N-乙酰氨基葡萄糖轉移酶V高表達細胞株的構建及其對神經生長因子受體TrKA作用影響的研究
發(fā)布時間：2018-07-24 19:01
【摘要】： N-糖鏈在細胞通訊、生長、分化中起重要的作用,文獻報道有證據表明N-糖鏈在神經發(fā)育中也起重要作用。目前糖組學(Glycomics)是后后基因組的研究重點。糖基轉移酶是廣泛存在的一大類酶,參與了聚糖、糖苷和復合糖類中糖部分的生物合成,具有高度的底物專一性。目前國際上關于糖基轉移酶的研究較為初步,與其他類型的酶類相比,如參與代謝的酶類等,在酶的表達,受哪些因素調控等方面研究不夠透徹,而且其涉及的生物學效應則更為廣泛,復雜。糖基轉移酶生物功能的完全闡釋將揭示龐大的相關生物信息。 N-乙酰氨基葡萄糖基轉移酶V(N-acetylglucosaminyltransferase V,GnT-V/Mgat5,EC 2.4.1.155)作為重要的高爾基體糖基轉移酶,是糖蛋白N-糖鏈加工酶之一,具有決定N-糖鏈類型及復雜型糖鏈結構的重要作用,催化GlcNAc基團轉移至N-糖鏈核心α1,6臂的α-甘露糖的反應,形成N-糖鏈的β1,6分支結構,能形成2、3、4天線的N-糖鏈產物,在蛋白質翻譯后修飾中起重要作用,而蛋白糖基化則是生物活性蛋白發(fā)揮其生物功能的必要條件之一,其中包括了具有重要功能的生長因子的受體膜蛋白的修飾。N-乙酰氨基葡萄糖基轉移酶V是重要的高爾基體糖基轉移酶,參與了糖蛋白N-糖鏈的合成,具有對糖蛋白翻譯后糖基化共翻譯修飾的重要功能。而且內質網中的蛋白質糖基化共翻譯修飾過程能促使蛋白進行正確折疊,同時蛋白質糖基化共翻譯修飾也是真核細胞存活必需的。研究發(fā)現(xiàn)N-乙酰氨基葡萄糖基轉移酶V的缺失能引起受體N-糖鏈異常,糖基化異常能影響某些類型的細胞表面的受體功能,導致受體信號轉導的改變。因此本文構建GnT-V高表達細胞株研究糖蛋白糖鏈變化對受體功能的影響。 神經生長因子NGF是最早發(fā)現(xiàn)的神經生長因子,作為重要的神經營養(yǎng)因子,能調節(jié)神經的生長、發(fā)育、分化,通過高親和性的受體TrKA調節(jié)營養(yǎng)神經元,不僅在成神經細胞瘤中,NGF受體TrkA表達升高與良好的預后相關,而且在神經退行性病變中,如帕金森和阿爾茨海默氏癥(AD),NGF也是治療藥物研究的熱點。NGF通過高親和性的受體引起軸突生長細胞分化,而低親和性受體引起凋亡,微環(huán)境中NGF過低則不能引起軸突生長。PC12細胞是大鼠腎嗜鎘瘤細胞,具有神經細胞的特點,是研究NGF神經軸突生長良好的細胞模型,文獻報道,在正常狀態(tài)下50-100 ng/mL左右的NGF刺激幾天后能誘導PC12細胞軸突生長。因此選取該細胞作為研究細胞模型,觀察低濃度NGF刺激下(降低4-8倍)GnT-V高表達后對細胞的影響,而此低濃度下,NGF單獨不足以誘導軸突生長。 人神經生長因子受體(Trk A)含有4個潛在的N-糖鏈糖基化位點,該位點在神經營養(yǎng)因子受體Trk家族中高度保守,提示糖基化可能有重要的作用,另外還有9個不太保守的N-糖鏈糖基化位點。研究發(fā)現(xiàn),成熟的Trk A受體在保守和可變的N-糖鏈糖基化位點均有糖基化。這些位點的糖基化有兩種不同的功能,一是Trk A受體糖基化是阻止配體不依賴的激活所必需的,去糖基化的Trk A受體核心蛋白在沒有配體激活時也是磷酸化的并且顯示組成性激酶活性作用于信號分子Shc和PLC-γ;二是Trk A受體糖基化是受體定位于細胞膜表面所必需的,只有在細胞膜表面的受體能引發(fā)Ras/Raf/MAP激酶通路。用激光共聚焦顯微鏡發(fā)現(xiàn)沒有糖基化的活性Trk受體陷入在細胞內,不能定位細胞膜,而且沒有糖基化的活性Trk A受體不能激活Ras-MAP、MEK和ErK激酶通路,因此,沒有糖基化的Trk A受體核心蛋白不能誘導PC12細胞神經元的分化。而且文獻報道去糖基化的Trk不能應答其配體NGF,即衣霉素預處理穩(wěn)定轉染的高表達Trk的PC12細胞后,抑制Trk糖基化,改變了Trk蛋白上GM1(ganglioside,神經節(jié)苷脂,酸性糖鞘脂)結合位點,失去了與GM1形成復合物的能力。而且文獻報道細胞膜N-糖鏈在神經胚胎發(fā)育中有重要作用,值得注意的是GnT-V在神經元細胞分化時期此酶的表達升高2-3倍,而GnT-V高表達引起的糖基化修飾的Trk A受體核心蛋白對其功能的影響和對PC12細胞神經元的分化影響未見報道。 目前N-糖鏈與腫瘤侵襲轉移的關系近年來備受關注,尤其是與GnT-V相關的β1,6分支結構的研究。研究表明在惡性腫瘤中,N-乙酰氨基葡萄糖轉移酶V活性增高,其催化產物β1,6分支也增加,腫瘤細胞表面β1,6分支存在能促進其入侵基膜,β1,6分支已成為乳腺癌和結腸癌腫瘤惡化的標志。