灵芝免疫调节蛋白（rLZ-8）亚细胞作用位点和抗肿瘤功能结构域的研究
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摘要
本研究利用基因重组表达技术构建了rLZ-8真核表达载体,电转化毕赤酵母Gs115后,筛选出稳定高效分泌表达的rLZ-8工程菌株,结合阳离子交换色谱和凝胶色谱技术纯化得到了纯度很高的rLZ-8。在体外实验中发现灵芝免疫调节蛋白可以直接杀伤人白血病肿瘤细胞,这种机制并非是之前研究人员推测的免疫调节的间接杀伤方式。通过激光扫描共聚焦显微镜等研究手段,研究发现rLZ-8可富集在细胞核上,并可以触发胞内钙池的释放,引起肿瘤细胞发生程序性死亡,推测rLZ-8触发NB4人急性早幼粒白血病细胞发生程序性死亡的位点是细胞核。
本研究对rLZ-8进行了晶体培养,通过X-ray的方法得到了一套1.8(A|°)的数据,解析得到了rLZ-8的晶体结构,将之与同一家族氨基酸序列相近却不具有抗肿瘤活性的FIP-fve蛋白的晶体结构进行了比对,发现主要的不同在于两个方面:(1)rLZ-8形成二聚体时,与N端α螺旋相互作用的环状结构域;(2)C端的FNIII结构上有5个疏水性差异较大的氨基酸,它们可能会与肿瘤细胞表面过度卷曲的糖链结合。综合以上比对的结果,决定设计去掉N端α螺旋的蛋白突变体,可以使形成二聚体时的环状结构与α螺旋形成的结构域消失,并观察4个疏水性很强的氨基酸对抗肿瘤活性的影响,同时还可以研究对rLZ-8免疫调节功能很重要的二聚体结构是否也是其抗肿瘤活性的结构基础,实验结果表明这个蛋白突变体仍具有抗肿瘤活性。
本实验的创新之处在于:1)发现了rLZ-8诱导NB4人急性早幼粒白血病细胞发生程序性细胞死亡;2)发现了rLZ-8的晶体结构;3)发现了rLZ-8在肿瘤细胞内的杀伤位点和其自身的抗肿瘤结构域。
Malignant tumor is a great threat to human health and a frequently-occurring disease, there is no particular effective control strategies so far. In recent years, with the development of cell biology, biophysics, molecular pharmacology, as well as clarification to the nature of tumor, large-scale, rapid screening of combinatorial chemistry, genetic engineering and other advanced technologies speed up the study and application of drug development process. In the field of Cell Biology, start or activate the tumor cell apoptosis mechanism in different ways to influence the different gene expression in cells to induce apoptosis of tumor cells via specific organelle. In the biophysics research, crystal structures of many key protein factor in cell death process have been revealed and some anti-tumor domains have been identified, investigating on relationship of crystal structure and function has become the most hot and important biological field.
This study project will show and discuss all the experiment results about Fungal Immunoregulatory Protein family, nuclear medicated cell death and the relationship of crystal structure and function of Ganoderma lucidum Immunoregulatory Protein.
1. The study of recombinant Ganoderma Lucidum Immunoregulatory Protein (rLZ-8)
Ganoderma lucidum Immunoregulatory Protein, the separation and purification of small-molecule protein from the extractive of Ganoderma mycelium was done by a Japanese Kino in 1989 (Kohsuke Kino et al., J. Bio. Chem. 1989, 1:472-478), it was named LZ-8, its amino acid sequence and physiological activity of immunity was also tested which indicates that the sequence of protein of LZ-8 is made up of 110 amino acid, serine acetylation on N-terminal, the molecular weight is 12.4 KD, and the isoelectric point is 4.4. In this study, LZ-8 was recombinantly expressed in Pichia pastoris, and production of rLZ-8 was larged to fermentation scale. The major function of Ganoderma Lucidum Immune Regulatory Protein lies in that it promotes the hyperplasia of peripheral lymphocytes and spleen cells, induces the macrophage both in human and animals to secrete various kinds of cell factors (as in interleukin, necrosis factor in tumors and interferon, etc.), and defending and dispelling the infringement of the causative agent, safeguards and maintains the health and to achieve the immune regulatory function.
