Studies On N-acetylglucosaminyltransferase V Regulation TrKA Receptor Function By Over Expression

N-glycan plays an important role in cell communication, differentiation, and survival. Several lines of evidence have suggested that N-glycan involves nerve development, but its precise mechanism is yet to be elucidated. Glycomics is the most important part of post post-geneomics. The processing of N-glycans in the Golgi apparatus is the most important step in the biosynthesis of the complex type of Asn-linked glycoproteins. It is known that at least six N-acetylglucosaminyltransferases (GnTs), designated as GnT I-VI, are involved in the biosynthesis of a core structure of the complex type of N-glycans with specific enzyme substrate. The glycosytltranferases are a large family, which is the part of glycomics, and elucidation of the mechanism of them will be explained great informations to cells and proteins.N-Acetylglucosaminyltransferases V (GnT-V/Mgat5) plays a pivotal role in the processing of N-linked glycoproteins in the Golgi apparatus. N-acetylglucosaminyltransferase V (GnT-V /Mgat5, EC 2.4.1.155) is one of the pivotal glycosyltransferases of Golgi enzyme which participates in the branching of N-glycans (N-glycans), and produces an unique sugar chain structure, aβ1,6 branching GlcNAc structure (GlcNAcβ1,6Manα1,6-) in C2C2,6 tri- or C2,4C2,6 tetra-antennary N-glycan products (C2C2, C2,4C2, C2C2,6 and C2,4C2,6 express the linkages of the bi-, tri- and tetra-antennae to the carbon atom positions ofα1,3 andα1,6 mannosides in the N-glycan core). This enzyme can motify many active glycoprotein for the posttranslational modifications, which is essential for their function, and the most important proteins are the receptors of the growth factor family. Co-translational modification of proteins in the endoplasmic reticulum by N-glycosylation facilitates their folding and is essential in single-cell eukaryotes. Aberrant glycosylation can affect the functions of several types of cell surface receptors by altering the signaling pathways mediated by these receptors.The modulation of receptor transcript and protein levels by abnormal N-glycosylation resulting from deletion of GnT-V appears to be a novel observation.NGF was the first identified member of a family of neurotrophic factors that function both in vitro and in vivo to promote neuronal survival and differentiation. The biological effects of NGF are mediated by high-affinity binding to cell-surface glycoprotein receptors called TrKA (for tyrosine kinase receptor) encoded by the trk protooncogene [now designated Ntrk (NTRK) for neurotrophic tyrosine kinase receptor in the human (mouse) genome data base]. NGF induces differentiation of the rat pheochromocytoma cell line PC12 into cells resembling sympathetic neurons, thus providing a well-characterized model for the investigation of the mechanism of action of NGF. Following the application of NGF to PC12 cells, long-term transcription medicated events occur. These include the extension of neurites and the acquisition of a differentiated phenotype that is characterized by the development of electrical excitability and the biosynthesis of neurotransmitters.The human nerve growth factor receptor (TrkA) contains four potential N-glycosylation sites that are highly conserved within the Trk family of neurotrophin receptors, and nine additional sites that are less well conserved. Using a microscale deglycosylation assay, it is showed that both conserved and variable N-glycosylation sites are used during maturation of TrKA. Glycosylation at these sites serves two distinct functions. First, glycosylation is necessary to prevent ligand-independent activation of TrKA. Unglycosylated TrKA core protein is phosphorylated even in the absence of ligand stimulation and displays constitutive kinase activity as well as constitutive interaction with the signaling molecules Shc and PLC-γ. Second, glycosylation is required to localize TrKA to the cell surface, where it can trigger the Ras/Raf/MAP kinase cascade. Using confocal microscopy, it is showed that unglycosylated active TrK receptors are trapped intracellularly. Furthermore, the unglycosylated active TrKA receptors are unable to activate kinase in the Ras-MAP kinase pathway, MEK and ErK.Consistent with these biochemical observations, unglycosylated TrKA core protein does not promote neuronal differentiation in TrK PC12 cells even at high levels of constitutive catalytic activity. The highly conserved nature of these N-glycosylation sites across a broad spectrum of species suggests these sites have an important function. TrKA contains intrinsic tyrosine kinase activity that has been localized to the cytoplasmic domain, and it is thought that activation of this kinase represents the initial step in the intracellular signal transduction pathway of NGF. TrKA has 13 glycolated sites, when the addition of tunicamycin, a potent inhibitor of N-glycosylation into culture medium, TrKA cannot phosphated. These data suggested that glycosylated TrKA is required for the receptors function. However, it is no reported that GnT-V modifies TrKA receptor glycosylation and effect the receptors