細胞膜N-糖鏈在個體發(fā)育和腫瘤生成中有顯著變化提示N-糖鏈在細胞分化和細胞增殖轉移中有重要作用。而GnT-V在神經中的生物學作用還未見報道。在神經發(fā)育中,復合型N-糖鏈尤其是神經組織胚胎發(fā)育必須的。Taguchi et al.報道指出腫瘤的侵襲與神經軸突的應答過程相似的概念,提示在腫瘤轉移中起作用的糖基轉移酶GnT-V很可能在神經中也起重要作用。 神經軸突的生長與腫瘤的遷移有很多相似之處,包括細胞骨架的變化和參與的信號調節(jié)通路。至今關于GnT-V在神經中的作用尚無文獻報道,我們主要在此方面進行闡釋。 綜上所述,本文選NGF為靶點,研究GnT-V轉染PC12細胞后GnT-V高表達對NGF及受體誘導的軸突生長通路的變化�？紤]到目前GnT-V在神經中的生物學作用未見報道,因此本文首次在此方面進行初步的探索研究,首先成功構建轉染穩(wěn)定表達GnT-V的PC12細胞和mock組細胞,進行了糖鏈和蛋白鑒定。研究NGF誘導軸突生長的影響,結果表明,GnT-V高表達,能修飾神經生長因子受體,引起NGF低濃度下神經軸突的生長,轉染細胞軸突生長明顯增強,提示GnT-V表達增加對神經元分化具有調節(jié)作用。研究了GnT-V對PC12細胞的生物效應,這種促進PC12細胞突起生長的作用,是促分化作用。為了闡釋N-糖鏈的變化,將NGF受體TrKA免疫沉淀后,用凝集素PHA-L分析GnT-V酶作用產物β1,6分支結構糖鏈。同時免疫沉淀TrKA受體,測定NGF受體的信號通路的激活,研究了轉染穩(wěn)定表達GnT-V的細胞,mock組細胞TrKA受體磷酸化隨時間的變化,TrKA受體用抗TrKA抗體免疫沉淀垂釣分析磷酸化水平。結果表明,轉染穩(wěn)定表達GnT-V的細胞比mock組細胞磷酸化水平明顯升高。GnT-V轉染PC12細胞對NGF刺激軸突信號轉導通路的敏感性增強�；谇捌谘芯堪l(fā)現(xiàn)低濃度的NGF能引起PC12細胞軸突生長的現(xiàn)象,進一步研究GnT-V對TrKA受體作用的機理。 研究發(fā)現(xiàn)GnT-V高表達,免疫沉淀神經生長因子細胞膜受體TrKA糖鏈β1,6分支結構增加,說明此酶GnT-V能直接糖基化修飾神經生長因子高親和性的受體TrKA,同時發(fā)現(xiàn)糖基化修飾的高親和性的受體TrKA的磷酸化增強。為進一步解釋其機制,我們研究了不同時間點TrKA受體的內吞,NGF-β孵育不同的時間點,測定轉染穩(wěn)定表達GnT-V的細胞,mock組細胞NGF-β與受體TrKA結合內吞的情況。結果發(fā)現(xiàn)轉染穩(wěn)定表達GnT-V的PC12細胞與mock組相比,TrKA受體內吞延遲,提示GnT-V高表達引起的N-糖鏈變化,使得NGF受體TrKA內吞延遲,進而增強了受體信號轉導通路,導致神經軸突的生長。這些結果表明,GnT-V高表達,修飾神經生長因子受體,增強其功能,能引起低濃度NGF刺激下神經軸突的生長, GnT-V在神經中可能通過修飾糖蛋白受體調節(jié)NGF誘導的信號通路,可能在與神經生長因子受體相關的神經退行性病變中起作用。本文研究發(fā)現(xiàn)N-乙酰氨基葡萄糖基轉移酶V糖基化修飾神經生長因子受體TrKA,能調節(jié)TrKA受體的功能,激活TrKA受體介導的軸突生長的信號通路。 此論文的研究意義在于首次闡釋了N-乙酰氨基葡萄糖基轉移酶V對神經生長因子受體的糖基化修飾作用對TrKA受體功能的影響,其研究結果為研究N-糖鏈在神經中的作用提供了深層次的理論依據,拓展了對N-乙酰氨基葡萄糖基轉移酶V在神經中作用的認識。發(fā)現(xiàn)N-乙酰氨基葡萄糖基轉移酶V具有促神經分化作用,這種對神經生長因子受體TrKA的調節(jié)作用,可能與神經生長因子相關的神經退行性病癥的藥物設計提供了新的靶點,拓展了糖基轉移酶研究的領域。
[Abstract]:N- sugar chains play an important role in cell communication, growth and differentiation. There is evidence that N- sugar chains play an important role in neural development. Glycosylation (Glycomics) is the focus of post genome research. Glycosyltransferase is a widely distributed enzyme that participates in the biosynthesis of sugar parts in chitosan, glycosides and carbohydrates. There is a high degree of substrate specificity. The