2. The study of rLZ-8’s anti-tumor effects and subcellular localization
Previous studies have shown that, LZ-8 achieves the anti-tumor effect mainly through immune regulation. However, the positive effects of this study is that it discussed the rLZ-8 through specific binding with the tumor cell membrane for the first time, it induces apoptosis direct destruction or kills tumor cells to achieve the anti-tumor effects, at the same time, unlike reducing leukocytes chemotherapy drugs, without affecting normal cells.
It is found that rLZ-8 could localize in nuclear of Human Leukemia cells (NB4) and trigger cell death. Observing under Laser Scan Confocal Microscope (LSCM), FITC-RLZ-8 and nuclear occupied the same relative position or the same area in space of the cells, it means that nuclear may be subcellular binding site of rLZ-8.
In our research, it also was checked that rLZ-8 behaved as a Ca~(2+) release inducer, Ca~(2+) stores were mainly found in nuclear and ER, which all supported our previous studies. As we known, Ca~(2+) signal is one of important pathways in the cell death.
3. The study of rLZ-8’s crystal structure and anti-tumor domain
To elucidate mechanism of rLZ-8 inducing tumor cells death further, some crystal structure researches were carried out in this study, and designed and expressed mutants of LZ-8 on the basis of pharmacology and structure again, then examine the activities of mutants. All kinds of work were to search and identify the most important domain of rLZ-8 for killing tumor cells. In 2003 and 2008, Paaventhan and our group presented 1.7 ? X-ray structure of FIP-fve and 1.8 ? X-ray structure of LZ-8, determined by single anomalous diffraction (SAD) using the anomalous signal of bromide ions present in the crystal for phasing.
The overall fold of LZ-8 resembles the structure of FIP-fve from F. velutipes, which consists of an N-terminal dimerization domain and a C-terminal FNIII domain. The N-terminal domain is composed of anα-helix and aβ-strand that sustains the dimerization via domain swapping, forming a dumb-bell-shaped dimer. The C-terminal FNIII domain belongs to the immunoglobulin-like-sandwich fold and comprises a sandwich structure of twoβ-sheets (I and II) formed byβ-strands A-B-E and G-F-C-D, respectively To our knowledge, the crystal structure of LZ-8 is the second structure of fungal immunomodulatory proteins to date.
Whether the formation of homodimers would also attributed to LZ-8’s anti-tumor activity is worth to discuss, because FIP-fve has nearly the same N-terminalα-helix as LZ-8, but has no anti-tumor active. To survey the‘hotspots’for the anti-tumor activity, a structure-based multi-alignment was performed. The N-terminalα-helix of LZ-8 and FIP-fve are quite closely similar from alignment results, it prompts us that the N-terminalα-helix may not associate with anti-tumor. The conformations of the loops in the FNIII domain of LZ-8 may be a starting point for elucidating the mechanism of specific residues responsible it’s the anti-tumor activity. The comparative structural analysis also provided us some clues to improve.
Dealing with the relationship of structure and bio-chemistry have been widely studied and used, but associating structure with cell biology is still a problem. In further researches, we hope that more functional domain of FIPs will be found, it will lay a solid foundation for future studies.
