基因重组白眉蝮蛇去整合素Adinbitor及其周边序列定点突变体功能的研究
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摘要
本研究室从产于中国旅顺的白眉蝮蛇(Gloydius blomhoffi brevicaudus)毒腺中提取总RNA,利用自行设计的引物进行RT-PCR扩增,获得了219bp的白眉蝮蛇去整合素基因。将去整合素基因进行克隆、转化与诱导后,得到了该蛋白的可溶性高效表达。经组氨酸亲和层析纯化,获得了分子量为9kD的均质蛋白,并将其命名为Adinbitor。经研究证实;Adinbitor具有去整合素的典型生物活性,即可以显著抑制血小板聚集和血管新生。
本研究试图通过对去整合素Adinbitor结构的改造从而改变其功能。本研究室已经获得AdinbitorRGD模体突变成为KGD的突变型Adinbitor(以下简称KGD突变体),并证实其不具有抑制血管新生的功能而仅保留抑制血小板聚集的功能[1]。本研究采用引物延伸PCR的方法获得了Adinbitor的突变序列,经转化、表达和纯化后,得到RGD模体前序列RIA突变成RPT的突变型Adinbitor(以下简称RPT突变体)。经血小板聚集实验初步证实,在与野生型Adinbitor相同浓度下,突变型Adinbitor对血小板聚集的影响很小。而据本室报道,野生型Adinbitor可显著性抑制血小板聚集,其IC50为6μmol/L[2]。为进一步阐明该作用的分子机制,运用流式细胞技术分析野生型及两种突变型Adinbitor对CD41及CD62p抗体与血小板膜糖蛋白整合素αⅡbβ3结合的影响。CD41是αⅡbβ3受体的抗体,它可与静止及活化的αⅡbβ3受体发生特异性结合。流式细胞仪检测结果表明野生型和KGD突变型Adinbitor可抑制PE标记的CD41抗体与αⅡbβ3的结合。这种抑制作用呈现出剂量依赖性。说明野生型Adinbitor和KGD突变体可通过占据αⅡbβ3位点,从而发挥其抑制血小板聚集的功能。而在相同浓度下的RPT突变体的作用下,血小板聚集度几乎没有变化,流氏细胞仪检测结果显示该突变体未明显影响CD41抗体与αⅡbβ3的结合,说明突变后,推测该RPT突变可能引起了Adinbitor空间构象的改变,从而导致Adinbitor与αⅡbβ3的亲和力显著降低。而静止血小板与CD62p结合的检测表明:野生Adinbitor及两种突变型Adinbitor均具有活化血小板的功能。
为研究RIARGD突变成为RPTRGD后Adinbitor是否影响其抑制血管新生功能,用体内实验进一步验证。采用鸡胚绒毛尿囊膜(chicken chorioallantoic membrane,CAM)模型作为本实验的血管生成体内实验模型。运用Image pro plus6.0软件计算新生血管面积与CAM开窗面积的比值,该比值可以比较准确地表现血管新生的情况[3]。本研究室已完成野生型Adinbitor血管新生的研究,证实野生型Adinbitor可显著减少CAM模型新生血管数,血管发生断裂。故本文仅对突变型Adinbitor进行考察,研究结果显示:该突变体可以显著抑制CAM模型的血管新生,血管数明显减少,血管面积与CAM开窗面积的比值随着剂量的加大而减少,阴性对照为14.4%,而加入突变体5μg、20μg、50μg时该比值分别为11.4%、9.4%、6.3%,呈现出剂量依赖性,说明该突变体仍保留有抑制血管新生的功能。
本文还研究了三种Adinbitor对SSMC7721细胞株增殖、凋亡、黏附、迁移以及侵袭的影响。用Transwell检测Adinbitor对SSMC7721细胞迁移的作用;MTT法检测Adinbitor对SSMC7721细胞增殖的作用;Hoechst33258试剂盒检测Adinbitor对SSMC7721凋亡的影响.Transwell和Matrigel检测两种Adinbitor对SSMC7721细胞侵袭的影响。结果显示:三种Adinbitor均可以显著性抑制细胞的增殖、迁移和侵袭,抑制细胞的黏附,促进细胞的凋亡。说明RPT和KGD突变型Adinbitor均保留了野生型Adinbitor所具有的这些生物活性。
为深入研究Adinbitor生物活性的分子机制,我们检测了野生型和RPT突变型Adinbitor对Akt信号转导通路的影响。Western-blotting法检测去整合素Adinbitor对信号分子Akt、p-Akt、NF-κB阻遏蛋白IκB-α、RelA(p65)的影响,分光光度法检测其对caspase-3活性的影响.结果显示Adinbitor可显著抑制SSMC7721细胞的迁移和增殖(与对照组比较,p<0.05),促进凋亡。Akt的磷酸化受到抑制,而细胞浆内IκB-α表达增加,细胞核内RelA(p65)表达减少,说明Adinbitor可通过抑制Akt相关信号转导分子的作用而抑制与Akt相关信号分子相关的一系列生物功能,如增殖、迁移、侵袭、黏附等。
我们同时检测了野生型及RPT突变型Adinbitor促进细胞凋亡的分子机制,Western blotting分析其对SSMC7721细胞p-Caspase-3表达的影响;Caspase-3活性检测试剂盒检测其对p-Caspase-3活性的影响。结果显示:在野生型Adinbitor作用下,胞浆p-Caspase-3含量减少,说明p-Caspase-3被剪切活化成为活性形式。同时活化程度增高,活化倍数在2.24-3.85之间。而RPT突变型Adinbitor亦可使胞浆caspase-3含量减少,活化Caspase-3的倍数在2.22-4.11之间,说明三种Adinbitor均可以通过影响Caspase-3而促进凋亡。
总之,RPT突变型Adinbitor部分或者全部地去除了其对血小板聚集的抑制作用,同时保留了其对血管新生、细胞迁移、增殖、侵袭、黏附和凋亡的作用。以上生物学活性可能通过其对Akt信号通路的抑制而实验。若将其开发成抗肿瘤药物,其最重要的副作用已经去除。本研究为今后的研究奠定了理论和实验基础。