function to regulate the neuronal differentiation, such as PC12 cell differentiation. Further research is needed to delineate the precise mechanism of modification of TrKA glycoprotein. Most importantly, GnT-V activity increased to approximately two- to three-fold the initial level with increasing mRNA level during nerve cellular differentiation. We demonstrate in the present study that GnT-V role on TrKA enhancement of NGF induction of neurite outgrowth. These effects are due to enhancement of the TrKA-associated tyrosine kinase activity elicited by NGF.There is a growing body of in vivo and in vitro evidence indicating that GnT-V are highly involved in cancer progression and metastasis andβ1, 6 GlcNAc branching is a key structure associated with cancer metastasis. The marked changes in the sugar chain structures of cell surface membrane occurring during ontogenesis and oncogenesis suggest that they play pivotal roles in cell differentiation and proliferation. The resultant pathology showed that complex type N-glycans are required for normal embryonic development, especially of neural tissues. Similarities between tumor invasion and physiologic responses, such as neurite outgrowth, have been noted (Taguchi et al., Nature 2000).The axon outgrowth and cancer metastasis are similarities in many ways, including cytoskeleton changes and signal pathways. However, it is unclear that the effect of GnT-V in nerve. Therefore, we study on the role of GnT-V regulation NGF receptor TrKA glycoprotein by over expression. In the present study, we have successfully contraction of GnT-V gene-transfected PC12 Cells and mock cells. And the level of GnT-V expression was higher in GnT-V gene-transfectioned PC12 cells than mock and PC12 cells. We have investigated the biological effect of GnT-V gene transfection into PC12 cells and found that they were sensitively responsive to NGF as indicated by the axon outgrowth. Those findings suggest that the modification of glycan structure on cell surface receptors may alter their function. Our previous works show that over expression of GnT-V gene-transfected PC12 Cells strongly enhances axon outgrowth elicited by a low dose of NGF. The results show that GnT-V strongly promotion differentiation of the PC12 cell. To investigate the change of sugar component of glycoproteins from cell lysates of GnT-V gene-transfectioned PC12 cells, mock and PC12 cells, lectin blot analyses were performed using L-PHA and E-PHA. To explore this interaction, the effect of GnT-V on receptor tyrosine kinase activity was examined by immunoprecipitates TrKA. Here, we attempts have been made to elucidate the mechanism of GnT-V function in nerve.We report here that over expression N-acetylglucosaminyltransferase V directly modifies TrKA protein, the high-affinity tyrosine kinase-type receptor for NGF, and strongly enhances axon outgrowth in rat pheochromocytoma cells (PC12) elicited by a low dose of NGF that alone is insufficient to induce neuronal differetiation. Furthermore, modification of TrKA glycoprotein could directly enhance NGF-activated autophosphorylation of immunoprecipitated TrKA in vitro. Receptor density at the cell surface is influenced by rates of de novo production, endocytosis, recycling, and degradation. Lattice-dependent regulation of receptors occurs primarily at the cell surface, is dependent on Golgi enzyme activities and the number of N-glycans per receptor, and opposes receptor loss to endocytosis. We examine the possibility that GnT-V modified N-glycans on cytokine NGF receptor TrKA oppose constitutive endocytosis by retaining surface receptors where membrane remodeling is active. Thus, we have investigated the interaction of N-glycan of NGF receptor TrKA and endocytosis. To further elucidate the mechanism of GnT-V, we study the different time point of endocytosis of TrKA receptor, and the result show that TrKA of GnT-V gene-transfected PC12 Cells delayed their removal by constitutive endocytosis than mock cells, suggesting high expression of GnT-V may alter the N-glycan of NGF receptors, effect their endocytosis and let to the NGF receptor TrKA stay longer at the cell membrane, inceasing the effect of NGF and enhancing the down stream signal pathway.In this paper, we firstly investigate the biological effect of GnT-V on the NGF receptor TrKA, and the results show that glycosylation of TrKA by GnT-V have regulated the function of TrKA receptor. It is new idea for GnT-V function on axon outgrowth, which is helpful for further reseach of GnT-V function. Thus, these results strongly suggest that N-acetylglucosaminyltransferase V as a specific endogenous role of NGF receptor function, and these enhanced effects appear to be due, at least in part, to promotion differentiation and protection of neuronal damage. This work is a step toward intriguing innovative therapeutic strategies for trials currently in consider with glycosyltransferase in neurodegenerative disorders. It has been shown that GnT-V function as a neurotrophic factor. Still, the present observations suggest intriguing innovative therapeutic strategies for trials currently in progress with neurotrophicis in neurodegenerative disorders.