骆驼蓬种子凝集素的分离纯化及其结构分析和生物活性研究
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摘要
[背景及目的]凝集素是一类能选择性地、非共价可逆结合细胞表面糖链的蛋白质或糖结合蛋白。研究发现许多植物、动物等生物细胞膜上、细胞质内和细胞外基质中都存在凝集素。其最大的特点在于它们能识别糖蛋白和糖脂,特别是细胞膜中复杂结构的糖链,即细胞膜表面决定簇。目前,已从多种植物中分离到许多不同类型的凝集素。一种凝集素具有对一种糖链专一性结合的能力并具有促使细胞凝集、细胞有丝分裂、防御昆虫危害、消除入侵的微生物等生理功能。目前临床医学将它主要用于构成免疫毒素和作为有效的免疫佐剂以及靶向性运载工具等。
骆驼蓬(Peganum harmala L.)是蒺藜科骆驼蓬属的一种多年生草本有毒植物。主要分布于甘肃、新疆、陕西、山西等地区的荒漠、沙质、干旱草地等地带,自然资源相当丰富。维吾尔医学常将其用于关节骨痛、跌打损伤、震颤麻痹、半身不遂等病症的治疗。目前研究骆驼蓬的活性成分主要是生物碱、黄酮等小分子物质。对其蛋白质和凝集素的研究报道较少。
本研究在前期研究的基础上,以新疆地区特有的维药—骆驼蓬为研究对象,从其凝集素粗品提取到体外活性高的筛选作为起点,通过生物活性跟踪,分离纯化具有凝血、抑菌及癌活性的天然蛋白凝集素纯品,并对其进行结构分析和蛋白质性质检测。为抗菌及抗癌新药的研究开发提供科学依据和基础资料。
[方法]采用硫酸铵分级沉淀和Resource S阳离子交换层析和Superdex 75凝胶过滤层析相结合的方法分离纯化骆驼蓬种子凝集素,并对其进行凝血活性、抑菌活性及抑癌活性检测。纯化出的凝集素N-末端采用Edman降解法进行氨基酸测序,分别使用肽质量指纹图谱(PMF)和圆二色谱法(CD)进行一级和二级结构分析,并测定其表观分子量、糖含量、等电点等蛋白质性质及稳定性和糖专一性。
[结果]从骆驼蓬种子中得到一种纯度较高的凝集素(PHL-1),它由两条相同的链组成,其单链分子量为7.812kD,其N-端前7个氨基酸序列为:缬氨酸-苏氨酸-半胱氨酸-天冬酰胺-脯氨酸-苏氨酸-谷氨酸;它的糖含量为10.05%;等电点为8.43。它对小鼠红细胞的最低凝集浓度为:0.001mg/mL,并且其凝集活性在酸碱、温度及金属离子的作用下很稳定;在蔗糖和阿拉伯糖溶液中,其凝集活力最小。同时实验结果还显示,当PHL-1浓度为0.254mg/mL时对鲍曼不动杆菌和意大利青霉菌的抑菌环半径分别为6.3mm、5.8mm;0.5 mg/mL时对B16细胞作用48h,抑制率为76.06%,其IC50为0.22mg/mL;肽质量指纹图谱搜索到7个具有显著性意义的蛋白结果;CD图谱结果显示其β-折叠含量高于80%,无规则卷曲含量高于17%,α-螺旋和β-转角的含量很少。
[结论]建立了硫酸铵分级沉淀和Resource S阳离子交换层析和Superdex 75凝胶过滤层析相结合的三步法分离纯化骆驼蓬种子凝集素的技术路线。该蛋白有两条相同的亚基组成,其单链表观分子量为7.812kD,是一个凝集活性较好和纯度较高的蛋白产物。骆驼蓬种子凝集素PHL-1是一种碱性糖蛋白,它对小鼠红细胞有很强的凝集活性,同时具有一定的抗菌和抗癌活性。其二级结构具有较好的热稳定性和酸碱稳定性。
[Background and purpose] Lectin is one kind of protein or carbohydrate-binding protein which can selectivitily bind carbohydrate-chain of cell surface. It is generally reside in cell envelope, intracytoplasm, extracellular matrix of plants and animals, which can bind with carbohydrate specificitily, no-covalence and reversibly. The remarkably characteristic of lectin is able to recognize glucoprotein and glucolipid especially the complex structural of carbohydrate-chain in cell envelope. At present, some different kinds of plant lectins have been isolated from lots of plants. One lectin has the ability of binding one carbohydrate-chain specificitily and some physiological functions, for example: precipitate cell agglutination, caryocinesia, defend disservice of inserts, eliminate the invadition of microorganism and so on. At present lectins are used to construct immunotoxin, conduct available immunoadjuvant, and target vehicles in clinical medicine.