Adinbitor was cloned from Gloydius blomhoffi brevicaudus and character- rized as a novel disintergrin. Total RNA was extracted from venom gland and RT-PCR was used to generate a cDNA, which was 219 bp long. The sequence encoded a polypeptide with 73 amino acids including 12 cysteines and an RGD motif, the signature motif of disintergrin. Recombinant Adinbitor (rAdinbitor) was expressed in E.coli and purified by using the His-Bind affinity chromatography. In previous study, Adinbitor could dose-dependently inhibit ADP-induced human platelet aggregation in human platelet-rich plasma (PRP) system. The IC50 value of Adinbitor for platelet aggregation was 6μmol/L. Furthermore, Adinbitor significantly inhibited angiogenesis both in vivo and in vitro. Taken together, these results suggested that Adinbitor had typical functions of disintegrins.
In our previous study, we constructed the site-directed mutagenesis of RGD motif of Adinbitor to KGD (we called it KGD mutant for short). We found that the KGD mutant could inhibit platelet aggregation but couldn’t inhibit angiogenesis in CAM model. In the present study, we constructed the site-directed mutagenesis of Adibibitor. The sequences RIARGD of Adinbitor was mutated to RPTRGD (we called it RPT mutant for short) by the method based on PCR mutagenesis. We evaluated the platelet aggre- gation of the RPT mutant Adinbitor, the results suggested that it had lost the function of inhibiting platelet aggregation. In order to elucidate the molecular mechanism of three kinds of Adinbitor for platelet aggregation, we evaluated the rate of CD41 binding to GPⅡb/Ⅲa by flow cytometry. Our data demonstrated that both the wild type Adinbitor and KGD mutant could inhibit the binding of anti-CD41 antibody toαⅡbβ3 in a dose-dependent manner with an IC50 of 1.72μmol/L and an IC50 of 1.72μmol/L. The effect of RPT mutant Adinbitor on the subsequent binding of anti-CD41 antibody was negligible at the same concentration. We believed that both the wild type Adinbitor and KGD mutant could competitively occupy the binding site of CD41 which then did not allow the binding for CD41 antibody. By mutating the amino acid residues flanking the RGD motif to RPTRGD, a conforma- tional change in the spatial structure of this motif could be suggested, and which resulted in the remarkable decrease in the affinity for integrinαⅡbβ3.
Angiogenesis, the formation of new capillaries from pre-existing vessels, is involved in the pathogenesis of inflammatory diseases as well as in tumor progression. The CAM model provides an excellent method to investigate the role of a wide spectrum of pro- or antiangiogenic substances. In our study, the RPTRGD Adinbitor mutant inhibited angiogenesis in the CAM model significantly versus the control group, p<0.05. The ratio of the vessel numbers to the total CAM area exposed of the control group was 14.4%, the ratio of 5μg, 20μg and 50μg group were 11.4%, 9.4%, and 6.3%, respectivaly.