本研究对rLZ-8进行了晶体培养,通过X-ray的方法得到了一套1.8(A|°)的数据,解析得到了rLZ-8的晶体结构,将之与同一家族氨基酸序列相近却不具有抗肿瘤活性的FIP-fve蛋白的晶体结构进行了比对,发现主要的不同在于两个方面:(1)rLZ-8形成二聚体时,与N端α螺旋相互作用的环状结构域;(2)C端的FNIII结构上有5个疏水性差异较大的氨基酸,它们可能会与肿瘤细胞表面过度卷曲的糖链结合。综合以上比对的结果,决定设计去掉N端α螺旋的蛋白突变体,可以使形成二聚体时的环状结构与α螺旋形成的结构域消失,并观察4个疏水性很强的氨基酸对抗肿瘤活性的影响,同时还可以研究对rLZ-8免疫调节功能很重要的二聚体结构是否也是其抗肿瘤活性的结构基础,实验结果表明这个蛋白突变体仍具有抗肿瘤活性。
本实验的创新之处在于:1)发现了rLZ-8诱导NB4人急性早幼粒白血病细胞发生程序性细胞死亡;2)发现了rLZ-8的晶体结构;3)发现了rLZ-8在肿瘤细胞内的杀伤位点和其自身的抗肿瘤结构域。
Malignant tumor is a great threat to human health and a frequently-occurring disease, there is no particular effective control strategies so far. In recent years, with the development of cell biology, biophysics, molecular pharmacology, as well as clarification to the nature of tumor, large-scale, rapid screening of combinatorial chemistry, genetic engineering and other advanced technologies speed up the study and application of drug development process. In the field of Cell Biology, start or activate the tumor cell apoptosis mechanism in different ways to influence the different gene expression in cells to induce apoptosis of tumor cells via specific organelle. In the biophysics research, crystal structures of many key protein factor in cell death process have been revealed and some anti-tumor domains have been identified, investigating on relationship of crystal structure and function has become the most hot and important biological field.
This study project will show and discuss all the experiment results about Fungal Immunoregulatory Protein family, nuclear medicated cell death and the relationship of crystal structure and function of Ganoderma lucidum Immunoregulatory Protein.
1. The study of recombinant Ganoderma Lucidum Immunoregulatory Protein (rLZ-8)
Ganoderma lucidum Immunoregulatory Protein, the separation and purification of small-molecule protein from the extractive of Ganoderma mycelium was done by a Japanese Kino in 1989 (Kohsuke Kino et al., J. Bio. Chem. 1989, 1:472-478), it was named LZ-8, its amino acid sequence and physiological activity of immunity was also tested which indicates that the sequence of protein of LZ-8 is made up of 110 amino acid, serine acetylation on N-terminal, the molecular weight is 12.4 KD, and the isoelectric point is 4.4. In this study, LZ-8 was recombinantly expressed in Pichia pastoris, and production of rLZ-8 was larged to fermentation scale. The major function of Ganoderma Lucidum Immune Regulatory Protein lies in that it promotes the hyperplasia of peripheral lymphocytes and spleen cells, induces the macrophage both in human and animals to secrete various kinds of cell factors (as in interleukin, necrosis factor in tumors and interferon, etc.), and defending and dispelling the infringement of the causative agent, safeguards and maintains the health and to achieve the immune regulatory function.
2. The study of rLZ-8’s anti-tumor effects and subcellular localization
Previous studies have shown that, LZ-8 achieves the anti-tumor effect mainly through immune regulation. However, the positive effects of this study is that it discussed the rLZ-8 through specific binding with the tumor cell membrane for the first time, it induces apoptosis direct destruction or kills tumor cells to achieve the anti-tumor effects, at the same time, unlike reducing leukocytes chemotherapy drugs, without affecting normal cells.
It is found that rLZ-8 could localize in nuclear of Human Leukemia cells (NB4) and trigger cell death. Observing under Laser Scan Confocal Microscope (LSCM), FITC-RLZ-8 and nuclear occupied the same relative position or the same area in space of the cells, it means that nuclear may be subcellular binding site of rLZ-8.
In our research, it also was checked that rLZ-8 behaved as a Ca~(2+) release inducer, Ca~(2+) stores were mainly found in nuclear and ER, which all supported our previous studies. As we known, Ca~(2+) signal is one of important pathways in the cell death.