international research on glycosyltransferases is relatively preliminary. Compared with other types of enzymes, such as the enzymes involved in metabolism, the expression of enzymes and what factors are regulated, the biological effects of glycosyltransferase are more extensive and complex. The full interpretation of energy will reveal a large number of related biological information.
N- acetylglucosaminosyltransferase V (N-acetylglucosaminyltransferase V, GnT-V/Mgat5, EC 2.4.1.155), as an important Golgi body glycosyltransferase, is one of the glycoprotein N- sugar chain processing enzymes. It plays an important role in determining the type of N- chain and the complex structure of sugar chain. It catalyzes the transfer of the GlcNAc group to the alpha glycosaminophone of the alpha 1,6 arm of the N- sugar chain. The reaction of sugar and the formation of the beta 1,6 branching structure of N- sugar chain can form the N- sugar chain product of the 2,3,4 antenna, which plays an important role in the post-translational modification of the protein, and the protein glycosylation is one of the necessary conditions for the biological activity protein to play its biological function, including the modified.N- of the receptor membrane protein with the heavy function of the growth factor. Acetylglucosaminosyltransferase V is an important Golgi body glycosyltransferase, which participates in the synthesis of glycoprotein N- sugar chain, and has an important function for the co translation of glycosylation after glycoprotein translation. Translation modification is also necessary for the survival of eukaryotic cells. The study found that the deletion of N- acetylglucosaminosyltransferase V can cause the abnormal glucose chain of the receptor N-, and the abnormal glycosylation can affect the receptor function of certain types of cell surface and lead to the change of receptor signal transduction. Therefore, this paper constructs a GnT-V high expression cell line to study glycoprotein carbohydrate chain change. The effect of chemistry on the function of the receptor.
