日本刺沙蚕蛋白酶的纯化、鉴定及日本刺沙蚕纤溶酶的部分药效学研究
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摘要
血栓性疾病(thrombotic disease, TD)包括血栓形成(thrombosis)和血栓栓塞(thromboembolism)。血栓性疾病已经成为严重危害人类生命健康的“头号杀手”,不仅发病率高居各种疾病之首,而且致残率、致死率和复发率也很高。
目前,临床上常用的抗栓治疗方法主要是溶栓治疗。过去十年中,国内外临床常用的溶栓剂有尿激酶、链激酶、葡激酶、组织型纤溶酶原激活剂、蛇毒降纤酶类(东菱迪芙)及蚓激酶等。但是所有这些溶栓剂都有不可避免的副作用,包括需要加大剂量治疗、有限的纤维蛋白特异性、再阻塞和出血倾向等。所以,从各种天然生物资源中寻找和研发溶解纤维蛋白特异性高、出血副作用小及相对较便宜的溶栓剂一直是研究的热点。
日本刺沙蚕是一种海洋无脊椎动物,广泛分布于我国的渤海、黄海沿岸及长江口等地区,此外还有日本太平洋沿岸。目前日本刺沙蚕主要被用作鱼虾的优质饵料,同时也是海洋垂钓的优质钓饵。本实验室前期已经从日本刺沙蚕体内发现了一种新的名为日本刺沙蚕纤溶酶(NJF)的丝氨酸蛋白酶,该实验过程中还发现有另外一种特殊纤溶活性及性质的蛋白酶—日本刺沙蚕蛋白酶(NJP),有可能开发成新的溶栓剂,更好的实现其经济和社会价值。本论文主要包括两个部分的研究,第一部分包括前三章,是关于日本刺沙蚕蛋白酶(NJP)的分离纯化工艺的建立,分子特征及酶学性质的检测；第二部分包括第四章,是关于NJF的体内部分药效学实验。现将主要研究结果分述如下：
1.通过酶的粗提、硫酸铵分级盐析、苯柱疏水层析、阴离子交换层析和凝胶过滤层析等方法,最终得到单一成分的、纯度较高的酶制剂,命名为日本刺沙蚕蛋白酶(N.japonica protease, NJP),最终纯化了1556倍,回收率为13%；以S-2238为特异性底物时,生色底物法测得NJP的特异性酶活力为10113.9 U/mg。
2.通过MALDI-TOF MS和SDS-PAGE电泳检测NJP是一个相对分子量为28.6～33.5kDa的单链蛋白质；2-DE检测其等电点为9.2。
3.通过串联质谱MALDI-TOF/TOF MS测序并进行De Novo分析,获得3段共28个氨基酸序列,分别是：V-T-V-V-Q-Y-R; S-T-N-A-S-S-G-Y-L-N-L-R和V-Y-L-L-D-T-G-L-R.将此序列在NCBI的non-redundant protein sequences (nr)和Swiss-Prot数据库利用protein-protein BLAST (blastp)软件进行比对,发现NJP是一种新发现的蛋白酶；将此序列登陆UniProt knowledgebase数据库中,获得登录号P86834, EC 3.4.21.-。
4. Azocasein水解法测定NJP的最适温度为40℃,且在30℃-60℃比较稳定；其最适pH为9.0,且在pH7.0-pH11.0时能保持最大活性的80%以上,表明NJP是-种碱性蛋白酶。
5. Azocasein水解法测定的结果表明Hg2+几乎能完全抑制NJP的酶活性,Co2+和Mg2+也能部分抑制其酶活性,其它金属离子对酶活性的抑制和活化作用都不明显。
6. Azocasein水解法测定的结果表明NJP的酶活性可以完全被典型的丝氨酸蛋白酶抑制剂PMSF抑制,其他蛋白酶抑制剂的抑制作用均不明显,表明NJP属于典型的丝氨酸蛋白酶类。
7.纤维蛋白平板法表明,NJP可以直接降解纤维蛋白,基本上无激酶作用,不是纤溶酶原激活剂；NJP具有很强的纤溶活性,以UK标准品为对照,NJP的纤溶活力为12000U/mg；
8.采用SDS-PAGE电泳法分析NJP水解纤维蛋白原的活性,结果表明NJP水解纤维蛋白原的先后顺序是：Aα链>Bp链>γ链。
9.生色底物法表明,NJP对凝血酶特异性底物S-2238特异性较高,其特异性水解活性为10.11±2.7 mmol/min/mg;以上结果表明NJP是一种新的碱性凝血酶样丝氨酸蛋白酶。
10.采用纤维蛋白平板法,以UK为对照,本实验所用的NJF酶制剂的纤溶活力为30000U/mg,我们将NJF的纤溶活性定为10BU/mg。。
11.本实验采用管腔内线栓法成功建立了大鼠大脑中动脉缺血再灌注模型。
12.通过神经功能损害评分和TTC染色,结果表明静脉输注NJF可以显著降低神经功能损害评分并剂量依赖性的降低缺血再灌注引起的脑梗死。
13.静脉输注NJF可以剂量依赖性的降低缺血再灌注引起的脑水肿。
14.静脉输注NJF可以显著降低MDA水平,提高SOD的活性,对抗脂质过氧化,提高内源性的抗氧化功能。
15.静脉输注5BU/kg的NJF和临床等效剂量的15000U/kg的UK在减少脑梗死和降低脑水肿,以及对抗脂质过氧化和增强抗氧化活性等方面有几乎相等的功效；NJF对缺血再灌注大鼠大脑引起的局灶性脑缺血有潜在的神经保护功能；NJF具有神经保护功能的机制可能是：抑制脂质过氧化和增强内源性的抗氧化酶类。
综上所述,本论文从日本刺沙蚕体内分离纯化并鉴定出一种明显不同于NJF和其他纤溶酶的新蛋白酶(NJP)。NJP是一种具有很高纤溶活性的碱性凝血酶样丝氨酸蛋白酶,可以直接降解纤维蛋白而不同于纤溶酶原激活剂。以上结果表明NJP具有成为防治血栓的新的溶栓剂的潜能；同时,本论文首次证明NJF对大鼠大脑中动脉阻断再灌注引起的局灶性脑缺血有很强的神经保护作用,此结果为NJF的进一步药效学和临床实验研究奠定了坚实的基础。
