力生长因子C端E肽的理化性质和功能研究
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摘要
力生长因子(mechano growth factor, MGF)是力效应细胞在力刺激下,胰岛素样生长因子Ⅰ(insulin-like growth factor, IGF-I)基因选择性剪接产生的由110个氨基酸组成的碱性多肽。MGF不仅是一种生长因子,也是一种修复因子。MGF在维持局部组织质量和修复组织的过程中起重要作用。
IGF-I基因通过选择性剪切产生两种异构体：IGF-I Ea和MGF。MGF的序列与成熟IGF-I和IGF-I Ea相比,除都拥有A,B,C和D结构域外,其主要差别是C端的E结构域。MGF比IGF-I C端多一个含40个氨基酸的E结构域；MGF和IGF-IEa的主要区别在于E结构域C端的24个氨基酸。研究发现MGF与成熟IGF-I和IGF-IEa的促细胞增殖和分化的功能不同,它的作用是促细胞增殖,但是抑制细胞分化,这一区别可能与它的C端E结构域有关。本研究拟通过基因工程手段对MGF和MGF的C端E结构域(又称E肽)进行体外表达纯化,并对它们的聚合性质和功能进行研究。研究内容包括以下三部分：
第一部分：MGF及其E肽的表达和纯化。分别构建pRSETc-MGF、pRSETc-MGFc40和pGEX-6P-1-MGFc24重组表达质粒。将重组质粒转化于E. coli BL21(DE3)中进行诱导表达。经Tris-Tricine SDS PAGE分析,融合蛋白His-MGF, His-MGFc40和GST-MGFc24均以可溶形式表达。表达产物分别通过His60 Ni SuperflowTM Resin和Glutathione sepharose 4B亲和层析纯化得到了纯的His-MGF, His-MGFc40和MGFc24蛋白。
第二部分：MGF自我聚合的特征研究。在上述实验中我们发现纯化的MGF及E肽易发生聚合现象,本部分实验利用体外pull down技术研究MGF与MGF, MGF的N端和MGF的C端E肽的相互作用。结果表明MGF能够和自身发生相互作用；MGF C端的E肽自身能发生相互作用；MGF的N端能与MGF C端24个氨基酸多肽发生相互作用；MGF的N端之间没有相互作用。因此,MGF的聚合很可能通过其C端E肽之间以及C端E肽与N端之间的相互作用进行。
第三部分：MGF及其E肽对C2C12成肌细胞的作用研究。本部分实验用纯化得到的His-MGF, His-MGFc40和MGFc24作用小鼠C2C12成肌细胞,分别用CCK-8法和肌酸激酶(Creatine kinase, CK)法检测它们对小鼠成肌细胞C2C12增殖和分化的作用；然后通过检测它们作用后细胞内游离钙离子浓度及胞外信号调节激酶(ERK1/2)的激活作用。结果表明His-MGF, His-MGFc40和MGFc24具有促进C2C12细胞增殖和抑制其分化的活性。His-MGF, His-MGFc40和MGFc24作用于C2C12细胞能够提高细胞内游离钙离子的浓度。His-MGF和His-MGFc40具有激活胞外信号调节激酶(ERK1/2)的作用。因此,MGF及其E肽对于C2C12细胞的作用可能是通过激活细胞内的钙信号及ERK信号通路来实现的。
Mechano growth factor (MGF) is one of alternative splicing variants of Insulin-like growth factor. MGF is comprised of 110 amino acid residues. MGF, as a growth factor and a repair factor, is strongly expressed in order to promoting local muscle regeneration and restoration.
There are two isoforms of IGF-I, known as MGF and IGF-I Ea. They contain a E domain besides the B、C、A、D domains of mature IGF-I.The difference of amino acid sequence between MGF and IGF-I Ea is the C-terminal 24 amino acids of E domain. Previous research indicated that IGF-I and IGF-I Ea could promote cell proliferation and differentiation, but MGF inhibited terminal differentiation. The main purpose of this study was to investigate the characterization and function of MGF and MGF E domain.
This thesis consists of three parts:
Part one:The expression and purification of MGF and MGF E peptides. MGF and MGFc40 gene were cloned into plasmid pRSETc and MGFc24 was cloned into plasmid pGEX-6P-1. Then these recombinant expression plasmids were transformed with E. coli BL21(DE3). The fusion proteins were expressed at 37℃induced by IPTG. His-MGF and His-MGFc40 were solubilization and purified by His60 Ni SuperflowTM Resin. GST-MGFc24 was purified by Glutathione sepharose 4B.