Peganum harmala L. is poisonous plant of perennial herb which belongs to Zygophyllaceae, Peganum L. It is mainly distribute over deserta, sand, drought and other areas of GanSu, XingJing, ShanXi of China. The natural Peganum harmala L. resources are very plentiful. It can be used to treat the pain of ossa articularia、injury from fall, chorea festinans, hemiparalysis in Vygur’s medicine. At present, the main study of active component from Peganum harmala L. is to discuss its active component, such as alkaloid, flavone which are small muscular materials. However, there is few report about its protein and lectin study.
The purpose of this study was to isolate and purificate one lectin with hemagglutination, antibacterial activity and anti-tumor activity by tracking the biological activity on the basic of the prior study from the Peganum harmala L. which is the especial Uygur medicine. Then analysis it,s structure and approach the protein physical chemistry. That would be provide science evidence and fundament date for the exploitation of new antibacterial and anti-tumor medicine.
[Method] The lectin of Peganum harmala L. seeds had been isolated and purificated through the stepwise addition of saturated ammonium sulfate, Resource S cation-exchange chromatography and Superdex 75 gel filtration chromatography and then it,s hemagglutination, antimicrobial and anti-tumor activity had been detected. The amino acid sequence of N-extrem of pure lectin were determined with Edman degradation; It,s primary structure and secondary structure had been identified separately use finger printfinger printing and circular dichroism spectra. At the same time the apparent molecular weight of subunit, saccharinity,isoelectric point and stability, sugar specificity had been determined.
[Result] We got a pure lectin from Peganum harmala L. seeds(PHL-1) that was homodimer with the apparent molecular weight of subunit is 7.812kD; the sequence of first 7 amino acid was Val-Thr-Cys-Asn-Pro-Thr-Glu. It,s saccharinity was 10.05% and isoelectric poin were 8.43. The lowest concentration of hemagglutination was 0.001mg/mL towared mouse red blood cell. The agglutination was very stable under the effect of acid base、temperature and metal ion. In the solution of saccharobiose and arabopyranose it has the smallest agglutination. The experimental result also showed the radii of antibacterial ring on Acinetobacter baumannii and Penicillium italicum were up to 6.3mm and 5.8mm respectively when the concentration of PHL-1 was 0.254mg/mL; After treat 48h the inhibition ratio of chromatophoroma B16 is 76.06% with 0.5mg/mL and the IC50 value is 0.22mg/mL; The seven significative proteins were got by PMF. The result of circular dichroism analysis showed the content ofβ-folding and random was up to 80% and 17% separately, while with little content ofα-helico andβ-turn.
[Conclusion] Established the technique line of stepwise addition of saturated ammonium sulfate, Resource S cation-exchange chromatography and Superdex 75 gel filtration chromatography which purificated one lectin from the Peganum harmala L.seeds. The protein has good agglutination and high purity, it was compact with two same subunits, the apparent molecular weight of single strand was 7.812kD. PHL-1 was one kind of alkalinity glycosidoprotein and has strong agglutination towards mouse red blood cell, it would be inhibit the growth of microbion and chromatophoroma B16. The secondary structure has high stability base toward thermal and acid.
骆驼蓬(Peganum harmala L.)是蒺藜科骆驼蓬属的一种多年生草本有毒植物。主要分布于甘肃、新疆、陕西、山西等地区的荒漠、沙质、干旱草地等地带,自然资源相当丰富。维吾尔医学常将其用于关节骨痛、跌打损伤、震颤麻痹、半身不遂等病症的治疗。目前研究骆驼蓬的活性成分主要是生物碱、黄酮等小分子物质。对其蛋白质和凝集素的研究报道较少。
本研究在前期研究的基础上,以新疆地区特有的维药—骆驼蓬为研究对象,从其凝集素粗品提取到体外活性高的筛选作为起点,通过生物活性跟踪,分离纯化具有凝血、抑菌及癌活性的天然蛋白凝集素纯品,并对其进行结构分析和蛋白质性质检测。为抗菌及抗癌新药的研究开发提供科学依据和基础资料。
[方法]采用硫酸铵分级沉淀和Resource S阳离子交换层析和Superdex 75凝胶过滤层析相结合的方法分离纯化骆驼蓬种子凝集素,并对其进行凝血活性、抑菌活性及抑癌活性检测。纯化出的凝集素N-末端采用Edman降解法进行氨基酸测序,分别使用肽质量指纹图谱(PMF)和圆二色谱法(CD)进行一级和二级结构分析,并测定其表观分子量、糖含量、等电点等蛋白质性质及稳定性和糖专一性。
[结果]从骆驼蓬种子中得到一种纯度较高的凝集素(PHL-1),它由两条相同的链组成,其单链分子量为7.812kD,其N-端前7个氨基酸序列为:缬氨酸-苏氨酸-半胱氨酸-天冬酰胺-脯氨酸-苏氨酸-谷氨酸;它的糖含量为10.05%;等电点为8.43。它对小鼠红细胞的最低凝集浓度为:0.001mg/mL,并且其凝集活性在酸碱、温度及金属离子的作用下很稳定;在蔗糖和阿拉伯糖溶液中,其凝集活力最小。同时实验结果还显示,当PHL-1浓度为0.254mg/mL时对鲍曼不动杆菌和意大利青霉菌的抑菌环半径分别为6.3mm、5.8mm;0.5 mg/mL时对B16细胞作用48h,抑制率为76.06%,其IC50为0.22mg/mL;肽质量指纹图谱搜索到7个具有显著性意义的蛋白结果;CD图谱结果显示其β-折叠含量高于80%,无规则卷曲含量高于17%,α-螺旋和β-转角的含量很少。
[结论]建立了硫酸铵分级沉淀和Resource S阳离子交换层析和Superdex 75凝胶过滤层析相结合的三步法分离纯化骆驼蓬种子凝集素的技术路线。该蛋白有两条相同的亚基组成,其单链表观分子量为7.812kD,是一个凝集活性较好和纯度较高的蛋白产物。骆驼蓬种子凝集素PHL-1是一种碱性糖蛋白,它对小鼠红细胞有很强的凝集活性,同时具有一定的抗菌和抗癌活性。其二级结构具有较好的热稳定性和酸碱稳定性。
