臺灣鱟血清中半乳糖結合蛋白和脂多醣結合蛋白之研究

Abstract

生物體免疫系統的演化會受到微生物入侵的影響。因此，所有多細胞的生物均會發展某種形式的防禦系統，藉著摧毀入侵者或者是中和有毒的物質來達到保護自身的目的。先天性免疫系統是一個在演化上出現較早的免疫系統，它不像適應性免疫系統一樣的擁有許多不同專一性的抗原受體及抗體，而是利用一些能夠辨認病原體上共同結構的受體。之前，科學家們一直認為此種免疫系統是演化上的殘留物，它的目的只在於暫時性的限制病原體對宿主的傷害。最近的研究顯示，先天性免疫系統除了能夠迅速的辨別入侵的病原，並且對於正確的調控生物體內其他防禦機制也扮演重要的角色。在這篇論文中，我以台灣種的鱟當作實驗的模式。由於台灣種的鱟只擁有先天性免疫系統，但是它卻能在地球上存活五億年，並且在形體上沒有任何的改變，因此它的免疫系統必定非常特別。藉由研究其血清中的外源凝集素來試圖釐清在病原入侵的早期，生物體會採取何種防禦機制。 經由親和層析管柱，我在鱟血清中分離出兩個新的外源凝集素，並且從肝胰腺中成功的將其基因找出來，分別將它們命名為半乳糖結合蛋白（galactose-binding protein）和脂多醣結合蛋白（LPS-binding protein）。半乳糖結合蛋白和脂多醣結合蛋白分別由232和128個胺基酸所構成的，兩者均為醣蛋白。由電泳分析可看出這兩個蛋白在血清中是以聚合體的方式存在，這暗示著這兩個蛋白在免疫系統上扮演的角色，可能是經由其聚合體的結構來將入侵者限制住，進而引導其他的蛋白或酵素將其摧毀。

The immune system of animals has evolved under the pressure imposed by infectious microorganisms. As a result, all multicellular organisms have developed some kind of defense mechanisms that can be triggered by infection to protect the host by destroying the invading microbes and neutralizing the virulence factors. In contrast to the adaptive immune system in having a repertoire of specific antigen receptors and antibodies, the phylogenetically ancient defense mechanism, known as innate immune system, use germline-encoded receptors for recognition of common antigens on the surface of microbial pathogens. This feature distinguishes the innate immune system found in invertebrate from the adaptive immune system of the vertebrate. In this study, horseshoe crab, Tachypleus tridentatus, also known as living fossil, was used as a model to investigate the innate immune system. Because horseshoe crabs have existed for over 500 million years without any significant change in morphology, their defense mechanism must be a quite extraordinary one. Two proteins with lectin-like activities, the galactose-binding protein (GBP), and the lipopolysaccharide-binding protein (LBP), and the respective genes were isolated and characterized. Each family of multi-genes that are highly homologous, codes for GBP and LBP. GBP binds tightly to Sepharose CL-4B, and was eluted with buffer containing 0.4M GlcNAc. The deduced amino acid sequence of GBP consisted of 232 amino acids with an N-glycosylation site, Asn-Gly-Ser at residues 74-76 and shared a 65% identity and similar internal repeats of about 20 amino acid motif punctuated with Trp to tachylectin-1. Tachylectin-1 was identified as an agarose and dextran-binding non-glycosylated intracellular protein from the amebocyte granules of Tachypleus tridentatus. LBP was eluted from the LPS-column in buffer containing 0.4M GlcNAc and 2M KCl. The deduced amino acid sequence of LBP consisted of 128 amino acids with an N-glycosylation site Asn-Cys-Tbr, at positions 3-5. It shares 80% sequence identity with tachylectin-3, isolated from the amebocyte of Tachypleus tridentatus. While LBP is a glycoprotein with an apparent molecular mass of about 36 kDa under SDS-PAGE, Tachylectin-3 is an intracellular non-glycosylated protein with an apparent molecular mass about 14 kDa. It recognizes the O-antigen of LPS and the blood group A antigen. The results obtained in this study support the view that pattern recognition molecules such as the GBP and the LBP have the propensity to form intermolecular dimer/oligomer and possibly heteromultimer to entrap the invading pathogens, which could rival the diversity of the immunoglobulins of the adaptive immune system.