Thrombotic disease (TD) including thrombosis and thromboembolism. Thrombotic disease is the largest cause of mortality in human life; it not only ranks the highest incidence of various diseases, and has a high morbidity, mortality and recurrence rate. Currently, thrombolytic therapy is the the most effective method for clinical treatment for thrombotic disease. During the past decade, thrombolytic agents such as urokinase (UK), streptokinase (SK), staphylokinase (SaK), tissue-type plasminogen activator (t-PA). snake venom fibrinolytic enzymes (Batroxobin) and lumbrokinase (LK) have been widely used in the treatment of thrombosis. However, all these enzymes have undesired side effects, including need of large therapeutic doses, limited fibrin-specificity, re-occlusion and bleeding tendency. Therefore, the search for other thrombolytic agents with high specificity for fibrin, low bleeding tendency and relatively inexpensive from various nature sources are always the hot topic of the research aeras.
Neanthes japonica (Izuka) is a marine invertebrates, which widely distributed in the Bohai Sea, Yellow Sea and the Yangtze River Estuary, it also specific to the Pacific coast of Japan. Nowadays, N. japonica is usually used as high-quality fish foods and marine fishing bait. A new serine protease named Neanthes japonica fibrinolytic enzyme (NJF) has been isolated from N. japonica by our laboratory. During the purification steps, we found another fraction named N. japonica protease (NJP) with specific fibrinolytic activity and characteristics, which might be developed into a novel thrombolytic agent, to achieve their most economic and social values. This thesis consist of two parts, the first part includes the first three chapters, which are about the process establishment of the separation, purification, the measurements of molecular characteristics and enzymatic properties of N. japonica protease (NJP); The second part includes the fourth chapter, which is about the in vivo pharmacodynamics experiments of NJF. Now the main results are presented as follows:
1. By combination of crude enzyme extraction, ammonium sulfate fractionation, Phenyl Sepharose hydrophobic chromatography, anion-exchange chromatography and gel filtration, we obtained a single chain protein with high purity, named N. japonica protease (NJP). NJP was purified 1556-fold with a final yield of 13% after five purification steps. When S-2238 was used as the specific substrate, the specific enzyme activity of NJP was 10113.9 U/mg according to chromogenic substrate assay.
