Sequences at the interface of the fifth immunoglobulin domain and first fibronectin type III repeat of the neural cell adhesion molecule are critical for its polysialylation

Polysialic acid is an anti-adhesive glycan that modifies a select group of mammalian proteins. The primary substrate of the polysialyltransferases (polySTs) is the neural cell adhesion molecule (NCAM). Polysialic acid negatively regulates cell adhesion, is required for proper brain development, and is expressed in specific areas of the adult brain where it promotes on-going cell migration and synaptic plasticity. The first fibronectin type III repeat (FN1) of NCAM is required for polysialylation of the N-glycans on the adjacent immunoglobulin-like domain (Ig5), and acidic residues on the surface of FN1 play a role in polyST recognition. Recent work demonstrated that the FN1 domain from the unpolysialylated olfactory cell adhesion molecule (OCAM) was able to partially replace NCAM FN1 (Foley, D. A., Swartzentruber, K. G., Thompson, M. G., Mendiratta, S. S., and Colley, K. J. (2010) J. Biol. Chem. 285, 35056-35067). Here we demonstrate that individually replacing three identical regions shared by NCAM and OCAM FN1, (500)PSSP(503) (PSSP), (526)GGVPI(530) (GGVPI), and (580)NGKG(583) (NGKG), dramatically reduces NCAM polysialylation. In addition, we show that the polyST, ST8SiaIV/PST, specifically binds NCAM and that this binding requires the FN1 domain. Replacing the FN1 PSSP sequences and the acidic patch residues decreases NCAM-polyST binding, whereas replacing the GGVPI and NGKG sequences has no effect. The location of GGVPI and NGKG in loops that flank the Ig5-FN1 linker and the proximity of PSSP to this linker suggest that GGVPI and NGKG sequences may be critical for stabilizing the Ig5-FN1 linker, whereas PSSP may play a dual role maintaining the Ig5-FN1 interface and a polyST recognition site.     

Sequences from the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Allow O-Glycan Polysialylation of an Adhesion Molecule Chimera

Polysialic acid is a developmentally regulated, anti-adhesive polymer that is added to N-glycans on the fifth immunoglobulin domain (Ig5) of the neural cell adhesion molecule (NCAM). We found that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of N-glycans on the adjacent Ig5 domain, and we proposed that the polysialyltransferases recognize specific sequences in FN1 to position themselves for Ig5 N-glycan polysialylation. Other studies identified a novel FN1 acidic surface patch and α-helix that play roles in NCAM polysialylation. Here, we characterize the contribution of two additional FN1 sequences, Pro⁵¹⁰-Tyr⁵¹¹-Ser⁵¹² (PYS) and Gln⁵¹⁶-Val⁵¹⁷-Gln⁵¹⁸ (QVQ). Replacing PYS or the acidic patch dramatically decreases the O-glycan polysialylation of a truncated NCAM protein, and replacing the α-helix or QVQ shifts polysialic acid to FN1 O-glycans in full-length NCAM. We also found that the FN1 domain of the olfactory cell adhesion molecule, a homologous but unpolysialylated protein, could partially replace NCAM FN1. Inserting Pro⁵¹⁰-Tyr⁵¹¹ eliminated N-glycan polysialylation and enhanced O-glycosylation of an NCAM- olfactory cell adhesion molecule chimera, and inserting other FN1 sequences unique to NCAM, predominantly the acidic patch, created a new polysialyltransferase recognition site. Taken together, our results highlight the role of the FN1 α-helix and QVQ sequences in N-glycan polysialylation and demonstrate that the acidic patch primarily functions in O-glycan polysialylation.     

Crystal Structure of the cis-Dimer of Nectin-1: implications for the architecture of cell-cell junctions

In multicellular organisms, cells are interconnected by cell adhesion molecules. Nectins are immunoglobulin (Ig)-like cell adhesion molecules that mediate homotypic and heterotypic cell-cell adhesion, playing key roles in tissue organization. To mediate cell-cell adhesion, nectin molecules dimerize in cis on the surface of the same cell, followed by trans-dimerization of the cis-dimers between the neighboring cells. Previous cell biological studies deduced that the first Ig-like domain of nectin and the second Ig-like domain are involved in trans-dimerization and cis-dimerization, respectively. However, to understand better the steps involved in nectin adhesion, the structural basis for the dimerization of nectin must be determined. In this study, we determined the first crystal structure of the entire extracellular region of nectin-1. In the crystal, nectin-1 formed a V-shaped homophilic dimer through the first Ig-like domain. Structure-based site-directed mutagenesis of the first Ig-like domain identified four essential residues that are involved in the homophilic dimerization. Upon mutating the four residues, nectin-1 significantly decreased cis-dimerization on the surface of cultured cells and abolished the homophilic and heterophilic adhesion activities. These results indicate that, in contrast with the previous notion, our structure represents a cis-dimer. Thus, our findings clearly reveal the structural basis for the cis-dimerization of nectins through the first Ig-like domains.     

