|Thesis Advisor||李鹏程 研究员 ; 于华华 研究员|
|Place of Conferral||北京|
|Keyword||水母毒素 酶活性 金属蛋白酶 磷脂酶a2 酶抑制剂|
|Other Abstract|| 近年来世界范围内水母暴发频繁,造成诸多环境问题，同时导致水母蜇伤事件增多，严重影响公共健康。水母毒素是水母蜇伤症状的化学基础，主要是由蛋白、多肽等构成的蛋白混合物，具有多种生物活性。水母毒素中的蛋白类成分可以分为具有催化活性的酶类蛋白与不具有酶学活性的非酶类蛋白。水母毒素的组学研究表明水母毒素中含有丰富的酶类成分。然而，目前对水母毒素中酶类成分的构成及其生物学功能尚不清楚。因此，本文以我国常见的两种大型蜇人水母，沙海蜇（Nemopilema nomurai）和白色霞水母（Cyanea nozakii Kishinouye）为研究对象，系统研究其刺丝囊毒素的酶学性质与生物活性，并研究海洋共附生真菌次级代谢产物对水母毒素酶学活性的抑制作用，主要研究结果如下：|
1. 基于生物化学和酶动力学方法，系统分析、比较沙海蜇毒素与白色霞水母毒素的酶学性质。研究发现，两种水母毒素均具有显著的酶学活性，对多种蛋白底物表现出催化活性。最适条件下，沙海蜇毒素的金属蛋白酶比活性为1470.0 ± 63.5 U/mg，是白色霞水母毒素的7.1倍。而且，沙海蜇毒素对蛋白酶底物azocasein的水解行为符合米氏动力学方程，其表观速率常数KM为17.0 ± 6.9 μmol/L，最大反应速度Vmax为11.4 ± 1.2 U/mg/min。沙海蜇毒素和白色霞水母毒素均可水解磷脂酶A2的单合成底物NOBA，且具有浓度依赖性。沙海蜇毒素催化水解NOBA的一级速度常数Kcat为7.5 ˟ 10-3，略高于白色霞水母毒素。沙海蜇毒素的金属蛋白酶活性、磷脂酶A2活性受多种理化条件的影响，如温度、pH、二价阳离子、抑制剂类型等，其中金属蛋白酶的最适pH为9.94 ± 0.04，磷脂酶A2的最适pH为8.0。此外，两种水母毒素还表现出丝氨酸蛋白酶活性、纤维蛋白原降解活性等。
2. 通过胶内酶谱偶联LC-MS/MS分析，鉴定沙海蜇毒素与白色霞水母毒素中的酶类成分。胶内酶谱分析表明，沙海蜇毒素中存在丰富的蛋白酶类成分，其分子量范围在26 ~ 200 kDa，可以降解明胶、干酪素、纤维蛋白等底物。磷脂酶酶谱分析表明，沙海蜇毒素及白色霞水母毒素在14.4 ~ 18.4 kDa可以降解蛋黄卵磷脂，说明其毒素中存在磷脂酶A2样成分。分析透明质酸酶谱发现沙海蜇毒素可能存在两种透明质酸酶，其分子量分别为~ 48 kDa、~ 105 kDa。LC-MS/MS分析发现沙海蜇毒素与霞水母毒素中鉴定出的蛋白酶主要为zinc metalloproteinase-disintegrin-like和astacin-like金属蛋白酶。此外， 还可能存在磷脂酶（PLA1，PLA2，PLDs）、透明质酸酶、丝氨酸蛋白酶和LAAOs，这些结果与酶谱分析及酶学活性测定结果基本相符。利用凝胶柱HiPrep 26/60 Sephacryl S-200对沙海蜇毒素酶类成分进行初步分离，发现peak D在非还原性条件下为一条蛋白条带，分子量约为60 kDa，且具有强金属蛋白酶活性。还原性条件下该蛋白进一步还原为两个蛋白条带，推测该蛋白可能由两个亚基构成。
3. 通过特异性酶抑制剂研究水母毒素酶类成分的生物学功能。金属蛋白酶抑制剂EDTA、1,10-菲啰啉、batimastat等均可以抑制沙海蜇毒素的溶血活性，其中40 μmol/L batimastat可使水母毒素的溶血活性降低约62%，说明沙海蜇毒素金属蛋白酶在溶血中起作用。而磷脂酶A2抑制剂varespladib在较低浓度下（> 1.6 μmol/L）可有效抑制沙海蜇毒素的溶血活性，这说明磷脂酶A2样毒素也溶血活性中也起重要作用。体内实验发现，沙海蜇毒素对小草鱼具有致死活性，可以引起小鼠足趾急性肿胀、小鼠皮肤、肾脏微血管通透性增大，以及导致小鼠大腿肌肉组织肿胀。然而，沙海蜇毒素经金属蛋白酶抑制剂batimastat预处理后，伊文思蓝染料在局部皮肤组织的渗漏显著减少（p < 0.05），说明沙海蜇毒素金属蛋白酶在血管通透性增大活性中起重要作用。胶原IV型及层粘蛋白是血管基底膜的重要结构成分，在维持血管完整性方面起重要作用，实验中发现沙海蜇毒素金属蛋白酶对这两种细胞外基质（ECM）成分都具有催化水解作用，因此推测水母毒素的降解细胞外基质作用可能是导致实验小鼠微血管通透性增大的主要原因。此外，沙海蜇毒素经金属蛋白酶抑制剂预处理后能够延长小草鱼的生存时间，但并不改变小草鱼在4 h内的死亡率。同时，也观察到小鼠足底注射50 μmol/L、200 μmol/L batimastat或varespladib预处理的沙海蜇毒素并不能显著影响4 h内小鼠足趾的急性肿胀，但是明显减轻200 μmol/L batimastat或varespladib处理组小鼠足趾在12 h后的肿胀程度，说明沙海蜇毒素金属蛋白酶、磷脂酶A2并非急性致炎成分，但是可能通过其酶学活性水解局部组织而加重炎性症状。
4. 利用已建立起来的水母毒素酶学活性模型，对来自沙海蜇、海葵共附生真菌进行发酵培养，并研究其次级代谢产物对沙海蜇毒素酶学活性的抑制作用。研究中发现，沙海蜇共附生真菌Aspergillus versicolor SmT07与Tilletiopsis sp. SmU05的共培养产物可以显著抑制沙海蜇毒素的金属蛋白酶活性，其中乙酸乙酯相与氯仿相最为显著。由于乙酸乙酯相与氯仿相总酚含量明显高于其它萃取组分，因此推测这两种有机相中的酚类化合物在抑制沙海蜇毒素蛋白酶活性中起关键作用。从海葵共附生真菌Emericella sp. SMA01和沙海蜇共附生真菌Aspergillus versicolor SmT07的次级代谢产物中均分离得到吩嗪-1-羧酸，并首次发现其能够明显抑制沙海蜇毒素金属蛋白酶活性，IC50为708.26 μg/mL。此外，当水母毒素与酚嗪-1-羧酸的质量比为1：0.6 - 1：10时，吩嗪-1-羧酸还可以有效保护纤维蛋白原Bβ链不被沙海蜇毒素降解。