Nerve growth factor NGF is the earliest discovered nerve growth factor. As an important neurotrophic factor, it can regulate nerve growth, development, differentiation, and regulate nutrient neurons through a highly compatible receptor TrKA. Not only in the neurocytoma, the elevated expression of NGF receptor TrkA is associated with a good prognosis, but also in neurodegenerative diseases. For example, Parkinson and Alzheimer's disease (AD), NGF is also a hot spot in the research of therapeutic drugs,.NGF can cause differentiation of axon growth cells through high affinity receptor, while low affinity receptor causes apoptosis, and low NGF in microenvironment can not cause axonal growth.PC12 cells are rat kidney eosinophil cells, and have the characteristics of neural cells, which is a study of NG. F neurite growth good cell model, the literature reported that the NGF stimulation of about 50-100 ng/mL in normal state can induce the growth of PC12 cell axon in a few days. Therefore, the cell model was selected as the study cell model, and the effect of the high expression of GnT-V on the cell was observed under the low concentration of NGF stimulation (reduced by 4-8 times), and at this low concentration, NGF was not alone. Enough to induce axon growth.
The human nerve growth factor receptor (Trk A) contains 4 potential N- glycosylation sites, which are highly conserved in the neurotrophic factor receptor Trk family, suggesting that glycosylation may play an important role. There are also 9 less conservative N- glycosylation sites. The study found that the mature Trk A receptor is conservative and variable in the sugar chain sugar chain sugar. The glycosylation sites are glycosylated. Glycosylation of these sites has two different functions. One is that the glycosylation of Trk A receptor is necessary to prevent the activation of ligands without dependence. The deglycosylated Trk A receptor core protein is phosphorylated without ligand activation and shows the activity of the constituent kinase activity to the signal molecule Shc and PLC- gamma; two It is the Trk A receptor glycosylation is necessary for the receptor to locate on the surface of the cell membrane. Only the receptor on the surface of the cell membrane can trigger the Ras/Raf/MAP kinase pathway. The laser confocal microscope shows that the non glycosylated active Trk receptor is trapped in the cell and can not locate the cell membrane, and the non glycosylated active Trk A receptor does not activate the Ras- MAP, MEK and ErK kinase pathways, therefore, the non glycosylated Trk A receptor core protein can not induce the differentiation of PC12 cell neurons. Moreover, it is reported that the glycosylated Trk can not respond to its ligand NGF, that is, after preconditioning the stable transfected PC12 cells with high expression of Trk, the Trk glycosylation is suppressed and the Trk protein is changed. The binding site of ganglioside, acid sphingolipid) loses the ability to form complex with GM1. Moreover, it is reported that the N- sugar chain of cell membrane plays an important role in the development of neural embryos. It is worth noting that the expression of this enzyme increased by 2-3 times of GnT-V during the differentiation period of neuron cells, and the glycosylated Trk A receptor nucleus caused by the high expression of GnT-V The effect of cardiac protein on its function and the differentiation of neurons in PC12 cells has not been reported.
The relationship between N- sugar chain and tumor invasion and metastasis has attracted much attention in recent years, especially the study of the GnT-V related beta 1,6 branch structure. The study shows that in malignant tumors, N- acetylglucosaminosamine transferase V activity increases, its catalytic product beta 1,6 branch is also increased, the tumor cell surface beta 1,6 branch exists to promote its invasion of the basement membrane, beta 1,6 The branching has become a sign of the deterioration of breast and colon cancer. The significant changes in the cell membrane N- sugar chain in ontogenesis and tumor formation suggest that the N- sugar chain plays an important role in cell differentiation and cell proliferation and metastasis. The biological role of GnT-V in the nerve has not been reported. In the development of nerve, the complex N- sugar chain is especially the nerve. .Taguchi et al., which is necessary for tissue development, indicates that the invasion of the tumor is similar to that of the neurite response process, suggesting that the glycosyltransferase GnT-V, which plays a role in tumor metastasis, is likely to play an important role in the nerve.
There are many similarities between the growth of the axon and the migration of the tumor, including the changes in the cytoskeleton and the signaling pathways involved. There has been no literature on the role of GnT-V in the nerve, and we are mainly explaining it in this respect.
