【作者】 李兆杰；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2009， 博士

【摘要】 卵黄蛋白原(Vitellogenin, Vg)是卵生脊椎动物和非脊椎动物卵黄蛋白的前体,它是受雌性激素调控的一种大分子的糖磷脂蛋白。Vg为人们所熟知的主要功能是为胚胎和幼体生长发育提供主要的营养物质。然而,新近一些研究表明Vg参与多个生理过程,具有多种功能。例如,Zhang等人(2005)和Shi等人(2006)分别证明玫瑰无须鲃和文昌鱼Vg具有抗菌和凝血功能,说明其参与免疫反应。然而,关于Vg参与免疫作用的机理完全不了解。本论文主要研究了Vg的免疫作用机理。本论文第一部分首先从六线鱼(Hexagrammos otakii)中纯化得到了Vg并对其进行了鉴定。通过DEAE-23离子交换柱层析和Sephadex G-200凝胶过滤柱层析首次从经17β-雌二醇(E2)诱导的六线鱼体内纯化得到了Vg,并通过二级质谱对其进行了鉴定;native-PAGE显示Vg分子量约为450 kDa;native-PAGE后的凝胶经特异性染色证明六线鱼Vg也是一种富含糖、脂、磷的蛋白。本论文第二部分通过实验证明了Vg是一种典型的模式识别受体,并且具有调理素功能,能够促进巨噬细胞的噬菌作用。将异硫氰酸荧光素(FITC)标记的Vg与各种菌孵育后荧光显微镜观察发现Vg可以分别与E. coli,S. aureus和P. pastoris结合;另外通过酶联免疫吸附实验(ELISA)证明Vg可以与病原相关分子模式(PAMPs)脂多糖(LPS)、脂磷壁酸(LTA)、肽聚糖(PGN)、β-1,3-葡聚糖(β-1,3-glucan)和海带多糖(laminarin)等发生特异性结合,并且结合呈现浓度依赖性。上述结果表明Vg是一种多价的模式识别受体。我们还研究了Vg对巨噬细胞噬菌作用的影响。实验表明Vg可以明显促进巨噬细胞对细菌的吞噬。硬骨鱼的头肾在功能和结构上被认为与哺乳动物的骨髓相当,其含有大量的巨噬细胞。我们利用Percoll密度梯度离心法分离得到六线鱼头肾巨噬细胞,通过吞噬实验统计两个参数——吞噬能力(PA)和吞噬指数(PI)评价巨噬细胞的噬菌水平。结果发现,与对照相比,经Vg处理的实验组的PA和PI都有显著性高(p<0.05),说明Vg能够促进巨噬细胞的噬菌作用;另外还发现FITC标记的Vg可以与巨噬细胞表面特异性结合。上述结果表面Vg可能具有类似调理素的功能。本论文第三部分主要对Vg的杀菌机制和Vg发挥杀菌作用的活性部位进行了研究。实验表明Vg是通过分别与革兰氏阴性菌细胞壁上的LPS和革兰氏阳性菌细胞壁上的LTA结合来发挥对E. coli和S. aureus的杀菌功能,并且发现Vg分子肽链的完整性以及糖基化对于Vg发挥杀菌作用是必需的。抗菌蛋白的抗菌机制有多种,大多数蛋白是通过与细菌细胞膜作用,而也有一些蛋白是通过与细菌细胞壁上的特定成分结合直接破坏细胞壁引起细菌溶胀破裂。为阐明Vg的杀菌机制,首先我们通过扫描电镜技术和细菌/原生质体裂解实验发现Vg能够引起E. coli和S. aureus的裂解,但不能引起无细胞壁的原生质体的裂解,说明Vg通过破坏细菌细胞壁来发挥对E. coli和S. aureus的杀菌作用,而并非以细胞膜为靶部位;其次通过抑菌圈实验发现LPS和LTA可以分别抑制Vg对E. coli和S. aureus的杀菌作用,这与LPS和LTA能与Vg结合结果相一致。因此上述结果可以说明Vg是通过与细菌细胞壁上的LPS或LTA结合来发挥杀菌作用的。另外本部分对Vg发挥杀菌作用的活性部位进行了研究。蛋白质的翻译后加工和修饰对其功能作用至关重要。为鉴定Vg分子发挥杀菌作用的活性部位,我们首先对Vg分别进行了肽链破坏、氧化糖基、去磷酸化以及去脂等处理,将上述处理的Vg分别进行抑菌圈实验,结果显示Vg脱脂和去磷酸化对其抑菌作用没有影响,而降解其肽链和氧化糖基却使Vg抑菌活性丧失。这说明Vg分子的糖蛋白部分在抑菌作用中发挥重要作用;另外发现Vg对E. coli的抑菌作用可以被D-甘露糖抑制,对S. aureus的抑菌作用可以被N-乙酰-D-氨基葡萄糖和D-海藻糖抑制,说明Vg可能具有类凝集素的活性。总之,本论文通过体外实验首次发现六线鱼Vg可以作为一种模式识别受体与多种PAMPs结合;首次报道了Vg具有调理素活性,可以促进巨噬细胞的噬菌作用;首次阐明了Vg是通过与细菌细胞壁上的LPS或LTA结合来发挥杀菌作用的;首次阐明了Vg分子肽链的完整性和糖基化与其杀菌活性的密切关系。更多还原

【Abstract】 Vitellogenin (Vg) is the egg yolk protein precursor in non-mammalian vertebrates and invertebrates. It is a high molecular mass phospholipoglycoprotein response to estrogen stimulation. Vg is known as its nutritional role to provide developing embryos and larvae nutrients. Many reports proved that Vg has evolved pleiotropic functions in many physiological processes. Vg has recently been demonstrated to possess both hemagglutinating and antibacterial activities in the protochordate amphioxus (Branchiostoma belcheri) as well as the bony fish rosy barb (Puntius conchonius). However, the mode of action by which Vg is involved in anti-infectious response remains unknown. The main objective of this study is to demonstrate the underlying mechanisms by which Vg is involved in anti-infectious response.First, fish Hexagrammos otakii (H. otakii) Vg was purified and characterized. A 17β-estradiol induced protein was purified by DEAE-23 anion exchange chromatography and Sephadex G-200 gel filtration chromatography from 17β-estradiol-injected intraperitoneally fish plasma. The purified protein was identified as Vg by MALDI TOF/TOF MS analysis. H. otakii Vg appeared to exist as a homogenous trimer of approximately 450 kDa in native-PAGE. Vg was proved as a phospholipoglycoprotein by staining using Sudan black B, periodic acid/Schiff reagent, and methyl green.Second, to test if Vg can bind to microbes, FITC-labeled Vg was incubated with Gram-negative bacterium Escherichia coli (E. coli), Gram-positive bacterium Staphylococcus aureus (S. aureus) and fungus Pichia pastoris (P. pastoris). It is found that Vg is able to bind both E. coli and S. aureus as well as P. pastoris. To better understand the mechanisms of binding activity, an enzyme-linked immunosorbent assay (ELISA) was carried out to investigate what molecules on the microbial surfaces are recognized by Vg, and it is found that Vg had a significantly stronger affinity to the immobilized ligands including LPS from Gram-negative bacteria, LTA from Gram-positive bacteria, PGN from both Gram-positive and Gram-negative bacteria,β-1, 3-glucan from fungi and laminarin from brown algae concentration-dependently than to BSA, the serum proteins (SPs) and total muscle proteins (TMPs) extracted from H. otakii itself. These results indicated that