; In recent years, jellyfish outbreak in the world frequently and cause various environmental problems. As one of the consequences, the number of jellyfish envenomations worldwide is increasing, which also raises the public health concern. In China, the situation is getting worse with numerous people stung by jellyfish Nemopilema nomurai (N. nomurai) and Cyanea nozakii (C. nozakii) in the summer. Jellyfish venoms underlie the chemical basis for human envenomations, which are composed of various proteins and peptides and possess extensively biological activities. Proteins species in jellyfish venom could be grouped into enzymatically active proteins and enzymatically inactive proteins. So far, understanding of the enzymatic constituents and their biological functions are critically needed. In the present study, two common scyphozoan jellyfish species, N. nomurai and C. nozakii, were chosen as research subjects to study the enzymatic properties and biological activities of the venom extracted from their respective nematocysts. And as another goal of the present study, the inhibitory effects of secondary metabolites from marine-derived fungi against the enzymatic properties of jellyfish venom were assayed for developing natural antidotes of jellyfish stings. Resuts are given as follows:
1. Based on the biochemical and kinetical profiling, the enzymatic properties of N. nomurai nematocyst venom (NnNV) and C. nozakii nematocyst venom (CnNV) were compared. The current data revealed that NnNV and CnNV exhibited various enzymatic activities, of which metalloproteinases activity and PLA2s-like activity were predominant. Under optimal conditions, the metalloproteinse activity of NnNV was 1470.0 ± 63.5 U/mg, which was 7.1-fold higher than that of CnNV. Moreover, the catalytic hydrolysis of azocasein by NnNV was in accordance with Michaelis-Menten equation with KM and Vmax values of 17.0 ± 6.9 μmol/L and 11.4 ± 1.2 U/mg/min. Results also indicated that NnNV and CnNV were found to degrade NOBA, a specific phospholipase A2 substrate, in a dose-dependent manner. The turnover numbers of the catalytic reactions of NnNV PLA2s-like, calculated as kcat, was 7.5 ˟ 10-3 min-1, which was slightly higher than that of CnNV PLA2s-like. Biochemical analysis revealed that the catalytic activities of metalloproteinases and PLA2s-like were dependent on different physiochemical conditions such as temperature, pH, divalent ions and inhibitors. The optimum pHs of NnNV metalloproteinase and PLA2s-like were 9.94 ± 0.04 and 8.0, respectively. In addition, NnNV and CnNV exhibited serine protease activity and fibrinogenolytic activity.
