張震東2006-07-252018-07-062006-07-252018-07-062004-07-31http://ntur.lib.ntu.edu.tw//handle/246246/10250本實驗室過去的研究發現鯉魚頭腎分泌蛋白 中含一蛋白分解酶，也成功地純化出該蛋白 分解酶及選殖到它的cDNA，由於它的分佈並 將它命名為腎泌分解酶（Nephrosin）。腎泌 分解酶的基本特性如下：（1）它屬 Zinc-metalloproteinase，活性可被金屬離子耦 合劑（Metal chelators）抑制，（2）僅出現在 頭腎、腎臟及脾臟（均含造血及免疫細胞） 及鰓，（3）從cDNA轉譯之氨基酸序列和己 知Zinc-metalloproteinase比對發現它屬Astacin family的成員，它們的鋅離子耦合屬五配位 （Penta-coordination）。Astacin family成員包 括crayfish astacin, medaka hatching enzymes, mammalian meprins, hydra HMP, Xenopus BMP-1, Drosophila tolloid等，功能各不相同， 但是有二共同點：它們的一級結構相似及它 們屬分泌蛋白分解酶或膜蛋白分解酶。我們 由腎泌分解酶的組織分佈及分泌的調節預測 它與免疫或造血功能有關。 鯉魚頭腎含一腎泌分解酶之內生性抑制蛋白 質(p40)。本實驗室以西方墨點法發現p40的前身 是一個血清蛋白p65分子﹐而且由cDNA cloning 發現它是胎兒蛋白(Fetuin, p65)的部份分子。胎 兒蛋白在哺乳動物功能有﹕調節骨鈣形成、抑制 Insulin Receptor活化、參與大腦皮質發育及抑 制巨噬細胞活化等﹐ 而抑制Astacin Metalloproteinase則屬首次發現。眾多證據指出 胎兒蛋白的許多功能應是透過細胞膜上受體來執 行﹐而目前卻對受體分子之生化特性完全缺乏﹒ 本計劃決定以蛋白質體學方法來找尋小鼠之胎兒 蛋白受體﹔純化依賴胎兒蛋白共價鍵結而成的親 和力膠體﹐鑑定依賴遠西方墨點法(Far-Western blotting)或Gel pull-down assay來確認其結合 活性﹑質譜儀(Mass Spectrometry)分析找尋蛋白 質身分與部份胜肰序列決定﹑最後將可能之受體 分子以PCR cloning方法選殖出cDNA﹒本實驗以老 鼠肺臟及脾臟等富含巨噬細胞的組織為材料，使 用detergent溶液萃取巨噬細胞的分子，再通以 fetuin親和性層析管柱，獲得與fetuin有強烈疏水性 作用力的結合分子群。鎖定這些結合分子，經由 蛋白質譜分析、比對資料庫，再製作抗體辨識。 在此分子群中鑑定出三個fetuin結合分子，分別為 二個粒線體內膜之蛋白質分子 Ubiquinol-cytochrome C reductase complex core protein 2、mitochondrial inner membrane protein （IMMT），以及一個ECM分子alpha 3 B chain of laminin-5。以Fetuin在細胞中的重要調節角色來評 估，其與粒線體內分子、ECM分子之結合應有重 要之生理意義，有待我們進一步地探討。The head kidney of bony fish is composed of chromaffin cells (similar to mammalian adrenal medulla), interrenal (similar to mammalian adrenal cortex), immune tissue and hematopoietic tissue (similar to mammalian bone marrow). We have studied secretory proteins from head kidney for some years and found that one of the secreted proteins is a zinc-metalloproteinase. We have successfully purified this zinc-metalloproteinase and also isolated one cDNA encoding this protein. Due to the discrete tissue distribution, we named this zinc-metalloproteinase as nephrosin. Nephrosin is a zinc-metalloproteinase and its activity can be inhibited by metal chelators. It is present only in the kidney, head kidney, spleen and gill , all of which are rich in lymphohematopoietic cells. From amino acid sequence comparison, nephrosin belongs to the astacin family. The astacin family includes crayfish astacin, medaka hatching enzymes, hydra HMP, Xenopus BMP-1, Drosophila tolloid and mammalian meprins. So far, nephrosin is the first member of this family involved in the immune or hematopoietic functions. Recently, we have purified a novel protein inhibitor of carp nephrosin (p40) from carp kidney. The nephrosin inhibitor forms a 1:1 tight complex with nephrosin and thus inhibits the enzyme activity. Interestingly, nephrosin/inhibitor complex seems to be present only in the lymphohematopoietic tissues. For the first time, an endogenous inhibitor of the astacin family has been identified. Immunoblotting analysis revealed that a serum protein p65 can be recognized by the anti-p40 antiserum. Cloning of cDNA encoding p65 reveals that p40 is derived from carp fetuin. Mammalian fetuins are involved in diverse functions; opsonization of cationic macrophage-deactivating molecules, tyrosine kinase inhibition of the insulin receptor, osteogenesis and bone resorption, and neocortex development. It is believed that some of the function of fetuin is mediated by putative membrane receptors. However, little is known about the putative fetuin receptors. Fetuin is a mammalian fetal protein present in fetal blood, liver, cerebrospinal fluid, and cerebral cortex. During the fetal development, it plays an essential role in regulating the tissue differentiation and transformation, especially in the nervous system and the osteogenesis. It also participates in the inflammatory response mediated by macrophages. It may function as a carrier to bring anti-inflammatory factors into the macrophages. In this study, we use mouse lung and spleen in which macrophages are enriched to purify fetuin binding proteins by fetuin affinity chromatography. We have found a group of proteins that bind strongly through hydrophobic interactions with fetuin. Using MALI-TOF, and LC-MS/MS spectrometry analysis, and PCR as well, we isolated partial cDNA sequences of these binding proteins. After antibody recognition, three molecules were identified as the putative binding proteins, ubiquinol-cytochrome C reductase complex core protein 2, mitochondrial inner membrane protein（IMMT）and alpha 3 B chain of laminin-5. The first two molecules are resided in the mitochondrial inner membrane and the third one is an ECM protein. The physiological significance of the interactions between fetuin and the three molecules is needed to be further elucidated.application/pdf262015 bytesapplication/pdfzh-TW國立臺灣大學生化科學研究所腎泌分解脢胎兒蛋白受體Nephrosinnephrosin inhibitorfetuinmetalloproteinasereceptorreporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/10250/1/922311B002104.pdf