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標題: | LMBD1蛋白質在協助維生素B12輸出溶酶體的角色 The role of LMBD1 protein in vitamin B12 transport out of lysosome |
作者: | Li-Yu Li 李麗玉 |
指導教授: | 張明富(Ming-Fu Chang) |
關鍵字: | 維生素B12,氰鈷胺,溶酶,體, LMBD1,NESI,LMBRD1,vitamin B12,cblF, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 維生素B12,又稱為氰鈷胺,在哺乳類動物細胞中做為酵素反應的輔因子,協助執行甲硫胺酸(methionine)生合成及methylmalonyl-CoA與succinyl-CoA之間的異構化。然而,維生素B12在細胞內的代謝路徑大部分仍未知,只能從代謝缺失的病人中取得纖維母細胞進行研究。目前,已有八種代謝缺失的互補型被發現,命名為cblA-G及mut。LMBR1 domain containing 1 (lmbrd1)基因被認為是cblF的對應基因,該病症使得維生素B12累積在溶酶體內而不能被細胞所利用。lmbrd1基因會轉譯二種主要的蛋白質:全長540個胺基酸的LMBD1蛋白質,及由另一個啟動子轉錄轉譯而得具有467個胺基酸的異體蛋白質NESI (nuclear export signal-interacting protein)。NESI蛋白質由本實驗室首度發現,並證實它參與D型肝炎病毒大型抗原的核輸出過程,與D型肝炎病毒的組合有關。另一方面的研究則指出LMBD1蛋白質可能是維生素B12於溶酶體的輸出蛋白質。LMBD1蛋白質可能透過其突出於溶酶體內側的區段和維生素B12結合,或者辨認B12的結合蛋白質。
本篇研究的實驗目的在於分析LMBD1蛋白質是否可以直接結合維生素B12,或者藉由溶酶體中可以與LMBD1蛋白質結合的B12攜帶蛋白質,與B12產生間接的結合。為了尋找溶酶體中與LMBD1有交互作用的蛋白質,使用OptiPrep濃度梯度及超高速離心的方法分離溶酶體。使用專一性辨認接在LMBD1上的V5-tag的抗體進行免疫共沉澱,發現至少有三個以上的蛋白質專一性地與LMBD1結合。經LC-MS/MS鑑定其身分,發現都未曾有與維生素B12相關的研究被提出。因此其與B12的相關性尚待進一步確認。另外,對於LMBD1與維生素B12直接結合的假說,使用GST pull-down assay進行測試。初步結果發現不同長度LMBD1的GST融合蛋白質中,只有包含C端第383-540個胺基酸的融合蛋白質與MeCbl的結合與對照組有顯著差異。藉由維生素B12於血液中的結合蛋白質transcobalamin (TCII)與B12結合後,會增加等電點約0.4 pH單位的特性,進一步使用GST-MMACHC融合蛋白質做為正控制組,從非變性蛋白質瓊脂膠電泳中看到其與MeCbl結合後,產生band-shift的現象,確認此系統可做為測試LMBD1與維生素B12直接結合的方法。此外,最近的文獻報告認為cblC的對應蛋白質MMACHC,可能在協助LMBD1蛋白質運送維生素B12移出溶酶體的過程中,扮演一定的角色。使用GST pull-down assay確認GST-MMACHC融合蛋白質與細胞中LMBD1蛋白質的結合關係。LMBD1亦可pull-down細胞中的MMACHC。而使用MeCbl處理細胞後,發現LMBD1與MMACHC的結合會進一步被增強。由於MMACHC並不存在於溶酶體中,因此我們認為MMACHC可能做為細胞質中的受體蛋白質,接收來自於溶酶體LMBD1所輸出的維生素B12。 Vitamin B12 (cobalamin) is a coenzyme that mediates enzymatic reactions for the methionine biosynthesis and the interconversion of methylmalonyl coenzyme A and succinyl coenzyme A in mammalian cells. However, the intracellular metabolism pathway of the cofactor is complicated and rarely revealed by clinical analyses of fibroblasts from patients with disorders of cobalamin metabolism. Currently, eight complementation groups, cblA-cblG and mut, which stand for different intracellular defects in vitamin B12 metabolism have been identified. LMBR1 domain containing 1 (lmbrd1) is the corresponding gene for cblF, a defect that causes accumulating free vitamin B12 in lysosomes. lmbrd1 gene encodes two major proteins, LMBD1 representing the full-length protein with 540 amino acid residues and the nuclear export signal-interacting protein (NESI) with 467 amino acid residues from an alternative promoter-drived transcript. NESI was first discovered in our laboratory and demonstrated to be involved in the nuclear export of the large hepatitis delta antigen (HDAg-L) and the assembly of hepatitis delta virus (HDV). On the other hand, others defined LMBD1 as a putative lysosomal cobalamin exporter. As an exporter, LMBD1 was proposed to either directly bind to cobalamin through its loops protruding into lysosomal compartment or to recognize a cobalamin-binding protein. The specific aims of this study are to examine whether LMBD1 can directly bind to cobalamin, and to identify cobalamin carrier proteins that interact with LMBD1. To identify potential LMBD1-associated cobalamin carrier proteins, lysosomes were isolated from LMBD1-V5 overexpressed 293T cells through ultracentrifugation in OptiprepTM gradient and subjected to co-immunoprecipitation using the V5 antibody. Several cellular proteins were identified to specifically interact with LMBD1, but their functions associated with cobalamin need to be further studied. To examine the direct binding hypothesis, GST pull-down assay was performed. The preliminary data demonstrated a significantly higher binding capacity of MeCbl to the C-terminal LMBD1 than the GST control and the other LMBD1 subdomains. Based on an earlier study that demonstrated an increase of the isoelectric point (pI) of transcobalamin (TCII) for 0.4 pH unit when it bound to cobalamin in the bloodstream, a band-shift assay was performed using GST-MMACHC as a positive control. This system would help us to confirm the direct interaction between LMBD1 and cobalamin. Furthermore, MMACHC that is responsible for cblC type disorder may play a role in helping LMBD1 to export cobalamin out of lysosomes. Indeed, GST-MMACHC pulled-down LMBD1-V5 at a low pH condition, and vice versa. In addition, the interaction between LMBD1 and MMACHC could be enhanced by MeCbl treatment in vivo. Nevertheless, MMACHC locates in the cytosol instead of lysosome. These results suggest MMACHC as a cytosolic receptor protein that gets cobalamin exported out from the lysosome via LMBD1. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35029 |
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顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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