Part two:The study about the self-assembly characterization of MGF. In the process of the purification of MGF and MGF E peptides, we found that they were prone to aggregate. Here, we investigated the interaction of individual domain of MGF by in vitro pull down technique. The results showed that MGF and MGF E peptides could interact by themselves, while the N-terminal of MGF could interact with MGFc24. The N-terminal of MGF did not interact by itself. These results suggested the aggregation of MGF may be due to the interaction of MGF E peptides itself and the N-terminal with the C-terminal 24 amino acid residues in MGF.
Part three:The study about the effect of MGF and MGF E peptide on C2C12 myoblasts. First, the C2C12 cells were treated with different concentration of His-MGF、His-MGFc40 and MGFc24. The activity of proliferation and differentiation was analyzed by CCK-8 and Creatine Kinase(CK) method, respectively. The results showed that His-MGF、His-MGFc40 and MGFc24 could promote the C2C12 cells proliferation and inhibited differentiation. Secondly, we detected the influence of MGF and MGF E peptide on Ca2+ concentration and ERK1/2 signal pathway in C2C12 cells. We found that the Ca2+ concentration was markedly increased and the ERK1/2 signal pathway was activated significantly when the C2C12 cells were treated with MGF and MGF E peptide. Thus, MGF and MGF E peptide could regulate the proliferation and differentiation of C2C12 cells though Ca2+ signal pathway and ERK1/2 signal pathway.
IGF-I基因通过选择性剪切产生两种异构体：IGF-I Ea和MGF。MGF的序列与成熟IGF-I和IGF-I Ea相比,除都拥有A,B,C和D结构域外,其主要差别是C端的E结构域。MGF比IGF-I C端多一个含40个氨基酸的E结构域；MGF和IGF-IEa的主要区别在于E结构域C端的24个氨基酸。研究发现MGF与成熟IGF-I和IGF-IEa的促细胞增殖和分化的功能不同,它的作用是促细胞增殖,但是抑制细胞分化,这一区别可能与它的C端E结构域有关。本研究拟通过基因工程手段对MGF和MGF的C端E结构域(又称E肽)进行体外表达纯化,并对它们的聚合性质和功能进行研究。研究内容包括以下三部分：
第一部分：MGF及其E肽的表达和纯化。分别构建pRSETc-MGF、pRSETc-MGFc40和pGEX-6P-1-MGFc24重组表达质粒。将重组质粒转化于E. coli BL21(DE3)中进行诱导表达。经Tris-Tricine SDS PAGE分析,融合蛋白His-MGF, His-MGFc40和GST-MGFc24均以可溶形式表达。表达产物分别通过His60 Ni SuperflowTM Resin和Glutathione sepharose 4B亲和层析纯化得到了纯的His-MGF, His-MGFc40和MGFc24蛋白。
第二部分：MGF自我聚合的特征研究。在上述实验中我们发现纯化的MGF及E肽易发生聚合现象,本部分实验利用体外pull down技术研究MGF与MGF, MGF的N端和MGF的C端E肽的相互作用。结果表明MGF能够和自身发生相互作用；MGF C端的E肽自身能发生相互作用；MGF的N端能与MGF C端24个氨基酸多肽发生相互作用；MGF的N端之间没有相互作用。因此,MGF的聚合很可能通过其C端E肽之间以及C端E肽与N端之间的相互作用进行。
第三部分：MGF及其E肽对C2C12成肌细胞的作用研究。本部分实验用纯化得到的His-MGF, His-MGFc40和MGFc24作用小鼠C2C12成肌细胞,分别用CCK-8法和肌酸激酶(Creatine kinase, CK)法检测它们对小鼠成肌细胞C2C12增殖和分化的作用；然后通过检测它们作用后细胞内游离钙离子浓度及胞外信号调节激酶(ERK1/2)的激活作用。结果表明His-MGF, His-MGFc40和MGFc24具有促进C2C12细胞增殖和抑制其分化的活性。His-MGF, His-MGFc40和MGFc24作用于C2C12细胞能够提高细胞内游离钙离子的浓度。His-MGF和His-MGFc40具有激活胞外信号调节激酶(ERK1/2)的作用。因此,MGF及其E肽对于C2C12细胞的作用可能是通过激活细胞内的钙信号及ERK信号通路来实现的。
Mechano growth factor (MGF) is one of alternative splicing variants of Insulin-like growth factor. MGF is comprised of 110 amino acid residues. MGF, as a growth factor and a repair factor, is strongly expressed in order to promoting local muscle regeneration and restoration.