[Background and purpose] Lectin is one kind of protein or carbohydrate-binding protein which can selectivitily bind carbohydrate-chain of cell surface. It is generally reside in cell envelope, intracytoplasm, extracellular matrix of plants and animals, which can bind with carbohydrate specificitily, no-covalence and reversibly. The remarkably characteristic of lectin is able to recognize glucoprotein and glucolipid especially the complex structural of carbohydrate-chain in cell envelope. At present, some different kinds of plant lectins have been isolated from lots of plants. One lectin has the ability of binding one carbohydrate-chain specificitily and some physiological functions, for example: precipitate cell agglutination, caryocinesia, defend disservice of inserts, eliminate the invadition of microorganism and so on. At present lectins are used to construct immunotoxin, conduct available immunoadjuvant, and target vehicles in clinical medicine.
Peganum harmala L. is poisonous plant of perennial herb which belongs to Zygophyllaceae, Peganum L. It is mainly distribute over deserta, sand, drought and other areas of GanSu, XingJing, ShanXi of China. The natural Peganum harmala L. resources are very plentiful. It can be used to treat the pain of ossa articularia、injury from fall, chorea festinans, hemiparalysis in Vygur’s medicine. At present, the main study of active component from Peganum harmala L. is to discuss its active component, such as alkaloid, flavone which are small muscular materials. However, there is few report about its protein and lectin study.
The purpose of this study was to isolate and purificate one lectin with hemagglutination, antibacterial activity and anti-tumor activity by tracking the biological activity on the basic of the prior study from the Peganum harmala L. which is the especial Uygur medicine. Then analysis it,s structure and approach the protein physical chemistry. That would be provide science evidence and fundament date for the exploitation of new antibacterial and anti-tumor medicine.
[Method] The lectin of Peganum harmala L. seeds had been isolated and purificated through the stepwise addition of saturated ammonium sulfate, Resource S cation-exchange chromatography and Superdex 75 gel filtration chromatography and then it,s hemagglutination, antimicrobial and anti-tumor activity had been detected. The amino acid sequence of N-extrem of pure lectin were determined with Edman degradation; It,s primary structure and secondary structure had been identified separately use finger printfinger printing and circular dichroism spectra. At the same time the apparent molecular weight of subunit, saccharinity,isoelectric point and stability, sugar specificity had been determined.
[Result] We got a pure lectin from Peganum harmala L. seeds(PHL-1) that was homodimer with the apparent molecular weight of subunit is 7.812kD; the sequence of first 7 amino acid was Val-Thr-Cys-Asn-Pro-Thr-Glu. It,s saccharinity was 10.05% and isoelectric poin were 8.43. The lowest concentration of hemagglutination was 0.001mg/mL towared mouse red blood cell. The agglutination was very stable under the effect of acid base、temperature and metal ion. In the solution of saccharobiose and arabopyranose it has the smallest agglutination. The experimental result also showed the radii of antibacterial ring on Acinetobacter baumannii and Penicillium italicum were up to 6.3mm and 5.8mm respectively when the concentration of PHL-1 was 0.254mg/mL; After treat 48h the inhibition ratio of chromatophoroma B16 is 76.06% with 0.5mg/mL and the IC50 value is 0.22mg/mL; The seven significative proteins were got by PMF. The result of circular dichroism analysis showed the content ofβ-folding and random was up to 80% and 17% separately, while with little content ofα-helico andβ-turn.
[Conclusion] Established the technique line of stepwise addition of saturated ammonium sulfate, Resource S cation-exchange chromatography and Superdex 75 gel filtration chromatography which purificated one lectin from the Peganum harmala L.seeds. The protein has good agglutination and high purity, it was compact with two same subunits, the apparent molecular weight of single strand was 7.812kD. PHL-1 was one kind of alkalinity glycosidoprotein and has strong agglutination towards mouse red blood cell, it would be inhibit the growth of microbion and chromatophoroma B16. The secondary structure has high stability base toward thermal and acid.
引文
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