To sum up, NGF is selected as the target to study the change of GnT-V high expression of NGF and receptor induced axon growth pathway after the transfection of GnT-V into PC12 cells. Considering that the biological role of GnT-V in the nerve is not reported, the first exploration and research in this paper is the first time to construct a PC1 that transfected the stable expression of GnT-V. 2 cells and mock cells were identified. The effects of NGF on the growth of axon were studied. The results showed that the high expression of GnT-V could modify the nerve growth factor receptor, cause the growth of the axon under the low concentration of NGF, and the growth of the axon in the transfected cells was obviously enhanced, suggesting that the expression of GnT-V has a regulatory effect on the differentiation of neurons. The biological effect of GnT-V on PC12 cells, which promotes the growth of PC12 cells, is the role of promoting differentiation. In order to explain the changes in the N- sugar chain, the NGF receptor TrKA is immune to the precipitation of the NGF receptor, and the agglutinin PHA-L is used to analyze the sugar chain of the beta 1,6 branching structure of the product of the GnT-V enzyme. At the same time, the NGF receptor is immune to the TrKA receptor and the signal pathway of the NGF receptor is determined. The cells transfected stably expressing GnT-V, the phosphorylation of TrKA receptor in mock group was changed with time, and the phosphorylation level of TrKA receptor was analyzed by anti TrKA antibody immunoprecipitation. The results showed that the transfected cells with stable expression of GnT-V significantly increased the phosphorylation level of.GnT-V transfected PC12 cells to NGF stimulated axon signal to NGF. The sensitivity of the transduction pathway is enhanced. Based on previous studies, a low concentration of NGF can cause the growth of PC12 cells, and the mechanism of the effect of GnT-V on the TrKA receptor is further studied.
The high expression of GnT-V and the increase of the branching structure of TrKA sugar chain beta 1,6 in the immunoprecipitation neural growth factor cell membrane receptor indicate that the enzyme GnT-V can directly glycosylate the highly compatible receptor TrKA of the nerve growth factor, and the phosphorylation of the glycosylated high affinity receptor TrKA is also found. The endocytosis of TrKA receptor at different time points was studied. NGF- beta incubated at different time points, the transfection of cells with stable expression of GnT-V and the endocytosis of NGF- beta and receptor TrKA in group mock were found. The results showed that the PC12 cells that transfected the stable expression of GnT-V were delayed in the TrKA receptor endocytosis compared with the mock group, suggesting the change of the N- sugar chain caused by the GnT-V high expression. The NGF receptor TrKA endocytosis is delayed and the receptor signal transduction pathway is enhanced, resulting in the growth of the axon. These results suggest that GnT-V is highly expressed, modifies the NGF receptor, enhances its function, and causes the growth of the axon under the low concentration of NGF stimulation, and GnT-V may regulate the NGF induced by modifying the glycoprotein receptor in the nerve. The signaling pathway may play a role in neurodegenerative diseases associated with NGF receptor. This study found that the N- acetylglucosaminyltransferase V glycosylated NGF receptor TrKA, which regulates the function of the TrKA receptor, and activates the signal pathway of the axon growth mediated by TrKA receptor.
The significance of this paper is for the first time to explain the effect of the glycosylation of N- acetylglucosaminotransferase V on the function of TrKA receptor, which provides a deep theoretical basis for the study of the role of N- sugar chain in the nerve and extends the N- acetylglucosaminosyltransferase (V). It is found that N- acetylglucosaminosyltransferase V has the role of promoting nerve differentiation, and this regulation of NGF receptor TrKA may provide new targets for the drug design of neurodegenerative diseases related to NGF, and expand the field of glycosyltransferase research.
【學位授予單位】：中國海洋大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R341
本文編號：2142338
[Abstract]:N- sugar chains play an important role in cell communication, growth and differentiation. There is evidence that N- sugar chains play an important role in neural development. Glycosylation (Glycomics) is the focus of post genome research. Glycosyltransferase is a widely distributed enzyme that participates in the biosynthesis of sugar parts in chitosan, glycosides and carbohydrates. There is a high degree of substrate specificity. The international research on glycosyltransferases is relatively preliminary. Compared with other types of enzymes, such as the enzymes involved in metabolism, the expression of enzymes and what factors are regulated, the biological effects of glycosyltransferase are more extensive and complex. The full interpretation of energy will reveal a large number of related biological information.