The results demonstrated that the mutant Adinbitor had maintained the biological function of inhibiting angiogenesis while it was no longer to inhibit platelet aggregation. This strategy may represent a novel potential anti-tumor agent devoid of the potential side effect on inhibiting blood clotting.
We also explored the effects of three kinds of Adinbitor on cell proliferation, apoptosis, migration, invasion and adhesion. MTT assay was used to detect the effect of the Adinbitors on proliferation. SSMC7721 cells were incubated with different concentrations of Adinbitor and stained by fluorescence dye Hoechst 33258 to observe apoptosis .Transwell was used to explore the effect of three kinds of Adinbitor on cell migration. Transwell and matrigel were used to observe the effect of Adinbitor on cell invasion. Our results showed that three kinds of Adinbitor could inhibit cell proliferation and enhance cell apoptosis. All of them could inhibit cell migration and invasion significantly.
In order to elucidate the mechanism of three kinds of Adinbitor for apoptosis, the amount of caspase-3 was detected by Western blotting. Spectrophotometric method was selected to explore the activity of caspase-3. The results showed that the amounts of caspase-3 in cytoplasm were decreased. The activity of caspase-3 was increased remarkably.
In order to elucidate some of the potential underlying mechanism by which Adinbitor inhibited angiogenesis, we explored the effects of the mutant Adinbitor on the signaling molecules Akt, p-Akt, NF-κB, IκB-α, RelA ( p65 ) NF-κB by Western blotting. NF-κB had previously been demonstrated to have critical role for endothelial cell migration, angio- genesis, and tumor metastasis. The results indicated that the phosphory- lation of Akt was inhibited, the expression of IκB-αin cytoplasm increased, the expression of RelA(p65)in nucleus decreased significantly. Our results suggested that these effects of both the wild type and RPT mutant Adinbitor were at least through Akt, which activated NF-κB. These results should provide clues for a better understanding of the mechanisms underlying angiogenesis.
In summary, the mutation of RIA residues at the flank of RGD to RPT decreased the binding affinity for integrinαⅡbβ3. Wild type Adinbitor’s function of inhibiting platelet aggregation had been eliminated in the mutant Adinbitor by this same mutation. However, the RPT mutant Adinbi- tor could still inhibit angiogenesis possibly through an Akt and NF-κB dependant pathway. Adinbitor as a potential anti-tumor agent would always suffer from the potential undesirable side effect of platelet aggregation inhibition. This experiment lays a ground work for understanding the influence between primary protein structure and the effects on spatial structure in proteins, and the interactions between proteins. In future studies, it may be possible to obtain molecules with a higher specificity for integrinαvβ3 or for integrinαⅡbβ3 by further mutagenesis.