3. The study of rLZ-8’s crystal structure and anti-tumor domain
To elucidate mechanism of rLZ-8 inducing tumor cells death further, some crystal structure researches were carried out in this study, and designed and expressed mutants of LZ-8 on the basis of pharmacology and structure again, then examine the activities of mutants. All kinds of work were to search and identify the most important domain of rLZ-8 for killing tumor cells. In 2003 and 2008, Paaventhan and our group presented 1.7 ? X-ray structure of FIP-fve and 1.8 ? X-ray structure of LZ-8, determined by single anomalous diffraction (SAD) using the anomalous signal of bromide ions present in the crystal for phasing.
The overall fold of LZ-8 resembles the structure of FIP-fve from F. velutipes, which consists of an N-terminal dimerization domain and a C-terminal FNIII domain. The N-terminal domain is composed of anα-helix and aβ-strand that sustains the dimerization via domain swapping, forming a dumb-bell-shaped dimer. The C-terminal FNIII domain belongs to the immunoglobulin-like-sandwich fold and comprises a sandwich structure of twoβ-sheets (I and II) formed byβ-strands A-B-E and G-F-C-D, respectively To our knowledge, the crystal structure of LZ-8 is the second structure of fungal immunomodulatory proteins to date.
Whether the formation of homodimers would also attributed to LZ-8’s anti-tumor activity is worth to discuss, because FIP-fve has nearly the same N-terminalα-helix as LZ-8, but has no anti-tumor active. To survey the‘hotspots’for the anti-tumor activity, a structure-based multi-alignment was performed. The N-terminalα-helix of LZ-8 and FIP-fve are quite closely similar from alignment results, it prompts us that the N-terminalα-helix may not associate with anti-tumor. The conformations of the loops in the FNIII domain of LZ-8 may be a starting point for elucidating the mechanism of specific residues responsible it’s the anti-tumor activity. The comparative structural analysis also provided us some clues to improve.
Dealing with the relationship of structure and bio-chemistry have been widely studied and used, but associating structure with cell biology is still a problem. In further researches, we hope that more functional domain of FIPs will be found, it will lay a solid foundation for future studies.
引文
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[2] Xiang Y, Zhu Z, Han G. JARID1B is a histone H3 lysine 4 demethylase up-regulated in prostate cancer[J]. Proc Natl Acad Sci . 2007 (49) 104:19226-31.
[3] Tanaka S. Kino. etal. Complete amino acid sequence of an immunomodulatory protein, Ling Zhi-8. An immunomodulator from a fungus, Ganoderma lucidum, having similarity to immunoglobulin variable regions [J]. Journal of Biochemistry, 1989; 264:16372.
[4] Murasugi A. et.al. Molecular cloning of a cDNA and a gene encoding an immunomodulatory protein, Ling-Zhi-8, from a fungus, Ganoderma lucidum [J]. J.Biol.Chem, 1991; 266:2486.
[5] Wang PH, Yang SF, Chen GD. Human nonmetastatic clone 23 type 1 gene suppresses migration of cervical cancer cells and enhances the migration inhibition of fungal immunomodulatory protein from Ganoderma tsugae[J]. Reprod Sci. 2007 (5) 14:475-85.
[6] Jinn TR, Wu CM, Tu WC. Functional expression of FIP-gts, a fungal immunomodulatory protein from Ganoderma tsugae in Sf21 insect cells[J]. Biosci Biotechnol Biochem. 2006 (11)70:2627-34.
[7] Kam Ming Ko and Hoi Yan Leung. Enhancement of ATP generation capacity, antioxidant activity and immunomodulatory activities by Chinese Yang and Yin tonifying herbs[J]. Chinese Medicine. 2007(2)3:1-10.
[8] Wu CM, Wu TY, Kao SS. Expression and purification of a recombinant Fip-fve protein from Flammulina velutipes in baculovirus-infected insect cells[J]. J Appl Microbiol. 2008 Feb 4 [Epub ahead of print].
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