N- acetylglucosaminosyltransferase V (N-acetylglucosaminyltransferase V, GnT-V/Mgat5, EC 2.4.1.155), as an important Golgi body glycosyltransferase, is one of the glycoprotein N- sugar chain processing enzymes. It plays an important role in determining the type of N- chain and the complex structure of sugar chain. It catalyzes the transfer of the GlcNAc group to the alpha glycosaminophone of the alpha 1,6 arm of the N- sugar chain. The reaction of sugar and the formation of the beta 1,6 branching structure of N- sugar chain can form the N- sugar chain product of the 2,3,4 antenna, which plays an important role in the post-translational modification of the protein, and the protein glycosylation is one of the necessary conditions for the biological activity protein to play its biological function, including the modified.N- of the receptor membrane protein with the heavy function of the growth factor. Acetylglucosaminosyltransferase V is an important Golgi body glycosyltransferase, which participates in the synthesis of glycoprotein N- sugar chain, and has an important function for the co translation of glycosylation after glycoprotein translation. Translation modification is also necessary for the survival of eukaryotic cells. The study found that the deletion of N- acetylglucosaminosyltransferase V can cause the abnormal glucose chain of the receptor N-, and the abnormal glycosylation can affect the receptor function of certain types of cell surface and lead to the change of receptor signal transduction. Therefore, this paper constructs a GnT-V high expression cell line to study glycoprotein carbohydrate chain change. The effect of chemistry on the function of the receptor.
Nerve growth factor NGF is the earliest discovered nerve growth factor. As an important neurotrophic factor, it can regulate nerve growth, development, differentiation, and regulate nutrient neurons through a highly compatible receptor TrKA. Not only in the neurocytoma, the elevated expression of NGF receptor TrkA is associated with a good prognosis, but also in neurodegenerative diseases. For example, Parkinson and Alzheimer's disease (AD), NGF is also a hot spot in the research of therapeutic drugs,.NGF can cause differentiation of axon growth cells through high affinity receptor, while low affinity receptor causes apoptosis, and low NGF in microenvironment can not cause axonal growth.PC12 cells are rat kidney eosinophil cells, and have the characteristics of neural cells, which is a study of NG. F neurite growth good cell model, the literature reported that the NGF stimulation of about 50-100 ng/mL in normal state can induce the growth of PC12 cell axon in a few days. Therefore, the cell model was selected as the study cell model, and the effect of the high expression of GnT-V on the cell was observed under the low concentration of NGF stimulation (reduced by 4-8 times), and at this low concentration, NGF was not alone. Enough to induce axon growth.
The human nerve growth factor receptor (Trk A) contains 4 potential N- glycosylation sites, which are highly conserved in the neurotrophic factor receptor Trk family, suggesting that glycosylation may play an important role. There are also 9 less conservative N- glycosylation sites. The study found that the mature Trk A receptor is conservative and variable in the sugar chain sugar chain sugar. The glycosylation sites are glycosylated. Glycosylation of these sites has two different functions. One is that the glycosylation of Trk A receptor is necessary to prevent the activation of ligands without dependence. The deglycosylated Trk A receptor core protein is phosphorylated without ligand activation and shows the activity of the constituent kinase activity to the signal molecule Shc and PLC- gamma; two It is the Trk A receptor glycosylation is necessary for the receptor to locate on the surface of the cell membrane. Only the receptor on the surface of the cell membrane can trigger the Ras/Raf/MAP kinase pathway. The laser confocal microscope shows that the non glycosylated active Trk receptor is trapped in the cell and can not locate the cell membrane, and the non glycosylated active Trk A receptor does not activate the Ras- MAP, MEK and ErK kinase pathways, therefore, the non glycosylated Trk A receptor core protein can not induce the differentiation of PC12 cell neurons. Moreover, it is reported that the glycosylated Trk can not respond to its ligand NGF, that is, after preconditioning the stable transfected PC12 cells with high expression of Trk, the Trk glycosylation is suppressed and the Trk protein is changed. The binding site of ganglioside, acid sphingolipid) loses the ability to form complex with GM1. Moreover, it is reported that the N- sugar chain of cell membrane plays an important role in the development of neural embryos. It is worth noting that the expression of this enzyme increased by 2-3 times of GnT-V during the differentiation period of neuron cells, and the glycosylated Trk A receptor nucleus caused by the high expression of GnT-V The effect of cardiac protein on its function and the differentiation of neurons in PC12 cells has not been reported.
The relationship between N- sugar chain and tumor invasion and metastasis has attracted much attention in recent years, especially the study of the GnT-V related beta 1,6 branch structure. The study shows that in malignant tumors, N- acetylglucosaminosamine transferase V activity increases, its catalytic product beta 1,6 branch is also increased, the tumor cell surface beta 1,6 branch exists to promote its invasion of the basement membrane, beta 1,6 The branching has become a sign of the deterioration of breast and colon cancer. The significant changes in the cell membrane N- sugar chain in ontogenesis and tumor formation suggest that the N- sugar chain plays an important role in cell differentiation and cell proliferation and metastasis. The biological role of GnT-V in the nerve has not been reported. In the development of nerve, the complex N- sugar chain is especially the nerve. .Taguchi et al., which is necessary for tissue development, indicates that the invasion of the tumor is similar to that of the neurite response process, suggesting that the glycosyltransferase GnT-V, which plays a role in tumor metastasis, is likely to play an important role in the nerve.