Butyrate regulation of glycosylation-related gene expression: evidence for galectin-1 upregulation in human intestinal epithelial goblet cells

Glycosylation of mucins produced by human intestinal goblet cells plays a crucial role in their functions: mucus gel physico-chemical protective properties, host-bacteria interactions, cell-cell adhesion, cell migration, and cell signaling. Colonic mucin glycosylation can be modified by luminal metabolites of fiber fermentation like butyrate. Our aim was to assess the effect of butyrate on the expression of a large panel of glycosylation-related genes in human intestinal epithelial goblet cells HT29-Cl.16E. We found that only a very scarce group of genes: 9 out of 252 were evidenced by microarray screening, and only three had their modulation significantly confirmed by real time PCR quantification. The most striking effect of butyrate was its 8- to 18-fold increase of galectin-1 gene expression, which was confirmed at the protein level, specifically with a central and apical intracellular localization. Significant butyrate effects will be discussed in regard to their possible link with mucins expressed by HT29-Cl.16E cells.     

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

Cross-talk between pRb/E2F and Wnt/beta-catenin pathways: E2F1 induces axin2 leading to repression of Wnt signalling and to increased cell death

The pRb/E2F and Wnt/beta-catenin pathways are two of the most frequently deregulated pathways in human cancers. In this study, we show that E2F1 up-regulates the expression of axin2. Further, we show that axin2 can repress Wnt signalling leading to reduced cell growth and increased cell death. This represents cross-talk between major pathways involved in the formation of tumours. We use our data to suggest a novel mechanism for tumour suppression.     

Wnt-4 activates the canonical beta-catenin-mediated Wnt pathway and binds Frizzled-6 CRD: functional implications of Wnt/beta-catenin activity in kidney epithelial cells

The Wnt signaling pathway is central to the development of all animals and to cancer progression, yet largely unknown are the pairings of secreted Wnt ligands to their respective Frizzled transmembrane receptors or, in many cases, the relative contributions of canonical (beta-catenin/LEF/TCF) versus noncanonical Wnt signals. Specifically, in the kidney where Wnt-4 is essential for the mesenchymal to epithelial transition that generates the tissue's collecting tubules, the corresponding Frizzled receptor(s) and downstream signaling mechanism(s) are unclear. In this report, we addressed these issues using Madin-Darby Canine Kidney (MDCK) cells, which are competent to form tubules in vitro. Employing established reporter constructs of canonical Wnt/beta-catenin pathway activity, we have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions, precisely reflecting functional findings from Wnt-4 null kidney mesenchyme ex vivo rescue studies. We have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/beta-catenin pathway using beta-Engrailed and dnTCF-4 constructs that suppress this pathway. We have further found that MDCK cells express the Frizzled-6 receptor and that Wnt-4 forms a biochemical complex with the Frizzled-6 CRD. Since Frizzled-6 did not appear to transduce Wnt-4's canonical signal, data supported recently by Golan et al., there presumably exists another as yet unknown Frizzled receptor(s) mediating Wnt-4 activation of beta-catenin/LEF/TCF. Finally, we report that canonical Wnt/beta-catenin signals cells help maintain cell growth and survival in MDCK cells but do not contribute to standard HGF-induced (nonphysiologic) tubule formation. Our results in combination with work from Xenopus laevis (not shown) lead us to believe that Wnt-4 binds both canonical and noncanonical Frizzled receptors, thereby activating Wnt signaling pathways that may each contribute to kidney tubulogenesis.     

Morphogenesis of chicken liver: identification of localized growth zones and the role of beta-catenin/Wnt in size regulation

During development and regeneration, new cells are added and incorporated to the liver parenchyma. Regulation of this process contributes to the final size and shape of the particular organs, including the liver. We identified the distribution of liver growth zones using an embryonic chicken model because of its accessibility to experimentation. Hepatocyte precursors were first generated all over the primordia surrounding the vitelline blood vessel at embryonic day 2 (E2), then became limited to the peripheral growth zones around E6. Differentiating daughter cells of the peripheral hepatocyte precursors were shown by DiI microinjection to be laid inward and were subsequently organized to form the hepatic architecture. At E8, hepatocyte precursor cells were further restricted to limited segments of the periphery, called localized growth zones (LoGZ). Adhesion and signaling molecules in the growth zone were studied. Among them, beta-catenin and Wnt 3a were highly enriched. We overexpressed constitutively active beta-catenin using replication competent avian sarcoma (RCAS) virus. Liver size increased about 3-fold with an expanded hepatocyte precursor cell population. In addition, blocking beta-catenin activity by either overexpression of dominant-negative LEF1 or overexpression of a secreted Wnt inhibitor Dickkopf (DKK) resulted in decreased liver size with altered liver shape. Our data suggest that (1) the duration of active growth zone activity modulates the size of the liver; (2) a shift in the position of the localized growth zone helps to shape the liver; and (3) beta-catenin/Wnt are involved in regulating growth zone activities during liver development.     

