台灣鱟血清中脂多醣結合蛋白結構與功能之研究

Abstract

先天性免疫反應位於生物體內第一防線，它必須快速辨別出入侵者是否為外來物、為何種物種，並進而決定生物體內應引用何種途徑來有效消滅外來物。此一步驟對於所有生物之防衛機制扮演非常重要的角色，然而這方面的調控尚未十分清楚。所以在本篇論文中，藉以尋找台灣種的鱟血清中與脂多醣具結合能力的脂多醣結合蛋白，試圖釐清當面對入侵物的早期，生物會採取何種防禦機制。脂多醣(Lipopolysaccharide)，又稱內毒素，它廣泛存在革蘭氏陰性菌。當革蘭氏陰性菌侵入體內時，先天性免疫反應迅速辨認出脂多醣，進一步刺激巨噬細胞、髓狀細胞等細胞活化，釋放細胞分裂素、附著蛋白質、或多種調節物質，傳遞訊息給後續的防衛機制。這種由脂多醣所誘導出的調節物質卻會引起生物體嚴重疾病，俗稱敗血症。敗血症乃我國十大死因之一，在世界各地也有許多人因感染細菌引發敗血症而喪命。所以科學家們一直希望能藉由找出脂多醣作用機制，來幫助減少敗血症的發生。我們利用鱟對脂多醣十分敏感的特性-當脂多醣接觸鱟血清的數秒間，鱟血球會迅速破裂並凝集-來研究脂多醣的訊息傳導。以往對鱟的研究大多著重其血球部分，對於脂多醣與鱟血清之間的關聯並未十分明瞭。故此論文中製作一脂多醣親和層析管柱，分離出存在於鱟血清中會與脂多醣結合的蛋白質，並對此蛋白質的結構與功能特性作分析。 經脂多醣親和層析管柱所分離出的結合蛋白，我們稱之為脂多醣結合蛋白(LPS－Binding protein）。將脂多醣結合蛋白對脂多醣作結合測試發現其解離常數為7.82×10-7，顯示兩者之間結合能力頗強。而由電泳膠片分析可看出此脂多醣結合蛋白含有許多不同分子量的蛋白質，主要有28kDa, 30kDa, 36kDa, 38kDa, 60kDa及66kDa六個蛋白質。這些不同組成雖具不同分子量，但以胺基酸序列、免疫轉印、電泳分析等實驗均顯示這些不同分子量的蛋白質極可能是源自於同一種蛋白質，或其間具有相互作用的特性。除此之外，尚發現脂多醣結合蛋白具有類似胰蛋白?的酵素之分解活性。原本未處理過的鱟血清並不具有胰蛋白?的酵素活性，唯有經過脂多醣親和層析管柱所取得之脂多醣結合蛋白具有顯著胰蛋白?活性。這些發現提供了初步的藍圖，讓我們可以進一步探尋酯多醣在生物體的作用途徑，以瞭解先天性免疫機制對外來細菌入侵所引導之防衛反應。

A fresh angle of study on whether the innate immunity is essential for the function of acquired immunity has emerged from studies of the ancient defense systems of invertebrates. Induction of the adaptive immune response depends on the expression of co-stimulatory molecules and cytokines by antigen-presenting cells. The mechanisms that control the initial induction of these signals upon infection are poorly understood. In this thesis, horseshoe crab was used as a model to study how endotoxin trigger its defense responses. Endotoxin, or LPS (lipopolysaccharide), is a major constituent of outer membranes of gram-negative bacteria. Endotoxin is the main target of innate immune response recognition, the so called pathogen-associated molecular patterns (PAMP). Not much is known about the protein molecules in the plasma of horseshoe crab that specifically recognize LPS and initiate host responses that either eliminate the pathogen or result in irreversible shock, multiorgan failure, and death. In this study, an LPS-affinity column was constructed to purify a family of novel lipopolysaccharide-binding protein (LBP) from the plasma of horseshoe crab. As an invertebrate, this animal has evolved only the innate immune response and lacks the adaptive immunity. It is extremely sensitive to LPS. In the plasma of this animal, a novel LBP proteins that bind to LPS with a dissociation constant of 7.82×10-7 was discovered. This family of LBP was found to consist of many proteins with different molecular weight. The SDS-PAGE analysis shows six major bands with apparent molecular weights of 28kDa, 30kDa, 36kDa, 38kDa, 60kDa, and 66kDa. Yet, amino-terminus and immunological analyses indicated that most likely, they evolved from the same precursor protein by post translational processes. Additionally, trypsin-like protease activity was found to be associated with LBPs. The specific trypsin-like activity of LBPs was about 1600 times that of the freshly collected horseshoe crab plasma. These results provided a link between the hemolymph to the amebocytes and hopefully will lead to the elucidation of the basic mechanism used by this ancient animal to distinguish self and non-self.