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研究生:陳瓊寶
研究生(外文):Keng-Poo Tan
論文名稱:藉由FUT1修飾的岩藻糖基化LAMP-1和LAMP-2與乳腺癌細胞的溶酶體定位和自噬潮流有關
論文名稱(外文):Fucosylation of LAMP-1 and LAMP-2 by FUT1 Correlates with the Lysosomal Positioning and Autophagic Flux of Breast Cancer Cells
指導教授:陳鈴津
指導教授(外文):Alice L Yu
學位類別:博士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:52
中文關鍵詞:乳腺癌細胞岩藻糖基化溶酶體定位自噬潮流
外文關鍵詞:breast cancer cellsalpha12-fucosylationlysosomal positioningautophagic flux
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α1,2-岩藻糖基转移酶,FUT1和FUT2,是負責將α1,2 連結之岩藻糖基催化轉移 到N-乙酰基-乳糖胺(LacNAc)末端的半乳糖的转移酶,此酶已經報導會高度表現於各種癌症細胞。透過FUT1受體的鑒定, 幾個研究已經證明了α1,2-岩藻糖基转移酶在腫瘤細胞增殖和遷移的重要性。已知LAMP-1蛋白在乳腺癌細胞裡是α1,2-岩藻糖基聚醣LeY 抗原的載體,但此抗原在LAMP-1生物學上的功能仍然有很大程度的不明。是否其家族成員LAMP-2亦有類似LAMP-1的α1,2-岩藻糖修飾和功能至今仍未報導。在這篇報導裡,我們透過了免疫沉澱技術和MALDI-TOF MS分析的結合證明了LAMP-1和LAMP-2蛋白是FUT1而非FUT2的目標受體。我們又藉由nanoLC-MS3的方法進一步驗證了H2和LeY抗原在LAMP-1的存在性。此外,我們發現了FUT1和LeY抗原會高表現於弱侵入性的ER+/ PR+/HER- 乳腺癌細胞(MCF-7和T47D),但較不會表現於高度侵入性的三陰性MDA-MB-231。另外,我們也發現了岩藻糖基化在LAMP-1和2的表現量與LeY抗原在此些癌細胞的表現量具有相聯性。值得注意的是我們透過免疫熒光染色觀察到LAMP-1和2的亞細胞分佈在減弱FUT1表現的細胞株有顯著變化。在對照組裡, LAMP-1 和 LAMP-2的分佈較趨近於細胞周邊;而在減弱FUT1表現的細胞組裡,LAMP-1 和 LAMP-2則趨向於聚集在細胞核周邊
。更重要的是, 相較於對照組,抑制FUT1的表現提高了自噬體形成與分解之速率。此現象可能與FUT1表現的減弱所造成的絕大部分溶酶體為核週分佈有關,因有報導指出核週分佈的溶酶體會抑制mTORC1的活性,但却能增強自噬體和溶酶體之融合。整體來說,我們的結果提議出:當FUT1的表現被抑制時
,LAMP-1和2的分佈會趨近於細胞核周邊,並進而藉由對mTOR的訊息和自噬溶酶體的形成之調控來提昇自噬潮流的速率。

Alpha1,2-fucosyltransferases, FUT1 and FUT2, which transfer fucoses onto the terminal galactose of N-acetyl-lactosamine via α1,2-linkage have been shown to be highly expressed in various types of cancers. A few studies have shown the involvement of FUT1 substrates in tumor cell proliferation and migration. LAMP-1, lysosome-associated membrane protein-1, has been reported to carry alpha1,2-fucosylated LeY antigens in breast cancer cells; however, the biological functions of LeY on LAMP-1 remain largely unknown. Whether or not its family member LAMP-2 displays similar modifications and functions as LAMP-1 has not yet been addressed. In this study, we have presented evidence supporting that both LAMP-1 and LAMP-2 are substrates for FUT1, but not FUT2. We have also demonstrated the presence of H2 and LeY antigens on LAMP-1 by a targeted nanoLC-MS3 and the decreased levels of fucosylation on LAMP-2 by MALDI-TOF analysis upon FUT1 knockdown. In addition, we found that the expression of LeY was substantial in less invasive ER+/PR+/HER- breast cancer cells (MCF-7 and T47D) but negligible in highly invasive triple negative MDA-MB-231 cells, of which LeY levels were correlated with the levels of LeY carried by LAMP-1 and 2. Intriguingly, we also observed a striking change in the subcellular localization of lysosomes upon FUT1 knockdown from peripheral distribution of LAMP-1 and 2 to a preferential perinuclear accumulation. Besides that, knockdown of FUT1 led to an increased rate of autophagic flux along with diminished activity of mTORC1 and enhanced autophagosome-lysosome fusion. This might be associated with the predominantly perinuclear distribution of lysosomes mediated by FUT1 knockdown as lysosomal positioning has been reported to regulate mTOR activity and autophagy. Taken together, our results suggest that downregulation of FUT1, which leads to the perinuclear localization of LAMP-1 and 2, is correlated with increased rate of autophagic flux by decreasing mTOR signaling and increasing autolysosome formation.
Contents

Acknowledgements……………………………………………………………………….I
Chinese Abstract ………………………………………………………………………….….II
English Abstract ………………………………………………………………………..III
Table of Contents ………………………………………………………………………IV
List of Figures ………………………………………………………………………………..V
List of Tables ……………………………………………………………………………....VI