There are two isoforms of IGF-I, known as MGF and IGF-I Ea. They contain a E domain besides the B、C、A、D domains of mature IGF-I.The difference of amino acid sequence between MGF and IGF-I Ea is the C-terminal 24 amino acids of E domain. Previous research indicated that IGF-I and IGF-I Ea could promote cell proliferation and differentiation, but MGF inhibited terminal differentiation. The main purpose of this study was to investigate the characterization and function of MGF and MGF E domain.
This thesis consists of three parts:
Part one:The expression and purification of MGF and MGF E peptides. MGF and MGFc40 gene were cloned into plasmid pRSETc and MGFc24 was cloned into plasmid pGEX-6P-1. Then these recombinant expression plasmids were transformed with E. coli BL21(DE3). The fusion proteins were expressed at 37℃induced by IPTG. His-MGF and His-MGFc40 were solubilization and purified by His60 Ni SuperflowTM Resin. GST-MGFc24 was purified by Glutathione sepharose 4B.
Part two:The study about the self-assembly characterization of MGF. In the process of the purification of MGF and MGF E peptides, we found that they were prone to aggregate. Here, we investigated the interaction of individual domain of MGF by in vitro pull down technique. The results showed that MGF and MGF E peptides could interact by themselves, while the N-terminal of MGF could interact with MGFc24. The N-terminal of MGF did not interact by itself. These results suggested the aggregation of MGF may be due to the interaction of MGF E peptides itself and the N-terminal with the C-terminal 24 amino acid residues in MGF.
Part three:The study about the effect of MGF and MGF E peptide on C2C12 myoblasts. First, the C2C12 cells were treated with different concentration of His-MGF、His-MGFc40 and MGFc24. The activity of proliferation and differentiation was analyzed by CCK-8 and Creatine Kinase(CK) method, respectively. The results showed that His-MGF、His-MGFc40 and MGFc24 could promote the C2C12 cells proliferation and inhibited differentiation. Secondly, we detected the influence of MGF and MGF E peptide on Ca2+ concentration and ERK1/2 signal pathway in C2C12 cells. We found that the Ca2+ concentration was markedly increased and the ERK1/2 signal pathway was activated significantly when the C2C12 cells were treated with MGF and MGF E peptide. Thus, MGF and MGF E peptide could regulate the proliferation and differentiation of C2C12 cells though Ca2+ signal pathway and ERK1/2 signal pathway.
引文
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[2]. McKoy, G., Ashley, W., Mander, J., et al. Expression of insulin growth factor-I splice variantas and structural genes in rabbit skeletal muscle induced by strech and stimulation. J Physiol 1999,516:583-592.
[3]. Barton, E.R., The ABCs of IGF-I isoforms:impact on muscle hypertrophy and implications for repair. Appl Physiol Nutr Metab.2006,31:791-797.
[4]. Shavlakadze, T., Winn, N., Rosentha, N., et al. Reconciling data from transgenic mice that overexpress IGF-I specifically in skeletal muscle. Growth Horm IGF Res.2005, 15:4-18.
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[2]. McKoy, G., Ashley, W., Mander, J., et al. Expression of insulin growth factor-I splice variantas and structural genes in rabbit skeletal muscle induced by strech and stimulation. J Physiol 1999,516:583-592.
[3]. Barton, E.R., The ABCs of IGF-I isoforms:impact on muscle hypertrophy and implications for repair. Appl Physiol Nutr Metab.2006,31:791-797.
[4]. Shavlakadze, T., Winn, N., Rosentha, N., et al. Reconciling data from transgenic mice that overexpress IGF-I specifically in skeletal muscle. Growth Horm IGF Res.2005, 15:4-18.