2. The molecular weight of NJP was about 28.6-33.5 kDa according to MALDI-TOF MS and SDS-PAGE, which indicated that NJP was a single chain protein; the isoelectric point of NJP determined by two-dimensional electrophoresis (2-DE) was 9.2.
3. We have obtained 3 peptides sequences of 28 amino acids by MALDI-TOF/TOF MS and De Novo Sequencing. The three peptides were presented as follows:VTVVQYR; STNASSGYLNLR and VYLLDTGLR. The homology with the amino acid sequences of NJP were searched through the non-redundant protein sequences (nr) and Swiss-Prot databases by using NCBI blastp program, the results indicated that NJP is a novel discovered protease;This protein sequence data will appear in the UniProt Knowledgebase under the accession number(s) P86834, EC 3.4.21.
4. Azocasein was used as the substrate in the assay of the effects of temperature and pH on the protease activity of NJP. The optimum temperature was at 40℃, the enzyme activity was stable between 30℃and 60℃; The optimum pH was found to be 9.0, NJP showed at least 80% of the maximum activity over a pH range from pH 7.0 to 11.0, which indicated that NJP is an alkaline protease.
5. The protease activity of NJP was completely inhibited by Hg2+ion according to azocasein asssy, and slightly inhibited by Co2+and Mg2+ions. However, other metal ions had no obvious effects on the inhibition or activation of the enzyme activity. 6. The protease activity of NJP was completely inhibited by the typical serine protease inhibitor PMSF using azocasein as a substrate, while other selected protease inhibitors were not effective or had weak inhibitory effects on NJP. These results suggest that NJF is a unique serine protease.
7. The fibrinolytic activity was assayed using the fibrin plate method, the results indicated that NJP could directly degrade fibrin, it had none kinase activity and it was not a plasminogen activator; NJP had stronger fibrinolytic activity compared with UK, the fibrinolytic activity of NJP was about 12000 U/mg proteins.
8. The fibrinogenolytic activity of NJP was analyzed by SDS-PAGE. The results indicated that the hydrolytic pattern of NJP on fibrinogen started from the Aa-chain, followed by the Bβ-chain, and the y-chain at last.
9. NJP showed a higher degree of specificity for the substrate S-2238 for thrombin according to chromogenic substrates methods, the amidolytic activity was 10.11+2.7 mmol/min/mg; these above results suggest that the newly discovered NJP is an alkaline thrombin-like serine protease.
10. The fibrinolytic activity of NJF was assayed using the fibrin plate method. The fibrinolytic activity of NJF was about 30000 U/mg proteins when UK was used as a control. We defined the fibrinolytic activity of NJF as 10 BU/mg.
11. In this study, the rat middle cerebral artery occlusion (MCAO) model was successfully produced by intraluminal suture method.
12. According to the results of neurological deficit scores and TTC stained, we can came to the conclusions that intravenous treatment of NJF could dose dependently reduce the cerebral infarction with significantly decreased the neurological deficit scores.
13. Ischemia/reperfusion induced cerebral edema was reduced in a dose dependent manner after intravenous treatment of NJF.
14. Intravenous treatment of NJF could significantly reduce the MDA levels and increase the SOD activities by attenuating lipid peroxidation and increasing endogenous antioxidation.
15. Intravenous treatment with NJF at 5 BU/kg was almost equivalent to UK at 15,000 U/kg dosage in the reduction of cerebral infarction and cerebral edema, lipid peroxidation and antioxidation; NJF could offer significant neuroprotection in rat models of MCAO induced focal cerebral ischemia; The neuroprotection mechanism shown by NJF may be attributed to inhibition of lipid peroxidation, increase endogenous antioxidant defense enzymes.
In conclusion, we have purified and characterized a novel protease (NJP) from Neanthes japonica (Izuka) which was clearly different from the previous isolated NJF and other fibrinolytic enzymes. NJP is an alkaline thrombin-like serine protease with much higher fibrinolytic activity. It can directly degrade fibrin distinct from the plasminogen activators. Taking account of these results, NJP might be a potential candidate for the thrombosis prevention and thrombolytic therapy; meanwhile, this work is the first evidence that NJF could offer significant neuroprotection in rat models of MCAO induced focal cerebral ischemia, which will provide a basis for further researches about pharmacodynamics and clinical trials of NJF application.