Chapter 1. Introduction and Motivation.………………………………………....1
1.1 Fucosylation…………………………………………………………………………..….1
1.2 Alpha1,2-fucosytransferases (FUT1 and FUT2).……………………………….…..1
1.3 Biological Roles and Importance of Alpha1,2-fucosytransferases……...2
1.4 Lysosomal Associated Membrane ptoreins (LAMPs).…………………….3
1.5 Glycosylation of LAMPs.………………………………………………....3
1.6 Specific Aims of this Study.…………………………………………………….4

Chapter 2. Methodology..……………………………………………………..5
2.1 Reagents and Antibodies………………………………………………………..5
2.2 Cell culture and siRNA transfection ………………………………………….5
2.3 Immunoprecipitation and Immunoblotting………………………………..6
2.4 On-Membrane Click Reaction…………………………………………….7
2.5 In-Gel PNGase F/ Tryptic Digestion…………………………………………….…7
2.6 N-glycan derivatization and MALDI-TOF MS analysis………………….…8
2.7 Nano LC-MS/MS analysis………………………………………………………9
2.8 Immunofluorescence microscopy…………………………………………….10
2.9 Quantification of lysosomal distribution………………………………………10
2.10 Quantification of LC3 and LAMP-1 co-localization…………………………10

Chapter 3. Results..………………………………………………………………….12
3.1 Both LAMP-1 and LAMP-2 are substrates of FUT1…………………………..12
3.2 Mass spectrometric analysis shows a decrease in terminal fucosylation on
both LAMP-1 and LAMP-2 upon FUT1 knockdown.…………………………14
3.3 Downregulation of FUT1 leads to accumulation of LAMP-1/2 (+)
vesicles at perinuclear area…………………………………..17
3.4 Downregulation of FUT1 is correlated with an increased rate of
autophagic flux..…………………………….………………………….18
3.5 Downregulation of FUT1 is associated with decreased mTORC1 activity……20

Chapter 4. Conclusions and Discussions.…………………………….....43
4.1 Problems and Challenges for Studying the α1,2-fucosylation of LAMPs…...44
4.2 Cellular Distribution of LAMPs.……………………………………...45
4.3 Factors Involved in Lysosomal Distribution or Autophagy.………..46
4.4 Molecular Mechanism of Tumorigenesis Mediated by FUT1…………..47
4.5 Future Prospect……………………………………………………47

References …………………………………………………………...48

List of Figures
Figure 1-1. Schematic representation of potential N-glycosylation sites on
LAMP-1 and LAMP-2……………………………………….…….22
Figure 1-2. The fucosylation of LAMP-1 was increased in FUT1
overexpressing cells.. ………………………………………........23
Figure 1-3. mRNA expression of FUT1, FUT2 and LAMP-1 in silencing
T47D cells……………………………………………………24
Figure 1-4. LAMP-1 and 2 are substrates of FUT1………………………………25
Figure 1-5. LeY levels were correlated with the levels of LeY carried by
LAMP-1 and LAMP-2……………………………………………..……26

Figure 2-1. Analyzing purified LAMP-1 by mass spectrometry on its
tryptic peptides…………………………………….…………………….27
Figure 2-2. Peptide mapping of purified LAMP-2 by LC-MS/MS……….…….28
Figure 2-3. MALDI-TOF profiles of permethylated N-glycans of LAMP-1
expressed in mock-treated T47D cells………………...…………...29
Figure 2-4. MALDI-TOF profiles of complex N-glycans of purified LAMP-1
from mock, control and FUT1 knockdown T47D cells…………….30
Figure 2-5. MALDI-TOF profiles of complex N-glycan from purified LAMP-2
expressed in control and FUT1 knockdown T47D cells…………….31
Figure 2-6. LC-MS3 analysis of bi-antennary N-glycans of LAMP-1 from
control and FUT1 knockdown T47D cells…………………..32

Figure 3-1. Downregulation of FUT1 leads to accumulation of LAMP-1/2 (+)
vesicles at perinulear area of MCF-7 and T47D cells………..34
Figure 3-2. Colocalization of LAMP-1 and 2 in perinuclear area of FUT1
knockdown MCF-7 and T47D cells……………………………….……….35
Figure 3-3. FUT1 knockdown leads to an increase in size of acidic compartments.....36

Figure 4-1. Knockdown of FUT1 is associated with an increase in
autophagic flux………………………………………………………...……..37
Figure 4-2. Measurement of LC3 levels under autophagy induction
and/or inhibition…………………………………………………….……..38
Figure 4-3. Downregulation of FUT1 enhanced the fusion of autophagosome
and lysosomes in MCF-7 cells.……………………………….………......39

Figure 5-1. Knockdown of FUT1 is associated with decreased mTORC1 activity…...40
Figure 5-2. Time course of mTORC1 activity in control and FUT1
knockdown MCF-7 cells.………………………….…….……….………..41
Figure 5-3. FUT1 knockdown facilitates the perinuclear accumulation of mTOR........42


List of Tables

Table 1-1. Summary of various analytical methods for the determination
of N-glycans of LAMPs……………………………………………...…33


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