[5]. Yang, H., Adamo, M.L., Koval, A.P., Yang, Y., et al. Alternative leader sequences in insulin-like growth factor I mRNAs modulate translational effi-ciency and encode multiple signal peptides. Mol Endocrinol.1995,9:1380-1395.
[6].鲜成玉,王远亮等.应力作用下成骨细胞内IGF-I的剪接变异及表达.科学通报.2006,20：2399-2043.
[7].唐丽灵等.外力作为信号诱导基因选择性剪接及力生长因子的表达.生物化学与生物物理进展,2007,34：454-459.
[8]. Hammeed, M., Orrell, R.W., Cobbold, M., et al. Express of IGF-I splice variants in young and old human skeletal muscle after high resistance exercise. J Physiol,2003, 34:247-254.
[9]. Nixon, A. J., Brower- Toland, B. D., Primary nucleotide structure of predominant and alternate splice forms of equine like growth factor I and their gene expression patterns in tissue. Am J Vet Res.1999,60:1234-1241.
[10]. Winn, N., Paul, A., Musaro, A., et al. Insulin-like growth factor isforms in skeletal muscle aging, regeneration and disease. Cold SpringHarb Symp Quantbio.2002,67: 507-518.
[11]. Jansen, E., Steenbergh, P. H., et al. Identification of multiple transcription start sites in the human insulin-like growth factor-I gene. Mol Cell Enodocrinol.1991,78: 115-125.
[12]. Bell, G.I., Stempien, M.M., Fong, N.M., et al. Sequences of liver cDNAsencoding two different mouse insulin-like growth factor Ⅰ precursors.1986,14:7873-7882.
[13]. Xian, C.Y., Wang, Y.L., Zhang, B.B., et al. Alternative and expression of the insulin-like growth factor gene in osteoblasts undermechanical stret.chin sci bul, 2006,51:2731-2736.
[14]. Hill, M.,Goldspink, G., Expression and splicing of insulin-like growth factor gene in rodent muscle is associated with muscle satellite(stem)cell activation following local tissue damage. J Physiol,2003,549:409-418.
[15]. Yang, S.Y., Goldspink, G., Different roles of the IGF-I Ec peptide(MGF) and mature IGF-I in myoblast proliferation and differentiation. FEBS Lett.2002,522:156-160.
[16]. Kuang, S., Gillespie, M.A., Rudnicki, M.A., Niche regulation of muscle satellite cell self-renewal and differentiation. Cell Stem Cell.2008,2:22-31.
[17]. Goldspink, G., Age-related loss of skeletal muscle function:impairment of gene expression. Musculoskelet Neuronal Interact.2004,4:143-147.
[18]. Kemp, T.J., Sadusky, T.J., Saltisi, F., et al. Identification of Ankrd,a novel skeletal muscle gene coding for a stretch-responsive ankyrin-repeat protein. Genomics,2000, 66:229-241.
[19]. Owino, V., Yang, S.Y., Goldspink, G.., Age-related loss of skeletal muscle function and the inability to express the autocrine form of insulin-like growth factor-Ⅰ (IGF-I) in response to mechanical overload. FEBS Lett,2001,505:259-263.
[20]. Goldspink, G. Impairment of IGF-I gene splicing and MGF expression associated with muscle wasting. The international Journal of Biochemistry & cell biology. 2006,38:481-489.
[21]. Skarli, M., Yang, S.Y., Bouloux, P., et al. Up-regulation and alternative splicing of the IGF-1 gene in the rabbit heart following a brief pressure volume overload. J. Physiol.1998,509:192-193.
[22]. Goldspink, G., and Goldspink, P., Use of the insulin-like-growth factor-i splice variant MGF for the prevention of myocardial damage. United tates Patent.2005, US 2005 0048028A1.
[23]. Stavropoulou, A., Halapas, A., Sourla, A., et al., IGF-1 expression in infracted yocardium and MGF E peptide actions on rat cardiomyocytes, in vitro, Mol. Med. 2009,15:127-135.
[24]. Carro, E., Trejo, J.L., Nunez, A., et al. Brain repair and neuroprotection by erum insulin-like growth factor I. Mol. Neurobiol.2003,27:153-162.
[25]. Neff, N.T., Prevette, D., Houenou, L.J., et al. Insulin-like rowth-factors-putative uscle-derived trophic agents that promote motoneuron survival. J. Neurobiol.1993, 24:1578-1588.
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