目前,临床上常用的抗栓治疗方法主要是溶栓治疗。过去十年中,国内外临床常用的溶栓剂有尿激酶、链激酶、葡激酶、组织型纤溶酶原激活剂、蛇毒降纤酶类(东菱迪芙)及蚓激酶等。但是所有这些溶栓剂都有不可避免的副作用,包括需要加大剂量治疗、有限的纤维蛋白特异性、再阻塞和出血倾向等。所以,从各种天然生物资源中寻找和研发溶解纤维蛋白特异性高、出血副作用小及相对较便宜的溶栓剂一直是研究的热点。
日本刺沙蚕是一种海洋无脊椎动物,广泛分布于我国的渤海、黄海沿岸及长江口等地区,此外还有日本太平洋沿岸。目前日本刺沙蚕主要被用作鱼虾的优质饵料,同时也是海洋垂钓的优质钓饵。本实验室前期已经从日本刺沙蚕体内发现了一种新的名为日本刺沙蚕纤溶酶(NJF)的丝氨酸蛋白酶,该实验过程中还发现有另外一种特殊纤溶活性及性质的蛋白酶—日本刺沙蚕蛋白酶(NJP),有可能开发成新的溶栓剂,更好的实现其经济和社会价值。本论文主要包括两个部分的研究,第一部分包括前三章,是关于日本刺沙蚕蛋白酶(NJP)的分离纯化工艺的建立,分子特征及酶学性质的检测；第二部分包括第四章,是关于NJF的体内部分药效学实验。现将主要研究结果分述如下：
1.通过酶的粗提、硫酸铵分级盐析、苯柱疏水层析、阴离子交换层析和凝胶过滤层析等方法,最终得到单一成分的、纯度较高的酶制剂,命名为日本刺沙蚕蛋白酶(N.japonica protease, NJP),最终纯化了1556倍,回收率为13%；以S-2238为特异性底物时,生色底物法测得NJP的特异性酶活力为10113.9 U/mg。
2.通过MALDI-TOF MS和SDS-PAGE电泳检测NJP是一个相对分子量为28.6～33.5kDa的单链蛋白质；2-DE检测其等电点为9.2。
3.通过串联质谱MALDI-TOF/TOF MS测序并进行De Novo分析,获得3段共28个氨基酸序列,分别是：V-T-V-V-Q-Y-R; S-T-N-A-S-S-G-Y-L-N-L-R和V-Y-L-L-D-T-G-L-R.将此序列在NCBI的non-redundant protein sequences (nr)和Swiss-Prot数据库利用protein-protein BLAST (blastp)软件进行比对,发现NJP是一种新发现的蛋白酶；将此序列登陆UniProt knowledgebase数据库中,获得登录号P86834, EC 3.4.21.-。
4. Azocasein水解法测定NJP的最适温度为40℃,且在30℃-60℃比较稳定；其最适pH为9.0,且在pH7.0-pH11.0时能保持最大活性的80%以上,表明NJP是-种碱性蛋白酶。
5. Azocasein水解法测定的结果表明Hg2+几乎能完全抑制NJP的酶活性,Co2+和Mg2+也能部分抑制其酶活性,其它金属离子对酶活性的抑制和活化作用都不明显。
6. Azocasein水解法测定的结果表明NJP的酶活性可以完全被典型的丝氨酸蛋白酶抑制剂PMSF抑制,其他蛋白酶抑制剂的抑制作用均不明显,表明NJP属于典型的丝氨酸蛋白酶类。
7.纤维蛋白平板法表明,NJP可以直接降解纤维蛋白,基本上无激酶作用,不是纤溶酶原激活剂；NJP具有很强的纤溶活性,以UK标准品为对照,NJP的纤溶活力为12000U/mg；
8.采用SDS-PAGE电泳法分析NJP水解纤维蛋白原的活性,结果表明NJP水解纤维蛋白原的先后顺序是：Aα链>Bp链>γ链。
9.生色底物法表明,NJP对凝血酶特异性底物S-2238特异性较高,其特异性水解活性为10.11±2.7 mmol/min/mg;以上结果表明NJP是一种新的碱性凝血酶样丝氨酸蛋白酶。
10.采用纤维蛋白平板法,以UK为对照,本实验所用的NJF酶制剂的纤溶活力为30000U/mg,我们将NJF的纤溶活性定为10BU/mg。。
11.本实验采用管腔内线栓法成功建立了大鼠大脑中动脉缺血再灌注模型。
12.通过神经功能损害评分和TTC染色,结果表明静脉输注NJF可以显著降低神经功能损害评分并剂量依赖性的降低缺血再灌注引起的脑梗死。
13.静脉输注NJF可以剂量依赖性的降低缺血再灌注引起的脑水肿。
14.静脉输注NJF可以显著降低MDA水平,提高SOD的活性,对抗脂质过氧化,提高内源性的抗氧化功能。
15.静脉输注5BU/kg的NJF和临床等效剂量的15000U/kg的UK在减少脑梗死和降低脑水肿,以及对抗脂质过氧化和增强抗氧化活性等方面有几乎相等的功效；NJF对缺血再灌注大鼠大脑引起的局灶性脑缺血有潜在的神经保护功能；NJF具有神经保护功能的机制可能是：抑制脂质过氧化和增强内源性的抗氧化酶类。
综上所述,本论文从日本刺沙蚕体内分离纯化并鉴定出一种明显不同于NJF和其他纤溶酶的新蛋白酶(NJP)。NJP是一种具有很高纤溶活性的碱性凝血酶样丝氨酸蛋白酶,可以直接降解纤维蛋白而不同于纤溶酶原激活剂。以上结果表明NJP具有成为防治血栓的新的溶栓剂的潜能；同时,本论文首次证明NJF对大鼠大脑中动脉阻断再灌注引起的局灶性脑缺血有很强的神经保护作用,此结果为NJF的进一步药效学和临床实验研究奠定了坚实的基础。
