and Polysialic acids (PolySia) were investigated by high performance liquid chromatography. Cell surface polySia were examined by flow-cytometry. Sia precursors treated cells were examined for the migration, invasion and sensitivity towards anticancer drugs and radiation treatment. RESULTS: Treatment of SH-SY5Y cells with ManNProp or ManNPent (referred as MSE) reduced their cell surface sialylation significantly. We found complete absence of polysialylation after treatment of SH-SY5Y cells with ManNPent. Loss of polysialylation results in a reduction of migration and invasion ability of these cells. Furthermore, radiation of Sia-engineered cells completely abolished their migration. In addition, MSE increases the cytotoxicity of anti-cancer drugs, such as 5-fluorouracil or cisplatin. CONCLUSIONS: Metabolic Sia Engineering (MSE) of neuroblastoma cells using modified Sia precursors reduces their sialylation, metastatic potential and increases their sensitivity towards radiation or chemotherapeutics. Therefore, MSE may serve as an effective method to treat neuroblastoma.

Fluorescent mimetics of CMP-Neu5Ac are highly potent, cell-permeable polarization probes of eukaryotic and bacterial sialyltransferases and inhibit cellular sialylation.

Oligosaccharides of the glycolipids and glycoproteins at the outer membranes of human cells carry terminal neuraminic acids, which are responsible for recognition events and adhesion of cells, bacteria, and virus particles. The synthesis of neuraminic acid containing glycosides is accomplished by intracellular sialyl transferases. Therefore, the chemical manipulation of cellular sialylation could be very important to interfere with cancer development, inflammations, and infections. The development and applications of the first nanomolar fluorescent inhibitors of sialyl transferases are described herein. The obtained carbohydrate-nucleotide mimetics were found to bind all four commercially available and tested eukaryotic and bacterial sialyl transferases in a fluorescence polarization assay. Moreover, it was observed that the anionic mimetics intruded rapidly and efficiently into cells in vesicles and translocated to cellular organelles surrounding the nucleus of CHO cells. The new compounds inhibit cellular sialylation in two cell lines and open new perspectives for investigations of cellular sialylation.

Analysis of tumor-associated mucin glycotopes by Western transfer methods.

In mucins, glycosylation is complex and the most predominant posttranslational modification. Since mucins exhibit differential glycosylation pattern under physiological and pathological conditions, analysis of mucin glycans is important for understanding their specific functions during pathological conditions like cancer. Given the complexity of mucin glycans, several sophisticated analytical tools such as HPLC, mass spectrometry, and lectin sandwich assays are employed for glyco-analysis of mucins. However the specialized expertise and instrumentation required for such analysis are beyond the reach of an average cancer biology laboratory. We described in this chapter the utility of the simple electrophoresis/immunoblotting method to examine the mucin glycan epitopes, using specific antibodies and lectins.

Novel INTeraction of MUC4 and galectin: potential pathobiological implications for metastasis in lethal pancreatic cancer.

PURPOSE: Several studies have reported aberrant expression of MUC4 in pancreatic cancer (PC), which is associated with tumorigenicity and metastasis. Mechanisms through which MUC4 promote metastasis of PC cells to distant organs are poorly defined. EXPERIMENTAL DESIGN: Identification of MUC4-galectin-3 interaction and its effect on the adhesion of cancer cells to endothelial cells were done by immunoprecipitation and cell-cell adhesion assays, respectively. Serum galectin-3 level for normal and PC patients were evaluated through ELISA. RESULTS: In the present study, we have provided clinical evidence that the level of galectin-3 is significantly elevated in the sera of PC patients with metastatic disease compared with patients without metastasis (P = 0.04) and healthy controls (P = 0.00001). Importantly, for the first time, we demonstrate that MUC4 present on the surface of circulating PC cells plays a significant role in the transient and reversible attachment (docking) of circulating tumor cells to the surface of endothelial cells. Further, exogenous galectin-3 at concentrations similar to that found in the sera of PC patients interacts with MUC4 via surface glycans such as T antigens, which results in the clustering of MUC4 on the cell surface and a stronger attachment (locking) of circulating tumor cells to the endothelium. CONCLUSIONS: Altogether, these findings suggest that PC cell-associated MUC4 helps in the docking of tumor cells on the endothelial surface. During cancer progression, MUC4-galectin-3 interaction-mediated clustering of MUC4 may expose the surface adhesion molecules, which in turn promotes a stronger attachment (locking) of tumor cells to the endothelial surface.