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標題: | 探討LMBD1及其結合蛋白質參與維生素B12輸送之機制 Mechanisms of LMBD1 and Its Associated Proteins Involved in the Vitamin B12 Transport |
作者: | Yng-Cun Ta 戴勇存 |
指導教授: | 張明富(Ming-Fu Chang) |
關鍵字: | LMBD1,維生素B12, LMBD1,vitamin B12, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 維生素B12是一個水溶性的維生素,又稱為氰鈷胺,在哺乳類動物細胞中主要做為酵素反應的輔因子,協助執行甲硫胺酸(methionine)的生合成及methylmalonyl- CoA與succinyl-CoA之間的異構化。至於維生素B12在細胞內經溶酶體釋出至細胞質再轉成酵素反應的輔因子的代謝路徑則瞭解相當有限。目前,藉由代謝缺失病人的纖維母細胞進行研究,發現共有九種維生素B12代謝缺失的互補型,命名為cblA-G,J及mut。其中LMBR1 domain containing 1 (lmbrd1)基因被認為是cblF的對應基因。lmbrd1基因發生frameshift之病人其細胞內維生素B12會累積在溶酶體內,無法被細胞所利用。lmbrd1基因會轉譯出不同的isoforms,其中全長含540個胺基酸之LMBD1蛋白質,被認為可能是在溶酶體上的維生素B12輸出蛋白質,負責將維生素B12送到細胞質中。本研究的目的在探討LMBD1蛋白質如何參與維生素B12從溶酶體運送至細胞質。首先將His/myc-tagged LMBD1表現質體轉染HEK293T細胞,48小時之後收取細胞蛋白質並與甲基氰鈷胺進行反應,再用nickel管柱進行純化分析,結果在LMBD1蛋白質存在的區段同時偵測到維生素B12吸光值的波峰。這個結果顯示LMBD1蛋白質可能與維生素B12結合。為了釐清LMBD1蛋白質是否直接與維生素B12的結合,進一步將大量表現的LMBD1經nickel管柱在2 M氯化鈉存在下純化得到較純的LMBD1後與甲基氰鈷胺進行反應,再利用ICP-MS分析法對鈷離子定量,以未加入甲基氰鈷胺的LMBD1當作對照組做比較,結果偵測到實驗組中的鈷離子有顯著上升,計算維生素B12與LMBD1蛋白質的結合莫耳數比為1-3:1。經由共軛焦螢光顯微鏡分析結果發現LMBD1與ABCD4及MMACHC蛋白質有共位現象。此外,使用myc-tag專一性抗體辨認myc-tagged LMBD1進行免疫共沉澱,發現ABCD4及MMACHC蛋白質專一性地與LMBD1結合,推斷ABCD4及MMACHC可能參與在LMBD1蛋白質運送維生素B12的過程。此外,為了探討LMBD1蛋白質與維生素B12的結合位,利用Stimulated emission depletion (STED) microscopy分析,確定LMBD1蛋白質在溶酶體上其C端朝向細胞質,進而推測LMBD1蛋白質面向溶酶體內側的片段329-DKALHSAG-336及452-DNHKG-456為維生素B12的結合位。以上研究結果支持LMBD1蛋白質與維生素B12直接結合的可能性,且經由ABCD4及MMACHC蛋白質的協助參與維生素B12的代謝過程。 Vitamin B12 (cobalamin) is a water-soluble vitamin. It functions as 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 metabolic pathway of cobalamin released from the lysosome to the cytosol and converted into cofactor is complicated and rarely revealed. Currently, nine complementation groups, cblA-G, J and mut, which stand for different defects in intracellular cobalamin metabolism have been identified from clinical analyses of fibroblasts isolated from patients with disorders of cobalamin metabolism. LMBR1 domain containing 1 (lmbrd1) is the corresponding gene for cblF, frameshift mutations in lmbrd1 cause accumulation of free cobalamin in lysosomes. lmbrd1 gene encodes several isoforms among which LMBD1 represents the full-length protein with 540 amino acid residues. Previous studies suggested that LMBD1 is a transpoter to translocate free cobalamin from the lysosome to the cytosol. As an exporter, LMBD1 was proposed to directly bind to cobalamin through its loops toward the lysosomal compartment. The specific aim of this study is to understand how LMBD1 involves in the export of cobalamin from the lysosome to the cytosol. HEK293T cells were transfected with an expression plasmid encoding a His/myc-tagged full length LMBD1 protein. Protein lysates were harvested 48 h posttransfection and incubated with methylcobalamin prior to purification of the LMBD1-His-myc protein by FPLC with nickel column. Unique absorption peaks of cobalamin were detected from the partially purified LMBD1 fractions, suggesting that cobalamin binds to LMBD1 protein. To examine whether cobalamin directly binds to LMBD1, LMBD1 protein was overexpressed and subjected to partial purification by FPLC with nickel column in the presence of 2 M NaCl that excludes non-specific binding proteins. Further ICP-MS analysis demonstrated that cobalamin bound to LMBD1 at a molar ratio of 1-3:1. Confocal microscopy analysis showed that LMBD1 colocalized with ABCD4 and MMACHC. Immunoprecipitation analysis further identified specific interaction of ABCD4 and MMACHC with LMBD1, suggesting that these two proteins play roles in helping LMBD1 to export cobalamin out of the lysosome. Stimulated emission depletion (STED) microscopy was performed to analyze the orientation of LMBD1 protein on the lysosome. Results showed that the C-terminus of LMBD1 was toward the cytosol, indicating that the LMBD1 motifs 329-DKALHSAG-336 and 452-DNHKG-456 with sequences similar to the consensus cobalamin binding sequence and faces to the lysosomal compartment. Our results support that LMBD1 can directly bind cobalamin and involves in its metabolism mediated by ABCD4 and MMACHC. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17203 |
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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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