本研究试图通过对去整合素Adinbitor结构的改造从而改变其功能。本研究室已经获得AdinbitorRGD模体突变成为KGD的突变型Adinbitor(以下简称KGD突变体),并证实其不具有抑制血管新生的功能而仅保留抑制血小板聚集的功能[1]。本研究采用引物延伸PCR的方法获得了Adinbitor的突变序列,经转化、表达和纯化后,得到RGD模体前序列RIA突变成RPT的突变型Adinbitor(以下简称RPT突变体)。经血小板聚集实验初步证实,在与野生型Adinbitor相同浓度下,突变型Adinbitor对血小板聚集的影响很小。而据本室报道,野生型Adinbitor可显著性抑制血小板聚集,其IC50为6μmol/L[2]。为进一步阐明该作用的分子机制,运用流式细胞技术分析野生型及两种突变型Adinbitor对CD41及CD62p抗体与血小板膜糖蛋白整合素αⅡbβ3结合的影响。CD41是αⅡbβ3受体的抗体,它可与静止及活化的αⅡbβ3受体发生特异性结合。流式细胞仪检测结果表明野生型和KGD突变型Adinbitor可抑制PE标记的CD41抗体与αⅡbβ3的结合。这种抑制作用呈现出剂量依赖性。说明野生型Adinbitor和KGD突变体可通过占据αⅡbβ3位点,从而发挥其抑制血小板聚集的功能。而在相同浓度下的RPT突变体的作用下,血小板聚集度几乎没有变化,流氏细胞仪检测结果显示该突变体未明显影响CD41抗体与αⅡbβ3的结合,说明突变后,推测该RPT突变可能引起了Adinbitor空间构象的改变,从而导致Adinbitor与αⅡbβ3的亲和力显著降低。而静止血小板与CD62p结合的检测表明:野生Adinbitor及两种突变型Adinbitor均具有活化血小板的功能。
为研究RIARGD突变成为RPTRGD后Adinbitor是否影响其抑制血管新生功能,用体内实验进一步验证。采用鸡胚绒毛尿囊膜(chicken chorioallantoic membrane,CAM)模型作为本实验的血管生成体内实验模型。运用Image pro plus6.0软件计算新生血管面积与CAM开窗面积的比值,该比值可以比较准确地表现血管新生的情况[3]。本研究室已完成野生型Adinbitor血管新生的研究,证实野生型Adinbitor可显著减少CAM模型新生血管数,血管发生断裂。故本文仅对突变型Adinbitor进行考察,研究结果显示:该突变体可以显著抑制CAM模型的血管新生,血管数明显减少,血管面积与CAM开窗面积的比值随着剂量的加大而减少,阴性对照为14.4%,而加入突变体5μg、20μg、50μg时该比值分别为11.4%、9.4%、6.3%,呈现出剂量依赖性,说明该突变体仍保留有抑制血管新生的功能。
本文还研究了三种Adinbitor对SSMC7721细胞株增殖、凋亡、黏附、迁移以及侵袭的影响。用Transwell检测Adinbitor对SSMC7721细胞迁移的作用;MTT法检测Adinbitor对SSMC7721细胞增殖的作用;Hoechst33258试剂盒检测Adinbitor对SSMC7721凋亡的影响.Transwell和Matrigel检测两种Adinbitor对SSMC7721细胞侵袭的影响。结果显示:三种Adinbitor均可以显著性抑制细胞的增殖、迁移和侵袭,抑制细胞的黏附,促进细胞的凋亡。说明RPT和KGD突变型Adinbitor均保留了野生型Adinbitor所具有的这些生物活性。
为深入研究Adinbitor生物活性的分子机制,我们检测了野生型和RPT突变型Adinbitor对Akt信号转导通路的影响。Western-blotting法检测去整合素Adinbitor对信号分子Akt、p-Akt、NF-κB阻遏蛋白IκB-α、RelA(p65)的影响,分光光度法检测其对caspase-3活性的影响.结果显示Adinbitor可显著抑制SSMC7721细胞的迁移和增殖(与对照组比较,p<0.05),促进凋亡。Akt的磷酸化受到抑制,而细胞浆内IκB-α表达增加,细胞核内RelA(p65)表达减少,说明Adinbitor可通过抑制Akt相关信号转导分子的作用而抑制与Akt相关信号分子相关的一系列生物功能,如增殖、迁移、侵袭、黏附等。
我们同时检测了野生型及RPT突变型Adinbitor促进细胞凋亡的分子机制,Western blotting分析其对SSMC7721细胞p-Caspase-3表达的影响;Caspase-3活性检测试剂盒检测其对p-Caspase-3活性的影响。结果显示:在野生型Adinbitor作用下,胞浆p-Caspase-3含量减少,说明p-Caspase-3被剪切活化成为活性形式。同时活化程度增高,活化倍数在2.24-3.85之间。而RPT突变型Adinbitor亦可使胞浆caspase-3含量减少,活化Caspase-3的倍数在2.22-4.11之间,说明三种Adinbitor均可以通过影响Caspase-3而促进凋亡。
总之,RPT突变型Adinbitor部分或者全部地去除了其对血小板聚集的抑制作用,同时保留了其对血管新生、细胞迁移、增殖、侵袭、黏附和凋亡的作用。以上生物学活性可能通过其对Akt信号通路的抑制而实验。若将其开发成抗肿瘤药物,其最重要的副作用已经去除。本研究为今后的研究奠定了理论和实验基础。
Adinbitor was cloned from Gloydius blomhoffi brevicaudus and character- rized as a novel disintergrin. Total RNA was extracted from venom gland and RT-PCR was used to generate a cDNA, which was 219 bp long. The sequence encoded a polypeptide with 73 amino acids including 12 cysteines and an RGD motif, the signature motif of disintergrin. Recombinant Adinbitor (rAdinbitor) was expressed in E.coli and purified by using the His-Bind affinity chromatography. In previous study, Adinbitor could dose-dependently inhibit ADP-induced human platelet aggregation in human platelet-rich plasma (PRP) system. The IC50 value of Adinbitor for platelet aggregation was 6μmol/L. Furthermore, Adinbitor significantly inhibited angiogenesis both in vivo and in vitro. Taken together, these results suggested that Adinbitor had typical functions of disintegrins.