Thrombotic disease (TD) including thrombosis and thromboembolism. Thrombotic disease is the largest cause of mortality in human life; it not only ranks the highest incidence of various diseases, and has a high morbidity, mortality and recurrence rate. Currently, thrombolytic therapy is the the most effective method for clinical treatment for thrombotic disease. During the past decade, thrombolytic agents such as urokinase (UK), streptokinase (SK), staphylokinase (SaK), tissue-type plasminogen activator (t-PA). snake venom fibrinolytic enzymes (Batroxobin) and lumbrokinase (LK) have been widely used in the treatment of thrombosis. However, all these enzymes have undesired side effects, including need of large therapeutic doses, limited fibrin-specificity, re-occlusion and bleeding tendency. Therefore, the search for other thrombolytic agents with high specificity for fibrin, low bleeding tendency and relatively inexpensive from various nature sources are always the hot topic of the research aeras.
Neanthes japonica (Izuka) is a marine invertebrates, which widely distributed in the Bohai Sea, Yellow Sea and the Yangtze River Estuary, it also specific to the Pacific coast of Japan. Nowadays, N. japonica is usually used as high-quality fish foods and marine fishing bait. A new serine protease named Neanthes japonica fibrinolytic enzyme (NJF) has been isolated from N. japonica by our laboratory. During the purification steps, we found another fraction named N. japonica protease (NJP) with specific fibrinolytic activity and characteristics, which might be developed into a novel thrombolytic agent, to achieve their most economic and social values. This thesis consist of two parts, the first part includes the first three chapters, which are about the process establishment of the separation, purification, the measurements of molecular characteristics and enzymatic properties of N. japonica protease (NJP); The second part includes the fourth chapter, which is about the in vivo pharmacodynamics experiments of NJF. Now the main results are presented as follows:
1. By combination of crude enzyme extraction, ammonium sulfate fractionation, Phenyl Sepharose hydrophobic chromatography, anion-exchange chromatography and gel filtration, we obtained a single chain protein with high purity, named N. japonica protease (NJP). NJP was purified 1556-fold with a final yield of 13% after five purification steps. When S-2238 was used as the specific substrate, the specific enzyme activity of NJP was 10113.9 U/mg according to chromogenic substrate assay.
