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Cloning and Identification of an Endogenous Nephrosin Inhibitor from Carp
|Publication Year :||2002|
|Abstract:||腎泌分解?是我們實驗室自鯉魚頭腎萃取液純化到的分泌性金屬耦合蛋白?，分子量約為23kD。其一級結構屬於astacin家族。腎泌分解?可與細胞間質中的肝制凝素（Heparin）等黏多醣類結合而滯留其中。主要分佈於鯉魚的頭腎、腎臟、及脾臟組織，是此家族成員中於淋巴造血器官中被發現的首例。之後我們利用肝制凝素親合力管柱在鯉魚腎臟純化到具有活性的腎泌分解?抑制蛋白，其分子量為40kDa且帶有醣類。在本篇論文裡，我們由蛋白質及其部分勝?片段的N端胺基酸序列設計聚合?連鎖反應的引子，進行抑制蛋白的基因選殖。得到兩種不同長度的cDNA株，其中序列較長的cDNA株，在3’端的附近包含一段特殊的重覆序列。兩者的轉譯蛋白質都包含signal peptide，顯示出此蛋白質屬於分泌性蛋白。轉譯的序列經比對後發現其一級結構和哺乳動物的胎兒蛋白家族相似。胎兒蛋白主要表現在胚胎的血液、肝臟、腦脊髓液和大腦中，參與組織的分化和轉型，尤其在神經系統發育和免疫反應的調控中扮演重要的角色。此外，我們還發現腎泌分解?抑制蛋白在初始合成是一個65kDa的前驅分子，分泌到細胞外後透過血液分佈到其他組織。當它進入淋巴造血器官時，會被蛋白?切割成40kDa的分子而成為具有抑制活性的蛋白質。 Fetuin 的 processing 現象，迄今尚未被報導過，在此我們所發表的腎泌分解?內生性抑制蛋白，是astacin蛋白?家族中第一個被發現的抑制蛋白，相信astacin家族中的其他成員亦可能有內生性抑制蛋白存在，負責調節其活性與功能。|
Nephrosin is a newly discovered member of the astacin family. It is a secreted proteinase and present in the head kidney, kidney, and spleen, all of which are responsible for the immune and haematopoietic function in fish.
We have recently purified the nephrosin inhibitor from carp kidney extract by heparin affinity chromatography. Its inhibitory activity has also been confirmed by reverse zymography. In this study, we have cloned the cDNA encoding for the nephrosin inhibitor. There are two different cDNA clones resulting from alternative splicing, and the long form contains unique tandem repeat sequences in the 3’end. Both of them have signal peptides suggesting that they are a secreted protein. The deduced primary structure of nephrosin inhibitor is similar to fetuin-A, a mammalian fetal protein present in fetal blood, liver, cerebrospinal fluid, and cerebral cortex during the fetal development. Although its real physiological function remains elusive, it is involved in tissue differentiation and transformation, especially in the nervous and immune systems. We also found that the nephrosin inhibitor was synthesized initially as a precursor in liver and then secreted to the blood. When it was distributed into the hematopoietic tissues, it could be processed from molecular weight 65kDa to 40kDa and acquired inhibitory activity. This processing phenomenon of fetuin has not been reported elsewhere. More importantly the presence of an endogenous inhibitor of nephrosin is the first report of this kind in astacin enzymes. It is very likely that endogenous inhibitors may also be present for the regulation of other astacin enzymes.
|Appears in Collections:||生化科學研究所|
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