In our previous study, we constructed the site-directed mutagenesis of RGD motif of Adinbitor to KGD (we called it KGD mutant for short). We found that the KGD mutant could inhibit platelet aggregation but couldn’t inhibit angiogenesis in CAM model. In the present study, we constructed the site-directed mutagenesis of Adibibitor. The sequences RIARGD of Adinbitor was mutated to RPTRGD (we called it RPT mutant for short) by the method based on PCR mutagenesis. We evaluated the platelet aggre- gation of the RPT mutant Adinbitor, the results suggested that it had lost the function of inhibiting platelet aggregation. In order to elucidate the molecular mechanism of three kinds of Adinbitor for platelet aggregation, we evaluated the rate of CD41 binding to GPⅡb/Ⅲa by flow cytometry. Our data demonstrated that both the wild type Adinbitor and KGD mutant could inhibit the binding of anti-CD41 antibody toαⅡbβ3 in a dose-dependent manner with an IC50 of 1.72μmol/L and an IC50 of 1.72μmol/L. The effect of RPT mutant Adinbitor on the subsequent binding of anti-CD41 antibody was negligible at the same concentration. We believed that both the wild type Adinbitor and KGD mutant could competitively occupy the binding site of CD41 which then did not allow the binding for CD41 antibody. By mutating the amino acid residues flanking the RGD motif to RPTRGD, a conforma- tional change in the spatial structure of this motif could be suggested, and which resulted in the remarkable decrease in the affinity for integrinαⅡbβ3.
Angiogenesis, the formation of new capillaries from pre-existing vessels, is involved in the pathogenesis of inflammatory diseases as well as in tumor progression. The CAM model provides an excellent method to investigate the role of a wide spectrum of pro- or antiangiogenic substances. In our study, the RPTRGD Adinbitor mutant inhibited angiogenesis in the CAM model significantly versus the control group, p<0.05. The ratio of the vessel numbers to the total CAM area exposed of the control group was 14.4%, the ratio of 5μg, 20μg and 50μg group were 11.4%, 9.4%, and 6.3%, respectivaly.
The results demonstrated that the mutant Adinbitor had maintained the biological function of inhibiting angiogenesis while it was no longer to inhibit platelet aggregation. This strategy may represent a novel potential anti-tumor agent devoid of the potential side effect on inhibiting blood clotting.
We also explored the effects of three kinds of Adinbitor on cell proliferation, apoptosis, migration, invasion and adhesion. MTT assay was used to detect the effect of the Adinbitors on proliferation. SSMC7721 cells were incubated with different concentrations of Adinbitor and stained by fluorescence dye Hoechst 33258 to observe apoptosis .Transwell was used to explore the effect of three kinds of Adinbitor on cell migration. Transwell and matrigel were used to observe the effect of Adinbitor on cell invasion. Our results showed that three kinds of Adinbitor could inhibit cell proliferation and enhance cell apoptosis. All of them could inhibit cell migration and invasion significantly.
In order to elucidate the mechanism of three kinds of Adinbitor for apoptosis, the amount of caspase-3 was detected by Western blotting. Spectrophotometric method was selected to explore the activity of caspase-3. The results showed that the amounts of caspase-3 in cytoplasm were decreased. The activity of caspase-3 was increased remarkably.
In order to elucidate some of the potential underlying mechanism by which Adinbitor inhibited angiogenesis, we explored the effects of the mutant Adinbitor on the signaling molecules Akt, p-Akt, NF-κB, IκB-α, RelA ( p65 ) NF-κB by Western blotting. NF-κB had previously been demonstrated to have critical role for endothelial cell migration, angio- genesis, and tumor metastasis. The results indicated that the phosphory- lation of Akt was inhibited, the expression of IκB-αin cytoplasm increased, the expression of RelA(p65)in nucleus decreased significantly. Our results suggested that these effects of both the wild type and RPT mutant Adinbitor were at least through Akt, which activated NF-κB. These results should provide clues for a better understanding of the mechanisms underlying angiogenesis.
In summary, the mutation of RIA residues at the flank of RGD to RPT decreased the binding affinity for integrinαⅡbβ3. Wild type Adinbitor’s function of inhibiting platelet aggregation had been eliminated in the mutant Adinbitor by this same mutation. However, the RPT mutant Adinbi- tor could still inhibit angiogenesis possibly through an Akt and NF-κB dependant pathway. Adinbitor as a potential anti-tumor agent would always suffer from the potential undesirable side effect of platelet aggregation inhibition. This experiment lays a ground work for understanding the influence between primary protein structure and the effects on spatial structure in proteins, and the interactions between proteins. In future studies, it may be possible to obtain molecules with a higher specificity for integrinαvβ3 or for integrinαⅡbβ3 by further mutagenesis.
引文
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