There are many similarities between the growth of the axon and the migration of the tumor, including the changes in the cytoskeleton and the signaling pathways involved. There has been no literature on the role of GnT-V in the nerve, and we are mainly explaining it in this respect.
To sum up, NGF is selected as the target to study the change of GnT-V high expression of NGF and receptor induced axon growth pathway after the transfection of GnT-V into PC12 cells. Considering that the biological role of GnT-V in the nerve is not reported, the first exploration and research in this paper is the first time to construct a PC1 that transfected the stable expression of GnT-V. 2 cells and mock cells were identified. The effects of NGF on the growth of axon were studied. The results showed that the high expression of GnT-V could modify the nerve growth factor receptor, cause the growth of the axon under the low concentration of NGF, and the growth of the axon in the transfected cells was obviously enhanced, suggesting that the expression of GnT-V has a regulatory effect on the differentiation of neurons. The biological effect of GnT-V on PC12 cells, which promotes the growth of PC12 cells, is the role of promoting differentiation. In order to explain the changes in the N- sugar chain, the NGF receptor TrKA is immune to the precipitation of the NGF receptor, and the agglutinin PHA-L is used to analyze the sugar chain of the beta 1,6 branching structure of the product of the GnT-V enzyme. At the same time, the NGF receptor is immune to the TrKA receptor and the signal pathway of the NGF receptor is determined. The cells transfected stably expressing GnT-V, the phosphorylation of TrKA receptor in mock group was changed with time, and the phosphorylation level of TrKA receptor was analyzed by anti TrKA antibody immunoprecipitation. The results showed that the transfected cells with stable expression of GnT-V significantly increased the phosphorylation level of.GnT-V transfected PC12 cells to NGF stimulated axon signal to NGF. The sensitivity of the transduction pathway is enhanced. Based on previous studies, a low concentration of NGF can cause the growth of PC12 cells, and the mechanism of the effect of GnT-V on the TrKA receptor is further studied.
The high expression of GnT-V and the increase of the branching structure of TrKA sugar chain beta 1,6 in the immunoprecipitation neural growth factor cell membrane receptor indicate that the enzyme GnT-V can directly glycosylate the highly compatible receptor TrKA of the nerve growth factor, and the phosphorylation of the glycosylated high affinity receptor TrKA is also found. The endocytosis of TrKA receptor at different time points was studied. NGF- beta incubated at different time points, the transfection of cells with stable expression of GnT-V and the endocytosis of NGF- beta and receptor TrKA in group mock were found. The results showed that the PC12 cells that transfected the stable expression of GnT-V were delayed in the TrKA receptor endocytosis compared with the mock group, suggesting the change of the N- sugar chain caused by the GnT-V high expression. The NGF receptor TrKA endocytosis is delayed and the receptor signal transduction pathway is enhanced, resulting in the growth of the axon. These results suggest that GnT-V is highly expressed, modifies the NGF receptor, enhances its function, and causes the growth of the axon under the low concentration of NGF stimulation, and GnT-V may regulate the NGF induced by modifying the glycoprotein receptor in the nerve. The signaling pathway may play a role in neurodegenerative diseases associated with NGF receptor. This study found that the N- acetylglucosaminyltransferase V glycosylated NGF receptor TrKA, which regulates the function of the TrKA receptor, and activates the signal pathway of the axon growth mediated by TrKA receptor.
The significance of this paper is for the first time to explain the effect of the glycosylation of N- acetylglucosaminotransferase V on the function of TrKA receptor, which provides a deep theoretical basis for the study of the role of N- sugar chain in the nerve and extends the N- acetylglucosaminosyltransferase (V). It is found that N- acetylglucosaminosyltransferase V has the role of promoting nerve differentiation, and this regulation of NGF receptor TrKA may provide new targets for the drug design of neurodegenerative diseases related to NGF, and expand the field of glycosyltransferase research.
【學位授予單位】：中國海洋大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R341
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