Vg was a multivalent pattern recognition receptor. In addition, we addressed the effects of Vg on the phagocytosis of H. otakii macrophages. It is demonstrated that Vg can promote macrophage phagocytosis. The head kidney of teleostean fish is considered to be the functional and structural homologue of mammalian bone marrow. H. otakii head kidney macrophages were isolated by discontinuous gradient centrigugation. Then phagocytosis assays were performed and two parameters phagocytic ability (PA) and phagocytic index (PI) were used to test the effects of Vg on phagocytosis. Compared with the microbes pre-incubated with BSA and PBS alone, those pre-incubated with Vg were readily phagocytosed by the macrophages. The PA and PI values of the macrophages phagocytosing Vg-treated E. coli, S. aureus and P. pastoris were significantly higher than those of cells phagocytosing BSA-treated and untreated microbes (p<0.05). These denoted that Vg was able to promote the macrophage phagocytosis. Furthermore, it is found that FITC-labeled Vg is able to bind to the surface of macrophages specifically. These results indicate that Vg maybe act as an opsonin.Finally, the way by which Vg plays antibacterial activities and its active components which are essential for the execution of immune activities were studied in this part. It is demonstrated that fish Vg is capable of killing E. coli and S. aureus whole cells via interaction with LPS and LTA existing in the bacterial cell walls, rather than targeting plasma membranes,and the integrity of the polypeptide chain and the carbohydrate residues of Vg are indispensible for its antibacterial activity. Antimicrobial proteins and peptides exert their bactericidal effects by several different pathways. Most proteins exert their antibacterial effects by interacting with and destabilizing the microbial cell membrane, but some proteins were demonstrated to kill the bacteria by damaging cell wall directly via interacting with components of cell walls. It is demonstrated by SEM and bacterial cell and protoplast lysis assays that Vg is able to cause lysis of both E. coli and S. aureus whole cells rather than their cell wall-lacking protoplasts. To test the effects of LPS, LTA and PGN on the antibacterial activities of Vg, these chemicals were preincubated with the protein, then antagonism assays were performed on a petri dish. It is found that the antibacterial activity of Vg against E.coli is able to be inhibited by LPS from E.coli, agreeing with the binding of LPS to Vg, and the antibacterial activity against S. aureus is able to be inhibited by LTA from S. aureus, according with the binding of LTA to Vg. It is believable that Vg acts as a direct microbial killing protein via interaction with LPS and LTA.The components of Vg active in bacteriostasis were indentified in this part. Post-translational processing and modifications of proteins are often important for their biological function. To identify the components of Vg active in bacteriostasis, the protein was degraded by trypsin, oxidated by NaIO4, delipidated by either and dephosphorylated by AP, and antibacterial assays were performed on a petri dish. It is found that neither delipidation nor dephosphorylation of Vg had any influence on its antibacterial activities, but degradation of polypeptide chain and oxidation of carbohydrate residues significantly reduced its antibacterial activities. These show that the glycoprotein component of Vg plays a crucial role in inhibiting the bacterial growth. This is further supported by the fact that the antibacial activities of Vg are inhibited by the sugars like D-mannose, GluNAc and D-fucose, suggesting that Vg functions like lectins.In summary, the present study demonstrates for the first time that Vg from fish H. otakii can act as a multivalent pattern recognition receptor binding to several PAMPs, and functions as an opsonin that can enhance macrophage phagocytosis; It is also demonstrated for the first time that Vg is capable of killing E. coli and S. aureus whole cells via interaction with LPS and LTA existing in the bacterial cell walls, and it highlights the correlation between the polypeptide integrity and glycosylation of Vg and its bactericidal activities.更多还原