2. The enzymatic components of NnNV and CnNV were functionally identified by combined in gel zymography and LC-MS/MS analysis. Various proteases were revealed by catalytic hydrolysis of gelatine, casein and fibrin in the zymograms of proteolytic enzymes, whose molecular weights ranged from 26 kDa to 200 kDa. The zymogram of PLA2 indicated lecithinolytic activity of NnNV and CnNV at 14.4 ~ 18.4 kDa. Moreover, marked hemolysis occurred at the location (14.4 ~ 18.4 kDa) where the lipase hydrolyzed the egg yolk substrate. Hyaluronic acid zymogram analysis further revealed two protein species exhibiting concentration-dependent lysis of hyaluronic acid, and their molecular weights were ~48 kDa and ~105 kDa, respectively. LC-MS/MS identification showed that the potential proteases responsible for the proteolytic activity observed in the zymography assays showed some homology to zinc metalloproteinase-disintegrin-like and astacin-like metalloproteinases. In addition, the LC-MS/MS identification also suggested that phospholipases (PLA1, PLA2, PLDs), hyaluronidases, serine proteases and L-amino acid oxidases were also potential components of jellyfish venom. Isolation of enzymatic components of NnNV was conducted with HiPrep 26/60 Sephacryl S-200 gel filtration column, fractions A ~ J were obtained from NnNV. And peak peak D were found to possess potent metalloproteinase activity. Moreover, under non-reducing conditions, SDS-PAGE analysis revealed that peak D was composed of one single protein band at 60 kDa. And under reducing conditions this protein band split into two bands with lower moleclar weights, indicating that the protein at 60 kDa has two subunits bridged by disulfide bond.
3. Biological functions of the enzymatic constituents of jellyfish venom were studied with specific inhibitors. Metalloproteinase inhibitors, EDTA, 1,10-phenanthroline and batimastat were found to significantly inhibit the metalloproteinase activity of NnNV. And among them, 40 μmol/L batimastat resulted in an approximate 62% reduction of jellyfish venom induced hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. At the same time, potent inhibition of sheep erythrocyte hemolysis was observed after venom pre-incubation with very low concentrations of varespladib (> 1.6 μmol/L). This observation indicated the possible involvement of NnNV PLA2-like in hemolytic activity to sheep erythrocytes. The potential roles of NnNV metalloproteinases and PLA2s in in vivo animal model of jellyfish envenomation were evaluated by pre-incubation NnNV with varying doses of batimastat or varespladib. Results showed that NnNV possessed lethal activity to juvenile grass carp Ctenopharyngodon idellus, could induce intensive paw edema within 30 min, elevated the extravasation of evans blue dye in dorsal or renal microvessel into outside tissues and led to thigh muscle tissue swelling of experimental animals. However, the extravasation of evans blue dye into local tissues was significantly reduced after pretreatement NnNV with 200 μmol/L of batimastat, indicating the important role of NnNV metalloproteinases in changing vascular permeability. Type IV collogen and laminin are important structural constitutents of extracellular matrix (ECM) and vascular basement membranes, playing an important role in maintaining vascular integrity. NnNV was found to degrade these two important structural constitutents in vitro within 12 h. This ECM-degrading capablity of NnNV may constitute one of the important factors, which leads to changing the vascular permeability. In addition, preincubation of NnNV with EDTA could extend the survival time of grass carp, but did not change the mortality of grass carp within 4 h. At the same time, it was also observed that preincubation of NnNV with 50 μmol/L, 200 μmol/L batimastat or varespladib did not significantly affect the acute swelling within 4 h, but preincubation of NnNV with 200 μmol/L batimastat or varespladib significantly reduced the degree of swelling after 12 h. These observations indicate that NnNV metalloproteinases and phospholipase A2 are not acute inflammatory factors, but may fuction through their enzymatic hydrolysis of the local tissue.
4. By means of the established enzymological model, marine-associated fungi from jellfish N. nomurai and unidentified sea anemone, were isolated and cultivated for obtaining their secondary metabolites. And then the NnNV metalloproteinase inhibitory activity of these metabolites or pure compounds were tested. The current data suggested that co-cultures from jellyfish-derived fungi Aspergillus versicolor SmT07 and Tilletiopsis sp. SmU05 showed remarkable inhibitory activity against NnNV metalloproteinase, and EtOAc, CHCl3 fractions from the cocultures exhibited significantly inhibitory potency. The current results also suggested that phenazine-1-carboxylic acid (PCA) isolated from sea anemone-associated fungus Emericella sp. SMA01 and jellyfish-associated fungi Aspergillus versicolor SmT07 was found to inhibit the metalloproteinase activity of NnNV for the first time, with the IC50 value of 708.26 μg/mL. Moreover, phenazine-1-carboxylic acid was able to inhibit the fibrinogenolytic activity of NnNV at the ratio of 1:0.6 ~ 1:10 (w/w, NnNV:PCA).
|Subject Area||多肽与蛋白质生物化学 ; 酶学 ; 微生物药物学|
|岳洋. 水母毒素酶类成分鉴定、生物活性及其抑制剂研究[D]. 北京. 中国科学院大学,2017.|
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