2. The molecular weight of NJP was about 28.6-33.5 kDa according to MALDI-TOF MS and SDS-PAGE, which indicated that NJP was a single chain protein; the isoelectric point of NJP determined by two-dimensional electrophoresis (2-DE) was 9.2.
3. We have obtained 3 peptides sequences of 28 amino acids by MALDI-TOF/TOF MS and De Novo Sequencing. The three peptides were presented as follows:VTVVQYR; STNASSGYLNLR and VYLLDTGLR. The homology with the amino acid sequences of NJP were searched through the non-redundant protein sequences (nr) and Swiss-Prot databases by using NCBI blastp program, the results indicated that NJP is a novel discovered protease;This protein sequence data will appear in the UniProt Knowledgebase under the accession number(s) P86834, EC 3.4.21.
4. Azocasein was used as the substrate in the assay of the effects of temperature and pH on the protease activity of NJP. The optimum temperature was at 40℃, the enzyme activity was stable between 30℃and 60℃; The optimum pH was found to be 9.0, NJP showed at least 80% of the maximum activity over a pH range from pH 7.0 to 11.0, which indicated that NJP is an alkaline protease.
5. The protease activity of NJP was completely inhibited by Hg2+ion according to azocasein asssy, and slightly inhibited by Co2+and Mg2+ions. However, other metal ions had no obvious effects on the inhibition or activation of the enzyme activity. 6. The protease activity of NJP was completely inhibited by the typical serine protease inhibitor PMSF using azocasein as a substrate, while other selected protease inhibitors were not effective or had weak inhibitory effects on NJP. These results suggest that NJF is a unique serine protease.
7. The fibrinolytic activity was assayed using the fibrin plate method, the results indicated that NJP could directly degrade fibrin, it had none kinase activity and it was not a plasminogen activator; NJP had stronger fibrinolytic activity compared with UK, the fibrinolytic activity of NJP was about 12000 U/mg proteins.
8. The fibrinogenolytic activity of NJP was analyzed by SDS-PAGE. The results indicated that the hydrolytic pattern of NJP on fibrinogen started from the Aa-chain, followed by the Bβ-chain, and the y-chain at last.
9. NJP showed a higher degree of specificity for the substrate S-2238 for thrombin according to chromogenic substrates methods, the amidolytic activity was 10.11+2.7 mmol/min/mg; these above results suggest that the newly discovered NJP is an alkaline thrombin-like serine protease.
10. The fibrinolytic activity of NJF was assayed using the fibrin plate method. The fibrinolytic activity of NJF was about 30000 U/mg proteins when UK was used as a control. We defined the fibrinolytic activity of NJF as 10 BU/mg.
11. In this study, the rat middle cerebral artery occlusion (MCAO) model was successfully produced by intraluminal suture method.
12. According to the results of neurological deficit scores and TTC stained, we can came to the conclusions that intravenous treatment of NJF could dose dependently reduce the cerebral infarction with significantly decreased the neurological deficit scores.
13. Ischemia/reperfusion induced cerebral edema was reduced in a dose dependent manner after intravenous treatment of NJF.
14. Intravenous treatment of NJF could significantly reduce the MDA levels and increase the SOD activities by attenuating lipid peroxidation and increasing endogenous antioxidation.
15. Intravenous treatment with NJF at 5 BU/kg was almost equivalent to UK at 15,000 U/kg dosage in the reduction of cerebral infarction and cerebral edema, lipid peroxidation and antioxidation; NJF could offer significant neuroprotection in rat models of MCAO induced focal cerebral ischemia; The neuroprotection mechanism shown by NJF may be attributed to inhibition of lipid peroxidation, increase endogenous antioxidant defense enzymes.
In conclusion, we have purified and characterized a novel protease (NJP) from Neanthes japonica (Izuka) which was clearly different from the previous isolated NJF and other fibrinolytic enzymes. NJP is an alkaline thrombin-like serine protease with much higher fibrinolytic activity. It can directly degrade fibrin distinct from the plasminogen activators. Taking account of these results, NJP might be a potential candidate for the thrombosis prevention and thrombolytic therapy; meanwhile, this work is the first evidence that NJF could offer significant neuroprotection in rat models of MCAO induced focal cerebral ischemia, which will provide a basis for further researches about pharmacodynamics and clinical trials of NJF application.
引文
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