The styrene-responsive StyS/StyR regulation system controls expression of an auxiliary phenylacetyl-coenzyme A ligase: implications for rapid metabolic coupling of the styrene upper- and lower-degradative pathways

Pseudomonas sp. strain Y2 degrades styrene through oxidation to phenylacetic acid via the styABCD operon-encoded enzymes, whose expression is induced in response to styrene by the StyS/StyR two-component regulatory system. Further transformation of phenylacetic acid to tricarboxylic acid cycle intermediates is mediated by the enzymes of paa catabolic genes, whose expression is regulated by the PaaX repressor. The first step of this paa degradation pathway is catalysed by paaF-encoded phenylacetyl-coenzyme A ligases that produce phenylacetyl-coenzyme A. This metabolic intermediate, upon being bound by PaaX, inactivates PaaX-mediated repression of both the paa genes and the styABCD operon. Strain Y2 is unique in having three paaF genes located within two complete copies of the paa gene clusters. Expression of both paaF and paaF3 is controlled by the PaaX repressor. Here we use specific mutants in combination with in vivo and in vitro assays to demonstrate that paaF2, adjacent to the StyS/StyR regulatory genes, belongs to the StyR regulon and is not subject to repression by PaaX. We propose that this unexpected styrene-responsive regulatory strategy for the otherwise metabolically redundant PaaF2 auxiliary enzyme provides a system for rapid co-ordinate de-repression of the two sets of catabolic genes required for styrene degradation.     

Widespread neuronal expression of branched-chain aminotransferase in the CNS: implications for leucine/glutamate metabolism and for signaling by amino acids

Transamination of the branched-chain amino acids produces glutamate and branched-chain alpha-ketoacids. The reaction is catalyzed by branched-chain aminotransferase (BCAT), of which there are cytosolic and mitochondrial isoforms (BCATc and BCATm). BCATc accounts for 70% of brain BCAT activity, and contributes at least 30% of the nitrogen required for glutamate synthesis. In previous work, we showed that BCATc is present in the processes of glutamatergic neurons and in cell bodies of GABAergic neurons in hippocampus and cerebellum. Here we show that this metabolic enzyme is expressed throughout the brain and spinal cord, with distinct differences in regional and intracellular patterns of expression. In the cerebral cortex, BCATc is present in GABAergic interneurons and in pyramidal cell axons and proximal dendrites. Axonal labeling for BCATc continues into the corpus callosum and internal capsule. BCATc is expressed by GABAergic neurons in the basal ganglia and by glutamatergic neurons in the hypothalamus, midbrain, brainstem, and dorsal root ganglia. BCATc is also expressed in hypothalamic peptidergic neurons, brainstem serotoninergic neurons, and spinal cord motor neurons. The results indicate that BCATc accumulates in neuronal cell bodies in some regions, while elsewhere it is exported to axons and nerve terminals. The enzyme is in a position to influence pools of glutamate in a variety of neuronal types. BCATc may also provide neurons with sensitivity to nutrient-derived BCAAs, which may be important in regions that control feeding behavior, such as the arcuate nucleus of the hypothalamus, where neurons express high levels of BCATc.     

Cell signaling pathways in the neuroprotective actions of the green tea polyphenol (-)-epigallocatechin-3-gallate: implications for neurodegenerative diseases

Accumulating evidence supports the hypothesis that brain iron misregulation and oxidative stress (OS), resulting in reactive oxygen species (ROS) generation from H2O2 and inflammatory processes, trigger a cascade of events leading to apoptotic/necrotic cell death in neurodegenerative disorders, such as Parkinson's (PD), Alzheimer's (AD) and Huntington's diseases, and amyotrophic lateral sclerosis (ALS). Thus, novel therapeutic approaches aimed at neutralization of OS-induced neurotoxicity, support the application of ROS scavengers, transition metals (e.g. iron and copper) chelators and non-vitamin natural antioxidant polyphenols, in monotherapy, or as part of antioxidant cocktail formulation for these diseases. Both experimental and epidemiological evidence demonstrate that flavonoid polyphenols, particularly from green tea and blueberries, improve age-related cognitive decline and are neuroprotective in models of PD, AD and cerebral ischemia/reperfusion injuries. However, recent studies indicate that the radical scavenger property of green tea polyphenols is unlikely to be the sole explanation for their neuroprotective capacity and in fact, a wide spectrum of cellular signaling events may well account for their biological actions. In this article, the currently established mechanisms involved in the beneficial health action and emerging studies concerning the putative novel molecular neuroprotective activity of green tea and its major polyphenol (-)-epigallocatechin-3-gallate (EGCG), will be reviewed and discussed.